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Jean-Yves Sgro
Inst. for Mol.Virology
731B Bock Labs
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Table of Contents for this page:

  • Current Issue
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    JVI Current Issue

  • Editorial Board [Masthead]

  • Discrete Dynamical Modeling of Influenza Virus Infection Suggests Age-Dependent Differences in Immunity [Pathogenesis and Immunity]

  • Immunosenescence, an age-related decline in immune function, is a major contributor to morbidity and mortality in the elderly. Older hosts exhibit a delayed onset of immunity and prolonged inflammation after an infection, leading to excess damage and a greater likelihood of death. Our study applies a rule-based model to infer which components of the immune response are most changed in an aged host. Two groups of BALB/c mice (aged 12 to 16 weeks and 72 to 76 weeks) were infected with 2 inocula: a survivable dose of 50 PFU and a lethal dose of 500 PFU. Data were measured at 10 points over 19 days in the sublethal case and at 6 points over 7 days in the lethal case, after which all mice had died. Data varied primarily in the onset of immunity, particularly the inflammatory response, which led to a 2-day delay in the clearance of the virus from older hosts in the sublethal cohort. We developed a Boolean model to describe the interactions between the virus and 21 immune components, including cells, chemokines, and cytokines, of innate and adaptive immunity. The model identifies distinct sets of rules for each age group by using Boolean operators to describe the complex series of interactions that activate and deactivate immune components. Our model accurately simulates the immune responses of mice of both ages and with both inocula included in the data (95% accurate for younger mice and 94% accurate for older mice) and shows distinct rule choices for the innate immunity arm of the model between younger and aging mice in response to influenza A virus infection.

    IMPORTANCE Influenza virus infection causes high morbidity and mortality rates every year, especially in the elderly. The elderly tend to have a delayed onset of many immune responses as well as prolonged inflammatory responses, leading to an overall weakened response to infection. Many of the details of immune mechanisms that change with age are currently not well understood. We present a rule-based model of the intrahost immune response to influenza virus infection. The model is fit to experimental data for young and old mice infected with influenza virus. We generated distinct sets of rules for each age group to capture the temporal differences seen in the immune responses of these mice. These rules describe a network of interactions leading to either clearance of the virus or death of the host, depending on the initial dosage of the virus. Our models clearly demonstrate differences in these two age groups, particularly in the innate immune responses.

  • Caenorhabditis elegans as an Emerging Model for Virus-Host Interactions [Gem]

  • Since 1999, Caenorhabditis elegans has been extensively used to study microbe-host interactions due to its simple culture, genetic tractability, and susceptibility to numerous bacterial and fungal pathogens. In contrast, virus studies have been hampered by a lack of convenient virus infection models in nematodes. The recent discovery of a natural viral pathogen of C. elegans and development of diverse artificial infection models are providing new opportunities to explore virus-host interplay in this powerful model organism.

  • Control of the HIV-1 DNA Reservoir Is Associated In Vivo and In Vitro with NKp46/NKp30 (CD335 CD337) Inducibility and Interferon Gamma Production by Transcriptionally Unique NK Cells [Virus-Cell Interactions]

  • The size of lentiviral DNA reservoirs reflects the effectiveness of immune responses against lentiviruses. So far, abundant information has been gathered on the control of HIV-1 replication. Understanding the innate mechanisms contributing to containment of the HIV DNA reservoir, however, are only partly clarified and are relevant to guiding interventions for reservoir containment or eradication. We studied the contribution of natural killer (NK) cell functional features in HIV patients controlling replication either spontaneously (HIV controllers [HIC]) or after progression and antiretroviral treatment (progressor patients [PP]). An inverse correlation between HIV DNA copy numbers (either total or integrated) in circulating CD4+ cells and NK cell function was observed. Induced interferon gamma (IFN-) production and NKp46/NKp30 activating receptor-induced expression correlated inversely with reservoir size. The correlation was present not only for a homogeneous cohort of HIC patients but also when PP were included in the analysis. Adaptive (NKG2C+ CD57+) NK cell features were not associated with reservoir size. However, a distinct set of 370 differentially expressed transcripts was found to underlie functional differences in NK cells controlling HIV DNA reservoir size. In proof-of-principle in vitro experiments of CD4+ cell infection with HIV-1, purified NK cells with the above-mentioned functional/transcriptional features displayed 10- and 30-fold higher abilities to control HIV replication and DNA burdens in vitro, respectively, than those of other NK cells. Thus, NK cells with a specific functional and transcriptional signature contribute to control of the HIV reservoir in CD4+ cells. Their selection, expansion, and/or adoptive transfer may support strategies to eradicate HIV-1 infection or to safely deescalate antiretroviral treatment.

    IMPORTANCE The most relevant feature of HIV-1 infection is represented by its DNA reservoir size in the body, which guarantees lifelong infection and resumption of virus replication after antiretroviral treatment interruption. So far, there has been little success in the identification of factors contributing to HIV-1 reservoir containment. In this study, by studying quantitative total and integrated HIV-1 DNA levels and NK cells in HIV-1 patients with either progressive or nonprogressive disease, we observed that inducible IFN- and natural cytotoxicity receptor (NCR) expression in a specific subset of NK cells with a characteristic transcriptional signature represents a correlate for HIV-1 reservoir control. This represents an advance in our understanding of the mechanism(s) that controls the lentivirus reservoir. Monitoring, selection, expansion, and adoptive transfer of these NK cells may allow monitoring of treatment efficacy and the likelihood of reservoir control and may support protocols for HIV-1 eradication.

  • Transcriptome Analysis of the Spodoptera frugiperda Ascovirus In Vivo Provides Insights into How Its Apoptosis Inhibitors and Caspase Promote Increased Synthesis of Viral Vesicles and Virion Progeny [Genome Replication and Regulation of Viral Gene Expression]

  • Ascoviruses are double-stranded DNA (dsDNA) viruses that attack caterpillars and differ from all other viruses by inducing nuclear lysis followed by cleavage of host cells into numerous anucleate vesicles in which virus replication continues as these grow in the blood. Ascoviruses are also unusual in that most encode a caspase or caspase-like proteins. A robust cell line to study the novel molecular biology of ascovirus replication in vitro is lacking. Therefore, we used strand-specific transcriptome sequencing (RNA-Seq) to study transcription in vivo in third instars of Spodoptera frugiperda infected with the type species, Spodoptera frugiperda ascovirus 1a (SfAV-1a), sampling transcripts at different time points after infection. We targeted transcription of two types of SfAV-1a genes; first, 44 core genes that occur in several ascovirus species, and second, 26 genes predicted in silico to have metabolic functions likely involved in synthesizing viral vesicle membranes. Gene cluster analysis showed differences in temporal expression of SfAV-1a genes, enabling their assignment to three temporal classes: early, late, and very late. Inhibitors of apoptosis (IAP-like proteins; ORF016, ORF025, and ORF074) were expressed early, whereas its caspase (ORF073) was expressed very late, which correlated with apoptotic events leading to viral vesicle formation. Expression analysis revealed that a Diedel gene homolog (ORF121), the only known "virokine," was highly expressed, implying that this ascovirus protein helps evade innate host immunity. Lastly, single-nucleotide resolution of RNA-Seq data revealed 15 bicistronic and tricistronic messages along the genome, an unusual occurrence for large dsDNA viruses.

    IMPORTANCE Unlike all other DNA viruses, ascoviruses code for an executioner caspase, apparently involved in a novel cytopathology in which viral replication induces nuclear lysis followed by cell cleavage, yielding numerous large anucleate viral vesicles that continue to produce virions. Our transcriptome analysis of genome expression in vivo by the Spodoptera frugiperda ascovirus shows that inhibitors of apoptosis are expressed first, enabling viral replication to proceed, after which the SfAV-1a caspase is synthesized, leading to viral vesicle synthesis and subsequent extensive production of progeny virions. Moreover, we detected numerous bicistronic and tricistronic mRNA messages in the ascovirus transcriptome, implying that ascoviruses use other noncanonical translational mechanisms, such as internal ribosome entry sites (IRESs). These results provide the first insights into the molecular biology of a unique coordinated gene expression pattern in which cell architecture is markedly modified, more than in any other known eukaryotic virus, to promote viral reproduction and transmission.

  • Distinct Mechanism for the Formation of the Ribonucleoprotein Complex of Tomato Spotted Wilt Virus [Structure and Assembly]

  • The Tomato spotted wilt virus (TSWV) belongs to the Tospovirus genus of the Bunyaviridae family and represents the sole plant-infecting group within bunyavirus. TSWV encodes a nucleocapsid protein (N) which encapsidates the RNA genome to form a ribonucleoprotein complex (RNP). In addition, the N has multiple roles during the infection of plant cells. Here, we report the crystal structure of the full-length TSWV N. The N features a body domain consisting of an N-lobe and a C-lobe. These lobes clamp a positively charged groove which may constitute the RNA binding site. Furthermore, the body domains are flanked by N- and C-terminal arms which mediate homotypic interactions to the neighboring subunits, resulting in a ring-shaped N trimer. Interestingly, the C terminus of one protomer forms an additional interaction with the protomer of an adjacent trimer in the crystal, which may constitute a higher-order oligomerization contact. In this way, this study provides insights into the structure and trimeric assembly of TSWV N, which help to explain previous functional findings, but also suggests distinct N interactions within a higher-order RNP.

    IMPORTANCE TSWV is one of the most devastating plant pathogens that cause severe diseases in numerous agronomic and ornamental crops worldwide. TSWV is also the prototypic member of the Tospovirus genus, which is the sole group of plant-infecting viruses in the bunyavirus family. This study determined the structure of full-length TSWV N in an oligomeric state. The structural observations explain previously identified biological properties of TSWV N. Most importantly, the additional homotypic interaction between the C terminus of one protomer with another protomer indicates that there is a distinct mechanism of RNP formation in the bunyavirus family, thereby enhancing the current knowledge of negative-sense single-stranded RNA virus-encoded N. TSWV N is the last remaining representative N with an unknown structure in the bunyavirus family. Combined with previous studies, the structure of TSWV N helps to build a complete picture of the bunyavirus-encoded N family and reveals a close evolutionary relationship between orthobunyavirus, phlebovirus, hantavirus, and tospovirus.

  • Heterogeneity of the Epstein-Barr Virus (EBV) Major Internal Repeat Reveals Evolutionary Mechanisms of EBV and a Functional Defect in the Prototype EBV Strain B95-8 [Genetic Diversity and Evolution]

  • Epstein-Barr virus (EBV) is a ubiquitous pathogen of humans that can cause several types of lymphoma and carcinoma. Like other herpesviruses, EBV has diversified through both coevolution with its host and genetic exchange between virus strains. Sequence analysis of the EBV genome is unusually challenging because of the large number and lengths of repeat regions within the virus. Here we describe the sequence assembly and analysis of the large internal repeat 1 of EBV (IR1; also known as the BamW repeats) for more than 70 strains. The diversity of the latency protein EBV nuclear antigen leader protein (EBNA-LP) resides predominantly within the exons downstream of IR1. The integrity of the putative BWRF1 open reading frame (ORF) is retained in over 80% of strains, and deletions truncating IR1 always spare BWRF1. Conserved regions include the IR1 latency promoter (Wp) and one zone upstream of and two within BWRF1. IR1 is heterogeneous in 70% of strains, and this heterogeneity arises from sequence exchange between strains as well as from spontaneous mutation, with interstrain recombination being more common in tumor-derived viruses. This genetic exchange often incorporates regions of llt;1 kb, and allelic gene conversion changes the frequency of small regions within the repeat but not close to the flanks. These observations suggest that IR1mmdash;and, by extension, EBVmmdash;diversifies through both recombination and breakpoint repair, while concerted evolution of IR1 is driven by gene conversion of small regions. Finally, the prototype EBV strain B95-8 contains four nonconsensus variants within a single IR1 repeat unit, including a stop codon in the EBNA-LP gene. Repairing IR1 improves EBNA-LP levels and the quality of transformation by the B95-8 bacterial artificial chromosome (BAC).

    IMPORTANCE Epstein-Barr virus (EBV) infects the majority of the world population but causes illness in only a small minority of people. Nevertheless, over 1% of cancers worldwide are attributable to EBV. Recent sequencing projects investigating virus diversity to see if different strains have different disease impacts have excluded regions of repeating sequence, as they are more technically challenging. Here we analyze the sequence of the largest repeat in EBV (IR1). We first characterized the variations in protein sequences encoded across IR1. In studying variations within the repeat of each strain, we identified a mutation in the main laboratory strain of EBV that impairs virus function, and we suggest that tumor-associated viruses may be more likely to contain DNA mixed from two strains. The patterns of this mixing suggest that sequences can spread between strains (and also within the repeat) by copying sequence from another strain (or repeat unit) to repair DNA damage.

  • Molecular Evolution of Herpes Simplex Virus 2 Complete Genomes: Comparison between Primary and Recurrent Infections [Genetic Diversity and Evolution]

  • Herpes simplex virus 1 (HSV-1) and HSV-2 are large, double-stranded DNA viruses that cause lifelong persistent infections characterized by periods of quiescence and recurrent disease. How HSV evolves within an infected individual experiencing multiple episodes of recurrent disease over time is not known. We determined the genome sequences of viruses isolated from two subjects in the Herpevac Trial for Women who experienced primary HSV-2 genital disease and compared them with sequences of viruses isolated from the subsequent fifth or sixth episode of recurrent disease in the same individuals. Each of the HSV-2 genome sequences was initially obtained using next-generation sequencing and completed with Sanger sequencing. Polymorphisms over the entire genomes were mapped, and amino acid variants resulting from nonsynonymous changes were analyzed based on the secondary and tertiary structures of a previously crystallized protein. A phylogenetic reconstruction was used to assess relationships among the four HSV-2 samples, other North American sequences, and reference sequences. Little genetic drift was detected in viruses shed by the same subjects following repeated reactivation events, suggesting strong selective pressure on the viral genome to maintain sequence fidelity during reactivations from its latent state within an individual host. Our results also demonstrate that some primary HSV-2 isolates from North America more closely resemble the HG52 laboratory strain from Scotland than the low-passage-number clinical isolate SD90e from South Africa or laboratory strain 333. Thus, one of the sequences reported here would be a logical choice as a reference strain for inclusion in future studies of North American HSV-2 isolates.

    IMPORTANCE The extent to which the HSV-2 genome evolves during multiple episodes of reactivation from its latent state within an infected individual is not known. We used next-generation sequencing techniques to determine whole-genome sequences of four viral samples from two subjects in the Herpevac Trial. The sequence of each subject's well-documented primary isolate was compared with the sequence of the isolate from their fifth or sixth episode of recurrent disease. Only 19 genetic polymorphisms unique to the primary or recurrent isolate were identified, 10 in subject A and 9 in subject B. These observations indicate remarkable genetic conservation between primary and recurrent episodes of HSV-2 infection and imply that strong selection pressures exist to maintain the fidelity of the viral genome during repeated reactivations from its latent state. The genome conservation observed also has implications for the potential success of a therapeutic vaccine.

  • Type I Interferon Signaling to Dendritic Cells Limits Murid Herpesvirus 4 Spread from the Olfactory Epithelium [Pathogenesis and Immunity]

  • Murid herpesvirus 4 (MuHV-4) is a B cell-tropic gammaherpesvirus that can be studied in vivo. Despite viral evasion, type I interferons (IFN-I) limit its spread. After MuHV-4 inoculation into footpads, IFN-I protect lymph node subcapsular sinus macrophages (SSM) against productive infection; after peritoneal inoculation, they protect splenic marginal zone macrophages, and they limit MuHV-4 replication in the lungs. While invasive infections can be used to test specific aspects of host colonization, it is also important to understand natural infection. MuHV-4 taken up spontaneously by alert mice enters them via olfactory neurons. We determined how IFN-I act in this context. Blocking IFN-I signaling did not increase neuronal infection but allowed the virus to spread to the adjacent respiratory epithelium. In lymph nodes, a complete IFN-I signaling block increased MuHV-4 lytic infection in SSM and increased the number of dendritic cells (DC) expressing viral green fluorescent protein (GFP) independently of lytic infection. A CD11c+ cell-directed signaling block increased infection of DC only. However, this was sufficient to increase downstream infection, consistent with DC providing the main viral route to B cells. The capacity of IFN-I to limit DC infection indicated that viral IFN-I evasion was only partly effective. Therefore, DC are a possible target for IFN-I-based interventions to reduce host colonization.

    IMPORTANCE Human gammaherpesviruses infect B cells and cause B cell cancers. Interventions to block virus binding to B cells have not stopped their infection. Therefore, we must identify other control points that are relevant to natural infection. Human infections are difficult to analyze. However, gammaherpesviruses colonize all mammals. A related gammaherpesvirus of mice reaches B cells not directly but via infected dendritic cells. We show that type I interferons, an important general antiviral defense, limit gammaherpesvirus B cell infection by acting on dendritic cells. Therefore, dendritic cell infection is a potential point of interferon-based therapeutic intervention.

  • Differentiated Human SH-SY5Y Cells Provide a Reductionist Model of Herpes Simplex Virus 1 Neurotropism [Virus-Cell Interactions]

  • Neuron-virus interactions that occur during herpes simplex virus (HSV) infection are not fully understood. Neurons are the site of lifelong latency and are a crucial target for long-term suppressive therapy or viral clearance. A reproducible neuronal model of human origin would facilitate studies of HSV and other neurotropic viruses. Current neuronal models in the herpesvirus field vary widely and have caveats, including incomplete differentiation, nonhuman origins, or the use of dividing cells that have neuropotential but lack neuronal morphology. In this study, we used a robust approach to differentiate human SH-SY5Y neuroblastoma cells over 2.5 weeks, producing a uniform population of mature human neuronal cells. We demonstrate that terminally differentiated SH-SY5Y cells have neuronal morphology and express proteins with subcellular localization indicative of mature neurons. These neuronal cells are able to support a productive HSV-1 infection, with kinetics and overall titers similar to those seen in undifferentiated SH-SY5Y cells and the related SK-N-SH cell line. However, terminally differentiated, neuronal SH-SY5Y cells release significantly less extracellular HSV-1 by 24 h postinfection (hpi), suggesting a unique neuronal response to viral infection. With this model, we are able to distinguish differences in neuronal spread between two strains of HSV-1. We also show expression of the antiviral protein cyclic GMP-AMP synthase (cGAS) in neuronal SH-SY5Y cells, which is the first demonstration of the presence of this protein in nonepithelial cells. These data provide a model for studying neuron-virus interactions at the single-cell level as well as via bulk biochemistry and will be advantageous for the study of neurotropic viruses in vitro.

    IMPORTANCE Herpes simplex virus (HSV) affects millions of people worldwide, causing painful oral and genital lesions, in addition to a multitude of more severe symptoms such as eye disease, neonatal infection, and, in rare cases, encephalitis. Presently, there is no cure available to treat those infected or prevent future transmission. Due to the ability of HSV to cause a persistent, lifelong infection in the peripheral nervous system, the virus remains within the host for life. To better understand the basis of virus-neuron interactions that allow HSV to persist within the host peripheral nervous system, improved neuronal models are required. Here we describe a cost-effective and scalable human neuronal model system that can be used to study many neurotropic viruses, such as HSV, Zika virus, dengue virus, and rabies virus.

  • Epstein-Barr Virus BKRF4 Gene Product Is Required for Efficient Progeny Production [Genome Replication and Regulation of Viral Gene Expression]

  • Epstein-Barr virus (EBV), a member of human gammaherpesvirus, infects mainly B cells. EBV has two alternative life cycles, latent and lytic, and is reactivated occasionally from the latent stage to the lytic cycle. To combat EBV-associated disorders, understanding the molecular mechanisms of the EBV lytic replication cycle is also important. Here, we focused on an EBV lytic gene, BKRF4. Using our anti-BKRF4 antibody, we revealed that the BKRF4 gene product is expressed during the lytic cycle with late kinetics. To characterize the role of BKRF4, we constructed BKRF4-knockout mutants using the bacterial artificial chromosome (BAC) and CRISPR/Cas9 systems. Although disruption of the BKRF4 gene had almost no effect on viral protein expression and DNA synthesis, it significantly decreased progeny virion levels in HEK293 and Akata cells. Furthermore, we show that BKRF4 is involved not only in production of progeny virions but also in increasing the infectivity of the virus particles. Immunoprecipitation assays revealed that BKRF4 interacted with a virion protein, BGLF2. We showed that the C-terminal region of BKRF4 was critical for this interaction and for efficient progeny production. Immunofluorescence analysis revealed that BKRF4 partially colocalized with BGLF2 in the nucleus and perinuclear region. Finally, we showed that BKRF4 is a phosphorylated, possible tegument protein and that the EBV protein kinase BGLF4 may be important for this phosphorylation. Taken together, our data suggest that BKRF4 is involved in the production of infectious virions.

    IMPORTANCE Although the latent genes of EBV have been studied extensively, the lytic genes are less well characterized. This study focused on one such lytic gene, BKRF4, which is conserved only among gammaherpesviruses (ORF45 of Kaposi's sarcoma-associated herpesvirus or murine herpesvirus 68). After preparing the BKRF4 knockout virus using B95-8 EBV-BAC, we demonstrated that the BKRF4 gene was involved in infectious progeny particle production. Importantly, we successfully generated a BKRF4 knockout virus of Akata using CRISPR/Cas9 technology, confirming the phenotype in this separate strain. We further showed that BKRF4 interacted with another virion protein, BGLF2, and demonstrated the importance of this interaction in infectious virion production. These results shed light on the elusive process of EBV progeny maturation in the lytic cycle. Notably, this study describes a successful example of the generation and characterization of an EBV construct with a disrupted lytic gene using CRISPR/Cas9 technology.

  • Host Noncoding Retrotransposons Induced by DNA Viruses: a SINE of Infection? [Gem]

  • Our genomes are dominated by repetitive elements. The majority of these elements derive from retrotransposons, which expand throughout the genome through a process of reverse transcription and integration. Short interspersed nuclear elements, or SINEs, are an abundant class of retrotransposons that are transcribed by RNA polymerase III, thus generating exclusively noncoding RNA (ncRNA) that must hijack the machinery required for their transposition. SINE loci are generally transcriptionally repressed in somatic cells but can be robustly induced upon infection with multiple DNA viruses. Recent research has focused on the gene expression and signaling events that are modulated by SINE ncRNAs, particularly during gammaherpesvirus infection. Here, we review the biology of these SINE ncRNAs, explore how DNA virus infection may lead to their induction, and describe how novel gene regulatory and immune-related functions of these ncRNAs may impact the viral life cycle.

  • Mutations in the Fusion Protein of Measles Virus That Confer Resistance to the Membrane Fusion Inhibitors Carbobenzoxy-D-Phe-L-Phe-Gly and 4-Nitro-2-Phenylacetyl Amino-Benzamide [Virus-Cell Interactions]

  • The inhibitors carbobenzoxy (Z)-d-Phe-l-Phe-Gly (fusion inhibitor peptide [FIP]) and 4-nitro-2-phenylacetyl amino-benzamide (AS-48) have similar efficacies in blocking membrane fusion and syncytium formation mediated by measles virus (MeV). Other homologues, such as Z-d-Phe, are less effective but may act through the same mechanism. In an attempt to map the site of action of these inhibitors, we generated mutant viruses that were resistant to the inhibitory effects of Z-d-Phe-l-Phe-Gly. These 10 mutations were localized to the heptad repeat B (HRB) region of the fusion protein, and no changes were observed in the viral hemagglutinin, which is the receptor attachment protein. Mutations were validated in a luciferase-based membrane fusion assay, using transfected fusion and hemagglutinin expression plasmids or with syncytium-based assays in Vero, Vero-SLAM, and Vero-Nectin 4 cell lines. The changes I452T, D458N, D458G/V459A, N462K, N462H, G464E, and I483R conferred resistance to both FIP and AS-48 without compromising membrane fusion. The inhibitors did not block hemagglutinin protein-mediated binding to the target cell. Edmonston vaccine/laboratory and IC323 wild-type strains were equally affected by the inhibitors. Escape mutations were mapped upon a three-dimensional (3D) structure modeled from the published crystal structure of parainfluenzavirus 5 fusion protein. The most effective mutations were situated in a region located near the base of the globular head and its junction with the alpha-helical stalk of the prefusion protein. We hypothesize that the fusion inhibitors could interfere with the structural changes that occur between the prefusion and postfusion conformations of the fusion protein.

    IMPORTANCE Due to lapses in vaccination worldwide that have caused localized outbreaks, measles virus (MeV) has regained importance as a pathogen. Antiviral agents against measles virus are not commercially available but could be useful in conjunction with MeV eradication vaccine programs and as a safeguard in oncolytic viral therapy. Three decades ago, the small hydrophobic peptide Z-d-Phe-l-Phe-Gly (FIP) was shown to block MeV infections and syncytium formation in monkey kidney cell lines. The exact mechanism of its action has yet to be determined, but it does appear to have properties similar to those of another chemical inhibitor, AS-48, which appears to interfere with the conformational change in the viral F protein that is required to elicit membrane fusion. Escape mutations were used to map the site of action for FIP. Knowledge gained from these studies could help in the design of new inhibitors against morbilliviruses and provide additional knowledge concerning the mechanism of virus-mediated membrane fusion.

  • Pathogenic Correlates of Simian Immunodeficiency Virus-Associated B Cell Dysfunction [Pathogenesis and Immunity]

  • We compared and contrasted pathogenic (in pig-tailed macaques [PTMs]) and nonpathogenic (in African green monkeys [AGMs]) SIVsab infections to assess the significance of the B cell dysfunction observed in simian (SIV) and human immunodeficiency virus (HIV) infections. We report that the loss of B cells is specifically associated with the pathogenic SIV infection, while in the natural hosts, in which SIV is nonpathogenic, B cells rapidly increase in both lymph nodes (LNs) and intestine. SIV-associated B cell dysfunction associated with the pathogenic SIV infection is characterized by loss of naive B cells, loss of resting memory B cells due to their redistribution to the gut, increases of the activated B cells and circulating tissue-like memory B cells, and expansion of the B regulatory cells (Bregs). While circulating B cells are virtually restored to preinfection levels during the chronic pathogenic SIV infection, restoration is mainly due to an expansion of the "exhausted," virus-specific B cells, i.e., activated memory cells and tissue-like memory B cells. Despite of the B cell dysfunction, SIV-specific antibody (Ab) production was higher in the PTMs than in AGMs, with the caveat that rapid disease progression in PTMs was strongly associated with lack of anti-SIV Ab. Neutralization titers and the avidity and maturation of immune responses did not differ between pathogenic and nonpathogenic infections, with the exception of the conformational epitope recognition, which evolved from low to high conformations in the natural host. The patterns of humoral immune responses in the natural host are therefore more similar to those observed in HIV-infected subjects, suggesting that natural hosts may be more appropriate for modeling the immunization strategies aimed at preventing HIV disease progression. The numerous differences between the pathogenic and nonpathogenic infections with regard to dynamics of the memory B cell subsets point to their role in the pathogenesis of HIV/SIV infections and suggest that monitoring B cells may be a reliable approach for assessing disease progression.

    IMPORTANCE We report here that the HIV/SIV-associated B cell dysfunction (defined by loss of total and memory B cells, increased B regulatory cell [Breg] counts, and B cell activation and apoptosis) is specifically associated with pathogenic SIV infection and absent during the course of nonpathogenic SIV infection in natural nonhuman primate hosts. Alterations of the B cell population are not correlated with production of neutralizing antibodies, the levels of which are similar in the two species. Rapid progressive infections are associated with a severe impairment in SIV-specific antibody production. While we did not find major differences in avidity and maturation between the pathogenic and nonpathogenic SIV infections, we identified a major difference in conformational epitope recognition, with the nonpathogenic infection being characterized by an evolution from low to high conformations. B cell dysfunction should be considered in designing immunization strategies aimed at preventing HIV disease progression.

  • A Selective Bottleneck Shapes the Evolutionary Mutant Spectra of Enterovirus A71 during Viral Dissemination in Humans [Genetic Diversity and Evolution]

  • RNA viruses accumulate mutations to rapidly adapt to environmental changes. Enterovirus A71 (EV-A71) causes various clinical manifestations with occasional severe neurological complications. However, the mechanism by which EV-A71 evolves within the human body is unclear. Utilizing deep sequencing and haplotype analyses of viruses from various tissues of an autopsy patient, we sought to define the evolutionary pathway by which enterovirus A71 evolves fitness for invading the central nervous system in humans. Broad mutant spectra with divergent mutations were observed at the initial infection sites in the respiratory and digestive systems. After viral invasion, we identified a haplotype switch and dominant haplotype, with glycine at VP1 residue 31 (VP1-31G) in viral particles disseminated into the integumentary and central nervous systems. In vitro viral growth and fitness analyses indicated that VP1-31G conferred growth and a fitness advantage in human neuronal cells, whereas VP1-31D conferred enhanced replication in human colorectal cells. A higher proportion of VP1-31G was also found among fatal cases, suggesting that it may facilitate central nervous system infection in humans. Our data provide the first glimpse of EV-A71 quasispecies from oral tissues to the central nervous system within humans, showing broad implications for the surveillance and pathogenesis of this reemerging viral pathogen.

    IMPORTANCE EV-A71 continues to be a worldwide burden to public health. Although EV-A71 is the major etiological agent of hand, foot, and mouth disease, it can also cause neurological pulmonary edema, encephalitis, and even death, especially in children. Understanding selection processes enabling dissemination and accurately estimating EV-A71 diversity during invasion in humans are critical for applications in viral pathogenesis and vaccine studies. Here, we define a selection bottleneck appearing in respiratory and digestive tissues. Glycine substitution at VP1 residue 31 helps viruses break through the bottleneck and invade the central nervous system. This substitution is also advantageous for replication in neuronal cells in vitro. Considering that fatal cases contain enhanced glycine substitution at VP1-31, we suggest that the increased prevalence of VP1-31G may alter viral tropism and aid central nervous system invasion. Our findings provide new insights into a dynamic mutant spectral switch active during acute viral infection with emerging viral pathogens.

  • Structurally Guided Removal of DeISGylase Biochemical Activity from Papain-Like Protease Originating from Middle East Respiratory Syndrome Coronavirus [Pathogenesis and Immunity]

  • Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging human pathogen that is the causative agent for Middle East respiratory syndrome (MERS). With MERS outbreaks resulting in over 35% fatalities and now spread to 27 countries, MERS-CoV poses a significant ongoing threat to global human health. As part of its viral genome, MERS-CoV encodes a papain-like protease (PLpro) that has been observed to act as a deubiquitinase and deISGylase to antagonize type I interferon (IFN-I) immune pathways. This activity is in addition to its viral polypeptide cleavage function. Although the overall impact of MERS-CoV PLpro function is observed to be essential, difficulty has been encountered in delineating the importance of its separate functions, particularly its deISGylase activity. As a result, the interface of MERS-CoV and human interferon-stimulated gene product 15 (hISG15) was probed with isothermal calorimetry, which suggests that the C-terminal domain of hISG15 is principally responsible for interactions. Subsequently, the structure of MERS-CoV PLpro was solved to 2.4 AAring; in complex with the C-terminal domain of hISG15. Utilizing this structural information, mutants were generated that lacked appreciable deISGylase activity but retained wild-type deubiquitinase and peptide cleavage activities. Hence, this provides a new platform for understanding viral deISGylase activity within MERS-CoV and other CoVs.

    IMPORTANCE Coronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV), encode a papain-like protease (PLpro) that possesses the ability to antagonize interferon immune pathways through the removal of ubiquitin and interferon-stimulated gene product 15 (ISG15) from target proteins. The lack of CoV proteases with attenuated deISGylase activity has been a key obstacle in delineating the impact between deubiquitinase and deISGylase activities on viral host evasion and pathogenesis. Here, biophysical techniques revealed that MERS-CoV PLpro chiefly engages human ISG15 through its C-terminal domain. The first structure of MERS-CoV PLpro in complex with this domain exposed the interface between these two entities. Employing these structural insights, mutations were employed to selectively remove deISGylase activity with no appreciable impact on its other deubiquitinase and peptide cleavage biochemical properties. Excitingly, this study introduces a new tool to probe the pathogenesis of MERS-CoV and related viruses through the removal of viral deISGylase activity.

  • Porcine Hemagglutinating Encephalomyelitis Virus Enters Neuro-2a Cells via Clathrin-Mediated Endocytosis in a Rab5-, Cholesterol-, and pH-Dependent Manner [Virus-Cell Interactions]

  • Porcine hemagglutinating encephalomyelitis virus (PHEV) is a highly neurovirulent coronavirus that invades the central nervous system (CNS) in piglets. Although important progress has been made toward understanding the biology of PHEV, many aspects of its life cycle remain obscure. Here we dissected the molecular mechanism underlying cellular entry and intracellular trafficking of PHEV in mouse neuroblastoma (Neuro-2a) cells. We first performed a thin-section transmission electron microscopy (TEM) assay to characterize the kinetics of PHEV, and we found that viral entry and transfer occur via membranous coating-mediated endo- and exocytosis. To verify the roles of distinct endocytic pathways, systematic approaches were used, including pharmacological inhibition, RNA interference, confocal microscopy analysis, use of fluorescently labeled virus particles, and overexpression of a dominant negative (DN) mutant. Quantification of infected cells showed that PHEV enters cells by clathrin-mediated endocytosis (CME) and that low pH, dynamin, cholesterol, and Eps15 are indispensably involved in this process. Intriguingly, PHEV invasion leads to rapid actin rearrangement, suggesting that the intactness and dynamics of the actin cytoskeleton are positively correlated with viral endocytosis. We next investigated the trafficking of internalized PHEV and found that Rab5- and Rab7-dependent pathways are required for the initiation of a productive infection. Furthermore, a GTPase activation assay suggested that endogenous Rab5 is activated by PHEV and is crucial for viral progression. Our findings demonstrate that PHEV hijacks the CME and endosomal system of the host to enter and traffic within neural cells, providing new insights into PHEV pathogenesis and guidance for antiviral drug design.

    IMPORTANCE Porcine hemagglutinating encephalomyelitis virus (PHEV), a nonsegmented, positive-sense, single-stranded RNA coronavirus, invades the central nervous system (CNS) and causes neurological dysfunction. Neural cells are its targets for viral progression. However, the detailed mechanism underlying PHEV entry and trafficking remains unknown. PHEV is the etiological agent of porcine hemagglutinating encephalomyelitis, which is an acute and highly contagious disease that causes numerous deaths in suckling piglets and enormous economic losses in China. Understanding the viral entry pathway will not only advance our knowledge of PHEV infection and pathogenesis but also open new approaches to the development of novel therapeutic strategies. Therefore, we employed systematic approaches to dissect the internalization and intracellular trafficking mechanism of PHEV in Neuro-2a cells. This is the first report to describe the process of PHEV entry into nerve cells via clathrin-mediated endocytosis in a dynamin-, cholesterol-, and pH-dependent manner that requires Rab5 and Rab7.

  • Vector Order Determines Protection against Pathogenic Simian Immunodeficiency Virus Infection in a Triple-Component Vaccine by Balancing CD4+ and CD8+ T-Cell Responses [Vaccines and Antiviral Agents]

  • An effective AIDS vaccine should elicit strong humoral and cellular immune responses while maintaining low levels of CD4+ T-cell activation to avoid the generation of target cells for viral infection. The present study investigated two prime-boost regimens, both starting vaccination with single-cycle immunodeficiency virus, followed by two mucosal boosts with either recombinant adenovirus (rAd) or fowlpox virus (rFWPV) expressing SIVmac239 or SIVmac251 gag/pol and env genes, respectively. Finally, vectors were switched and systemically administered to the reciprocal group of animals. Only mucosal rFWPV immunizations followed by systemic rAd boost significantly protected animals against a repeated low-dose intrarectal challenge with pathogenic SIVmac251, resulting in a vaccine efficacy (i.e., risk reduction per exposure) of 68%. Delayed viral acquisition was associated with higher levels of activated CD8+ T cells and Gag-specific gamma interferon (IFN-)-secreting CD8+ cells, low virus-specific CD4+ T-cell responses, and low Env antibody titers. In contrast, the systemic rFWPV boost induced strong virus-specific CD4+ T-cell activity. rAd and rFWPV also induced differential patterns of the innate immune responses, thereby possibly shaping the specific immunity. Plasma CXCL10 levels after final immunization correlated directly with virus-specific CD4+ T-cell responses and inversely with the number of exposures to infection. Also, the percentage of activated CD69+ CD8+ T cells correlated with the number of exposures to infection. Differential stimulation of the immune response likely provided the basis for the diverging levels of protection afforded by the vaccine regimen.

    IMPORTANCE A failed phase II AIDS vaccine trial led to the hypothesis that CD4+ T-cell activation can abrogate any potentially protective effects delivered by vaccination or promote acquisition of the virus because CD4+ T helper cells, required for an effective immune response, also represent the target cells for viral infection. We compared two vaccination protocols that elicited similar levels of Gag-specific immune responses in rhesus macaques. Only the animal group that had a low level of virus-specific CD4+ T cells in combination with high levels of activated CD8+ T cells was significantly protected from infection. Notably, protection was achieved despite the lack of appreciable Env antibody titers. Moreover, we show that both the vector and the route of immunization affected the level of CD4+ T-cell responses. Thus, mucosal immunization with FWPV-based vaccines should be considered a potent prime in prime-boost vaccination protocols.

  • Role of Herpes Simplex Virus 1 {gamma}34.5 in the Regulation of IRF3 Signaling [Pathogenesis and Immunity]

  • During viral infection, pattern recognition receptors (PRRs) and their associated adaptors recruit TANK-binding kinase 1 (TBK1) to activate interferon regulatory factor 3 (IRF3), resulting in production of type I interferons (IFNs). ICP0 and ICP34.5 are among the proteins encoded by herpes simplex virus 1 (HSV-1) that modulate type I IFN signaling. We constructed a recombinant virus (XX) that lacks amino acids 87 to 106, a portion of the previously described TBK1-binding domain of the 34.5 gene (D. Verpooten, Y. Ma, S. Hou, Z. Yan, and B. He, J Biol Chem 284:1097nndash;1105, 2009, https://doi.org/10.1074/JBC.M805905200). These 20 residues are outside the 34.5 beclin1-binding domain (BBD) that interacts with beclin1 and regulates autophagy. Unexpectedly, XX showed no deficit in replication in vivo in a variety of tissues and showed virulence comparable to that of wild-type and marker-rescued viruses following intracerebral infection. XX was fully capable of mediating the dephosphorylation of eIF2aalpha;, and the virus was capable of controlling the phosphorylation of IRF3. In contrast, a null mutant in 34.5 failed to control IRF3 phosphorylation due to an inability of the mutant to sustain expression of ICP0. Our data show that while 34.5 regulates IRF3 phosphorylation, the TBK1-binding domain itself has no impact on IRF3 phosphorylation or on replication and pathogenesis in mice.

    IMPORTANCE Interferons (IFNs) are potent activators of a variety of host responses that serve to control virus infections. The Herpesviridae have evolved countermeasures to IFN responses. Herpes simplex virus 1 (HSV-1) encodes the multifunctional neurovirulence protein ICP34.5. In this study, we investigated the biological relevance of the interaction between ICP34.5 and TANK-binding kinase 1 (TBK1), an activator of IFN responses. Here, we establish that although ICP34.5 binds TBK1 under certain conditions through a TBK1-binding domain (TBD), there was no direct impact of the TBD on viral replication or virulence in mice. Furthermore, we showed that activation of IRF3, a substrate of TBK1, was independent of the TBD. Instead, we provided evidence that the ability of ICP34.5 to control IRF3 activation is through its ability to reverse translational shutoff and sustain the expression of other IFN inhibitors encoded by the virus. This work provides new insights into the immunomodulatory functions of ICP34.5.

  • Three Conserved Regions in Baculovirus Sulfhydryl Oxidase P33 Are Critical for Enzymatic Activity and Function [Structure and Assembly]

  • Baculoviruses encode a conserved sulfhydryl oxidase, P33, which is necessary for budded virus (BV) production and multinucleocapsid occlusion-derived virus (ODV) formation. Here, the structural and functional relationship of P33 was revealed by X-ray crystallography, site-directed mutagenesis, and functional analysis. Based on crystallographic characterization and structural analysis, a series of P33 mutants within three conserved regions, i.e., the active site, the dimer interface, and the R127-E183 salt bridge, were constructed. In vitro experiments showed that mutations within the active site and dimer interface severely impaired the sulfhydryl oxidase activity of P33, while the mutations in the salt bridge had a relatively minor influence. Recombinant viruses containing mutated P33 were constructed and assayed in vivo. Except for the active-site mutant AXXA, all other mutants produced infectious BVs, although certain mutants had a decreased BV production. The active-site mutant H114A, the dimer interface mutant H227D, and the salt bridge mutant R127A-E183A were further analyzed by electron microscopy and bioassays. The occlusion bodies (OBs) of mutants H114A and R127A-E183A had a ragged surface and contained mostly ODVs with a single nucleocapsid. The OBs of all three mutants contained lower numbers of ODVs and had a significantly reduced oral infectivity in comparison to control virus. Crystallographic analyses further revealed that all three regions may coordinate with one another to achieve optimal function of P33. Taken together, our data revealed that all the three conserved regions are involved in P33 activity and are crucial for virus morphogenesis and peroral infectivity.

    IMPORTANCE Sulfhydryl oxidase catalyzes disulfide bond formation of substrate proteins. P33, a baculovirus-encoded sulfhydryl oxidase, is different from other cellular and viral sulfhydryl oxidases, bearing unique features in tertiary and quaternary structure organizations. In this study, we found that three conserved regions, i.e., the active site, dimer interface, and the R127-E183 salt bridge, play important roles in the enzymatic activity and function of P33. Previous observations showed that deletion of p33 results in a total loss of budded virus (BV) production and in morphological changes in occlusion-derived virus (ODV). Our study revealed that certain P33 mutants lead to occlusion bodies (OBs) with a ragged surface, decreased embedded ODVs, and reduced oral infectivity. Interestingly, some P33 mutants with impaired ODV/OB still retained BV productivity, indicating that the impacts on BV and on ODV/OB are two distinctly different functions of P33, which are likely to be performed via different substrate proteins.

  • Insights into the Impact of CD8+ Immune Modulation on Human Immunodeficiency Virus Evolutionary Dynamics in Distinct Anatomical Compartments by Using Simian Immunodeficiency Virus-Infected Macaque Models of AIDS Progression [Genetic Diversity and Evolution]

  • A thorough understanding of the role of human immunodeficiency virus (HIV) intrahost evolution in AIDS pathogenesis has been limited by the need for longitudinally sampled viral sequences from the vast target space within the host, which are often difficult to obtain from human subjects. CD8+ lymphocyte-depleted macaques infected with simian immunodeficiency virus (SIV) provide an increasingly utilized model of pathogenesis due to clinical manifestations similar to those for HIV-1 infection and AIDS progression, as well as a characteristic rapid disease onset. Comparison of this model with SIV-infected non-CD8+ lymphocyte-depleted macaques also provides a unique opportunity to investigate the role of CD8+ cells in viral evolution and population dynamics throughout the duration of infection. Using several different phylogenetic methods, we analyzed viral gp120 sequences obtained from extensive longitudinal sampling of multiple tissues and enriched leukocyte populations from SIVmac251-infected macaques with or without CD8+ lymphocyte depletion. SIV evolutionary and selection patterns in non-CD8+ lymphocyte-depleted animals were characterized by sequential population turnover and continual viral adaptation, a scenario readily comparable to intrahost evolutionary patterns during human HIV infection in the absence of antiretroviral therapy. Alternatively, animals that were depleted of CD8+ lymphocytes exhibited greater variation in population dynamics among tissues and cell populations over the course of infection. Our findings highlight the major role for CD8+ lymphocytes in prolonging disease progression through continual control of SIV subpopulations from various anatomical compartments and the potential for greater independent viral evolutionary behavior among these compartments in response to immune modulation.

    IMPORTANCE Although developments in combined antiretroviral therapy (cART) strategies have successfully prolonged the time to AIDS onset in HIV-1-infected individuals, a functional cure has yet to be found. Improvement of drug interventions for a virus that is able to infect a wide range of tissues and cell types requires a thorough understanding of viral adaptation and infection dynamics within this target milieu. Although it is difficult to accomplish in the human host, longitudinal sampling of multiple anatomical locations is readily accessible in the SIV-infected macaque models of neuro-AIDS. The significance of our research is in identifying the impact of immune modulation, through differing immune selective pressures, on viral evolutionary behavior in a multitude of anatomical compartments. The results provide evidence encouraging the development of a more sophisticated model that considers a network of individual viral subpopulations within the host, with differing infection and transmission dynamics, which is necessary for more effective treatment strategies.

  • IKK{gamma}-Mimetic Peptides Block the Resistance to Apoptosis Associated with Kaposi's Sarcoma-Associated Herpesvirus Infection [Virus-Cell Interactions]

  • Primary effusion lymphoma (PEL) is a lymphogenic disorder associated with Kaposi's sarcoma-associated herpesvirus (KSHV) infection. Key to the survival and proliferation of PEL is the canonical NF-B pathway, which becomes constitutively activated following overexpression of the viral oncoprotein KSHV vFLIP (ks-vFLIP). This arises from its capacity to form a complex with the modulatory subunit of the IB kinase (IKK) kinase, IKK (or NEMO), resulting in the overproduction of proteins that promote cellular survival and prevent apoptosis, both of which are important drivers of tumorigenesis. Using a combination of cell-based and biophysical assays together with structural techniques, we showed that the observed resistance to cell death is largely independent of autophagy or major death receptor signaling pathways and demonstrated that direct targeting of the ks-vFLIPnndash;IKK interaction both in cells and in vitro can be achieved using IKK-mimetic peptides. Our results further reveal that these peptides not only induce cell killing but also potently sensitize PEL to the proapoptotic agents tumor necrosis factor alpha and etoposide and are the first to confirm ks-vFLIP as a tractable target for the treatment of PEL and related disorders.

    IMPORTANCE KSHV vFLIP (ks-vFLIP) has been shown to have a crucial role in cellular transformation, in which it is vital for the survival and proliferation of primary effusion lymphoma (PEL), an aggressive malignancy associated with infection that is resistant to the majority of chemotherapeutic drugs. It operates via subversion of the canonical NF-B pathway, which requires a physical interaction between ks-vFLIP and the IKK kinase modulatory subunit IKK. While this interaction has been directly linked to protection against apoptosis, it is unclear whether the suppression of other cell death pathways implicated in ks-vFLIP pathogenesis is an additional contributor. We demonstrate that the interaction between ks-vFLIP and IKK is pivotal in conferring resistance to apoptosis. Additionally, we show that the ks-vFLIPnndash;IKK complex can be disrupted using peptides leading to direct killing and the sensitization of PEL cells to proapoptotic agents. Our studies thus provide a framework for future therapeutic interventions.

  • Comparative Transcriptomics Highlights the Role of the Activator Protein 1 Transcription Factor in the Host Response to Ebolavirus [Cellular Response to Infection]

  • Ebolavirus and Marburgvirus comprise two genera of negative-sense single-stranded RNA viruses that cause severe hemorrhagic fevers in humans. Despite considerable research efforts, the molecular events following Ebola virus (EBOV) infection are poorly understood. With the view of identifying host factors that underpin EBOV pathogenesis, we compared the transcriptomes of EBOV-infected human, pig, and bat kidney cells using a transcriptome sequencing (RNA-seq) approach. Despite a significant difference in viral transcription/replication between the cell lines, all cells responded to EBOV infection through a robust induction of extracellular growth factors. Furthermore, a significant upregulation of activator protein 1 (AP1) transcription factor complex members FOS and JUN was observed in permissive cell lines. Functional studies focusing on human cells showed that EBOV infection induces protein expression, phosphorylation, and nuclear accumulation of JUN and, to a lesser degree, FOS. Using a luciferase-based reporter, we show that EBOV infection induces AP1 transactivation activity within human cells at 48 and 72 h postinfection. Finally, we show that JUN knockdown decreases the expression of EBOV-induced host gene expression. Taken together, our study highlights the role of AP1 in promoting the host gene expression profile that defines EBOV pathogenesis.

    IMPORTANCE Many questions remain about the molecular events that underpin filovirus pathophysiology. The rational design of new intervention strategies, such as postexposure therapeutics, will be significantly enhanced through an in-depth understanding of these molecular events. We believe that new insights into the molecular pathogenesis of EBOV may be possible by examining the transcriptomic response of taxonomically diverse cell lines (derived from human, pig, and bat). We first identified the responsive pathways using an RNA-seq-based transcriptomics approach. Further functional and computational analysis focusing on human cells highlighted an important role for the AP1 transcription factor in mediating the transcriptional response to EBOV infection. Our study sheds new light on how host transcription factors respond to and promote the transcriptional landscape that follows viral infection.

  • An Envelope-Modified Tetravalent Dengue Virus-Like-Particle Vaccine Has Implications for Flavivirus Vaccine Design [Vaccines and Antiviral Agents]

  • Dengue viruses (DENV) infect 50 to 100 million people each year. The spread of DENV-associated infections is one of the most serious public health problems worldwide, as there is no widely available vaccine or specific therapeutic for DENV infections. To address this, we developed a novel tetravalent dengue vaccine by utilizing virus-like particles (VLPs). We created recombinant DENV1 to -4 (DENV1-4) VLPs by coexpressing precursor membrane (prM) and envelope (E) proteins, with an F108A mutation in the fusion loop structure of E to increase the production of VLPs in mammalian cells. Immunization with DENV1-4 VLPs as individual, monovalent vaccines elicited strong neutralization activity against each DENV serotype in mice. For use as a tetravalent vaccine, DENV1-4 VLPs elicited high levels of neutralization activity against all four serotypes simultaneously. The neutralization antibody responses induced by the VLPs were significantly higher than those with DNA or recombinant E protein immunization. Moreover, antibody-dependent enhancement (ADE) was not observed against any serotype at a 1:10 serum dilution. We also demonstrated that the Zika virus (ZIKV) VLP production level was enhanced by introducing the same F108A mutation into the ZIKV envelope protein. Taken together, these results suggest that our strategy for DENV VLP production is applicable to other flavivirus VLP vaccine development, due to the similarity in viral structures, and they describe the promising development of an effective tetravalent vaccine against the prevalent flavivirus.

    IMPORTANCE Dengue virus poses one of the most serious public health problems worldwide, and the incidence of diseases caused by the virus has increased dramatically. Despite decades of effort, there is no effective treatment against dengue. A safe and potent vaccine against dengue is still needed. We developed a novel tetravalent dengue vaccine by using virus-like particles (VLPs), which are noninfectious because they lack the viral genome. Previous attempts of other groups to use dengue VLPs resulted in generally poor yields. We found that a critical amino acid mutation in the envelope protein enhances the production of VLPs. Our tetravalent vaccine elicited potent neutralizing antibody responses against all four DENV serotypes. Our findings can also be applied to vaccine development against other flaviviruses, such as Zika virus or West Nile virus.

  • Calcitonin Gene-Related Peptide Induces HIV-1 Proteasomal Degradation in Mucosal Langerhans Cells [Virus-Cell Interactions]

  • The neuroimmune dialogue between peripheral neurons and Langerhans cells (LCs) within mucosal epithelia protects against incoming pathogens. LCs rapidly internalize human immunodeficiency virus type 1 (HIV-1) upon its sexual transmission and then trans-infect CD4+ T cells. We recently found that the neuropeptide calcitonin gene-related peptide (CGRP), secreted mucosally from peripheral neurons, inhibits LC-mediated HIV-1 trans-infection. In this study, we investigated the mechanism of CGRP-induced inhibition, focusing on HIV-1 degradation in LCs and its interplay with trans-infection. We first show that HIV-1 degradation occurs in endolysosomes in untreated LCs, and functionally blocking such degradation with lysosomotropic agents results in increased trans-infection. We demonstrate that CGRP acts via its cognate receptor and at a viral postentry step to induce faster HIV-1 degradation, but without affecting the kinetics of endolysosomal degradation. We reveal that unexpectedly, CGRP shifts HIV-1 degradation from endolysosomes toward the proteasome, providing the first evidence for functional HIV-1 proteasomal degradation in LCs. Such efficient proteasomal degradation significantly inhibits the first phase of trans-infection, and proteasomal, but not endolysosomal, inhibitors abrogate CGRP-induced inhibition. Together, our results establish that CGRP controls the HIV-1 degradation mode in LCs. The presence of endogenous CGRP within innervated mucosal tissues, especially during the sexual response, to which CGRP contributes, suggests that HIV-1 proteasomal degradation predominates in vivo. Hence, proteasomal, rather than endolysosomal, HIV-1 degradation in LCs should be enhanced clinically to effectively restrict HIV-1 trans-infection.

    IMPORTANCE During sexual transmission, HIV-1 is internalized and degraded in LCs, the resident antigen-presenting cells in mucosal epithelia. Yet during trans-infection, infectious virions escaping degradation are transferred to CD4+ T cells, the principal HIV-1 targets. We previously found that the neuroimmune dialogue between LCs and peripheral neurons, innervating mucosal epithelia, significantly inhibits trans-infection via the action of the secreted neuropeptide CGRP on LCs. In this study, we investigated whether CGRP-induced inhibition of trans-infection is linked to CGRP-controlled HIV-1 degradation in LCs. We show that in untreated LCs, HIV-1 is functionally degraded in endolysosomes. In sharp contrast, we reveal that in CGRP-treated LCs, HIV-1 is diverted toward and degraded via another cytosolic protein degradative pathway, namely, the proteasome. These results establish that CGRP regulates HIV-1 degradation in LCs. As CGRP contributes to the sexual response and present within mucosal epithelia, HIV-1 proteasomal degradation in LCs might predominate in vivo and should be enhanced clinically.

  • Novel Role for Interleukin-17 in Enhancing Type 1 Helper T Cell Immunity in the Female Genital Tract following Mucosal Herpes Simplex Virus 2 Vaccination [Pathogenesis and Immunity]

  • It is well established that interferon gamma (IFN-) production by CD4+ T cells is critical for antiviral immunity against herpes simplex virus 2 (HSV-2) genital infection. However, the role of interleukin-17A (IL-17A) production by CD4+ T cells in HSV-2 antiviral immunity is yet to be elucidated. Here we demonstrate that IL-17A plays an important role in enhancing antiviral T helper type 1 (Th1) responses in the female genital tract (FGT) and is essential for effective protection conferred by HSV-2 vaccination. While IL-17A did not play a critical role during primary genital HSV-2 infection, seen by lack of differences in susceptibility between IL-17A-deficient (IL-17Anndash;/nndash;) and wild-type (WT) C57BL/6 mice, it was critical for mediating antiviral responses after challenge/reexposure. Compared to WT mice, IL-17Anndash;/nndash; mice (i) infected intravaginally and reexposed or (ii) vaccinated intranasally and challenged intravaginally demonstrated poor outcomes. Following intravaginal HSV-2 reexposure or challenge, vaccinated IL-17Anndash;/nndash; mice had significantly higher mortality, greater disease severity, higher viral shedding, and higher levels of proinflammatory cytokines and chemokines in vaginal secretions. Furthermore, IL-17Anndash;/nndash; mice had impaired Th1 cell responses after challenge/reexposure, with significantly lower proportions of vaginal IFN-+ CD4+ T cells. The impaired Th1 cell responses in IL-17Anndash;/nndash; mice coincided with smaller populations of IFN-+ CD4+ tissue resident memory T (TRM) cells in the genital tract postimmunization. Taken together, these findings describe a novel role for IL-17A in regulating antiviral IFN-+ Th1 cell immunity in the vaginal tract. This strategy could be exploited to enhance antiviral immunity following HSV-2 vaccination.

    IMPORTANCE T helper type 1 (Th1) immunity, specifically interferon gamma (IFN-) production by CD4+ T cells, is critical for protection against genital herpesvirus (HSV-2) infection, and enhancing this response can potentially help improve disease outcomes. Our study demonstrated that interleukin-17A (IL-17A) plays an essential role in enhancing antiviral Th1 responses in the female genital tract (FGT). We found that in the absence of IL-17A, preexposed and vaccinated mice showed poor disease outcomes and were unable to overcome HSV-2 reexposure/challenge. IL-17A-deficient mice (IL-17Anndash;/nndash;) had smaller populations of IFN-+ CD4+ tissue resident memory T (TRM) cells in the genital tract postimmunization than did wild-type (WT) mice, which coincided with attenuated Th1 responses postchallenge. This has important implications for developing effective vaccines against HSV-2, as we propose that strategies inducing IL-17A in the genital tract may promote more effective Th1 cell immunity and better overall protection.

  • Epstein-Barr Virus Fusion with Epithelial Cells Triggered by gB Is Restricted by a gL Glycosylation Site [Virus-Cell Interactions]

  • Epstein-Barr virus (EBV) entry into epithelial cells is mediated by the conserved core fusion machinery, composed of the fusogen gB and the receptor-binding complex gH/gL. The heterodimeric gH/gL complex binds to the EBV epithelial cell receptor or gp42, which binds to the B-cell receptor, triggering gB-mediated fusion of the virion envelope with cellular membranes. Our previous study found that the gL glycosylation mutant N69L/S71V had an epithelial cell-specific hyperfusogenic phenotype. To study the influence of this gL mutant on the initiation and kinetics of gB-driven epithelial cell fusion, we established a virus-free split-green fluorescent protein cell-cell fusion assay that enables real-time measurements of membrane fusion using live cells. The gL_N69L/S71V mutant had a large increase in epithelial cell fusion activity of up to 300% greater than that of wild-type gL starting at early time points. The hyperfusogenicity of the gL mutant was not a result of alterations in complex formation with gH or alterations in cellular localization. Moreover, the hyperfusogenic phenotype of the gL mutant correlated with the formation of enlarged syncytia. In summary, our present findings highlight an important role of gL in the kinetics of gB-mediated epithelial cell fusion, adding to previous findings indicating a direct interaction between gL and gB in EBV membrane fusion.

    IMPORTANCE EBV predominantly infects epithelial cells and B lymphocytes, which are the cells of origin for the EBV-associated malignancies Hodgkin and Burkitt lymphoma as well as nasopharyngeal carcinoma. Contrary to the other key players of the core fusion machinery, gL has the most elusive role during EBV-induced membrane fusion. We found that the glycosylation site N69/S71 of gL is involved in restricting epithelial cell fusion activity, strongly correlating with syncytium size. Interestingly, our data showed that the gL glycosylation mutant increases the fusion activity of the hyperfusogenic gB mutants, indicating that this gL mutant and the gB mutants target different steps during fusion. Our studies on how gL and gB work together to modulate epithelial cell fusion kinetics are essential to understand the highly tuned tropism of EBV for epithelial cells and B lymphocytes and may result in novel strategies for therapies preventing viral entry into target host cells. Finally, making our results of particular interest is the absence of gL syncytial mutants in other herpesviruses.

  • Differential Inhibitory Receptor Expression on T Cells Delineates Functional Capacities in Chronic Viral Infection [Pathogenesis and Immunity]

  • Inhibitory receptors have been extensively described for their importance in regulating immune responses in chronic infections and cancers. Blocking the function of inhibitory receptors such as PD-1, CTLA-4, 2B4, Tim-3, and LAG-3 has shown promise for augmenting CD8 T cell activity and boosting pathogen-specific immunity. However, the prevalence of inhibitory receptors on CD4 T cells and their relative influence on CD4 T cell functionality in chronic HIV infection remains poorly described. We therefore determined and compared inhibitory receptor expression patterns of 2B4, CTLA-4, LAG-3, PD-1, and Tim-3 on virus-specific CD4 and CD8 T cells in relation to their functional T cell profile. In chronic HIV infection, inhibitory receptor distribution differed markedly between cytokine-producing T cell subsets with, gamma interferon (IFN-)- and tumor necrosis factor alpha (TNF-aalpha;)-producing cells displaying the highest and lowest prevalence of inhibitory receptors, respectively. Blockade of inhibitory receptors differentially affected cytokine production by cells in response to staphylococcal enterotoxin B stimulation. CTLA-4 blockade increased IFN- and CD40L production, while PD-1 blockade strongly augmented IFN-, interleukin-2 (IL-2), and TNF-aalpha; production. In a Friend retrovirus infection model, CTLA-4 blockade in particular was able to improve control of viral replication. Together, these results show that inhibitory receptor distribution on HIV-specific CD4 T cells varies markedly with respect to the functional subset of CD4 T cells being analyzed. Furthermore, the differential effects of receptor blockade suggest novel methods of immune response modulation, which could be important in the context of HIV vaccination or therapeutic strategies.

    IMPORTANCE Inhibitory receptors are important for limiting damage by the immune system during acute infections. In chronic infections, however, their expression limits immune system responsiveness. Studies have shown that blocking inhibitory receptors augments CD8 T cell functionality in HIV infection, but their influence on CD4 T cells remains unclear. We assessed the expression of inhibitory receptors on HIV-specific CD4 T cells and their relationship with T cell functionality. We uncovered differences in inhibitory receptor expression depending on the CD4 T cell function. We also found differences in functionality of CD4 T cells following blocking of different inhibitory receptors, and we confirmed our results in a Friend virus retroviral model of infection in mice. Our results show that inhibitory receptor expression on CD4 T cells is linked to CD4 T cell functionality and could be sculpted by blockade of specific inhibitory receptors. These data reveal exciting possibilities for the development of novel treatments and immunotherapeutics.

  • NLRX1 Mediates MAVS Degradation To Attenuate the Hepatitis C Virus-Induced Innate Immune Response through PCBP2 [Pathogenesis and Immunity]

  • Activation of innate immunity is essential for host cells to restrict the spread of invading viruses and other pathogens. However, attenuation or termination of signaling is also necessary for preventing immune-mediated tissue damage and spontaneous autoimmunity. Here, we identify nucleotide binding oligomerization domain (NOD)-like receptor X1 (NLRX1) as a negative regulator of the mitochondrial antiviral signaling protein (MAVS)-mediated signaling pathway during hepatitis C virus (HCV) infection. The depletion of NLRX1 enhances the HCV-triggered activation of interferon (IFN) signaling and causes the suppression of HCV propagation in hepatocytes. NLRX1, a HCV-inducible protein, interacts with MAVS and mediates the K48-linked polyubiquitination and subsequent degradation of MAVS via the proteasomal pathway. Moreover, poly(rC) binding protein 2 (PCBP2) interacts with NLRX1 to participate in the NLRX1-induced degradation of MAVS and the inhibition of antiviral responses during HCV infection. Mutagenic analyses further revealed that the NOD of NLRX1 is essential for NLRX1 to interact with PCBP2 and subsequently induce MAVS degradation. Our study unlocks a key mechanism of the fine-tuning of innate immunity by which NLRX1 restrains the retinoic acid-inducible gene I-like receptor (RLR)-MAVS signaling cascade by recruiting PCBP2 to MAVS for inducing MAVS degradation through the proteasomal pathway. NLRX1, a negative regulator of innate immunity, is a pivotal host factor for HCV to establish persistent infection.

    IMPORTANCE Innate immunity needs to be tightly regulated to maximize the antiviral response and minimize immune-mediated pathology, but the underlying mechanisms are poorly understood. In this study, we report that NLRX1 is a proviral host factor for HCV infection and functions as a negative regulator of the HCV-triggered innate immune response. NLRX1 recruits PCBP2 to MAVS and induces the K48-linked polyubiquitination and degradation of MAVS, leading to the negative regulation of the IFN signaling pathway and promoting HCV infection. Overall, this study provides intriguing insights into how innate immunity is regulated during viral infection.

  • Genesis and Spread of Newly Emerged Highly Pathogenic H7N9 Avian Viruses in Mainland China [Genetic Diversity and Evolution]

  • The novel low-pathogenic avian influenza A H7N9 viruses (LPAI H7N9 viruses) have been a threat to public health since their emergence in 2013 because of the high rates of mortality and morbidity that they cause. Recently, highly pathogenic variants of these avian influenza A H7N9 viruses (HPAI H7N9 viruses) have emerged and caused human infections and outbreaks among poultry in mainland China. However, it is still unclear how the HPAI H7N9 virus was generated and how it evolved and spread in China. Here, we show that the ancestor virus of the HPAI H7N9 viruses originated in the Yangtze River Delta region and spread southward to the Pearl River Delta region, possibly through live poultry trade. After introduction into the Pearl River Delta region, the origin LPAI H7N9 virus acquired four amino acid insertions in the hemagglutinin (HA) protein cleavage site and mutated into the HPAI H7N9 virus in late May 2016. Afterward, the HPAI H7N9 viruses further reassorted with LPAI H7N9 or H9N2 viruses locally and generated multiple different genotypes. As of 14 July 2017, the HPAI H7N9 viruses had spread from Guangdong Province to at least 12 other provinces. The rapid geographical expansion and genetic evolution of the HPAI H7N9 viruses pose a great challenge not only to public health but also to poultry production. Effective control measures, including enhanced surveillance, are therefore urgently needed.

    IMPORTANCE The LPAI H7N9 virus has caused five outbreak waves in humans and was recently reported to have mutated into highly pathogenic variants. It is unknown how the HPAI H7N9 virus originated, evolved, and disseminated in China. In this study, we comprehensively analyzed the sequences of HPAI H7N9 viruses from 28 human and 21 environmental samples covering eight provinces in China that were taken from November 2016 to June 2017. The results show that the ancestor virus of the HPAI H7N9 viruses originated in the Yangtze River Delta region. However, the insertion of four amino acids into the HA protein cleavage site of an LPAI H7N9 virus occurred in late May 2016 in the Pearl River Delta region. The mutated HPAI H7N9 virus further reassorted with LPAI H7N9 or H9N2 viruses that were cocirculating in poultry. Considering the rapid geographical expansion of the HPAI H7N9 viruses, effective control measures are urgently needed.

  • APOBEC3A Is Upregulated by Human Cytomegalovirus (HCMV) in the Maternal-Fetal Interface, Acting as an Innate Anti-HCMV Effector [Pathogenesis and Immunity]

  • Human cytomegalovirus (HCMV) is the leading cause of congenital infection and is associated with a wide range of neurodevelopmental disabilities and intrauterine growth restriction. Yet our current understanding of the mechanisms modulating transplacental HCMV transmission is poor. The placenta, given its critical function in protecting the fetus, has evolved effective yet largely uncharacterized innate immune barriers against invading pathogens. Here we show that the intrinsic cellular restriction factor apolipoprotein B editing catalytic subunit-like 3A (APOBEC3A [A3A]) is profoundly upregulated following ex vivo HCMV infection in human decidual tissuesmmdash;constituting the maternal aspect of the placenta. We directly demonstrated that A3A severely restricted HCMV replication upon controlled overexpression in epithelial cells, acting by a cytidine deamination mechanism to introduce hypermutations into the viral genome. Importantly, we further found that A3 editing of HCMV DNA occurs both ex vivo in HCMV-infected decidual organ cultures and in vivo in amniotic fluid samples obtained during natural congenital infection. Our results reveal a previously unexplored role for A3A as an innate anti-HCMV effector, activated by HCMV infection in the maternal-fetal interface. These findings pave the way to new insights into the potential impact of APOBEC proteins on HCMV pathogenesis.

    IMPORTANCE In view of the grave outcomes associated with congenital HCMV infection, there is an urgent need to better understand the innate mechanisms acting to limit transplacental viral transmission. Toward this goal, our findings reveal the role of the intrinsic cellular restriction factor A3A (which has never before been studied in the context of HCMV infection and vertical viral transmission) as a potent anti-HCMV innate barrier, activated by HCMV infection in the authentic tissues of the maternal-fetal interface. The detection of naturally occurring hypermutations in clinical amniotic fluid samples of congenitally infected fetuses further supports the idea of the occurrence of A3 editing of the viral genome in the setting of congenital HCMV infection. Given the widely differential tissue distribution characteristics and biological functions of the members of the A3 protein family, our findings should pave the way to future studies examining the potential impact of A3A as well as of other A3s on HCMV pathogenesis.

  • Natural Reassortants of Potentially Zoonotic Avian Influenza Viruses H5N1 and H9N2 from Egypt Display Distinct Pathogenic Phenotypes in Experimentally Infected Chickens and Ferrets [Genetic Diversity and Evolution]

  • The cocirculation of zoonotic highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 and avian influenza virus (AIV) of subtype H9N2 among poultry in Egypt for at least 6 years should render that country a hypothetical hot spot for the emergence of reassortant, phenotypically altered viruses, yet no reassortants have been detected in Egypt. The present investigations proved that reassortants of the Egyptian H5N1 clade 2.2.1.2 virus and H9N2 virus of the G1-B lineage can be generated by coamplification in embryonated chicken eggs. Reassortants were restricted to the H5N1 subtype and acquired between two and all six of the internal segments of the H9N2 virus. Five selected plaque-purified reassortant clones expressed a broad phenotypic spectrum both in vitro and in vivo. Two groups of reassortants were characterized to have retarded growth characteristics in vitro compared to the H5N1 parent virus. One clone provoked reduced mortality in inoculated chickens, although the characteristics of a highly pathogenic phenotype were retained. Enhanced zoonotic properties were not predicted for any of these clones, and this prediction was confirmed by ferret inoculation experiments: neither the H5N1 parent virus nor two selected clones induced severe clinical symptoms or were transmitted to sentinel ferrets by contact. While the emergence of reassortants of Egyptian HPAIV of subtype H5N1 with internal gene segments of cocirculating H9N2 viruses is possible in principle, the spread of such viruses is expected to be governed by their fitness to outcompete the parental viruses in the field. The eventual spread of attenuated phenotypes, however, would negatively impact syndrome surveillance on poultry farms and might foster enzootic virus circulation.

    IMPORTANCE Despite almost 6 years of the continuous cocirculation of highly pathogenic avian influenza virus H5N1 and avian influenza virus H9N2 in poultry in Egypt, no reassortants of the two subtypes have been reported. Here, the principal compatibility of the two subtypes is shown by forcing the reassortment between copassaged H5N1 und H9N2 viruses in embryonated chicken eggs. The resulting reassortant viruses displayed a wide range of pathogenicity including attenuated phenotypes in chickens, but did not show enhanced zoonotic propensities in the ferret model.

  • Susceptibility of Human Endogenous Retrovirus Type K to Reverse Transcriptase Inhibitors [Vaccines and Antiviral Agents]

  • Human endogenous retroviruses (HERVs) make up 8% of the human genome. The HERV type K (HERV-K) HML-2 (HK2) family contains proviruses that are the most recent entrants into the human germ line and are transcriptionally active. In HIV-1 infection and cancer, HK2 genes produce retroviral particles that appear to be infectious, yet the replication capacity of these viruses and potential pathogenicity has been difficult to ascertain. In this report, we screened the efficacy of commercially available reverse transcriptase inhibitors (RTIs) at inhibiting the enzymatic activity of HK2 RT and HK2 genomic replication. Interestingly, only one provirus, K103, was found to encode a functional RT among those examined. Several nucleoside analogue RTIs (NRTIs) blocked K103 RT activity and consistently inhibited the replication of HK2 genomes. The NRTIs zidovudine (AZT), stavudine (d4T), didanosine (ddI), and lamivudine (3TC), and the nucleotide RTI inhibitor tenofovir (TDF), show efficacy in blocking K103 RT. HIV-1-specific nonnucleoside RTIs (NNRTIs), protease inhibitors (PIs), and integrase inhibitors (IIs) did not affect HK2, except for the NNRTI etravirine (ETV). The inhibition of HK2 infectivity by NRTIs appears to take place at either the reverse transcription step of the viral genome prior to HK2 viral particle formation and/or in the infected cells. Inhibition of HK2 by these drugs will be useful in suppressing HK2 infectivity if these viruses prove to be pathogenic in cancer, neurological disorders, or other diseases associated with HK2. The present studies also elucidate a key aspect of the life cycle of HK2, specifically addressing how they do, and/or did, replicate.

    IMPORTANCE Endogenous retroviruses are relics of ancestral virus infections in the human genome. The most recent of these infections was caused by HK2. While HK2 often remains silent in the genome, this group of viruses is activated in HIV-1-infected and cancer cells. Recent evidence suggests that these viruses are infectious, and the potential exists for HK2 to contribute to disease. We show that HK2, and specifically the enzyme that mediates virus replication, can be inhibited by a panel of drugs that are commercially available. We show that several drugs block HK2 with different efficacies. The inhibition of HK2 replication by antiretroviral drugs appears to occur in the virus itself as well as after infection of cells. Therefore, these drugs might prove to be an effective treatment by suppressing HK2 infectivity in diseases where these viruses have been implicated, such as cancer and neurological syndromes.

  • p21 Restricts HIV-1 in Monocyte-Derived Dendritic Cells through the Reduction of Deoxynucleoside Triphosphate Biosynthesis and Regulation of SAMHD1 Antiviral Activity [Virus-Cell Interactions]

  • HIV-1 infection of noncycling cells, such as dendritic cells (DCs), is impaired due to limited availability of deoxynucleoside triphosphates (dNTPs), which are needed for HIV-1 reverse transcription. The levels of dNTPs are tightly regulated during the cell cycle and depend on the balance between dNTP biosynthesis and degradation. SAMHD1 potently blocks HIV-1 replication in DCs, although the underlying mechanism is still unclear. SAMHD1 has been reported to be able to degrade dNTPs and viral nucleic acids, which may both hamper HIV-1 reverse transcription. The relative contribution of these activities may differ in cycling and noncycling cells. Here, we show that inhibition of HIV-1 replication in monocyte-derived DCs (MDDCs) is associated with an increased expression of p21cip1/waf, a cell cycle regulator that is involved in the differentiation and maturation of DCs. Induction of p21 in MDDCs decreases the pool of dNTPs and increases the antiviral active isoform of SAMHD1. Although both processes are complementary in inhibiting HIV-1 replication, the antiviral activity of SAMHD1 in our primary cell model appears to be, at least partially, independent of its dNTPase activity. The reduction in the pool of dNTPs in MDDCs appears rather mostly due to a p21-mediated suppression of several enzymes involved in dNTP synthesis (i.e., RNR2, TYMS, and TK-1). These results are important to better understand the interplay between HIV-1 and DCs and may inform the design of new therapeutic approaches to decrease viral dissemination and improve immune responses against HIV-1.

    IMPORTANCE DCs play a key role in the induction of immune responses against HIV. However, HIV has evolved ways to exploit these cells, facilitating immune evasion and virus dissemination. We have found that the expression of p21, a cyclin-dependent kinase inhibitor involved in cell cycle regulation and monocyte differentiation and maturation, potentially can contribute to the inhibition of HIV-1 replication in monocyte-derived DCs through multiple mechanisms. p21 decreased the size of the intracellular dNTP pool. In parallel, p21 prevented SAMHD1 phosphorylation and promoted SAMHD1 dNTPase-independent antiviral activity. Thus, induction of p21 resulted in conditions that allowed the effective inhibition of HIV-1 replication through complementary mechanisms. Overall, p21 appears to be a key regulator of HIV infection in myeloid cells.

  • A DNA Vaccine That Targets Hemagglutinin to Antigen-Presenting Cells Protects Mice against H7 Influenza [Vaccines and Antiviral Agents]

  • Zoonotic influenza H7 viral infections have a case fatality rate of about 40%. Currently, no or limited human to human spread has occurred, but we may be facing a severe pandemic threat if the virus acquires the ability to transmit between humans. Novel vaccines that can be rapidly produced for global distribution are urgently needed, and DNA vaccines may be the only type of vaccine that allows for the speed necessary to quench an emerging pandemic. Here, we constructed DNA vaccines encoding the hemagglutinin (HA) from influenza A/chicken/Italy/13474/99 (H7N1). In order to increase the efficacy of DNA vaccination, HA was targeted to either major histocompatibility complex class II molecules or chemokine receptors 1, 3, and 5 (CCR1/3/5) that are expressed on antigen-presenting cells (APC). A single DNA vaccination with APC-targeted HA significantly increased antibody levels in sera compared to nontargeted control vaccines. The antibodies were confirmed neutralizing in an H7 pseudotype-based neutralization assay. Furthermore, the APC-targeted vaccines increased the levels of antigen-specific cytotoxic T cells, and a single DNA vaccination could confer protection against a lethal challenge with influenza A/turkey/Italy/3889/1999 (H7N1) in mice. In conclusion, we have developed a vaccine that rapidly could contribute protection against a pandemic threat from avian influenza.

    IMPORTANCE Highly pathogenic avian influenza H7 constitute a pandemic threat that can cause severe illness and death in infected individuals. Vaccination is the main method of prophylaxis against influenza, but current vaccine strategies fall short in a pandemic situation due to a prolonged production time and insufficient production capabilities. In contrast, a DNA vaccine can be rapidly produced and deployed to prevent the potential escalation of a highly pathogenic influenza pandemic. We here demonstrate that a single DNA delivery of hemagglutinin from an H7 influenza could mediate full protection against a lethal challenge with H7N1 influenza in mice. Vaccine efficacy was contingent on targeting of the secreted vaccine protein to antigen-presenting cells.

  • Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion [Pathogenesis and Immunity]

  • Zika virus (ZIKV), a mosquito-transmitted flavivirus responsible for sporadic outbreaks of mild and febrile illness in Africa and Asia, reemerged in the last decade causing serious human diseases, including microcephaly, congenital malformations, and Guillain-Barreeacute; syndrome. Although genomic and phylogenetic analyses suggest that genetic evolution may have led to the enhanced virulence of ZIKV, experimental evidence supporting the role of specific genetic changes in virulence is currently lacking. One sequence motif, VNDT, containing an N-linked glycosylation site in the envelope (E) protein, is polymorphic; it is absent in many of the African isolates but present in all isolates from the recent outbreaks. In the present study, we investigated the roles of this sequence motif and glycosylation of the E protein in the pathogenicity of ZIKV. We first constructed a stable full-length cDNA clone of ZIKV in a novel linear vector from which infectious virus was recovered. The recombinant ZIKV generated from the infectious clone, which contains the VNDT motif, is highly pathogenic and causes lethality in a mouse model. In contrast, recombinant viruses from which the VNDT motif is deleted or in which the N-linked glycosylation site is mutated by single-amino-acid substitution are highly attenuated and nonlethal. The mutant viruses replicate poorly in the brains of infected mice when inoculated subcutaneously but replicate well following intracranial inoculation. Our findings provide the first evidence that N-linked glycosylation of the E protein is an important determinant of ZIKV virulence and neuroinvasion.

    IMPORTANCE The recent emergence of Zika virus (ZIKV) in the Americas has caused major worldwide public health concern. The virus appears to have gained significant pathogenicity, causing serious human diseases, including microcephaly and Guillain-Barreeacute; syndrome. The factors responsible for the emergence of pathogenic ZIKV are not understood at this time, although genetic changes have been shown to facilitate virus transmission. All isolates from the recent outbreaks contain an N-linked glycosylation site within the viral envelope (E) protein, whereas many isolates of the African lineage virus lack this site. To elucidate the functional significance of glycosylation in ZIKV pathogenicity, recombinant ZIKVs from infectious clones with or without the glycan on the E protein were generated. ZIKVs lacking the glycan were highly attenuated for the ability to cause mortality in a mouse model and were severely compromised for neuroinvasion. Our studies suggest glycosylation of the E protein is an important factor contributing to ZIKV pathogenicity.

  • Protective Humoral Immunity in the Central Nervous System Requires Peripheral CD19-Dependent Germinal Center Formation following Coronavirus Encephalomyelitis [Cellular Response to Infection]

  • B cell subsets with phenotypes characteristic of naive, non-isotype-switched, memory (Bmem) cells and antibody-secreting cells (ASC) accumulate in various models of central nervous system (CNS) inflammation, including viral encephalomyelitis. During neurotropic coronavirus JHMV infection, infiltration of protective ASC occurs after T cell-mediated viral control and is preceded by accumulation of non-isotype-switched IgD+ and IgM+ B cells. However, the contribution of peripheral activation events in cervical lymph nodes (CLN) to driving humoral immune responses in the infected CNS is poorly defined. CD19, a signaling component of the B cell receptor complex, is one of multiple regulators driving B cell differentiation and germinal center (GC) formation by lowering the threshold of antigen-driven activation. JHMV-infected CD19nndash;/nndash; mice were thus used to determine how CD19 affects CNS recruitment of B cell subsets. Early polyclonal ASC expansion, GC formation, and virus-specific ASC were all significantly impaired in CLN of CD19nndash;/nndash; mice compared to wild-type (WT) mice, consistent with lower and unsustained virus-specific serum antibody (Ab). ASC were also significantly reduced in the CNS, resulting in increased infectious virus during persistence. Nevertheless, CD19 deficiency did not affect early CNS IgD+ B cell accumulation. The results support the notion that CD19-independent factors drive early B cell mobilization and recruitment to the infected CNS, while delayed accumulation of virus-specific, isotype-switched ASC requires CD19-dependent GC formation in CLN. CD19 is thus essential for both sustained serum Ab and protective local Ab within the CNS following JHMV encephalomyelitis.

    IMPORTANCE CD19 activation is known to promote GC formation and to sustain serum Ab responses following antigen immunization and viral infections. However, the contribution of CD19 in the context of CNS infections has not been evaluated. This study demonstrates that antiviral protective ASC in the CNS are dependent on CD19 activation and peripheral GC formation, while accumulation of early-recruited IgD+ B cells is CD19 independent. This indicates that IgD+ B cells commonly found early in the CNS do not give rise to local ASC differentiation and that only antigen-primed, peripheral GC-derived ASC infiltrate the CNS, thereby limiting potentially harmful nonspecific Ab secretion. Expanding our understanding of activation signals driving CNS migration of distinct B cell subsets during neuroinflammatory insults is critical for preventing and managing acute encephalitic infections, as well as preempting reactivation of persistent viruses during immune-suppressive therapies targeting B cells in multiple sclerosis (MS), such as rituximab and ocrelizumab.

  • Vertical Transmission of Hepatitis C Virus: Variable Transmission Bottleneck and Evidence of Midgestation In Utero Infection [Pathogenesis and Immunity]

  • Hepatitis C virus (HCV) can be transmitted from mother to child during pregnancy and childbirth. However, the timing and precise biological mechanisms that are involved in this process are incompletely understood, as are the determinants that influence transmission of particular HCV variants. Here we report results of a longitudinal assessment of HCV quasispecies diversity and composition in 5 cases of vertical HCV transmission, including 3 women coinfected with human immunodeficiency virus type 1 (HIV-1). The population structure of HCV variant spectra based on E2 envelope gene sequences (nucleotide positions 1491 to 1787), including hypervariable regions 1 and 2, was characterized using next-generation sequencing and median-joining network analysis. Compatible with a loose transmission bottleneck, larger numbers of shared HCV variants were observed in the presence of maternal coinfection. Coalescent Bayesian Markov chain Monte Carlo simulations revealed median times of transmission between 24.9 weeks and 36.1 weeks of gestation, with some confidence intervals ranging into the 1st trimester, considerably earlier than previously thought. Using recombinant autologous HCV pseudoparticles, differences were uncovered in HCV-specific antibody responses between coinfected mothers and mothers infected with HCV alone, in whom generalized absence of neutralization was observed. Finally, shifts in HCV quasispecies composition were seen in children around 1 year of age, compatible with the disappearance of passively transferred maternal immunoglobulins and/or the development of HCV-specific humoral immunity. Taken together, these results provide insights into the timing, dynamics, and biologic mechanisms involved in vertical HCV transmission and inform preventative strategies.

    IMPORTANCE Although it is well established that hepatitis C virus (HCV) can be transmitted from mother to child, the manner and the moment at which transmission operates have been the subject of conjecture. By carrying out a detailed examination of viral sequences, we showed that transmission could take place comparatively early in pregnancy. In addition, we showed that when the mother also carried human immunodeficiency virus type 1 (HIV-1), many more HCV variants were shared between her and her child, suggesting that the mechanism and/or the route of transmission of HCV differed in the presence of coinfection with HIV-1. These results could explain why cesarean section is ineffective in preventing vertical HCV transmission and guide the development of interventions to avert pediatric HCV infection.

  • Conformational Changes in the 5' End of the HIV-1 Genome Dependent on the Debranching Enzyme DBR1 during Early Stages of Infection [Genome Replication and Regulation of Viral Gene Expression]

  • Previous studies in our laboratory showed that the RNA debranching enzyme (DBR1) is not required for early steps in HIV cDNA formation but is necessary for synthesis of intermediate and late cDNA products. To further characterize this effect, we evaluated the topology of the 5' end of the HIV-1 RNA genome during early infection with and without inhibition of DBR1 synthesis. Cells were transfected with DBR1 short hairpin RNA (shRNA) followed 48 h later by infection with an HIV-1-derived vector containing an RNase H-deficient reverse transcriptase (RT). RNA was isolated at several times postinfection and treated with various RNA-modifying enzymes prior to rapid amplification of 5' cDNA ends (5' RACE) for HIV-1 RNA and quantitative reverse transcriptase PCR (qRT-PCR). In infected cells, DBR1 knockdown inhibited detection of free HIV-1 RNA 5' ends at all time points. The difference in detection of free HIV-1 RNA 5' ends in infected DBR1 knockdown versus control cells was eliminated by in vitro incubation of infected cell RNAs with yeast or human DBR1 enzyme prior to 5' RACE and qRT-PCR. This was dependent on the 2'-5' phosphatase activity of DBR1, since it did not occur when we used the catalytically inactive DBR1(N85A) mutant. Finally, HIV-1 RNA from infected DBR1 knockdown cells was resistant to RNase R that degrades linear RNAs but not RNAs in circular or lariat-like conformations. These results provide evidence for formation of a lariat-like structure involving the 5' end of HIV-1 RNA during an early step in infection and the involvement of DBR1 in resolving it.

    IMPORTANCE Our findings support a new view of the early steps in HIV genome replication. We show that the HIV genomic RNA is rapidly decapped and forms a lariat-like structure after entering a cell. The lariat-like structure is subsequently resolved by the cellular enzyme DBR1, leaving a 5' phosphate. This pathway is similar to the formation and resolution of pre-mRNA intron lariats and therefore suggests that similar mechanisms may be used by HIV. Our work therefore opens a new area of investigation in HIV replication and may ultimately uncover new targets for inhibiting HIV replication and for preventing the development of AIDS.

  • The Exonuclease Activity of Herpes Simplex Virus 1 UL12 Is Required for Production of Viral DNA That Can Be Packaged To Produce Infectious Virus [Genome Replication and Regulation of Viral Gene Expression]

  • The herpes simplex virus (HSV) type I alkaline nuclease, UL12, has 5'-to-3' exonuclease activity and shares homology with nucleases from other members of the Herpesviridae family. We previously reported that a UL12-null virus exhibits a severe defect in viral growth. To determine whether the growth defect was a result of loss of nuclease activity or another function of UL12, we introduced an exonuclease-inactivating mutation into the viral genome. The recombinant virus, UL12 D340E (the D340E mutant), behaved identically to the null virus (AN-1) in virus yield experiments, exhibiting a 4-log decrease in the production of infectious virus. Furthermore, both viruses were severely defective in cell-to-cell spread and produced fewer DNA-containing capsids and more empty capsids than wild-type virus. In addition, DNA packaged by the viral mutants was aberrant, as determined by infectivity assays and pulsed-field gel electrophoresis. We conclude that UL12 exonuclease activity is essential for the production of viral DNA that can be packaged to produce infectious virus. This conclusion was bolstered by experiments showing that a series of natural and synthetic aalpha;-hydroxytropolones recently reported to inhibit HSV replication also inhibit the nuclease activity of UL12. Taken together, our results demonstrate that the exonuclease activity of UL12 is essential for the production of infectious virus and may be considered a target for development of antiviral agents.

    IMPORTANCE Herpes simplex virus is a major pathogen, and although nucleoside analogs such as acyclovir are highly effective in controlling HSV-1 or -2 infections in immunocompetent individuals, their use in immunocompromised patients is complicated by the development of resistance. Identification of additional proteins essential for viral replication is necessary to develop improved therapies. In this communication, we confirm that the exonuclease activity of UL12 is essential for viral replication through the analysis of a nuclease-deficient viral mutant. We demonstrate that the exonuclease activity of UL12 is essential for the production of viral progeny and thus provides an attractive, druggable enzymatic target.

  • Vaccinia Virus Encodes a Novel Inhibitor of Apoptosis That Associates with the Apoptosome [Virus-Cell Interactions]

  • Apoptosis is an important antiviral host defense mechanism. Here we report the identification of a novel apoptosis inhibitor encoded by the vaccinia virus (VACV) M1L gene. M1L is absent in the attenuated modified vaccinia virus Ankara (MVA) strain of VACV, a strain that stimulates apoptosis in several types of immune cells. M1 expression increased the viability of MVA-infected THP-1 and Jurkat cells and reduced several biochemical hallmarks of apoptosis, such as PARP-1 and procaspase-3 cleavage. Furthermore, ectopic M1L expression decreased staurosporine-induced (intrinsic) apoptosis in HeLa cells. We then identified the molecular basis for M1 inhibitory function. M1 allowed mitochondrial depolarization but blocked procaspase-9 processing, suggesting that M1 targeted the apoptosome. In support of this model, we found that M1 promoted survival in Saccharomyces cerevisiae overexpressing human Apaf-1 and procaspase-9, critical components of the apoptosome, or overexpressing only conformationally active caspase-9. In mammalian cells, M1 coimmunoprecipitated with Apaf-1nndash;procaspase-9 complexes. The current model is that M1 associates with and allows the formation of the apoptosome but prevents apoptotic functions of the apoptosome. The M1 protein features 14 predicted ankyrin (ANK) repeat domains, and M1 is the first ANK-containing protein reported to use this inhibitory strategy. Since ANK-containing proteins are encoded by many large DNA viruses and found in all domains of life, studies of M1 may lead to a better understanding of the roles of ANK proteins in virus-host interactions.

    IMPORTANCE Apoptosis selectively eliminates dangerous cells such as virus-infected cells. Poxviruses express apoptosis antagonists to neutralize this antiviral host defense. The vaccinia virus (VACV) M1 ankyrin (ANK) protein, a protein with no previously ascribed function, inhibits apoptosis. M1 interacts with the apoptosome and prevents procaspase-9 processing as well as downstream procaspase-3 cleavage in several cell types and under multiple conditions. M1 is the first poxviral protein reported to associate with and prevent the function of the apoptosome, giving a more detailed picture of the threats VACV encounters during infection. Dysregulation of apoptosis is associated with several human diseases. One potential treatment of apoptosis-related diseases is through the use of designed ANK repeat proteins (DARPins), similar to M1, as caspase inhibitors. Thus, the study of the novel antiapoptosis effects of M1 via apoptosome association will be helpful for understanding how to control apoptosis using either natural or synthetic molecules.

  • Highly Efficacious Novel Vaccine, Humoral Immunity, and Ocular Herpes Simplex Virus 1: Reality or Myth? [Letters to the Editor]

  • Genome-Wide Mutagenesis of Dengue Virus Reveals Plasticity of the NS1 Protein and Enables Generation of Infectious Tagged Reporter Viruses [Genome Replication and Regulation of Viral Gene Expression]

  • Dengue virus (DENV) is a major global pathogen that causes significant morbidity and mortality in tropical and subtropical areas worldwide. An improved understanding of the regions within the DENV genome and its encoded proteins that are required for the virus replication cycle will expedite the development of urgently required therapeutics and vaccines. We subjected an infectious DENV genome to unbiased insertional mutagenesis and used next-generation sequencing to identify sites that tolerate 15-nucleotide insertions during the virus replication cycle in hepatic cell culture. This revealed that the regions within capsid, NS1, and the 3' untranslated region were the most tolerant of insertions. In contrast, prM- and NS2A-encoding regions were largely intolerant of insertions. Notably, the multifunctional NS1 protein readily tolerated insertions in regions within the Wing, connector, and bbeta;-ladder domains with minimal effects on viral RNA replication and infectious virus production. Using this information, we generated infectious reporter viruses, including a variant encoding the APEX2 electron microscopy tag in NS1 that uniquely enabled high-resolution imaging of its localization to the surface and interior of viral replication vesicles. In addition, we generated a tagged virus bearing an mScarlet fluorescent protein insertion in NS1 that, despite an impact on fitness, enabled live cell imaging of NS1 localization and traffic in infected cells. Overall, this genome-wide profile of DENV genome flexibility may be further dissected and exploited in reporter virus generation and antiviral strategies.

    IMPORTANCE Regions of genetic flexibility in viral genomes can be exploited in the generation of reporter virus tools and should arguably be avoided in antiviral drug and vaccine design. Here, we subjected the DENV genome to high-throughput insertional mutagenesis to identify regions of genetic flexibility and enable tagged reporter virus generation. In particular, the viral NS1 protein displayed remarkable tolerance of small insertions. This genetic flexibility enabled generation of several novel NS1-tagged reporter viruses, including an APEX2-tagged virus that we used in high-resolution imaging of NS1 localization in infected cells by electron microscopy. For the first time, this analysis revealed the localization of NS1 within viral replication factories known as "vesicle packets" (VPs), in addition to its acknowledged localization to the luminal surface of these VPs. Together, this genetic profile of DENV may be further refined and exploited in the identification of antiviral targets and the generation of reporter virus tools.

  • Exploring the Human-Nipah Virus Protein-Protein Interactome [Virus-Cell Interactions]

  • Nipah virus is an emerging, highly pathogenic, zoonotic virus of the Paramyxoviridae family. Human transmission occurs by close contact with infected animals, the consumption of contaminated food, or, occasionally, via other infected individuals. Currently, we lack therapeutic or prophylactic treatments for Nipah virus. To develop these agents we must now improve our understanding of the host-virus interactions that underpin a productive infection. This aim led us to perform the present work, in which we identified 101 human-Nipah virus protein-protein interactions (PPIs), most of which (88) are novel. This data set provides a comprehensive view of the host complexes that are manipulated by viral proteins. Host targets include the PRP19 complex and the microRNA (miRNA) processing machinery. Furthermore, we explored the biologic consequences of the interaction with the PRP19 complex and found that the Nipah virus W protein is capable of altering p53 control and gene expression. We anticipate that these data will help in guiding the development of novel interventional strategies to counter this emerging viral threat.

    IMPORTANCE Nipah virus is a recently discovered virus that infects a wide range of mammals, including humans. Since its discovery there have been yearly outbreaks, and in some of them the mortality rate has reached 100% of the confirmed cases. However, the study of Nipah virus has been largely neglected, and currently we lack treatments for this infection. To develop these agents we must now improve our understanding of the host-virus interactions that underpin a productive infection. In the present work, we identified 101 human-Nipah virus protein-protein interactions using an affinity purification approach coupled with mass spectrometry. Additionally, we explored the cellular consequences of some of these interactions. Globally, this data set offers a comprehensive and detailed view of the host machinery's contribution to the Nipah virus's life cycle. Furthermore, our data present a large number of putative drug targets that could be exploited for the treatment of this infection.

  • Reply to "Highly Efficacious Novel Vaccine, Humoral Immunity, and Ocular Herpes Simplex Virus 1: Reality or Myth?" [Letters to the Editor]

  • Correction for Avey et al., "Discovery of a Coregulatory Interaction between Kaposi's Sarcoma-Associated Herpesvirus ORF45 and the Viral Protein Kinase ORF36" [Author Correction]

  • Correction for Ansari et al., "Constitutive Interferon-Inducible Protein 16-Inflammasome Activation during Epstein-Barr Virus Latency I, II, and III in B and Epithelial Cells" [Author Correction]

  • Correction for Wang et al., "Mitogen-Activated Protein Kinase Kinase 2, a Novel E2-Interacting Protein, Promotes the Growth of Classical Swine Fever Virus via Attenuation of the JAK-STAT Signaling Pathway" [Author Correction]

  • Correction for Perez et al., "A Short cis-Acting Motif in the M112-113 Promoter Region Is Essential for IE3 To Activate M112-113 Gene Expression and Is Important for Murine Cytomegalovirus Replication" [Author Correction]

  • Correction for Yoon et al., "p53-Derived Host Restriction of HIV-1 Replication by Protein Kinase R-Mediated Tat Phosphorylation and Inactivation" [Author Correction]

  • Correction for Cousins et al., "Human Herpesvirus 8 Viral Interleukin-6 Signaling through gp130 Promotes Virus Replication in Primary Effusion Lymphoma and Endothelial Cells" [Author Correction]

  • Classic Spotlight, 2012 and 2013: Articles of Significant Interest Selected from the Journal of Virology Archives by the Editors [Editorial]

  • Vaccination with Recombinant Parainfluenza Virus 5 Expressing Neuraminidase Protects against Homologous and Heterologous Influenza Virus Challenge [Vaccines and Antiviral Agents]

  • Seasonal human influenza virus continues to cause morbidity and mortality annually, and highly pathogenic avian influenza (HPAI) viruses along with other emerging influenza viruses continue to pose pandemic threats. Vaccination is considered the most effective measure for controlling influenza; however, current strategies rely on a precise vaccine match with currently circulating virus strains for efficacy, requiring constant surveillance and regular development of matched vaccines. Current vaccines focus on eliciting specific antibody responses against the hemagglutinin (HA) surface glycoprotein; however, the diversity of HAs across species and antigenic drift of circulating strains enable the evasion of virus-inhibiting antibody responses, resulting in vaccine failure. The neuraminidase (NA) surface glycoprotein, while diverse, has a conserved enzymatic site and presents an appealing target for priming broadly effective antibody responses. Here we show that vaccination with parainfluenza virus 5 (PIV5), a promising live viral vector expressing NA from avian (H5N1) or pandemic (H1N1) influenza virus, elicited NA-specific antibody and T cell responses, which conferred protection against homologous and heterologous influenza virus challenges. Vaccination with PIV5-N1 NA provided cross-protection against challenge with a heterosubtypic (H3N2) virus. Experiments using antibody transfer indicate that antibodies to NA have an important role in protection. These findings indicate that PIV5 expressing NA may be effective as a broadly protective vaccine against seasonal influenza and emerging pandemic threats.

    IMPORTANCE Seasonal influenza viruses cause considerable morbidity and mortality annually, while emerging viruses pose potential pandemic threats. Currently licensed influenza virus vaccines rely on the antigenic match of hemagglutinin (HA) for vaccine strain selection, and most vaccines rely on HA inhibition titers to determine efficacy, despite the growing awareness of the contribution of neuraminidase (NA) to influenza virus vaccine efficacy. Although NA is immunologically subdominant to HA, and clinical studies have shown variable NA responses to vaccination, in this study, we show that vaccination with a parainfluenza virus 5 recombinant vaccine candidate expressing NA (PIV5-NA) from a pandemic influenza (pdmH1N1) virus or highly pathogenic avian influenza (H5N1) virus elicits robust, cross-reactive protection from influenza virus infection in two animal models. New vaccination strategies incorporating NA, including PIV5-NA, could improve seasonal influenza virus vaccine efficacy and provide protection against emerging influenza viruses.

  • Correction for Dixit et al., "FUSE Binding Protein 1 Facilitates Persistent Hepatitis C Virus Replication in Hepatoma Cells by Regulating Tumor Suppressor p53" [Author Correction]

  • Correction for Gutierrez et al., "Ets-1 Is Required for the Activation of VEGFR3 during Latent Kaposi's Sarcoma-Associated Herpesvirus Infection of Endothelial Cells" [Author Correction]

  • Correction for Gjyshi et al., "Activated Nrf2 Interacts with Kaposi's Sarcoma-Associated Herpesvirus Latency Protein LANA-1 and Host Protein KAP1 To Mediate Global Lytic Gene Repression" [Author Correction]

  • Articles of Significant Interest Selected from This Issue by the Editors [Spotlight]

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    JVI Accepts: Articles Published Ahead of Print

  • Identification of a small molecule that compromises the structural integrity of viroplasms and rotavirus double-layered particles [PublishAheadOfPrint]

  • Despite the availability of two attenuated vaccines, rotavirus (RV) gastroenteritis remains an important cause of mortality among children in developing countries causing about 215,000 infant deaths annually. Currently, there are no specific antiviral therapies available. RV is a non-enveloped virus with a segmented double-stranded RNA genome. Viral genome replication and assembly of transcriptionally active double-layered particles (DLPs) take place in cytoplasmic viral structures called viroplasms. In this study, we describe strong impairment of the early stages of RV replication induced by a small molecule known as RNA polymerase III inhibitor, ML-60218 (ML). This compound was found to disrupt already assembled viroplasms and hamper the formation of new ones without the need of de novo transcription of cellular RNAs. This phenotype correlated with reduction in accumulated viral proteins and newly made viral genome segments, disappearance of the hyperphosphorylated isoforms of the viroplasm-resident protein NSP5 and inhibition of infectious progeny virus production. In in vitro transcription assays with purified DLPs, ML showed a dose-dependent inhibitory activity indicating the viral nature of its target. ML was found to interfere with the formation of higher order structures of VP6, the protein forming the DLP outer layer, without compromising its ability to trimerize. Electron microscopy of ML-treated DLPs showed a dose-dependent structural damage. Our data suggest that interactions between VP6 trimers are essential not only for DLP stability but also for the structural integrity of viroplasms in infected cells.

    IMPORTANCE Rotavirus gastroenteritis is responsible for a large number of infant deaths in developing countries. Unfortunately, in those countries where effective vaccines are urgently needed, the efficacy of the available vaccines is particularly low. Therefore, the development of antivirals is an important goal, as they might complement the available vaccines or represent an alternative option. Moreover, they may be decisive in fighting the acute phase of infection. This work describes the inhibitory effect on rotavirus replication of a small molecule initially reported as an RNA polymerase III inhibitor. The molecule is the first chemical compound identified able to disrupt viroplasms, the viral replication machinery, and to compromise the stability of DLPs by targeting the viral protein VP6. This molecule thus represents a starting point towards the development of more potent and less cytotoxic compounds against rotavirus infection.

  • Entry of human coronavirus NL63 to the cell. [PublishAheadOfPrint]

  • First steps of human coronavirus NL63 (HCoV-NL63) infection were previously described. The virus binds to target cells by heparan sulfate proteoglycans, and interacts with the ACE2 protein. Subsequent events, including virus internalization and trafficking, remain to be elucidated. In this study, we mapped the process of HCoV-NL63 entry into LLC-Mk2 cell line and ex vivo 3D tracheobronchial tissue.

    Using a variety of techniques we have shown that HCoV-NL63 virions require endocytosis for successful entry to the LLC-MK2 cells, and interaction between the virus and the ACE2 molecule triggers recruitment of clathrin. Subsequent vesicle scission by dynamin results in virus internalization, and the newly formed vesicle passes the actin cortex, what requires active cytoskeleton rearrangement. Finally, acidification of the endosomal microenvironment is required for successful fusion and release of viral genome into the cytoplasm. Also for 3D tracheobronchial tissue cultures we observed that the virus enters the cell by clathrin-mediated endocytosis, but obtained results suggest that this pathway may be bypassed.

    IMPORTANCE Available data on coronavirus' entry originate frequently from studies employing immortalized cell lines or undifferentiated cells. Here, using the most advanced 3D tissue culture system mimicking the epithelium of conductive airways, we systematically mapped HCoV-NL63 entry into susceptible cell. Obtained data allow for better understanding of the infection process and may support development of novel treatment strategies.

  • Epstein-Barr virus protein EB2 stimulates translation initiation of mRNAs through direct interactions with both PABP and eIF4G [PublishAheadOfPrint]

  • Epstein-Barr virus (EBV) expresses several mRNAs produced from intronless genes that could potentially be unfavorably translated compared to cellular spliced mRNAs. To overcome this situation, the virus encodes an RNA-binding protein (RBP) called EB2, previously found to both facilitate the export of nuclear mRNAs and increase their translational yield. Here, we show that EB2 binds both nuclear and cytoplasmic cap-binding complexes (respectively, CBC and eIF4F) as well as the poly(A)-binding protein (PABP) to enhance translation initiation of a given mRNP. Interestingly, such an effect can only be obtained if EB2 is initially bound to the native mRNPs in the nucleus. We also demonstrate that the EB2-eIF4F-PABP association renders translation of these mRNPs less sensitive to translation initiation inhibitors. Taken together, our data suggest that EB2 binds and stabilizes cap-binding complexes in order to increase mRNP translation and furthermore demonstrates the importance of the mRNP assembly process in the nucleus to promote protein synthesis in the cytoplasm.

    IMPORTANCE Most herpesvirus early and late genes are devoid of introns. However, it is now well documented that mRNA splicing facilitates recruitment on the mRNA of cellular factors involved in nuclear mRNA export and translation efficiency. To overcome the absence of splicing of herpesvirus mRNAs, a viral protein - EB2 in the case of Epstein-Barr virus - is produced to facilitate the cytoplasmic accumulation of viral mRNAs. Although we previously showed that EB2 also specifically enhances translation of its target mRNAs, the mechanism was unknown. Here, we show that EB2 is first recruited to the mRNA cap structure in the nucleus, then interacts with the eIF4G and PABP proteins to enhance the initiation step of translation.

  • HPV Replication Regulation by Acetylation of a Conserved Lysine in the E2 Protein [PublishAheadOfPrint]

  • The Papillomavirus (PV) E2 protein is a sequence specific DNA binding protein that recruits cellular factors to its genome in infected epithelial cells. E2 also binds to and loads the viral E1 DNA helicase at the origin of replication. Post-translational modifications (PTMs) of PV E2 have been identified as potential regulators E2 functions. We recently reported lysine (K) 111 as a target of p300 acetylation in bovine (B)PV. The di-lysines at 111 and 112 are conserved in almost all papillomaviruses. We pursued a mutational approach to query the functional significance of lysine in human (H)PV E2. Amino acid substitutions that prevent acetylation, including arginine, were unable to stimulate transcription and E1 mediated DNA replication. The arginine K111 mutant retained E2 transcriptional repression, nuclear localization, DNA and chromatin binding, and association with E2 binding partners involved in PV transcription and replication. While the replication defective E2 K111R mutant recruited E1 to the viral replication origin, surprisingly, unwinding of the duplex DNA did not occur. In contrast, the glutamine (Q) K111 mutant increased origin melting and stimulated replication compared to wild type E2. These experiments reveal a novel activity of E2 necessary for denaturing the viral origin that likely depends on acetylation of highly conserved lysine 111.

    IMPORTANCE HPV is one of the most common sexually transmitted infections in the United States. Over 200 HPVs have been described and they manifest in a variety of ways; they can be asymptomatic, or can result in benign lesions (papillomas) or progress to malignancy. Although 90% of infections are asymptomatic and resolve easily, HPV 16 and 18 alone are responsible for 70% of all cervical cancers, which are almost entirely caused by HPV infection. Interestingly, 60-90% of other cancers have been linked to HPV. The goal of this research is to further elucidate the mechanisms that regulate and mediate viral replication.

  • Interaction between a unique minor protein and a major capsid protein of Bluetongue virus controls virus infectivity [PublishAheadOfPrint]

  • Among the Reoviridae family of double-stranded RNA viruses, only members of the Orbivirus genus possess a unique structural protein, termed VP6, within their particles. Bluetongue virus (BTV), an important livestock pathogen, is the prototype Orbivirus. The BTV VP6 is an ATP-dependent RNA helicase and it is indispensable for virus replication. In this report, we investigated how VP6 might be recruited to the virus capsid and whether the BTV structural protein VP3, which forms the internal layer of the virus capsid core, is involved in VP6 recruitment. We first demonstrated that VP6 interacts with VP3 and co-localized with VP3 during capsid assembly. A series of VP6 mutants were then generated and, in combination with immunoprecipitation and size-exclusion chromatographic analyses, we demonstrated that VP6 directly interacts with VP3 via a specific region of the C-terminal portion of VP6. Finally, using our reverse genetics system, mutant VP6 proteins were introduced into the BTV genome and interactions between VP6 and VP3 were shown in a live cell system. We demonstrated that BTV strains, possessing a mutant VP6, were replication-deficient in wild-type BSR cells and failed to recruit the viral replicase complex into the virus particle core. Taken together, these data suggest that the interaction between VP3 and VP6 could be important in the packaging of the viral genome and early stages of particle formation.

    IMPORTANCE The orbivirus bluetongue virus (BTV) is the causative agent of bluetongue disease of livestock, often causing significant economical and agricultural impact in the livestock industry. In this report, we have identified the essential region and residues of the unique orbivirus capsid protein VP6, which are responsible for its interaction with other BTV proteins and its subsequent recruitment into the virus particle. The nature and mechanism of these interactions suggest VP6 has a key role in packaging of the BTV genome into the virus particle. As such, this is a highly significant finding, as this new understanding of BTV assembly could be exploited to design novel vaccines and antivirals against bluetongue disease.

  • Initiation, elongation and realignment during influenza virus mRNA synthesis [PublishAheadOfPrint]

  • The RNA-dependent RNA polymerase (RdRp) of the influenza A virus replicates and transcribes the viral genome segments in the nucleus of the host cell. To transcribe these viral genome segments, the RdRp llsquo;snatches' capped RNA oligonucleotides from nascent host cell mRNAs and aligns these primers to the ultimate or penultimate nucleotide of the segments for the initiation of viral mRNA synthesis. It has been proposed that this initiation process is not processive and that the RdRp uses a prime-realign mechanism during transcription. Here we provide in vitro evidence for the existence of this transcriptional prime-realign mechanism, but show that it only functions efficiently for primers that are short or can not stably base pair with the template. In addition, we demonstrate that transcriptional elongation is dependent on the priming loop of the PB1 subunit of the RdRp. We propose that the prime-realign mechanism may be used to rescue abortive transcription initiation events or cope with sequence variation among primers. Overall, these observations advance our mechanistic understanding of how the influenza A virus initiates transcription correctly and efficiently.

    Importance

    The influenza A virus causes severe disease in humans and is considered a major global health threat. The virus replicates and transcribes its genome using an enzyme called the RNA polymerase. To ensure that the genome is amplified faithfully and abundant viral mRNAs are made for viral protein synthesis, the viral RNA polymerase must transcribe the viral genome efficiently. In this report, we characterise a structure inside the polymerase that contributes to the efficiency of viral mRNA synthesis.

  • Adaptive immune responses following Senecavirus A infection in pigs [PublishAheadOfPrint]

  • Senecavirus A (SVA), an emerging picornavirus of swine, causes vesicular disease (VD) that is clinically indistinguishable from foot-and-mouth disease (FMD) in pigs. Many aspects of SVA interactions with the host and the host immune responses to infection, however, remain unknown. In the present study, humoral and cellular immune responses to SVA were evaluated following infection in pigs. We show that SVA infection elicited an early and robust neutralizing antibody (NA) response, which coincided with and was strongly correlated with VP2- and VP3-specific IgM responses. Notably, NA responses paralleled with reduction of viremia and resolution of the disease. Analysis of the major porcine T cell subsets revealed that during the acute/clinical phase of SVA infection (14 days post-infection; pi) T cell responses were characterized by an increased frequency of aalpha;bbeta; T cells, especially CD4+ T cells which were initially detected by day 7 pi and increased in frequency until day 14 pi. Additionally, the frequency of CD8+ and double-positive CD4+CD8+ T cells (effector/memory T cells) expressing IFN- or proliferating in response to SVA-antigen stimulation increased after day 10 pi. Results here show that SVA elicits B and T cell activation early upon infection, with IgM antibody levels being associated with early neutralizing activity against the virus and peak B and T-cell responses paralleling clinical resolution of the disease. The work provides important insights on the immunological events that follow SVA infection in the natural host.

    Importance

    Senecavirus A (SVA) has recently emerged in swine causing outbreaks of vesicular disease (VD) in major swine producing countries around the world, including the US, Brazil, China, Thailand and Colombia. Notably, SVA-induced disease is clinically indistinguishable from other high consequence VDs of swine such as FMD, swine vesicular disease, vesicular stomatitis and vesicular exanthema of swine. Despite the clinical relevance of SVA caused VD, many aspects of the virus infection biology remain unknown. Here, we assessed host immune responses to SVA infection. Results show that SVA infection elicits early B and T cell responses, with the levels of NA and CD4+ T cell responses paralleling with reduction of viremia and resolution of the disease. SVA-specific CD8+ T cells are detected later during infection. A better understanding of SVA interactions with the host immune system may allow the design and implementation of improved control strategies for this important pathogen of swine.

  • Autographa californica nucleopolyhedrovirus AC141 (Exon0), a potential E3 ubiquitin ligase, interacts with viral ubiquitin and AC66 to facilitate nucleocapsid egress [PublishAheadOfPrint]

  • During the infection cycle of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) two forms of virions are produced, budded virus (BV) and occlusion derived virus (ODV). Nucleocapsids that form BV have to egress from the nucleus whereas nucleocapsids that form ODVs remain inside the nucleus. The molecular mechanism that determines whether nucleocapsids remain inside or egress from the nucleus is unknown. AC141 (a predicted E3 ubiquitin ligase) and viral ubiquitin (vUbi) have both been shown to be required for efficient BV production. In this study, it was hypothesized that vUbi interacts with AC141 and in addition, this interaction was required for BV production. Deletion of both ac141 and vubi restricted viral infection to a single cell and BV production was completely eliminated. AC141 was ubiquitinated by either vUbi or cellular Ubi and this interaction was required for optimal BV production. Nucleocapsids in BV but not ODV were shown to be specifically ubiquitinated by vUbi including a 100 kDa protein as well as high molecular weight conjugates. The viral ubiquitinated 100 kDa BV specific nucleocapsid protein was identified as AC66, which is known to be required for BV production, and was shown by co-immunoprecipitation and mass spectrometry to interact with AC141. Confocal microscopy also showed that AC141, AC66 and vUbi interact at the nuclear periphery. These results suggest ubiquitination of nucleocapsid proteins by vUbi functions as a signal for determining if a nucleocapsid will egress from the nucleus and form BV or remain in the nucleus to form ODV.

    IMPORTANCE Baculoviruses produce two types of virions called occlusion derived virus (ODV) and budded virus (BV). ODV are required for oral infection whereas BV enables the systemic spread of virus to all host tissues which are critical for killing insects. One of the important steps for BV production is the export of nucleocapsids out of the nucleus. This study investigated the molecular mechanisms that enable the selection of nucleocapsids for nuclear export instead of being retained within the nucleus where they would become ODV. Our data show that ubiquitination, a universal cellular process, specifically tags nucleocapsids of BV but not those found in ODV using a virus encoded ubiquitin (vUbi). Therefore ubiquitination may be the molecular signal that determines if a nucleocapsid is destined to form a BV and thus ensuring a lethal infection of the host.

  • Truncating the Gp41 Cytoplasmic Tail of SIV Decreases Sensitivity to Neutralizing Antibodies without Increasing Envelope Content of Virions [PublishAheadOfPrint]

  • An incomplete understanding of native HIV and SIV envelope glycoprotein (Env) impedes the development of structural models of Env and vaccine design. This shortcoming is due in part to the low number of Env trimers on virus particles. For SIV, this low expression can be counteracted by truncating the cytoplasmic tail (CT) of Env. CT truncation has been shown to increase Env incorporation into the virion and is commonly used in vaccine and imaging studies, but its effects on viral antigenicity have not been fully elucidated. To study the effects of CT truncation of Env in viruses with similar genetic contexts, we introduced stop codons into the CT of a SIVsmE660 molecular clone and two neutralizing antibody (NAb) escape variants. These viruses shared 98% sequence identity in Env but were characterized as either Tier-1 (sensitive to neutralization), Tier-2 (moderately resistant), or Tier-3 (resistant). However, introduction of premature stop codons in Env at position Q741/Q742 converted all three transfection-derived viruses to a Tier-3-like phenotype that were uniformly resistant to neutralization by sera from infected macaques and monoclonal antibodies (mAbs). These changes in neutralization sensitivity were not accompanied by an increase in either virion Env content of infection-derived viruses or infectivity of transfection-derived viruses in human cells, suggesting that CT mutations may result in global changes to Env conformation. Our results demonstrate that some CT truncations can affect viral antigenicity, and as such may not be suitable surrogate models of the native HIV/SIV Env.

    IMPORTANCE

    Modifications to the SIV envelope protein (Env) are commonly used in structural and vaccine studies to stabilize and increase the expression of Env, often without consideration for effects on antigenicity. One such widespread modification is truncation of the Env C-terminal tail. Here, we studied the effects of a particular cytoplasmic tail truncation in three SIVsm strains that have highly similar Env sequence but exhibit different sensitivities to neutralizing antibodies. After truncation of the Env CT, these viruses were all very resistant to neutralization by sera from infected macaques and monoclonal antibodies. The viruses with truncated Env CT also did not exhibit the desired and typical increase in Env expression. These results underscore the importance of carefully evaluating the use of truncated Env as a model in HIV/SIV vaccine and imaging studies, and of the continued need to find better models of native Env that contain fewer modifications.

  • TNF-mediated survival of CD169+ cells promotes immune activation during vesicular stomatitis virus infection [PublishAheadOfPrint]

  • Innate immune activation is essential to mount an effective antiviral response and to prime adaptive immunity. Although a crucial role of CD169+ cells during vesicular stomatitis virus (VSV) infections is increasingly recognized, factors regulating CD169+ cells during viral infections remain unclear. Here we show that tumor necrosis factor is produced by CD11b+ Ly6C+Ly6G+ cells following infection with VSV. The absence of TNF or TNF receptor 1 (TNFR1) resulted in reduced numbers of CD169+ cells and in reduced IFN-I production during VSV infection, with a severe disease outcome. Specifically, TNF triggered RelA translocation into the nucleus of CD169+ cells; this translocation was inhibited when paracaspase MALT-1 was absent. Consequently, MALT1 deficiency resulted in reduced VSV replication, defective innate immune activation, and severe disease development. These findings indicate that TNF mediates the maintenance of CD169+ cells and innate and adaptive immune activation during VSV infection.

    IMPORTANCE Over the last decade, strategically placed CD169+ metallophilic macrophages in the marginal zone of the murine spleen and LN have been shown to play a very important role in host defense against viral pathogens. CD169+ macrophages are shown to activate innate and adaptive immunity via "enforced virus replication" a controlled amplification of virus particles. However, factors regulating the CD169+ macrophages remain to be studied. In this paper, we show that after Vesicular stomatitis virus infection, phagocytes produce tumor necrosis factor (TNF) which signals via TNFR1 and promote "enforced virus replication" in CD169+ macrophages. Consequently, lack of TNF or TNFR1 resulted in defective immune activation and VSV clearance.

  • Size, Composition, and Evolution of HIV DNA Populations during Early Antiretroviral Therapy and Intensification with Maraviroc [PublishAheadOfPrint]

  • Residual viremia is common during antiretroviral therapy (ART), and could be caused by ongoing low-level virus replication or by release of viral particles from infected cells. ART Intensification should impact ongoing viral propagation but not virion release. Eighteen acutely infected men were enrolled in a randomized controlled trial, and followed for a median of 107 weeks. Participants started ART with (n=9) or without (n=9) intensification with maraviroc (MVC) within 90 days of infection. Levels of HIV DNA and cell-free RNA were quantified by droplet digital PCR. Deep sequencing of C2-V3 env, gag and pol (454-Roche) was performed on longitudinally collected plasma and PBMC samples while on ART. Sequence data were analyzed for evidence of evolution by: 1) molecular diversity analysis, 2) non-parametric test for panmixia, 3) tip-date randomization within Bayesian framework. There was a longitudinal decay of HIV DNA after initiation of ART with no difference between MVC intensification groups (-0.08pplusmn;0.01 vs -0.09pplusmn;0.01 log10 copies/week in MVC+ vs MVC-, p=0.62). All participants had low-level residual viremia (median: 2.8 RNA copies/mL). Across participants a median of 56 (IQR:36-74), 29 (IQR:25-35) and 40 (IQR:31-54) haplotypes were generated for env, gag and pol regions, respectively. There was no clear evidence of viral evolution during ART and no difference in viral diversity or population structure from individuals with or without MVC intensification. Further efforts focusing on elucidating the mechanism(s) of viral persistence in various compartments using recent sequencing technologies are still needed and potential low-level viral replication should always be considered in cure strategies.

    IMPORTANCE Residual viremia is common among HIV-infected people on antiretroviral therapy (ART). It remains controversial if this viremia is a consequence of propagating infection. We hypothesized that molecular evolution would be detectable during viral propagation, and that therapy intensified with the entry inhibitor maraviroc would demonstrate less evolution. We performed a randomized double-blinded treatment trial with 18 acutely infected men (standard ART versus standard ART+maraviroc). From longitudinally collected blood plasma and cells, levels of HIV DNA and cell-free HIV RNA were quantified by droplet digital PCR, and HIV DNA (env, gag and pol coding regions) was deep sequenced (454 Roche). Investigating people who started ART during the earliest stages of their HIV infection, when viral diversity is small, provides an opportunity to detect evidence of viral evolution. Despite using a battery of analytical techniques, no clear and consistent evidence of viral propagation for over 90 weeks of observation could be discerned.

  • Effective priming of HSV-specific CD8+ T cells in vivo does not require infected dendritic cells [PublishAheadOfPrint]

  • Resolution of virus infections depends on the priming of virus-specific CD8+ T cells by dendritic cells (DC). While this process requires MHC class I-restricted antigen presentation by DC, the relative contribution to CD8+ T cell priming by infected DC is less clear. We have addressed this question in the context of a peripheral infection with herpes simplex virus type 1 (HSV). Assessing the endogenous, polyclonal HSV-specific CD8+ T cell response, we found that effective in vivo T cell priming depended on the presence of DC subsets specialized in cross-presentation while Langerhans cells and plasmacytoid DC were dispensable. Utilizing a novel mouse model that allows for the in vivo elimination of infected DC, we also demonstrate in vivo that this requirement for cross-presenting DC was not related to their infection, but instead reflected their capacity to cross-present HSV-derived antigen. Taken together, this study shows that infected DC are not required for effective CD8+ T cell priming during a peripheral virus infection.

    IMPORTANCE The ability of some DC to present viral antigen to CD8+ T cells without being infected is thought to enable the host to induce killer T cells even when viruses evade or kill infected DC. However, direct experimental in vivo proof for this notion has remained elusive. Work described in this study characterizes the role that different DC play in the induction of virus-specific killer T cell responses and, critically, introduces a novel mouse model that allows for the selective elimination of infected DC in vivo. Our finding that HSV-specific CD8+ T cells can be fully primed in the absence of DC infection shows that cross-presentation by DC is indeed sufficient for effective CD8+ T cell priming during a peripheral virus infection.

  • The Epstein-Barr virus episome maneuvers between nuclear chromatin compartments during reactivation [PublishAheadOfPrint]

  • The human genome is structurally organized in three-dimensional space to facilitate functional partitioning of transcription. We learned that the latent episome of the human Epstein-Barr virus (EBV) preferentially associates with gene-poor chromosomes and avoids gene-rich chromosomes. Kaposi's sarcoma-associated herpesvirus behaves similarly, but human papillomavirus does not. Contacts localize on the EBV side to OriP, the latent origin of replication. This genetic element, and the EBNA1 protein that binds there, are sufficient to reconstitute chromosome association preferences of the entire episome. Contacts localize on the human side to gene-poor and AT-rich regions of chromatin distant from transcription start sites. Upon reactivation from latency, however, the episome moves away from repressive heterochromatin and toward active euchromatin. Our work adds three-dimensional relocalization to the molecular events that occur during reactivation. Involvement of a myriad of interchromosomal associations also suggests a role for this type of long-range association in gene regulation.

    IMPORTANCE The human genome is structurally organized in three-dimensional space, and this structure functionally affects transcriptional activity. We set out to investigate whether a double stranded DNA virus, Epstein-Barr virus (EBV), uses similar mechanisms as the human genome to regulate transcription. We found that the EBV genome associates with repressive compartments of the nucleus during latency and active compartments during reactivation. This study is advances our knowledge of the EBV life cycle, adding three-dimensional re-localization as a novel component to the molecular events that occur during reactivation. Furthermore, the data adds to our understanding of nuclear compartments, showing that disperse interchromosomal interactions may be important for regulating transcription.

  • Emergence of double- and triple-gene reassortant G1P[8] rotaviruses possessing a DS-1-like backbone post rotavirus vaccine introduction in Malawi [PublishAheadOfPrint]

  • To combat the high burden of rotavirus gastroenteritis, multiple African countries have introduced rotavirus vaccines into their childhood immunisation programmes. Malawi incorporated a G1P[8] rotavirus vaccine (Rotarixttrade;) into its immunisation schedule in 2012. Utilising a surveillance platform of hospitalised rotavirus gastroenteritis cases, we examined the phylodynamics of G1P[8] rotavirus strains that circulated in Malawi before (1998 nndash; 2012) and after (2013 nndash; 2014) vaccine introduction. Analysis of whole genomes obtained through next generation sequencing revealed that all randomly-selected pre-vaccine G1P[8] strains sequenced (n=32) possessed a Wa-like genetic constellation, whereas post-vaccine G1P[8] strains (n=18) had a DS-1-like constellation. Phylodynamic analyses indicated that post-vaccine G1P[8] strains emerged through reassortment events between human Wa- and DS-1-like rotaviruses that circulated in Malawi from the 1990's, hence classified as atypical DS-1-like reassortants. The time to the most recent common ancestor for G1P[8] strains was from 1981-1994; their evolutionary rates ranged from 9.7 x 10-4nndash;4.1 x 10-3 nucleotide/substitutions/site/year. Three distinct G1P[8] lineages chronologically replaced each other between 1998 and 2014. Genetic drift was the likely driver for lineage turnover in 2005, whereas replacement in 2013 was due to reassortment. Amino acid substitution within the outer glycoprotein VP7 of G1P[8] strains had no impact on the structural conformation of the antigenic regions, suggesting that it is unlikely that they would affect recognition by vaccine-induced neutralizing antibodies. While the emergence of DS-1-like G1P[8] rotavirus reassortants in Malawi was therefore likely due to natural genotype variation, vaccine effectiveness against such strains needs careful evaluation.

    Importance

    The error-prone RNA-dependent RNA polymerase and the segmented RNA genome predispose rotaviruses to genetic mutation and genome reassortment, respectively. These evolutionary mechanisms generate novel strains and have the potential to lead to the emergence of vaccine-escape mutants. While multiple African countries have introduced rotavirus vaccine, there are few data describing the evolution of rotaviruses that circulated before and after vaccine introduction. We report the emergence of atypical DS-1-like G1P[8] strains during the post-vaccine era in Malawi. Three distinct G1P[8] lineages circulated chronologically from 1998nndash;2014; mutation and reassortment drove lineage turnover in 2005 and 2013, respectively. Amino acid substitutions within the outer capsid VP7 glycoprotein did not affect the structural conformation of mapped antigenic sites, suggesting limited effect in recognition of G1 specific vaccine-derived antibodies. The genes that constitute the remaining genetic backbone may play important roles in immune evasion, and vaccine effectiveness against such atypical strains needs careful evaluation.

  • Single Dose Trivalent Vesiculovax Vaccine Protects Macaques from Lethal Ebolavirus and Marburgvirus Challenge [PublishAheadOfPrint]

  • Previous studies demonstrated that a single intramuscular (IM) dose of an attenuated vesicular stomatitis virus vector (Vesiculovaxttrade;, rVSV-N4CT1) expressing the glycoprotein (GP) from the Mayinga strain of Zaire ebolavirus (EBOV) protected nonhuman primates (NHP) from lethal challenge with EBOV Kikwit and Makona strains. Here we studied the immunogenicity of an expanded range of attenuated rVSV vectors expressing filovirus GP in mice. Based on data from those studies an optimal attenuated tri-valent rVSV vector formulation was identified which included rVSV vectors expressing EBOV, Sudan ebolavirus (SUDV) or Angola strain of Marburg marburgvirus (MARV) GPs. NHPs were then vaccinated with a single dose of the tri-valent formulation, followed by lethal challenge 28 days later with each of the three corresponding filoviruses. At day 14 post vaccination, a serum IgG response specific for all three GPs was detected in all vaccinated macaques. A modest and balanced cell-mediated immune response specific for each GP protein was also detected in a majority of vaccinated macaques. No matter the level of total GP-specific immune response detected post vaccination, all vaccinated macaques were protected from disease and death following lethal challenge with each of the three filoviruses. These findings indicate that vaccination with a single dose of attenuated rVSV-N4CT1 vectors each expressing a single filovirus GP may provide protection against those filoviruses most commonly responsible for outbreaks of hemorrhagic fever in sub-Saharan Africa.

    IMPORTANCE The West African Ebola Zaire outbreak in 2013 showed that this disease was not only a regional concern, but a worldwide problem and highlighted the need for a safe and efficacious vaccine to be administered to the populace. However, other endemic pathogens like Ebola Sudan and Marburg also pose an important health risk to the public and therefore require development of a vaccine prior to the occurrence of an outbreak. The significance of our research was the development of a blended tri-valent filovirus vaccine that would elicit a balanced immune response when administered as a single dose and provide complete protection against a lethal challenge of all three filovirus pathogens.

  • Human adenovirus infection causes the cellular MKRN1 E3 ubiquitin ligase degradation involving the viral core protein pVII [PublishAheadOfPrint]

  • Human adenoviruses (HAdVs) are common human pathogens encoding a highly abundant histone-like core protein VII, which is involved in nuclear delivery and protection of viral DNA as well as in sequestering immune danger signals in infected cells. The molecular details of how protein VII acts as a multifunctional protein have remained to a large extent enigmatic. Here we report the identification of several cellular proteins interacting with the precursor pVII protein. We show that the cellular E3 ubiquitin ligase MKRN1 is a novel precursor pVII interacting protein in HAdV-C5-infected cells. Surprisingly, the endogenous MKRN1 protein underwent proteasomal degradation during the late phase of HAdV-C5 infection in various human cell lines. The MKRN1 protein degradation occurred independently of the HAdV E1B55K and E4orf6 proteins. We provide experimental evidence that the precursor pVII protein binding enhances MKRN1 self-ubiquitination, whereas the processed mature VII protein is deficient in this function. Based on these data, we propose that the pVII protein binding promotes MKRN1 self-ubiquitination followed by proteasomal degradation of the MKRN1 protein in HAdV-C5-infected cells. In addition, we show that measles virus and vesicular stomatitis virus infections reduce the MKRN1 protein accumulation in the recipient cells. Taken together, our results expand the functional repertoire of the HAdV-C5 precursor pVII protein in lytic virus infection and highlight MKRN1 as a potential common target during different virus infections.

    IMPORTANCE Human adenoviruses (HAdVs) are common pathogens causing a wide range of diseases. To achieve pathogenicity HAdVs have to counteract a variety of host cell antiviral defense systems, which would otherwise hamper virus replication. In this study, we show that the HAdV-C5 histone-like core protein pVII binds to and promotes self-ubiquitination of a cellular E3 ubiquitin ligase named as MKRN1. This mutual interaction between the pVII and MKRN1 proteins may prime MKRN1 for proteasomal degradation because the MKRN1 protein is efficiently degraded during the late phase of HAdV-C5 infection. Since the MKRN1 protein accumulation is also reduced in measles virus and vesicular stomatitis virus infected cells, our results signify the general strategy of viruses to target MKRN1.

  • The analysis of KV mimivirus major capsid gene and its transcript highlights a distinct pattern of gene evolution and splicing among mimiviruses [PublishAheadOfPrint]

  • The inclusion of Mimiviridae members in the putative monophyletic NCLDV group is based on genomic and phylogenomic patterns. This shows that, along with other viral families, they share a set of genes known as core or "hallmark genes", including the major capsid protein (MCP). Although previous studies have suggested that the maturation of mimivirus MCP transcripts is dependent on splicing, there is little information about the processing of this transcript in other mimivirus isolates. In this work, we report the characterization of a new mimivirus isolate, called KV mimivirus. Analysis of the structure, syntheny and phylogenetic relationships of the MCP gene in many mimivirus isolates revealed a remarkable variation at position and types of intronic and exonic regions, even for mimiviruses belonging to the same lineage. In addition, sequencing of KV and APMV MCP transcripts has shown that, inside the family, even related giant viruses may present different ways to process the MCP mRNA. These results contribute to the understanding of the genetic organization and evolution of the MCP gene in mimiviruses.

    Importance

    Mimivirus isolates have been obtained by prospecting studies since 2003. Based on genomic and phylogenomic studies of conserved genes, these viruses have been clustered together with members of six other viral families. Although being an important part of the so called "hallmark genes", there is little information about the processing and structure of the major capsid protein (MCP) gene in many mimivirus isolates. In this work, we have analyzed the structure, syntheny and phylogenetic relationships of the MCP gene in many mimivirus isolates, showing remarkable variation at position and types of intronic and exonic regions, even for mimiviruses belonging to the same lineage. These results contribute to the understanding of the genetic organization and evolution of the MCP gene in mimiviruses.

  • A mechanism for prime-realignment during influenza A virus replication [PublishAheadOfPrint]

  • The influenza A virus genome consists of eight segments of single-stranded RNA. These segments are replicated and transcribed by a viral RNA-dependent RNA polymerase (RdRp) that is made up of the influenza virus proteins PB1, PB2 and PA. To copy the viral RNA (vRNA) genome segments and the complementary RNA (cRNA) segments, the replicative intermediate of viral replication, the RdRp must use two promoters and two different de novo initiation mechanisms. On the vRNA promoter, the RdRp initiates on the 3rrsquo; terminus, while on the cRNA promoter the RdRp initiates internally and subsequently realigns the nascent vRNA product to ensure that the template is copied in full. In particular the latter process, which is also used by other RNA viruses, is not understood. Here we provide mechanistic insight into prime-realignment during influenza virus replication and show that it is controlled by the priming loop and a helix-loop-helix motif of the PB1 subunit of the RdRp. Overall, these observations advance our understanding of how the influenza A virus initiates viral replication and amplifies the genome correctly.

    IMPORTANCE Influenza A viruses cause severe disease in humans and are considered a major threat to our economy and health. The viruses replicate and transcribe their genome using an enzyme called the RNA polymerases. To ensure that the genome is amplified faithfully and abundant viral mRNAs are made for viral protein synthesis, the RNA polymerase must work correctly. In this report, we provide insight into the mechanism that the RNA polymerase employs to ensure that the viral genome is copied correctly.

  • ALT-803 transiently reduces SIV replication in the absence of antiretroviral treatment [PublishAheadOfPrint]

  • Developing biological interventions to control HIV replication in the absence of antiretroviral therapy (ART) could contribute to the development of a functional cure. As a potential alternative to ART, the IL-15 superagonist ALT-803 has been shown to boost the number and function of HIV-specific CD8+ T and NK cell populations, in vitro. Four SIV+ rhesus macaques, three of whom possessed MHC alleles associated with control of SIV, and all of whom had received SIV vaccine vectors that had the potential to elicit CD8+ T cell responses, were given ALT-803 in three treatment cycles. The first and second cycles of treatment were separated by two weeks, while the third cycle was administered after a 29-week break. ALT-803 transiently elevated the total CD8+ effector and central memory T cell and NK cell populations in peripheral blood, while viral loads transiently decreased by ~2 logs in all animals. Virus suppression was not sustained as T cells became less responsive to ALT-803 and waned in numbers. No effect on viral loads was observed in the second cycle of ALT-803, concurrent with downregulation of the IL-2/15 common -C and bbeta; chain receptors on both CD8+ T cells and NK cells. Furthermore, populations of immunosuppressive T cells increased during the second cycle of ALT-803. During the third treatment cycle, responsiveness to ALT-803 was restored. CD8+ T cells and NK cells increased again 3-5 fold, and viral loads transiently decreased again, by 1-2 logs.

    Importance:

    Overall, our data suggests that ALT-803 has the potential to be used as an immunomodulatory agent to elicit effective immune control of HIV/SIV replication. We identify mechanisms to explain why virus control is transient, so that this model can be used to define a clinically appropriate treatment regimen.

  • Estimating initial viral levels during HIV/SIV reactivation from latency [PublishAheadOfPrint]

  • HIV viremia rebounds rapidly after treatment interruption, and a variety of strategies are being explored to reduce or control viral reactivation post-treatment. This viral rebound arises from reactivation of individual latently infected cells, which spread during ongoing rounds of productive infection. The level of virus produced by the initial individual reactivating cells is not known, although it may have major implications for the ability of different immune interventions to control viral rebound. Here we use data from both HIV and SIV treatment interruption studies to estimate the initial viral load post-interruption and thereby the initial individual reactivation event. Using a barcoded virus (SIVmac239M) to track reactivation from individual latent cells, we use the observed viral growth rates and frequency of reactivation to model the dynamics of reactivation to estimate that a single reactivated latent cell can produce an average viral load equivalent to 0.1-0.5 vRNA copies/ml. Modelling of treatment interruption in HIV suggests an initial viral load equivalent of 0.6-1 vRNA copies/ml. These low viral loads immediately following latent cell reactivation provide a window of opportunity for viral control by host immunity, before further replication allows viral spread. This work shows initial levels of viral production that must be controlled in order to successfully suppress HIV reactivation following treatment interruption.

    Importance:

    Current treatment for HIV is able to suppress viral replication and prevent disease progression. However, treatment cannot eradicate infection, because the virus lies silent within latently infected cells. If treatment is stopped, the virus usually rebounds above the level of detection within a few weeks. There are a number of approaches being tested aimed at either eradicating latently infected cells, or controlling the virus if it returns.

    Studying both the small pool of latently infected cells and the early events during viral reactivation are difficult, because these involve very small levels of virus that are difficult to measure directly. Here, we combine experimental data and mathematical modelling to understand the very early events during viral reactivation from latency in both HIV infection of humans and SIV infection of monkeys. We find that the initial levels of virus are low, which may help in designing therapies to control early viral reactivation.

  • Peripheral blood biomarkers of disease outcome in a monkey model of Rift Valley Fever encephalitis [PublishAheadOfPrint]

  • Rift Valley Fever (RVF) is an emerging arboviral disease of livestock and people. Although a mosquito-borne virus, humans are infected through contact with or inhalation of virus-laden particles from contaminated animal carcasses. Some individuals infected with RVF virus (RVFV) develop meningoencephalitis resulting in morbidity and mortality. Little is known about the pathogenic mechanisms that lead to neurologic sequelae, and thus animal models that represent human disease are needed. African green monkeys (AGM) exposed to aerosols containing RVFV develop a reproducibly lethal neurological disease that resembles human illness. To understand the disease process and identify biomarkers of lethality, two groups of 5 AGM were infected by inhalation with either a lethal or sub-lethal dose of RVFV. Divergence between lethal and sub-lethal infections occurred as early as 2 days post-infection (dpi) at which point CD8+ T cells from lethally-infected AGM expressed activated caspase-3 and simultaneously failed to increase levels of MHC Class II molecules compared to surviving animals. At 4 dpi, lethally-infected animals failed to demonstrate proliferation of total CD4+ and CD8+ T cells compared to survivors. These marked changes in peripheral blood cells occur much earlier than more established indicators of severe RVF disease such as granulocytosis and fever. In addition, an early pro-inflammatory (IFN-, IL-6, IL-8, MCP-1) and anti-viral (IFN-aalpha;) response was seen in survivors, while very late cytokine expression was found in the lethal infections. By characterizing immunological markers of lethal disease, this study furthers our understanding of RVF pathogenesis and will allow testing of therapeutics and vaccines in the AGM model.

    Importance

    Rift Valley Fever (RVF) is an important emerging viral disease for which we lack both an effective human vaccine and treatment. Encephalitis and neurological disease resulting from RVF leads to death or significant long-term disability in infected people. African green monkeys (AGM) develop lethal neurological disease when infected with RVF virus by inhalation. Here, we report the similarities in disease course in infected AGM compared to humans. For the first time, we examine the peripheral immune response during the course of infection in AGM and show that there are very early differences in the immune response between animals that survive infection and those that succumb. We conclude that AGM are a novel and suitable monkey model for studying the neuropathogenesis of RVF and for testing vaccines and therapeutics against this emerging viral pathogen.

  • A Y161F Hemagglutinin Substitution Increases Thermostability and Improves Yields of 2009 H1N1 influenza A Virus in Cells [PublishAheadOfPrint]

  • Vaccination is the primary strategy for influenza prevention and control. Yet egg-based vaccines, the predominant production platform, have several disadvantages including the emergence of viral antigenic variants that can be induced during egg passage. These limitations have prompted development of cell-based vaccines which themselves are not without issue. Most importantly, vaccine seed viruses often do not grow efficiently in mammalian cell lines. Here we aimed to identify novel high-yield signatures for influenza viruses in continuous Madin-Darby canine kidney (MDCK) and Vero cells. Using influenza A(H1N1)pdm09 virus as the testing platform and an integrating error-prone PCR strategy, we identified a Y161F mutation in the hemagglutinin (HA) that not only enhanced the infectivity of the resultant virus by more than 300-fold, but also increased its thermostability without changing its original antigenic properties. Vaccine produced from the Y161F mutant fully protected mice against wild-type A(H1N1)pdm09 lethal challenge. Compared with A(H1N1)pdm09, the Y161F mutant had significantly higher avidity for avian-like and human-like receptor analogs. Of note, introduction of the Y161F mutation into the HA of seasonal H3N2 influenza A virus (IAV) and canine H3N8 IAV also increased yields and thermostability in MDCK cells and chicken embryotic eggs. Thus, residue F161 plays an important role in determining viral growth and thermostability, which could be harnessed to optimize IAV vaccine seed viruses.

    IMPORTANCE Although a promising complement to current egg-based influenza vaccines, cell-based vaccines have one big challenge - high-yield vaccine seeds for production. In this study, we identified a molecular signature- Y161F in hemagglutinin (HA) that resulted in increased virus growth in Madin-Darby canine kidney and Vero cells, two commonly used cell lines in influenza vaccine manufacturing. This Y161F mutation not only increased HA thermostability, but also enhanced its binding affinity to aalpha;2,6 and aalpha;2,3-linked Neu5Ac. These results suggest that a vaccine strain bearing the Y161F mutation in HA could potentially increase vaccine yields in mammalian cell culture systems.

  • Unique directional motility of influenza C virus controlled by its filamentous morphology and short-range motions [PublishAheadOfPrint]

  • Influenza virus motility is based on cooperation between two viral spike proteins, hemagglutinin (HA) and neuraminidase (NA), and is a major determinant of virus infectivity. To translocate a virus particle on the cell surface, HA molecules exchange viral receptors and NA molecules accelerate the receptor exchange of HA. This type of virus motility was recently identified in influenza A virus (IAV). To determine if other influenza virus types have a similar receptor-exchange mechanism-driven motility, we investigated influenza C virus (ICV) motility on a receptor-fixed glass surface. This system excludes receptor mobility, which makes it more optimal than a cell surface for demonstrating virus motility by receptor-exchange. Like IAV, ICV was observed to move across the receptor-fixed surface. However, in contrast with the random movement of IAV, a filamentous ICV strain, Ann Arbor/1/50 (AA), moved straight, in a directed manner, and at a constant rate, whereas a spherical ICV strain, Taylor/1233/47 (Taylor), moved randomly, similar to IAV. The AA and Taylor viruses each moved with a combination of gradual (crawling) and rapid (gliding) motions, but the lengths of crawling and gliding for the AA virus were shorter than those of the Taylor virus. Our findings indicate that, like IAV, ICV also has a motility that is driven by the receptor-exchange mechanism. However, compared with IAV movement, filamentous ICV movement is highly regulated in both direction and speed. Control of ICV movement is based on its specific motility employing short crawling and gliding motions as well as its own filamentous morphology.

    IMPORTANCE Influenza virus enters into a host cell for infection via cellular endocytosis. Human influenza virus infects epithelial cells of the respiratory tract, the surfaces of which are hidden by abundant cilia that are inactive in endocytosis. An open question is the manner by which the virus migrates to endocytosis-active domains. In analyzing individual virus behaviors through single-virus tracking, we identified a novel function of the hemagglutinin and esterase of influenza C virus (ICV) as motility machinery. Hemagglutinin iteratively exchanges a viral receptor, causing virus movement. Esterase degrades the receptors along the trajectory traveled by the virus and prevents the virus from moving backward, causing directional movement. We propose that ICV has unique motile machinery directionally controlled via hemagglutinin sensing the receptor density manipulated by esterase.

  • Respiratory Syncytial Virus (RSV): Targeting the G Protein Provides a New Approach for an Old Problem [PublishAheadOfPrint]

  • Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection (LRTI) annually affecting ggt;2 million children in the US llt;5 years old. In the elderly (ggt;65 years old), RSV results in ~175,000 hospitalizations annually in the US with worldwide incidence ~34 million. There is no approved RSV vaccine and treatments are limited. Recently, a Phase 3 trial in the elderly using a recombinant RSV F protein vaccine failed to meet its efficacy objectives, namely prevention of moderate-to-severe RSV-associated LRTI and reduced incidence of acute respiratory disease. Moreover, a recent Phase 3 trial evaluating suptavumab (REGN2222), an antibody to RSV F protein, did not meet its primary endpoint of preventing medically attended RSV infections in pre-term infants. Despite these setbacks, numerous efforts targeting the RSV F protein with vaccines, antibodies, and small molecules continue based on the commercial success of a monoclonal antibody (mAb) against the RSV F protein (palivizumab). As the understanding of RSV biology has improved, the other major coat protein, the RSV G protein, has re-emerged as an alternative target reflecting progress in understanding its roles in infecting bronchial epithelial cells and in altering the host immune response. In mouse models, a high-affinity, strain-independent human mAb to the RSV G protein has shown potent direct antiviral activity combined with the alleviation of virus-induced immune system effects that contribute to disease pathology. This mAb, being prepared for clinical trials, provides a qualitatively new approach to managing RSV for populations not eligible for prophylaxis with palivizumab.

  • c-Myc Represses Transcription of the Epstein-Barr Virus Latent Membrane Protein 1 Early After Primary B Cell Infection [PublishAheadOfPrint]

  • Recent evidence has shown that the EBV oncogene LMP1 is not expressed at high levels early after EBV-infection of primary B cells, despite its being essential for the long-term outgrowth of immortalized lymphoblastoid cell lines (LCLs). In this study, we found that expression of LMP1 increased fifty-fold between seven days post infection and the LCL state. Metabolic labeling of nascently transcribed mRNA indicated this was primarily a transcription-mediated event. EBNA2, the key viral transcription factor regulating LMP1, and CTCF, an important chromatin insulator, were recruited to the LMP1 locus similarly early and late after infection. However, the activating histone H3K9Ac mark was enriched at the LMP1 promoter in LCLs relative to early-infected B cells. We found that high c-Myc activity in EBV-infected lymphoma cells as well as overexpression of c-Myc in an LCL model system repressed LMP1 transcription. Finally, we found that chemical inhibition of c-Myc both in LCLs and early after primary B-cell infection increased LMP1 expression. These data support a model in which high levels of endogenous c-Myc activity induced early after primary B-cell infection directly represses LMP1 transcription.

    IMPORTANCE

    EBV is a highly successful pathogen that latently infects greater than 90% of adults worldwide and is also causally associated with a number of B-cell malignancies. EBV expresses a set of viral oncoproteins and non-coding RNAs during the latent life cycle with the potential to promote cancer. Critical among these is the viral latent membrane protein, LMP1. Prior work suggests that LMP1 is essential for EBV to immortalize B cells, but our recent work indicates that LMP1 is not produced at high levels during the first few weeks after infection. Here, we show that the transcription of LMP1 can be negatively regulated by a host transcription factor, c-Myc. Ultimately, understanding the regulation of EBV-encoded oncogenes will allow us to better treat cancers that rely on these viral products for survival.

  • N-Myc Downstream-Regulated Gene 1 Restricts Hepatitis C Virus Propagation by Regulating Lipid Droplet Biogenesis and Viral Assembly [PublishAheadOfPrint]

  • Host cells harbor various intrinsic mechanisms to restrict viral infections as a first line of antiviral defense. Viruses have evolved various countermeasures against these antiviral mechanisms. Here we show that N-Myc Downstream-Reguated Gene 1 (NDRG1) limits productive HCV infection by inhibiting viral assembly. Interestingly, HCV infection down-regulates NDRG1 protein and mRNA expression. Loss of NDRG1 increases the size and number of lipid droplets, which are the sites of HCV assembly. HCV suppresses NDRG1 expression by up-regulating MYC, which directly inhibits the transcription of NDRG1. Up-regulation of MYC also leads to reduced expression of NDRG1-specific kinase SGK1, resulting in markedly diminished phosphorylation of NDRG1. Knockdown of MYC during HCV infection rescues NDRG1 expression and phosphorylation, suggesting that MYC regulates NDRG1 at both transcriptional and post-translational levels. Overall, our results suggest that NDRG1 restricts HCV assembly by limiting lipid droplet formation. HCV counteracts this intrinsic antiviral mechanism by down-regulating NDRG1 via a MYC-dependent mechanism.

    IMPORTANCE Hepatitis C virus (HCV) is an enveloped single-stranded RNA virus that targets hepatocytes in the liver. HCV is a leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma and estimates suggest a global prevalence of 2.35%. Up to 80% of acutely infected individuals will develop chronic infection and as many as 5% eventually progress to liver cancer. Understanding of the mechanisms behind virus-host interaction and viral carcinogenesis is still lacking. The significance of our research is that it identifies a previously unknown relationship between HCV and a known tumor-associated gene. Further our data point to a new role for this gene in the liver and lipid metabolism. Thus HCV infection serves as a great biological model to advance our knowledge of liver functions and the development of liver cancer.

  • HIV-1 R5 macrophage-tropic envelope glycoprotein trimers bind CD4 with high affinity, while the CD4 binding site on non-macrophage-tropic, T- tropic R5 envelopes is occluded [PublishAheadOfPrint]

  • HIV-1 R5 viruses exploit CCR5 as a coreceptor to infect both T-cells and macrophages. R5 viruses that are transmitted or derived from immune tissue and peripheral blood are mainly inefficient at mediating infection of macrophages. In contrast, highly macrophage-tropic R5 viruses predominate in brain tissue and can be detected in cerebral spinal fluid, but are infrequent in immune tissue or blood even in late disease. These mac-tropic R5 variants carry envelope glycoproteins (Envs) adapted to exploit low levels of CD4 on macrophages to induce infection. However, it is unclear whether this adaptation is conferred by an increased affinity of the Env trimer for CD4 or is mediated by post-binding structural rearrangements in the trimer that enhance the exposure of the coreceptor binding site and facilitate events leading to fusion and virus entry. Here, we investigated CD4 binding to mac-tropic and non-mac-tropic Env trimers and show that CD4-IgG binds efficiently to mac-tropic R5 Env trimers, while binding to non-mac-tropic trimers was undetectable.

    Our data indicated that the CD4bs is highly occluded on Env trimers of non-mac-tropic R5 viruses. Such viruses may therefore infect T-cells via viral synapses where Env and CD4 become highly concentrated. This environment will enable high avidity interactions that overcome extremely low Env:CD4 affinities.

    Importance

    HIV R5 viruses bind to CD4 and CCR5 receptors on T-cells and macrophages to initiate infection. Transmitted HIV viruses infect T-cells but not macrophages and these viral strains persist in immune tissue even in late disease. Here, we show that the binding site for CD4 present on HIV's envelope protein is occluded on viruses replicating in immune tissue. This occlusion likely prevents antibodies binding to this site and neutralizing the virus, but makes it difficult for virus:CD4 interactions to occur. Such viruses probably pass from T-cell to T-cell via cell contacts where CD4 is highly concentrated and allows infection via inefficient envelope:CD4 binding. Our data is highly relevant for vaccines that aim to induce antibodies targeting the CD4 binding site on the envelope protein.

  • The ubiquitin-proteasome system is necessary for the efficient replication of human astrovirus [PublishAheadOfPrint]

  • Astroviruses, members of the family Astroviridae, represent an important cause of human gastroenteritis in the world. The cellular factors required for astrovirus replication have been poorly studied. In this work, we evaluated the relevance of the ubiquitin-proteasome system (UPS) in the replication of Yuc8, a human astrovirus serotype 8 strain. We found that proteasome inhibitors decrease the production of infectious viral progeny at a step in the replication cycle subsequent to virus entry. The inhibition of the proteasome activity decreases viral RNA levels and viral protein synthesis; similarly, the inhibition of ubiquitination by chemical inhibitors or RNAi reduces the production of viral progeny as well as viral protein synthesis. The effect on viral progeny production induced by proteasome inhibitors is not explained by a reduction in the pool of monoubiquitin or the induction of early apoptosis or autophagy. Our observations are consistent with the need of the proteolytic activity of the UPS for the efficient replication of the virus and suggest that UPS is necessary for the production of genomic and subgenomic RNA but not for antigenomic RNA.

    IMPORTANCE Astroviruses are a major cause of gastroenteritis in young humans and animals and recently it has been associated to fatal encephalitis in humans. The role of ubiquitin proteasome system in the replication of these viruses has not been studied before. In this work, we present evidence that supports that the proteolytic activity of proteasome is necessary for efficient viral progeny production, and that this proteolytic system is required for the accumulation of both, the genomic and subgenomic viral RNAs.

  • microRNA-based attenuation of influenza virus across susceptible hosts [PublishAheadOfPrint]

  • Influenza A virus drives significant morbidity and mortality in humans and livestock. Annual circulation of the virus in livestock and waterfowl contributes to severe economic disruption and increases the risk of zoonotic transmission of novel strains into the human population where there is no preexisting immunity. Seasonal vaccinations in humans help prevent infection and can reduce symptoms when infection does occur. However, current vaccination regimens available for livestock are limited in part due to safety concerns regarding reassortment/recombination with circulating strains. Therefore, inactivated vaccines are used instead of the more immunostimulatory live attenuated vaccines. microRNAs have been previously used to generate attenuated influenza A viruses for use as a vaccine. Here we systematically targeted individual influenza gene mRNAs using the same miRNA to determine the segment(s) that yield maximal attenuation potential. This analysis demonstrated that targeting of NP mRNA most efficiently ablates replication. We further increased the plasticity of miRNA-mediated attenuation of influenza A virus by exploiting a miRNA, miR-21, that is ubiquitously expressed across influenza-susceptible hosts. In order to construct this targeted virus we used CRISPR/Cas9 to eliminate the universally expressed miR-21 from MDCK cells. miR-21 targeted viruses were attenuated in human, mouse, canine, and avian cells and drove protective immunity in mice. This strategy has the potential to enhance the safety of live attenuated vaccines in humans and zoonotic reservoirs.

    IMPORTANCE Influenza A virus circulates annually in both avian and human populations causing significant morbidity, mortality, and economic burden. High incidence of zoonotic infections greatly increases the potential for transmission to humans, where no preexisting immunity or vaccine exists. There is a critical need for new vaccine strategies to combat emerging influenza outbreaks. microRNAs have been previously used to attenuate influenza A viruses. We propose the development of a novel platform to produce live attenuated vaccines that are highly customizable, efficacious across a broad species range, and exhibit enhanced safety over traditional vaccination methods. This strategy exploits a microRNA that is expressed abundantly in influenza susceptible hosts. By eliminating this ubiquitous microRNA from a cell line targeted viruses that are attenuated across susceptible strains can be generated. This approach greatly increases the plasticity of the microRNA targeting approach and enhancing vaccine safety.

  • Functional insights into the adjacent stem-loop in honey bee dicistroviruses that promotes IRES-mediated translation and viral infection [PublishAheadOfPrint]

  • All viruses must successfully harness the host translational apparatus and divert it towards viral protein synthesis. Dicistroviruses use an unusual internal ribosome entry site (IRES) mechanism whereby the IRES adopts a three-pseudoknot structure that accesses the ribosome tRNA binding sites to directly recruit the ribosome and initiate translation from a non-AUG start site. A subset of dicistroviruses, including the honey bee Israeli acute paralysis virus (IAPV), encode an extra stem-loop (SLVI) 5rrsquo; -adjacent to the IGR IRES. Previously, the function of this additional stem-loop is unknown. Here, we provide mechanistic and functional insights into the role of SLVI in IGR IRES translation and in virus infection. Biochemical analyses of a series of mutant IRESs demonstrated that SLVI does not function in ribosome recruitment but is required for proper ribosome positioning on the IRES to direct translation. Using a chimeric infectious clone derived from the related Cricket paralysis virus, we showed that the integrity of SLVI is important for optimal viral translation and viral yield. Based on structural models of ribosome-IGR IRES complexes, the SLVI is predicted to be in the vicinity of the ribosome E site. We propose that SLVI of IAPV IGR IRES functionally mimics interactions of an E-site tRNA with the ribosome to direct positioning of the tRNA-like domain of the IRES in the A site.

    IMPORTANCE

    Viral internal ribosome entry sites are RNA elements and structures that allow some positive-sense monopartite RNA viruses to hijack the host ribosome to start viral protein synthesis. We demonstrate that a unique stem-loop structure is essential for optimal viral protein synthesis and for virus infection. Biochemical evidence shows that this viral stem-loop RNA structure impacts a fundamental property of the ribosome to start protein synthesis.

  • Isolation and Characterization of v{Delta}I3 Confirms that the Vaccinia Virus SSB Plays an Essential Role in Viral Replication [PublishAheadOfPrint]

  • Vaccinia virus is unusual among DNA viruses in replicating exclusively in the cytoplasm of infected cells. The ssDNA binding protein (SSB), I3, is among the replication machinery encoded by the 195 kb genome, although direct genetic analysis of I3 has been lacking. Herein, we describe a complementing cell line (CV1-I3) that fully supports the replication of a null virus lacking the I3 ORF (vI3). In non-complementing CV1-CAT cells, vI3 shows a severe defect in the production of infectious virus (gge;200-fold reduction). Early protein synthesis and core disassembly occur normally. However, DNA replication is profoundly impaired (lle; 0.2% of WT levels) and late proteins do not accumulate. When several other non-complementing cell lines are infected with vI3, the yield of infectious virus is also dramatically reduced (168-1776-fold reduction). Surprisingly, the residual levels of DNA accumulation vary from 1 to 12% in the different cell lines (CV1-CATllt;A549llt;BSC40llt;HeLa); however, any nascent DNA that can be detected is subgenomic in size. Although this sub-genomic DNA supports late protein expression, it does not support the production of infectious virions. EM analysis of vI3-infected BSC40 cells reveals that immature virions are abundant but no mature virions are observed. Aberrant virions characteristic of a block to genome encapsidation are seen instead. Finally, we demonstrate that a CV1 cell line encoding a previously described I3 variant with impaired ssDNA binding activity is unable to complement vI3. This report provides definitive evidence that the vaccinia I3 protein is the replicative SSB and is essential for productive viral replication.

    IMPORTANCE Poxviruses are of historical and contemporary importance as infectious agents, vaccines, and oncolytic therapeutics. The cytoplasmic replication of poxviruses is unique among DNA viruses of mammalian cells, and necessitates that the dsDNA genome encode the viral replication machinery. This study focuses on the I3 protein. As a ssDNA binding protein (SSB), I3 has been presumed to play essential roles in genome replication, recombination and repair, although genetic analysis has been lacking. Herein, we report the characterization of an I3 deletion virus. In the absence of I3 expression, DNA replication is severely compromised and viral yield profoundly decreased. The production of infectious virus can be restored in a cell line expressing WT I3, but not in a cell line expressing an I3 mutant that is defective in ssDNA binding activity. These data show conclusively that I3 is an essential viral protein and functions as the viral replicative SSB.

  • Polyploidy And Mitotic Cell Death Are Two Distinct HIV-1 Vpr-Driven Outcomes In Renal Tubule Epithelial Cells [PublishAheadOfPrint]

  • Prior studies found that HIV, through the Vpr protein, promotes genome reduplication (polyploidy) in infection-surviving epithelial cells within renal tissue. However, the temporal progression and molecular regulation through which Vpr promotes polyploidy remained unclear. Here, we define a sequential progression to Vpr-mediated polyploidy in human renal tubule epithelial cells (RTECs). As in many cell types, we find that Vpr first initiates a G2 cell cycle arrest in RTECs. We then identified a previously unreported cascade of Vpr-dependent events that lead to renal cell survival and polyploidy. Specifically, we found that a fraction of G2-arrested RTECs re-enter the cell cycle. Following this cell cycle re-entry, two distinct outcomes occur. Cells that enter complete mitosis undergo mitotic cell death due to extra centrosomes and aberrant division. Conversely, cells that abort mitosis undergo endoreplication to become polyploid. We further show that multiple small molecule inhibitors of the phosphatidylinositol-3 kinase related kinase (PIKK) family, including those that target ATR, ATM, and mTOR, indirectly prevent Vpr-mediated polyploidy by preventing G2 arrest. In contrast, an inhibitor that targets DNA-dependent protein kinase (DNA-PK) specifically blocks the Vpr-mediated transition from G2 arrest to polyploidy. These findings outline a temporal, molecularly-regulated path to polyploidy in HIV+ renal cells.

    IMPORTANCE Current cure-focused efforts in HIV research aim to elucidate the mechanisms of long term persistence of HIV in compartments. The kidney is recognized as one such compartment, as viral DNA and mRNA persist in renal tissue of HIV+ patients. Further, renal disease is a long-term co-morbidity in the setting of HIV. Thus, understanding the regulation and impact of HIV infection on renal cell biology will provide important insights into this unique HIV compartment. Our work identifies mechanisms that distinguish between HIV+ cell survival and death in a known HIV compartment, as well as pharmacological agents that alter these outcomes.

  • Dynamics of Sendai virus spread, clearance, and immunotherapeutic efficacy after hematopoietic cell transplant imaged non-invasively in mice [PublishAheadOfPrint]

  • There are no approved vaccines or virus-specific treatments for human parainfluenza viruses (PIVs), which have recently been reclassified into species human respirovirus 1 and 3 and human rubulavirus 2 and 4. These viruses cause morbidity and mortality in immunocompromised patients including those undergoing hematopoietic cell transplant (HCT). No small-animal models exist for non-invasive imaging of respiratory viral infection in the HCT host despite the utility such a system would offer to monitor prolonged infection, its clearance, and treatment options. We used a luciferase-expressing reporter virus to non-invasively image in mice the infection of murine respirovirus (strain: Sendai virus, SeV), the murine counterpart of HPIV1. Independent of disease severity, clearance of infection began approximately 21 days after HCT, largely due to recovery of CD8+ T cells. Immunotherapy with granulocyte colony-stimulating factor (G-CSF) and adoptive transfer of natural killer (NK) cells provided limited therapeutic benefit. Treatment with a fusion (F) protein-specific monoclonal antibody arrested the spread of lung infection and reduced disease severity even when treatment was delayed up to 10 days post infection but had little observable effect on upper respiratory tract infection. Adoptive transfer of virus-specific T cells 10 days post infection accelerated clearance by 5 days, reduced the extent of infection throughout the respiratory tract, and reduced disease severity. Overall, the results support investigation of clinical treatment of respiratory virus infection in the HCT host with monoclonal antibodies and adoptive T-cell transfer; the imaging system should be extendable to other respiratory viruses such as RSV and influenza virus.

    IMPORTANCE Parainfluenza viruses are a major cause of disease and death due to respiratory virus infection in the immunocompromised host including those undergoing Bone Marrow Transplantation. There are currently no effective treatment measures. We non-invasively imaged mice undergoing bone marrow transplant and infected with Sendai virus, a murine parainfluenza virus (respirovirus). For the first time, we show the therapeutic windows of adoptive T-cell therapy and monoclonal antibody to the fusion (F) protein in clearing Sendai virus from the respiratory tract and reducing disease severity. Mice tolerated these treatments without any detectable toxicity. This paves the way for studies assessing the safety of T-cell therapy against parainfluenza virus in humans. Adoptive T-cell therapy in humans against other blood-borne viruses has been shown to be safe and effective. Our model of non-invasive imaging in transplanted mice may be well suited to track other respiratory virus infections and develop novel preventive and therapeutic strategies.

  • Characterization of Elements Regulating the Nuclear to Cytoplasmic Translocation of ICP0 in Late HSV-1 Infection [PublishAheadOfPrint]

  • Infected cell protein 0 (ICP0) of HSV-1 is an immediate early protein containing a RING-type E3 ubiquitin ligase. It targets several host factors for proteasomal degradation and subsequently activates viral expression. ICP0 has a nuclear localization sequence and functions in the nucleus at early infection. However, later in infection, ICP0 is found solely in the cytoplasm. The molecular mechanism and biological function of ICP0 nuclear to cytoplasmic translocation is not well understood. In this study, we sought to characterize elements important for this translocation. We found that (i) in HEL cells, ICP0 C-terminal residues 741-775 were necessary but not sufficient for the nuclear to cytoplasmic translocation; (ii) loss of ICP0 E3 ubiquitin ligase activity, which led to defective viral replication in nonpermissive cells, also caused the mutant ICP0 to be retained in the nucleus of HEL cells; (iii) in the permissive U2OS cells, however, ICP0 lacking E3 ligase activity was translocated to the cytoplasm at a pace faster than the wild type ICP0, suggesting a nuclear retention of ICP0 exists in an ICP0 E3 ligase dependent manner; and (iv) ICP0 C-terminus and late viral proteins cooperate in order to overcome the nuclear retention and stimulate the ICP0 cytoplasmic translocation. Taken together, lesser ICP0 nuclear retention may contribute to the permissiveness of U2OS cells to HSV-1 in the absence of a functional ICP0.

    IMPORTANCE A distinct characteristic for eukaryotes is the compartmentalization of cell metabolic pathways, which allows greater efficiency and specificity to cellular functions. ICP0 of HSV-1 is a multifunctional viral protein that travels through different compartments as infection progresses. Its main regulatory functions are carried out in the nucleus but it is translocated to the cytoplasm in late HSV-1 infection. To understand the biological significance of cytoplasmic ICP0 in HSV-1 infection, we investigated the potential players involved in this nuclear to cytoplasmic translocation. We found that a nuclear retention force exists in an ICP0 E3 ubiquitin ligase dependent manner. In addition, we identified the C-terminus of ICP0 as a cis-element cooperating with late viral proteins to overcome the nuclear retention and stimulate the nuclear to cytoplasmic translocation of ICP0.

  • Identification of a Critical and Conformational Neutralizing Epitope in Human Adenovirus Type 4 Hexon [PublishAheadOfPrint]

  • Human adenovirus type 4 (HAdV-4) is an epidemic virus that contributes to serious acute respiratory disease (ARD) in both pediatric and adult patients. However, no licensed drug or vaccine is currently available to the civilian population. The identification of the neutralizing epitopes of HAdV-4 should allow the development of a novel antiviral vaccine and novel gene transfer vector, and an effective neutralizing monoclonal antibody (MAb) will be useful in developing appropriate antiviral drugs. In this study, the MAb MN4b is reported to show strong neutralizing activity against HAdV-4. MN4b recognizes a conformational epitope (418AGSEK422) within hypervariable region 7 (HVR7). Mutations within this site permitted HAdV-4 mutants to escape neutralization by MN4b and to resist neutralization by animal and human anti-HAdV-4 sera. A recombinant virus, rAd3-A4R7-1 containing the identified neutralizing epitope in the HVR7 region of HAdV-3 hexon, successfully induced antiserum that inhibited HAdV-4 infection. These results indicate that a small surface loop of HAdV-4 hexon is a critical neutralization epitope for this virus. The generation of MN4b and the identification of this neutralizing epitope may be useful in developing a therapeutic treatment, subunit vaccine, and novel vector that can escape preexisting neutralization for HAdV-4.

    IMPORTANCE

    Neutralizing antibodies are considered good tools for the prevention of human adenovirus type 4 (HAdV-4) infections. The identification of the epitopes recognized by such neutralizing antibodies is important in the generation of recombinant antiviral vaccines. However, until now, no neutralizing epitope has been reported for HAdV-4. Here, we developed a serotype-specific neutralizing MAb directed against HAdV-4, MN4b. We provide evidence that MN4b recognizes a conformational epitope within HVR7 of HAdV-4 hexon. Antisera generated to this conformational epitope displayed on HAdV-3 hexon inhibited the infection of AD293 cells by HAdV-4. Our findings are very important for the development of a therapeutic treatment, subunit vaccine, and novel vector for HAdV-4.

  • Glycan shield and fusion activation of a deltacoronavirus spike glycoprotein fine-tuned for enteric infections [PublishAheadOfPrint]

  • Coronaviruses recently emerged as major human pathogens causing outbreaks of severe acute respiratory syndrome and Middle-East respiratory syndrome. They utilize the spike (S) glycoprotein anchored in the viral envelope to mediate host attachment and fusion of the viral and cellular membranes to initiate infection. The S protein is a major determinant of the zoonotic potential of coronaviruses and is also the main target of the host humoral immune response. We report here the 3.5 AAring; resolution cryo-electron microscopy structure of the S glycoprotein trimer from the pathogenic porcine deltacoronavirus (PDCoV), which belongs to the recently identified delta genus. Structural and glycoproteomics data indicate that the glycans of PDCoV S are topologically conserved when compared with the human respiratory coronavirus HCoV-NL63 S, resulting in similar surface areas being shielded from neutralizing antibodies and implying that both viruses are under comparable immune pressure in their respective hosts. The structure further reveals a shortened S2' activation loop, containing a reduced number of basic amino acids, which participates to rendering the spike largely protease-resistant. This property distinguishes PDCoV S from recently characterized betacoronavirus S proteins and suggests that the S protein of enterotropic PDCoV has evolved to tolerate the protease-rich environment of the small intestine and to fine-tune its fusion activation to avoid premature triggering and reduction of infectivity.

    IMPORTANCE Coronaviruses use transmembrane spike (S) glycoprotein trimers to promote host attachment and fusion of the viral and cellular membranes. We determined a near-atomic resolution cryo-electron microscopy structure of the S ectodomain trimer from the pathogenic porcine deltacoronavirus (PDCoV), which is responsible for diarrhea in piglets and has had devastating consequences for the swine industry worldwide. Structural and glycoproteomics data reveal that PDCoV S is decorated with 78 N-linked glycans obstructing the protein surface to limit accessibility to neutralizing antibodies in a way reminiscent of what has recently been described for a human respiratory coronavirus. PDCoV S is largely protease-resistant which distinguishes it from most other characterized coronavirus S glycoproteins and suggests that enteric coronaviruses have evolved to fine-tune fusion activation in the protease-rich environment of the small intestine of infected hosts.

  • Inducible MicroRNA-3570 Feedback Inhibits the RIG-I-Dependent Innate Immune Response to Rhabdovirus in Teleost Fish by Targeting MAVS/IPS-1 [PublishAheadOfPrint]

  • Effectively recognizing invading viruses and subsequently inducing the innate antiviral immunity are essential for the host antiviral defense. Although these processes are closely regulated by the host to maintain the immune balance, viruses have evolved the abilities to downregulate or upregulate these processes for their survival. MicroRNAs (miRNAs) are a family of small non-coding RNAs that play vital roles in modulating host immune response. Accumulating evidence demonstrates that host miRNAs as mediators are involved in regulating viral replication and host anti-viral immunity in mammals. However, the underlying regulatory mechanisms in fish species are still poorly understood. Here we found that rhabdovirus infection significantly upregulated host miR-3570 expression in miiuy croaker macrophages. Induced miR-3570 negatively modulated RNA virus-triggered type I interferon (IFN) and antiviral gene production, thus facilitating viral replication. Furthermore, miR-3570 was found to target and post-transcriptionally downregulate mitochondrial antiviral-signaling protein (MAVS), which functions as platforms for innate antiviral signal transduction. Moreover, we demonstrated that miR-3570 suppressed the expression of MAVS thereby inhibiting MAVS-mediated NF-B and IRF3 signaling. The collective results demonstrated a novel regulation mechanism of MAVS-mediated immunity during RNA viral infection by miRNA.

    IMPORTANCE RNA viral infection could upregulate host miR-3570 expression in miiuy croaker macrophages. Induced miR-3570 negatively modulates RNA virus-triggered type I IFN and antiviral gene production, thus facilitating viral replication. Remarkably, miR-3570 could target and inhibit MAVS expression which thus modulates MAVS-mediated NF-B and IRF3 signaling. The collective results of this study suggest a novel regulation mechanism of MAVS-mediated immunity during RNA viral infection by miR-3570. Thus, a novel mechanism for virus evasion in fish is proposed.

  • Characterization of recombinant Flaviviridae viruses possessing a small reporter-tag [PublishAheadOfPrint]

  • The family Flaviviridae consists of four genera, Flavivirus, Pestivirus, Pegivirus, and Hepacivirus, and comprises important pathogens of human and animals. Although the construction of recombinant viruses carrying reporter genes including fluorescent and bioluminescent proteins has been reported, the stable insertion of foreign genes into viral genomes retaining infectivity remains difficult. Here, we applied the 11-amino-acid subunit derived from NanoLuc luciferase to the engineering of the Flaviviridae viruses, and then examined the biological characteristics of the viruses. We successfully generated recombinant viruses carrying the split-luciferase gene including dengue virus, Japanese encephalitis virus, hepatitis C virus (HCV), and bovine viral diarrhea virus. The stability of the viruses was confirmed by five rounds of serial passages in respective susceptible cell lines. The propagation of the recombinant luciferase viruses in each cell line was comparable to that of the parental viruses. By using a purified counterpart luciferase protein, this split-luciferase assay can be applicable in various cell lines, even when it is difficult to transduce the counterpart gene. The efficacy of antiviral reagents against the recombinant viruses could be monitored by the reduction of luciferase expression, which was correlated with that of viral RNA, and the recombinant HCV was also useful to examine viral dynamics in vivo. Taken together, our findings indicate that the recombinant Flaviviridae viruses possessing the split NanoLuc luciferase gene generated herein provide powerful tools to understand viral life cycle and pathogenesis, and a robust platform to develop novel antivirals against Flaviviridae viruses.

    IMPORTANCE The construction of reporter viruses possessing a stable transgene capable of expressing specific signals is crucial to investigations of viral life cycle and pathogenesis and the development of antivirals. However, it is difficult to maintain the stability of a large foreign gene such as those for fluorescent and bioluminescent proteins after insertion into a viral genome. Here, we successfully generated recombinant Flaviviridae viruses carrying the 11-amino-acid subunit derived from NanoLuc luciferase, and demonstrated that these viruses are applicable to in vitro and in vivo experiments, suggesting that these recombinant Flaviviridae viruses are powerful tools for increasing our understanding of viral life cycle and pathogenesis, and that these recombinant viruses will provide a robust platform to develop antivirals against Flaviviridae viruses.

  • Analysis of Select HSV-1 Proteins for Restriction of Human Immunodeficiency Virus Type 1: The HSV-1 gM Protein Potently Restricts HIV-1 by Preventing the Intracellular Transport and Processing of Env gp160 [PublishAheadOfPrint]

  • Proteins encoded by viruses that impair or shutdown specific host cell functions during replication can be used as probes to identify potential proteins/pathways used in the replication of viruses from other families. We screened nine proteins from herpes simplex virus type 1 (HSV-1) for the ability to enhance or restrict human immunodeficiency virus type 1 (HIV-1) replication. We show that several HSV-1 proteins (glycoprotein M (gM), US3 and UL24) potently restricted the replication of HIV-1. Unlike UL24 and US3, which reduced viral protein synthesis, we observed that gM restriction of HIV-1 occurred through interference of the processing and transport of gp160, resulting in a significantly reduced level of mature gp120/gp41 released from cells. Finally, we show that an HSV-1 gM mutant lacking the majority of the C-terminal domain (HA-gM[345-473]) restricted neither gp160 processing nor the release of infectious virus. These studies identify and show that proteins from heterologous viruses restrict viruses through novel pathways.

    IMPORTANCE HIV-1 infection of humans' results in acquired immunodeficiency syndrome (AIDS) characterized by the loss of CD4+ T cells and increased susceptibility to opportunistic infections. Both HIV-1 and HSV-1 can infect astrocytes and microglial of the central nervous system (CNS). Thus, the identification of proteins that directly restrict HIV-1 or interfere with pathways required for HIV-1 replication could lead to novel anti-retroviral strategies. The results of this study show that select viral proteins from HSV-1 can potently restrict HIV-1. Further, our results indicate that the gM protein of HSV-1 restricts HIV-1 through a novel pathway by interfering in the processing of gp160 and incorporation into virus maturing from the cell.

  • Early EBV genomic diversity and convergence towards the B95.8 genome in primary infection [PublishAheadOfPrint]

  • Over 90% of the world's population is persistently infected with Epstein-Barr virus. While EBV does not cause disease in most individuals, it is the common cause of acute infectious mononucleosis (AIM), has been associated with several cancers and autoimmune diseases, highlighting a need for a preventive vaccine. At present, very few primary, circulating EBV genomes have been sequenced directly from infected individuals. While low levels of diversity and viral evolution rates have been predicted for dsDNA viruses, recent studies have demonstrated appreciable diversity in common dsDNA pathogens (e.g., cytomegalovirus). Here we report 40 full length EBV genome sequences obtained from matched oral wash and B cell fractions from a cohort of 10 AIM patients. Both intra- and interpatient diversity were observed across the length of the entire viral genome. Diversity was most pronounced in viral genes required for establishing latent infection and persistence, with appreciable levels of diversity also detected in structural genes, including envelope glycoproteins. Interestingly, intra-patient diversity declined significantly over time (pllt;0.01), and this was particularly evident on comparison of viral genomes sequenced from B cell fractions in early primary infection and convalescence (pllt;0.001). B cell-associated viral genomes were observed to converge, becoming nearly identical to the B95.8 reference genome over time (Spearman rank-order correlation test; r = -0.5589, p=0.0264). The reduction in diversity was most marked in the EBV latency genes. In summary, our data suggest independent convergence of diverse viral genome sequences toward a reference-like strain within a relatively short period following primary EBV infection.

    Importance: Identification of viral proteins with low variability and high immunogenicity is important for the development of a protective vaccine. Knowledge of genome diversity within circulating viral populations is a key step in this process, as is the expansion of intra-host genomic variation during infection. We report full length EBV genomes sequenced from the blood and oral wash of ten individuals early in primary infection and during convalescence. Our data demonstrate considerable diversity within the pool of circulating EBV, as well as within individual patients. Overall viral diversity decreased from early to persistent infection, particularly in latently infected B cells, which serve as the viral reservoir. Reduction in B cell-associated viral genome diversity coincided with a convergence towards a reference-like EBV genotype. Greater convergence positively correlated with time after infection, suggesting that the reference-like genome is the result of selection.

  • According to HCV infection stage, IL-7 plus 4-1BB triggering alone or combined with PD-1 blockade increases TRAF1low HCV-specific CD8+ cell reactivity [PublishAheadOfPrint]

  • Hepatitis C virus (HCV)-specific CD8+ T cells suffer a progressive exhaustion during persistent HCV infection (PI). This process could involve the positive immune checkpoint 4-1BB/4-1BBL, through the loss of its signal transducer TRAF1. To address this issue, peripheral HCV-specific CD8+ T cells (Pentamer+/CD8+) from patients with PI and resolved infection after treatment (RI) were studied. Duration of HCV infection and liver fibrosis progression rate inversely correlated with the likelihood of detecting peripheral pentamer+/CD8+ cells. In PI, pentamer+/CD8+ cells had impaired antigen-specific reactivity that worsened when these cells were not detectable ex-vivo. Short/mid-lasting PI was characterized by detectable peripheral PD-1+ CD127low TRAF1low cells. After TCR triggering, TRAF1 level positively correlated with CD127, Mcl-1, CD107a and proliferation intensity, but negatively with PD-1, linking TRAF1low to exhaustion. IL-7 in-vitro treatment up-regulated TRAF1, while TGF-bbeta;1 did the opposite, suggesting that IL-7/TGF-bbeta;1 balance, besides TCR stimulation, could be involved in TRAF1 regulation. In fact, TGF-bbeta;1 serum concentration was higher in PI than in RI patients, and it negatively correlated with TRAF1 expression. In line with IL-7 increasing TRAF1 level, IL-7/4-1BBL in-vitro treatment enhanced T cell reactivity in short/mid-lasting infection. However, in long-lasting PI, anti-PD-L1 in addition to IL-7/4-1BBL combination was necessary to re-establish T cell proliferation in slow fibrosers, but had no effect in rapid fibrosers. In conclusion, a peripheral hypo-reactive TRAF1low HCV-specific CD8+ T cell response, restorable by IL-7/4-1BBL treatment, characterizes short/mid-length PI. In long-lasting disease, HCV-specific CD8+ T cells are rarely detectable ex-vivo, but IL-7/4-1BBL/anti-PD-L1 treatment recovers their reactivity in-vitro in slow fibrosers.

    IMPORTANCE Hepatitis C virus (HCV) infects 71 million people worldwide. Two thirds develop a chronic disease that can lead to cirrhosis and hepatocellular carcinoma. Direct-acting antivirals clear the infection but there are still patients who relapse. In these cases, additional immunotherapy could play a vital role. A successful anti-HCV immune response depends on virus-specific CD8+ T cells. During chronic infection, these cells are functionally impaired, which could be due to co-stimulation failure. This study describes exhausted specific T cells, characterized by low expression of the signal transducer TRAF1 of the positive co-stimulatory pathway 4-1BB/4-1BBL. IL-7 up-regulated TRAF1 and improved T cell reactivity in short/mid duration disease, while in long-lasting infection, it was also necessary to block the negative checkpoint PD-1/PD-L1. Taken together, this work supports novel ways of restoring specific CD8+ T cell response, shedding light on the importance of TRAF1 signaling. This could be a promising target for future immunotherapy.

  • Rabbit haemorrhagic disease virus 2 (GI.2) is replacing endemic strains of RHDV in the Australian landscape within 18 months of its arrival [PublishAheadOfPrint]

  • Rabbit haemorrhagic disease virus 2 (RHDV2; Lagovirus GI.2) is a pathogenic calicivirus that affects European rabbits (Oryctolagus cuniculus) and various hare (Lepus) species. GI.2 was first detected in France in 2010 and subsequently caused epidemics in wild and domestic lagomorph populations throughout Europe. In May 2015 GI.2 was detected in Australia. Within 18 months of its initial detection GI.2 had spread to all Australian states and territories and rapidly became the dominant circulating strain, replacing Rabbit haemorrhagic disease virus (RHDV/GI.1) in mainland Australia. Reconstruction of the evolutionary history of 127 Australian GI.2 isolates revealed that the virus arrived in Australia at least several months before its initial description and likely circulated unnoticed in wild rabbit populations in the east of the continent prior to its detection. GI.2 sequences isolated from five hares clustered with sequences from sympatric rabbit populations sampled contemporaneously, indicating multiple spillover events into hares rather than an adaptation of the Australian GI.2 to a new host. Since the presence of GI.2 in Australia may have wide ranging consequences for rabbit biocontrol, particularly with the release of the novel biocontrol agent GI.1a/RHDVa-K5 in March 2017, ongoing surveillance is critical to understanding the interactions of the various lagoviruses in Australia, and their impact on host populations.

    IMPORTANCE This study describes the spread and distribution of Rabbit Haemorrhagic Disease virus 2 (GI.2) in Australia since its first detection in May 2015. Within the first 18 months following its detection, RHDV2 spread from east to west across the continent and became the dominant strain in all mainland states of Australia. This has important implications for pest animal management and for owners of pet and farmed rabbits, as there is currently no effective vaccine available in Australia for GI.2. The closely related RHDV (GI.1) is used to control overabundant wild rabbits, a serious environmental and agricultural pest in this country, and it is currently unclear how widespread circulation of GI.2 will impact ongoing targeted wild rabbit management operations.

  • VP1 and VP3 are required and sufficient for translation initiation of uncapped IBDV genomic dsRNA [PublishAheadOfPrint]

  • Infectious bursal disease virus (IBDV) is a bi-segmented double-strand RNA (dsRNA) virus of the Birnaviridae family. While IBDV genomic dsRNA lacks a 5rrsquo; cap, the means by which the uncapped IBDV genomic RNA is translated effectively is unknown. In this study, we describe a cap-independent pathway of translation initiation of IBDV uncapped RNA that relies on VP1 and VP3. We show that neither purified IBDV genomic dsRNA nor the uncapped viral plus-sense RNA transcripts was directly translated and rescued into infectious viruses in host cells. This defect in translation of the uncapped IBDV genomic dsRNA was rescued by trans-supplementation of the viral proteins VP1 and VP3, which was dependent on both the intact polymerase activity of VP1 and the dsRNA binding activity of VP3. Deletion analysis showed that both 5rrsquo; - and 3rrsquo; -UTRs of IBDV dsRNA were essential for the VP1/VP3-dependent translation initiation. Significantly, VP1 and VP3 could also mediate the recovery of infectious IBDV from the authentic minus-sense strand of IBDV dsRNA. Moreover, down-regulation or inhibition of the cap-binding protein eIF4E did not decrease, but rather enhanced the VP1/VP3-mediated translation of the uncapped IBDV RNA. Collectively, our findings for the first time reveal that VP1 and VP3 compensate for the deficiency of 5rrsquo; cap and replace eIF4E to confer upon the uncapped IBDV RNA the ability to be translated and rescued into infectious viruses.

    IMPORTANCE

    A key point of control for virus replication is the viral translation initiation. The current study shows that the uncapped IBDV RNA cannot be translated into viral proteins directly by host translation machinery, and is thus noninfectious. Our results constitute the first direct experimental evidence that the VP1 and VP3 are required and sufficient to initiate translation of uncapped IBDV genomic RNA by acting as a substitute of cap and replacing the cap-binding protein eIF4E. Significantly, the VP1/VP3 mediate the recovery of infectious IBDV not only from the plus-sense but also from the minus-sense strand of the IBDV dsRNA. These findings provide not only new insights into the molecular mechanisms of the life cycle of IBDV, but also a new tool for an alternative strategy for the recovery of IBDV from both the plus- and the minus-sense strand of the viral genomic dsRNA.

  • A 3.0-Angstrom Resolution Cryo-Electron Microscopy Structure and Antigenic Sites of Coxsackievirus A6-Like Particles [PublishAheadOfPrint]

  • Coxsackievirus A6 (CVA6) has recently emerged as one of the predominant causative agents of hand, foot, and mouth disease (HFMD). Structure of CVA6 mature viral particle has not been solved thus far. Our previous work shows that recombinant virus-like particles (VLPs) of CVA6 represent a promising CVA6 vaccine candidate. Here, we report the first cryo-electron microscopy (cryo-EM) structure of CVA6 VLP at 3.0 AAring; resolution. CVA6 VLP exhibits the characteristic features of enteroviruses, but presents an open channel at the two-fold axis and an empty, collapsed VP1 pocket, which is broadly similar to the structures of enterovirus 71 (EV71) VLP and coxsackievirus A16 (CVA16) 135S expanded particle, indicating that CVA6 VLP is in an expanded conformation. Structural comparisons reveal that two common salt bridges within protomers are maintained in CVA6 VLP and other viruses of the Enterovirus genus, implying that these salt bridges may play a critical role in enteroviral protomer assembly. However, there are apparent structural differences among CVA6 VLP, EV71 VLP and CVA16 135S particle in the surface-exposed loops and C termini of subunit proteins, which are often antigenic sites for enteroviruses. By immunological assays, we identified two CVA6-specific linear B-cell epitopes (designated P42 and P59) located at the GH loop and the C-terminal region of VP1, respectively, in agreement with the structure-based prediction of antigenic sites. Our findings elucidate the structural basis and important antigenic sites of CVA6 VLP as a strong vaccine candidate and also provide insight into enteroviral protomer assembly.

    IMPORTANCE Coxsackievirus A6 (CVA6) is becoming one of the major pathogens of hand, foot, and mouth disease (HFMD), causing significant morbidity and mortality in children and adults. However, no vaccine is currently available to prevent CVA6 infection. Our previous work shows that recombinant virus-like particles (VLPs) of CVA6 are a promising CVA6 vaccine candidate. Here, we present a 3.0 AAring; structure of CVA6 VLP determined by cryo-electron microscopy. The overall architecture of CVA6 VLP is similar to those of the expanded structures of enterovirus 71 (EV71) and coxsackievirus A16 (CVA16), but careful structural comparisons reveal significant differences in the surface-exposed loops and C-termini of each capsid protein of these particles. In addition, we identified two CVA6-specific linear B-cell epitopes and mapped them to the GH loop and the C-terminal region of VP1, respectively. Collectively, our findings provide structural basis and important antigenic information for the CVA6 VLP vaccine development.

  • Analysis of Epstein-Barr Virus Genomes and Expression Profiles in Gastric Adenocarcinoma [PublishAheadOfPrint]

  • Epstein-Barr virus (EBV) is a causative agent of a variety of lymphomas, nasopharyngeal carcinoma (NPC) and ~9% of gastric (GC) carcinomas. An important question is whether particular EBV variants are more oncogenic than others but conclusions are currently hampered by the lack of sequenced EBV genomes. Here we contribute to this question by mining whole genome sequences of 201 GCs to identify13 EBV-positive GCs, and assembling 13 new EBV genome sequences; almost doubling the number of available GC-derived EBV genome sequences and providing the first non-Asian EBV genome sequences from GC. Whole genome sequence comparisons of all EBV isolates sequenced to date (85 from tumours and 57 from health individuals) showed that most GC and NPC EBV isolates were closely related, although American Caucasian GC samples were more distant, suggesting a geographical component. However, EBV GC isolates were found to contain some consistent changes in protein sequences regardless of geography. In addition, transcriptome data available for 8 of the EBV-positive GCs was analysed to determine which EBV genes are expressed in GC. In addition to the expected latency proteins (EBNA1, LMP1 and LMP2A), specific subsets of lytic genes were consistently expressed, that did not reflect a typical lytic or abortive lytic infection, suggesting a novel mechanism of EBV gene regulation in the context of GC. These results are consistent with a model in which a combination of specific latent and lytic EBV proteins promote tumourigenesis.

    Importance Epstein-Barr virus (EBV) is a widespread virus that causes cancer, including gastric carcinoma (GC), in a small subset of individuals. An important question is whether particular EBV variants are more cancer-associated than others, but more EBV sequences are required to address this question. Here we have generated 13 new EBV genome sequences from GC; almost doubling the number of EBV sequences from GC isolates and providing the first EBV sequences from non-Asian GC. We further identify sequence changes in some EBV proteins common to GC isolates. In addition, gene expression analysis of eight of the EBV-positive GCs showed consistent expression of both the expected latency proteins and a subset of lytic proteins, that was not consistent with typical lytic or abortive lytic expression. These results suggest novel mechanisms activating expression of some EBV lytic proteins and that their expression may contribute to oncogenesis.

  • Weaker HLA footprints on HIV in the unique and highly genetically admixed host population of Mexico [PublishAheadOfPrint]

  • HIV circumvents HLA class I-restricted CD8+ T cell responses through selection of escape mutations that leave characteristic mutational "footprints" - also known as HLA-associated polymorphisms (HAPs) - on HIV sequences at the population level. While many HLA footprints are universal across HIV subtypes and human populations, others can be region-specific as a result of the unique immunogenetic background of each host population. Using a published probabilistic phylogenetically-informed model, we compared HAPs in HIV Gag and Pol (PR-RT) in 1,612 subtype B-infected, antiretroviral treatment-naïve individuals from Mexico and 1,641 from Canada/USA. A total of 252 HLA class I allele subtypes were represented, including 140 observed in both cohorts, 67 unique to Mexico and 45 unique to Canada/USA. At the predefined statistical threshold of qllt;0.2, 358 HAPs (201 in Gag; 157 in PR-RT) were identified in Mexico, while 905 (534 in Gag and 371 in PR-RT) were identified in Canada/USA. HAP identified in Mexico included both "canonical" HLA-associated escape pathways and novel associations, in particular with HLA alleles enriched in Amerindian and mestizo populations. Remarkably, HLA footprints on HIV in Mexico were not only fewer but also on average significantly weaker than those in Canada/USA, though some exceptions were noted. Moreover, exploratory analyses suggested that the weaker HLA footprint on HIV in Mexico may be due, at least in part, to weaker and/or less reproducible HLA-mediated immune pressures on HIV in this population. The implications of these differences for natural and vaccine-induced anti-HIV immunity merit further investigation.

    IMPORTANCE

    HLA footprints on HIV identify viral regions under intense and consistent pressure by HLA-restricted immune responses and the common mutational pathways that HIV uses to evade them. In particular, HLA footprints can identify novel immunogenic regions and/or epitopes targeted by understudied HLA alleles; moreover, comparative analyses across immunogenetically distinct populations can illuminate the extent to which HIV immunogenic regions and escape pathways are shared versus population-specific, information which can in turn inform the design of universal or geographically-tailored HIV vaccines. We compared HLA-associated footprints on HIV in two immunogenetically distinct North American populations - Mexico and Canada/USA. We identify both shared and population-specific pathways of HIV adaptation, but also make the surprising observation that HLA footprints on HIV in Mexico are overall fewer and weaker than in Canada/USA, raising the possibility that HLA-restricted antiviral immune responses in Mexico may be weaker, and/or escape pathways somewhat less consistent, than in other populations.

  • Combination Adenovirus and Protein Vaccines Prevent Infection or Reduce Viral Burden after Heterologous Clade C SHIV Mucosal Challenge [PublishAheadOfPrint]

  • HIV vaccine development is focused on designing immunogens and delivery methods that elicit protective immunity. We evaluated a combination of Ad vectors expressing HIV 1086.C (Clade C) Envelope glycoprotein, SIV Gag p55, and human pegivirus GBV-C E2 glycoprotein. We compared replicating simian (SAd7) with non-replicating human (Ad4) adenovirus-vectored vaccines paired with recombinant proteins in a novel prime-boost regimen in rhesus macaques, with the goal of eliciting protective immunity against SHIV challenge. In both vaccine groups plasma and buccal Env-specific IgG, Tier 1 heterologous neutralizing antibodies, and ADCVI were readily generated. High Env-specific T cell responses elicited in all vaccinees were significantly greater than responses targeting Gag. Following three intrarectal exposures to heterologous Tier 1 Clade C SHIV, all 10 sham vaccinated controls were infected, while 4/10 SAd7 and 3/10 Ad4 vaccinated macaques remained uninfected or maintained tightly controlled plasma viremia. Time to infection was significantly delayed in SAd7-vaccinated macaques compared to the controls. Cell-associated and plasma virus levels were significantly lower in each group of vaccinated macaques compared with controls; the lowest plasma viral burden was found in those vaccinated with the SAd7 vectors, suggesting superior immunity conferred by the replicating simian vectors. Furthermore, higher V1V2-specific binding antibody titers correlated with viral control in the SAd7 vaccine group. Thus recombinant Ad plus protein vaccines generated humoral and cellular immunity that was effective in either protecting from SHIV acquisition or significantly reducing viremia in animals that became infected, consequently supporting additional development of replicating Ad vectors as HIV vaccines.

    IMPORTANCE There is a well-acknowledged need for an effective AIDS vaccine that protects against HIV infection and limits in vivo viral replication and associated pathogenesis. Although replicating virus vectors have been advanced as HIV-vaccine platforms, there have not been any direct comparisons of the replicating to the non-replicating format. The current study directly compared the replicating SAd7 to non-replicating Ad4 vectors in macaques and demonstrated that in the SAd7 vaccine group, time to infection was significantly delayed compared to the control group, and V1V2 Env-specific binding antibodies correlated with viral outcomes. Viral control was significantly enhanced in vaccinated macaques compared to controls, and in infected SAd7-vaccinated macaques compared to Ad4-vaccinated macaques, suggesting that this vector may have conferred more effective immunity. Because blocking infection is so difficult with current vaccines, development of a vaccine that can limit viremia if infection occurs would be valuable. These data support further development of replicating adenovirus vectors.

  • Merkel cell polyomavirus small T antigen drives cell motility via Rho-GTPase-induced filopodia formation [PublishAheadOfPrint]

  • Cell motility and migration is a complex, multi-step, and multi-component process, intrinsic to progression and metastasis. Motility is dependent on the activity of integrin receptors and Rho-family GTPases resulting in the remodelling of the actin cytoskeleton and formation of various motile actin-based protrusions. Merkel cell carcinoma (MCC) is an aggressive skin cancer with a high likelihood of recurrence and metastasis. Merkel cell polyomavirus (MCPyV) is associated with the majority of MCC cases, and MCPyV-induced tumourigenesis largely depends on the expression of the small tumour antigen (ST). Since the discovery of MCPyV, a number of mechanisms have been suggested to account for replication and tumourigenesis, but to date, little is known about potential links between MCPyV T antigen expression and the metastatic nature of MCC. Previously, we have described the action of MCPyV ST on the microtubule network and how this impacts on cell motility and migration. Here we demonstrate that MCPyV ST affects the actin cytoskeleton, to promote the formation of filopodia, through a mechanism involving the catalytic subunit of protein phosphatase 4 (PP4C). We also show that MCPyV ST-induced cell motility is dependent upon the activity of Rho-family GTPases Cdc42 and RhoA. In addition, our results indicate that the MCPyV ST-PP4C interaction results in the dephosphorylation of bbeta;1 integrin, likely driving the cell motility pathway. These findings describe a novel mechanism by which a tumour virus induces cell motility, which may ultimately lead to cancer metastasis and provides opportunities and strategies for targeted interventions for disseminated MCC.

    IMPORTANCE Merkel Cell Polyomavirus (MCPyV) is the most recently discovered human tumour virus. It causes the majority of cases of Merkel cell carcinoma (MCC), an aggressive skin cancer. However, the molecular mechanisms implicating MCPyV-encoded proteins in cancer development are yet to be fully elucidated. This manuscript builds upon our previous observations which demonstrate that the MCPyV ST antigen enhances cell motility, providing a potential link between MCPyV protein expression and the highly metastatic nature of MCC. Here we show that MCPyV ST remodels the actin cytoskeleton, promoting the formation of filopodia which is essential for MCPyV ST-induced cell motility and we also implicate the activity of specific Rho-family GTPases, cdc42 and RhoA in these processes. Moreover, we describe a novel mechanism for the activation of Rho GTPases and the cell motility pathway due to the interaction between MCPyV ST and the cellular phosphatase catalytic subunit PP4C, which leads to the specific dephosphorylation of bbeta;1 integrin. These findings may therefore provide novel strategies for therapeutic intervention for disseminated MCC.

  • Stability and expression levels of HLA-C on the cell membrane modulate HIV-1 infectivity [PublishAheadOfPrint]

  • HLA-C expression is associated with a differential ability to control HIV-1 infection. Higher HLA-C levels may lead to a better control of HIV-1 infection through both a higher efficiency of antigen presentation to cytotoxic T lymphocytes (CTLs), as well as the triggering of activating Killer Immunoglobulin like receptors (KIR) on NK-cells, whereas lower levels may provide a poor HIV-1 control and a rapid progression toward AIDS.

    We characterized the relative amount of HLA-C heterotrimers (heavy chain/bbeta;2m/peptide) and HLA-C free heavy chains on PBMC from healthy blood donors harboring both alleles with stable or unstable binding to bbeta;2m/peptide. We analyzed the stability of HLA-C heterotrimers of different allotypes and the infectivity of HIV-1 virions produced by PBMC with various allotypes.

    We observed significant differences in HLA-C heterotrimers stability and in expression levels. We found that R5 HIV-1 virions produced by PBMC harboring unstable HLA-C alleles were more infectious than those produced by PBMC carrying the stable variants.

    We propose that HIV-1 infectivity might depend both on the amounts of HLA-C molecules and on their stability as trimeric complex. According to this model, individuals with low expressed HLA-C alleles and unstable binding to bbeta;2m/peptide might have a worse control of HIV-1 infection and an intrinsically higher capacity to support viral replication.

    IMPORTANCE Following HIV-1 infection, some people advance rapidly toward AIDS while others have a slow disease progression. HLA-C, a molecule involved in immunity, is a key determinant of HIV-1 control.

    Here we reveal how HLA-C variants contribute to modulate viral infectivity. HLA-C is present on the cell surface in two different conformations: the immunologically active conformation is part of a complex that includes bbeta;2-microglobulin/peptide; the other conformation is not bound to bbeta;2-microglobulin/peptide and can associate with HIV-1, increasing its infectivity. Individuals with HLA-C variants with a predominance of immunologically active conformations would display a stronger immunity against HIV-1, a reduced viral infectivity and an effective control of HIV-1 infection, while subjects with HLA-C variants that easily dissociate from bbeta;2-microglobulin/peptide would have a reduced immunological response to HIV-1 and produce more infectious virions.

    This study provides new information that could be useful to design novel vaccine strategies and therapeutic approaches against HIV-1.

  • Epistastic interactions within the Junin virus envelope glycoprotein complex provide an evolutionary barrier to reversion in the live-attenuated Candid#1 vaccine [PublishAheadOfPrint]

  • The Candid#1 strain of Juniiacute;n virus was developed using a conventional attenuation strategy of serial passage in non-host animals and cultured cells. The live-attenuated Candid#1 vaccine is used in Argentina to protect at-risk individuals against Argentine hemorrhagic fever, but has not been licensed in the United States. Recent studies have revealed that Candid#1 attenuation is entirely dependent on a phenylalanine-to-isoleucine substitution at position 427 in the fusion subunit (GP2) of the viral envelope glycoprotein complex (GPC), thereby raising concerns regarding the potential for reversion to virulence. In this study, we report the identification and characterization of an intragenic epistatic interaction between the attenuating F427I mutation in GP2 and a lysine-to-serine mutation at position 33 in the stable signal peptide (SSP) subunit of GPC, and demonstrate the utility of this interaction in creating an evolutionary barrier against reversion to the pathogenic genotype. In the presence of the wild-type F427 residue, the K33S mutation abrogates the ability of ectopically expressed GPC to mediate membrane fusion at endosomal pH. This defect is rescued by the attenuating F427I mutation. We show that the recombinant Candid#1 (rCan) virus bearing K33S GPC is viable and retains its attenuated genotype under cell-culture conditions that readily select for reversion in the parental rCan virus. If back-mutation to F427 offers an accessible pathway to increase fitness in rCan, reversion in K33S-GPC rCan is likely to be lethal. The epistatic interaction between K33S and F427I may thus minimize the likelihood of reversion and enhance safety in a second-generation Candid#1 vaccine.

    IMPORTANCE The live-attenuated Candid#1 vaccine strain of Juniiacute;n virus is used to protect against Argentine hemorrhagic fever. Recent findings that a single missense mutation in the viral envelope glycoprotein complex (GPC) is responsible for attenuation raise the prospect of facile reversion to pathogenicity. Here we characterize a genetic interaction between GPC subunits that evolutionarily forces retention of the attenuating mutation. By incorporating this secondary mutation into Candid#1 GPC, we hope to minimize the likelihood of reversion and enhance safety in a second-generation Candid#1 vaccine. A similar approach may guide the design of live-attenuated vaccines against Lassa and other arenaviral hemorrhagic fevers.

  • Residues 28 to 39 of the Extracellular Loop 1 of Chicken Na+/H+ Exchanger Type I Mediate Cell Binding and Entry of Subgroup J Avian Leukosis Virus [PublishAheadOfPrint]

  • Chicken Na+/H+ exchanger type I (chNHE1), a multispan transmembrane protein, is a cellular receptor of the subgroup J avian leukosis virus (ALV-J). To identify the functional determinants of chNHE1 responsible for the ALV-J receptor activity, a series of chimeric receptors was created by exchanging the extracellular loops (ECL) of human NHE1 (huNHE1) and chNHE1, and by ECL replacement with a hemagglutinin (HA) tag. These chimeric receptors were then used in binding and entry assays to map the minimal ALV-J gp85-binding domain of chNHE1. We show that ECL1 of chNHE1 (chECL1) is the critical functional ECL that interacts directly with ALV-J gp85; ECL3 is also involved in ALV-J gp85 binding. Amino acid residues 28--39 of the N-terminal membrane-proximal region of chECL1 constitute the minimal domain required for chNHE1 binding of ALV-J gp85. These residues are sufficient to mediate viral entry into ALV-J non-permissive cells. Point-mutation analysis revealed that A30, V33, W38, and E39 of chECL1 are the key residues mediating the binding between chNHE1 and ALV-J gp85. Further, the replacement of residues 28--39 of huNHE1 with the corresponding chNHE1 residues converted the nonfunctional ALV-J receptor huNHE1 to a functional one. Importantly, soluble chECL1 and huECL1 harboring chNHE1 residues 28--39 could both effectively block ALV-J infection. Collectively, our findings indicate that residues 28--39 of chNHE1 constitute a domain that is critical for receptor function and mediate ALV-J entry.

    IMPORTANCE: ChNHE1 is a cellular receptor of ALV-J, a retrovirus that causes infections in chickens and serious economic losses in the poultry industry. Until now, the domains determining the chNHE1 receptor function remained unknown. We demonstrate that chECL1 is critical for receptor function, with residues 28--39 constituting the minimal functional domain responsible for chNHE1 binding of ALV-J gp85, and efficiently mediating ALV-J cell entry. These residues are located in the membrane-proximal region of the N-terminus of chECL1, suggesting that the binding site of ALV-J gp85 on chNHE1 is probably located on the apex of the molecule; the receptor-binding mode might be different from that of retroviruses. We also found that soluble chECL1, as well as huECL1 harboring chNHE1 residues 28--39, effectively blocked ALV-J infection. These findings contribute to a better understanding of the ALV-J infection mechanism, and also provide new insights into the control strategies for ALV-J infection.

  • The post-fusion structure of the Heartland virus Gc glycoprotein supports taxonomic separation of the bunyaviral families Phenuiviridae and Hantaviridae [PublishAheadOfPrint]

  • Heartland virus (HRTV) is an emerging human pathogen that belongs to the newly defined family Phenuiviridae, order Bunyavirales. Gn and Gc are two viral surface glycoproteins encoded by the M segment and are required for early events during infection. HRTV delivers its genome into the cytoplasm by fusion of the viral envelope and endosomal membranes under low pH conditions. Here, we describe the crystal structure of HRTV Gc in its post-fusion conformation. The structure shows that Gc displays a typical class II fusion protein conformation, and the overall structure is identical to severe fever with thrombocytopenia syndrome virus (SFTSV) Gc, which also belongs to the Phenuiviridae family. However, our structural analysis indicates that the hantavirus Gc presents distinct feature in the aspects of subdomain orientation, N-linked glycosylation, the interactions pattern between protomers, and the fusion loop conformation. This suggests their family-specific subunit arrangement during the fusogenic process and supports the recent taxonomic revision of bunyaviruses. Our results provide insights into the comprehensive comparison of class II membrane fusion proteins in two bunyavirus families, yielding valuable information for treatments against these human pathogens.

    IMPORTANCE HRTV is an insect-borne virus found in America that can infect humans. It belongs to the newly defined family Phenuiviridae, order Bunyavirales. HRTV contains three single-stranded RNA segments (L, M, and S). The M segment of the virus encodes a polyprotein precursor that is cleaved into two glycoproteins, Gn and Gc. Gc is a fusion protein facilitating virus entry into host cells. Here, we report the crystal structure of the HRTV Gc protein. The structure displays a typical class II fusion protein conformation. Comparison of HRTV Gc with a recently solved structure of another bunyavirus Gc revealed that these Gc structures display a newly defined family specificity, supporting the recent International Committee of Taxonomy of Viruses re-classification of the bunyaviruses. Our results expand the knowledge of bunyavirus fusion proteins and help us to understand bunyavirus characterizations. This study provides useful information to improve protection against and therapies for bunyavirus infections.

  • Cryo-EM structure of porcine delta coronavirus spike protein in the pre-fusion state [PublishAheadOfPrint]

  • Coronavirus spike proteins from different genera are divergent, although they all mediate coronavirus entry into cells by binding to host receptors and fusing viral and cell membranes. Here we determined the cryo-EM structure of porcine delta coronavirus (PdCoV) spike protein at 3.3-angstrom resolution. The trimeric protein contains three receptor-binding S1 subunits that tightly pack into a crown-like structure and three membrane-fusion S2 subunits that form a stalk. Each S1 subunit contains two domains, N-terminal domain (S1-NTD) and C-terminal domain (S1-CTD). PdCoV S1-NTD has the same structural fold as alpha- and beta-coronavirus S1-NTDs as well as host galectins, and it recognizes sugar as its potential receptor. PdCoV S1-CTD has the same structural fold as alpha-coronavirus S1-CTDs, but its structure differs from that of beta-coronavirus S1-CTDs. PdCoV S1-CTD binds to an unidentified receptor on host cell surfaces. PdCoV S2 is locked in the pre-fusion conformation by structural restraint of S1 from a different monomeric subunit. PdCoV spike possesses several structural features that may facilitate immune evasion by the virus, such as its compact structure, concealed receptor-binding sites, and shielded critical epitopes. Overall, this study reveals that delta-coronavirus spikes are structurally and evolutionally more closely related to alpha-coronavirus spikes than to beta-coronavirus spikes; it also has implications for the receptor recognition, membrane fusion, and immune evasion by delta-coronaviruses as well as coronaviruses in general.

    SIGNIFICANCE In this study we determined the cryo-EM structure of porcine delta coronavirus (PdCoV) spike protein at 3.3 angstrom. This is the first atomic structure of a spike protein from the delta coronavirus genus, which is divergent in amino acid sequences from the well-studied alpha- and beta-coronavirus spike proteins. In the current study, we described the overall structure of the PdCoV spike and the detailed structure of each of its structural elements. Moreover, we analyzed the functions of each of the structural elements. Based on the structures and functions of these structural elements, we discussed the evolution of PdCoV spike protein in relation to the spike proteins from other coronavirus genera. This study combines the structure, function, and evolution of coronavirus spike proteins, and provides many insights into the receptor recognition, membrane fusion, immune evasion, and evolution of PdCoV spike protein.

  • Structural basis for the escape of human astrovirus from antibody neutralization: broad implications for rational vaccine design [PublishAheadOfPrint]

  • Human astroviruses are recognized as a leading cause of viral diarrhea worldwide in children, immunocompromised patients, and the elderly. There are currently no vaccines available to prevent astrovirus infection, however antibodies developed by healthy individuals during previous infection correlate with protection from reinfection, suggesting that an effective vaccine could be developed. In this study, we investigated the molecular mechanism by which several strains of human astrovirus serotype 2 (HAstV-2) are resistant to the potent HAstV-2-neutralizing monoclonal antibody PL-2 (mAb PL-2). Sequencing of the HAstV-2 capsid genes reveals mutations in the PL-2 epitope within the capsid's spike domain. To understand the molecular basis for resistance from mAb PL-2 neutralization, we determined the 1.35 AAring;mmdash;resolution crystal structure of the capsid spike from one of these HAstV-2 strains. Our structure reveals a dramatic conformational change in a loop within the PL-2 epitope due to a serine-to-proline mutation, locking the loop in a conformation that sterically blocks binding and neutralization by mAb PL-2. We show that mutation to serine permits loop flexibility and recovers mAb PL-2 binding. Importantly, we find that HAstV-2 capsid spike containing a serine in this loop is immunogenic and elicits antibodies that neutralize all HAstV-2 strains. Taken together, our results have broad implications for rational selection of vaccine strains that do not contain prolines in antigenic loops, so as to elicit antibodies against diverse loop conformations.

    IMPORTANCE Human astroviruses (HAstVs) infect nearly every person in the world during childhood and cause diarrhea, vomiting, and fever. In this study, we investigated how several strains of HAstV are resistant to a virus-neutralizing monoclonal antibody. We determined the crystal structure of the capsid protein spike domain from one of these HAstV strains and found that a single amino acid mutation induces a structural change in a loop that is responsible for antibody binding. Our findings reveal how viruses can escape antibody neutralization and provide insight for the rational design of vaccines to elicit diverse antibodies that provide broader protection from infection.

  • The infectious laryngotracheitis virus (ILTV) viral chemokine-binding protein glycoprotein G alters the transcription of key inflammatory mediators in vitro and in vivo [PublishAheadOfPrint]

  • Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that infects chickens, causing upper respiratory tract disease and significant losses to poultry industries worldwide. Glycoprotein G (gG) is a broad range viral chemokine-binding protein conserved amongst most alphaherpesviruses, including ILTV. A number of studies comparing immunological parameters between infection with gG-expressing and gG-deficient ILTV strains have demonstrated that expression of gG is associated with increased virulence, modification of the amount and composition of the inflammatory response and modulation of immune responses towards antibody production and away from cell-mediated immune responses. The aims of the current study were to examine the establishment of infection and inflammation by ILTV and determine how gG influences that response to infection. In vitro infection studies using tracheal organ cultures and blood-derived monocytes, and in vivo infection studies in specific-pathogen-free chickens, showed that leukocyte recruitment to the site of infection is an important component of the induced pathology, and that this is influenced by the expression of ILTV gG and changes in the transcription of the chicken orthologues of mammalian CXCL8, chCXCLi1 and chCXCLi2, among other cytokines and chemokines. Results from this study demonstrate that ILTV gG interferes with chemokine and cytokine transcription at different steps of the inflammatory cascade, thus altering inflammation, virulence and the balance of the immune response to infection.

    IMPORTANCE Infectious laryngotracheitis virus is an alphaherpesvirus that expresses gG, a conserved broad range viral chemokine-binding protein known to interfere with host immune responses. However, little is known about how gG modifies virulence and influences the inflammatory signalling cascade associated with infection. Here, data from in vitro and in vivo infection studies are presented, showing that gG has a direct impact on the transcription of cytokines and chemokine ligands in vitro (such as chicken CXCL8 orthologues among others), which explains the altered balance of the inflammatory response that is associated with gG during ILTV infection of the upper respiratory tract of chickens. This is the first report to associate gG with dysregulation of cytokine transcription at different stages of the inflammatory cascade triggered by ILTV infection of the natural host.

  • Rotavirus degrades multiple type interferon receptors to inhibit IFN signaling and protects against mortality from endotoxin in suckling mice [PublishAheadOfPrint]

  • STAT1 phosphorylation in response to exogenous IFN administration can be inhibited by rotaviral replication both in vitro and in vivo. In addition many rotavirus strains are resistant to the actions of different IFN types . The regulation of multiple IFN type-mediated antiviral pathways by RVs is not well understood. In this study, we find that during infection in vitro and in vivo RV significantly depletes IFN-type I, II, and III receptors (IFNRs). Regulation of IFNRs occurred exclusively within RV-infected cells and could be abrogated by inhibiting the lysosomal-endosomal degradation pathway. In vitro, IFNR degradation was conserved across multiple RV strains that differ in their modes of regulating IFN induction. In suckling mice, exogenously administered types I, II, or III IFN each induced phosphorylation of STAT1-Y701 within intestinal epithelial cells (IECs) of suckling mice. Murine EW strain RV infection transiently activated intestinal STAT1 at 1dpi, but not subsequently at 2-3dpi. In response to injection of purified IFN-aalpha;/bbeta; or -, IECs in EW-infected mice exhibited impaired STAT1-Y701 phosphorylation, correlating with depletion of different intestinal IFNRs and impaired IFN-mediated transcription. The ability of EW murine RV to inhibit multiple IFN types led us to test protection of suckling mice from endotoxin-mediated shock, an outcome that is dependent on the host IFN response. Compared to controls, mice infected with EW murine RV were substantially protected against mortality following parenteral endotoxin administration. These studies identify a novel mechanism of IFN subversion by RV and reveal an unexpected protective effect of RV infection on endotoxin-mediated shock in suckling mice.

    IMPORTANCE Antiviral functions of types 1, 2, and 3 IFN are mediated by receptor-dependent activation of STAT1. Here we find that RV degrades the types 1, 2, and 3 IFN receptors (IFNR) in vitro. In a suckling mouse model, RV effectively blocked STAT1 activation and transcription following injection of different purified IFNs. This correlated with significantly decreased protein expression of intestinal types 1 and 2 IFNRs. Recent studies demonstrate that in mice, LPS-induced lethality is prevented by genetic ablation of IFN signaling genes such as IFNAR1 and STAT1. When suckling mice were infected with RV they were substantially protected from lethal exposure to endotoxin. These findings provide novel insights into the mechanisms underlying rotavirus regulation of different interferons and are likely to stimulate new research into both rotavirus pathogenesis and endotoxemia.

  • Structure, Immunogenicity and Protective Mechanism of an Engineered Enterovirus 71-Like Particle Vaccine Mimicking 80S Empty Capsid [PublishAheadOfPrint]

  • Enterovirus 71 (EV71) is the major causative agent of severe hand, foot and mouth disease which affects millions of young children in the Asia-Pacific region annually. In this study, we engineered a novel EV71 virus-like particle (VLP) that lacks VP4 (thereby designated "VLPVP4"), and investigated its structure, antigenicity and vaccine potential. The cryo-EM structure of VLPVP4 was reconstructed to 3.71 AAring; resolution. Results from structural and biochemical analyses reveal that VLPVP4 resembles the end product of the viral uncoating process, the 80S empty capsid. VLPVP4 is able to elicit high-titer neutralizing antibodies and fully protect mice against lethal viral challenge. Mechanistic studies show that, at the cellular level, the anti-VLPVP4 sera exert neutralization effects at both pre- and post-attachment stages by inhibiting both virus attachment and internalization, and at the molecular level, the antisera can block multiple interactions between EV71 and its key receptors. Our study gains a better understanding of EV71 capsid assembly and provides important information for the design and development of new generation vaccines for EV71 and perhaps for other enteroviruses as well.

    IMPORTANCE Enterovirus 71 (EV71) infection may lead to severe hand, foot and mouth disease with significant morbidity and mortality. Knowledge regarding EV71 particle assembly remains limited. Here, we report the generation and characterization of a novel EV71 virus-like particle that lacks the VP4 capsid subunit protein. This particle, termed VLPVP4, structurally mimics 80S empty capsid which is the end-stage of EV71 uncoating. We further show that VLPVP4 exhibits desirable immunogenicity and protective efficacy in proof-of-concept studies. In addition, the inhibitory mechanisms of the VLPVP4-induced antibodies are unraveled at both cellular and molecular levels. Our work provides the first evidence of picornaviral particle assembly in the complete absence of VP4 and identifies VLPVP4 as an improved EV71 vaccine candidate with desirable traits. These findings not only enhance our understanding of particle assembly and uncoating of picornaviruses, but also provide important information for structure-guided vaccine design for EV71 and other enteroviruses.

  • NS3 from HCV strain JFH-1 is an unusually robust helicase that is primed to bind and unwind viral RNA [PublishAheadOfPrint]

  • Hepatitis C viruses (HCV) encode a helicase enzyme that is essential for viral replication and assembly (NS3). This helicase has become the focus of extensive basic research on general helicase mechanism and it is also of interest as a novel drug target. Despite the importance of this protein, mechanistic work on NS3 has been conducted almost exclusively on variants from HCV genotype 1. Our understanding of NS3 from the highly active HCV strains that are used to study HCV genetics and mechanism in cell culture (such as JFH-1) is lacking. We therefore set out to determine whether NS3 from the replicatively efficient genotype 2a strain JFH-1 displays novel functional or structural properties. Using biochemical assays for RNA binding and duplex unwinding, we show that JFH-1 NS3 binds RNA much more rapidly than the previously-studied NS3 variants from genotype 1b. Unlike NS3 variants from other genotypes, JFH-1 NS3 binds RNA with high affinity in a functionally active form that is capable of immediately unwinding RNA duplexes without undergoing rate-limiting conformational changes that precede activation. Unlike other SF2 helicases, JFH-1 NS3 does not require long 3rrsquo; -overhangs and it unwinds duplexes that are flanked by only a few nucleotides, as in the folded HCV genome. To understand the physical basis for this, we solved the crystal structure of JFH-1 NS3, revealing a novel conformation that contains an open, positively charged RNA binding cleft that is primed for productive interaction with RNA targets, potentially explaining robust replication by HCV JFH-1.

    Importance Genotypes of HCV are as divergent as different types of flavivirus, and yet mechanistic features of HCV variants are presumed to be held in common. One of the most well-studied components of the HCV replication complex is a helicase known as nonstructural protein 3 (NS3). We set out to determine whether this important mechanical component possesses biochemical and structural properties that differ between common strains such as genotype 1b and a strain of HCV that replicates with exceptional efficiency (JFH-1, classified as genotype 2a). Indeed, unlike the inefficient gt1b NS3 that has been well-studied, JFH-1 NS3 is a super helicase with strong RNA affinity and high unwinding efficiency on a broad range of targets. Crystallographic analysis reveals architectural features that promote enhanced biochemical activity of JFH-1 NS3. These findings show that even within a single family of viruses, drift in sequence can result in the acquisition of radically new functional properties that enhance viral fitness.

  • Genotype 2 strains of porcine reproductive and respiratory syndrome virus dysregulate alveolar macrophage cytokine production via the unfolded protein response. [PublishAheadOfPrint]

  • Porcine reproductive and respiratory syndrome virus (PRRSV) infects alveolar macrophages (AM) causing dysregulated interferon (IFN)-aalpha; and tumor necrosis factor (TNF)-aalpha; production through a mechanism(s) yet to be resolved. Here, we show that AM infected with PRRSV secreted a reduced quantity of IFN-aalpha; following the cell exposure to synthetic dsRNA. This reduction did not correlate with reduced IFNA1 gene transcription. Rather, it coincided with two events that occurred late during infection and were indicative of translational attenuation, specifically, the activation of eukaryotic translation initiation factor 2aalpha; (eIF2aalpha;), and the appearance of stress granules. Notably, the typical rapid production of TNF-aalpha; by AM exposed to lipopolysaccharide (LPS) was suppressed or enhanced by PRRSV depending on when the LPS exposure occurred after virus infection. If exposure was delayed until 6 h post-infection (hpi) so that the development of the cytokine response coincided with the time in which phosphorylation of eIF2aalpha; by the stress sensor PERK (protein kinase RNA (PKR)-like ER kinase) occurred, inhibition of TNF-aalpha; production was observed. However, if LPS exposure occurred at 2 hpi, prior to a detectable onset of eIF2aalpha; phosphorylation, a synergistic response was observed due to the earlier NF-B activation via the stress sensor IRE1aalpha; (inositol-requiring kinase 1aalpha;). These results suggest that the asynchronous actions of two branches of the unfolded protein response (UPR), namely IRE1aalpha;, and PERK, activated by ER stress resulting from the virus infection, are associated with enhancement or suppression of TNF-aalpha; production, respectively.

    IMPORTANCE The activation of AM is controlled by its microenvironment to deter excessive pro-inflammatory cytokine responses to microbes that could impair lung function. However, viral pneumonias frequently become complicated by secondary bacterial infections triggering severe inflammation, lung dysfunction, and death. Although dysregulated cytokine production is considered an integral component of the exacerbated inflammatory response in viral-bacterial co-infections, the mechanism responsible for this event is unknown. Here, we show that PRRSV replication in porcine AM triggers activation of the IRE1aalpha; branch of the UPR, which causes a synergistic TNF-aalpha; response to LPS exposure. Thus, the severe pneumonias typically observed in pigs afflicted with PRRSV-bacterial co-infections could result from dysregulated, overly robust TNF-aalpha; production to opportunistic pathogens that is not commensurate with the typical restrained reaction by uninfected AM. This notion could help design therapies to mitigate the severity of viral and bacterial co-infections.

  • Both sphingomyelin and cholesterol in the host cell membrane are essential for Rubella virus entry [PublishAheadOfPrint]

  • Rubella virus (RuV) causes a systemic infection and transplacental fetal infection causes congenital rubella syndrome. In this study, we showed that treatment of cells with sphingomyelinase inhibited RuV infection. Assays using inhibitors of serine palmitoyl transferase and ceramide transport protein demonstrated the contribution of sphingomyelin (SM) to RuV infection. Compelling evidence for direct binding of RuV to lipid membranes at neutral pH was obtained using liposome co-flotation assays. The absence of either SM or cholesterol (Chol) abrogated the RuV--liposome interaction. SM and Chol (SM/Chol) were also critical for RuV binding to erythrocytes and lymphoid cells. Removal of Ca2+ from the assay buffer or mutation of RuV envelope E1 protein Ca2+-binding sites abrogated RuV binding to liposomes, erythrocytes, and lymphoid cells. However, RuV bound to various non-lymphoid adherent cell lines independently of extracellular Ca2+ or SM/Chol. Even in these adherent cell lines, both the E1 protein Ca2+-binding sites and cellular SM/Chol were essential for the early stage of RuV infection, possibly affecting envelope-membrane fusion in acidic compartments. Myelin oligodendrocyte glycoprotein (MOG) has recently been identified as a cellular receptor for RuV. However, RuV bound to MOG-negative cells in a Ca2+-independent manner. Collectively, our data demonstrate that RuV has two distinct binding mechanisms: one is Ca2+-dependent and the other Ca2+-independent. Ca2+-dependent binding observed in lymphoid cells occurs by the direct interaction between E1 protein fusion loops and SM/Chol-enriched membranes. Clarification of the mechanism of Ca2+-independent RuV binding is an important next step in understanding the pathology of RuV infection.

    IMPORTANCE Rubella has a significant impact on public health as infection during early pregnancy can result in babies being born with congenital rubella syndrome. Despite effective rubella vaccines being available, rubella outbreaks still occur in many countries. We studied the entry mechanism of Rubella virus (RuV) and found that RuV binds directly to the host plasma membrane in the presence of Ca2+ at neutral pH. This Ca2+-dependent binding is specifically directed to membranes enriched in sphingomyelin and cholesterol, and is critical for RuV infection. Importantly, RuV also binds to many cell lines in a Ca2+-independent manner. An unidentified RuV receptor(s) is involved in this Ca2+-independent binding. We believe that the data presented here may aid the development of the first anti-RuV drug.

  • Emergence and adaptation of a novel highly pathogenic H7N9 influenza virus in birds and humans from a 2013-human-infecting low pathogenic ancestor [PublishAheadOfPrint]

  • Since its emergence in 2013, the H7N9 low pathogenic avian influenza virus (LPAIV) has been circulating in domestic poultry in China, causing five waves of human infections. Recently, a novel H7N9 highly pathogenic avian influenza virus (HPAIV) variant possessing multiple basic amino acids at the cleavage site of the hemagglutinin protein was firstly reported in two human infection cases in January 2017. More seriously, those novel HPAI H7N9 variants have transmitted and caused poultry farms outbreaks in eight provinces. Herein, we demonstrate the presence of three different amino acid motifs at the cleavage sites of these HPAIV variants which were isolated from chickens and humans and likely evolved from the pre-existing LPAIVs. Animal experiments showed that these novel HPAI H7N9 variants are both highly pathogenic in chickens and lethal to mice. Notably, human origin viruses were more pathogenic in mice than avian viruses, and the mammalian adaptation associated E627K, A588V, and D701N mutations in the PB2 gene were identified in the infected mice using next-generation sequencing (NGS) and Sanger sequencing. In the key amino acid substitutions of PB2 and HA, no polymorphism was detected in the infected chickens lungs by NGS. In sum, these results highlight the highly pathogenicity and transmission in chickens, and the quickly adaptation in mammals of this new H7N9 variant, so the risk should be evaluated and payed more attention.

    IMPORTANCE Due to the recent increase zoonotic infections in poultry and persistent human infections in China, influenza A (H7N9) virus has remained a public health threat. Most of the A(H7N9) viruses previously reported have been of low pathogenicity. Now, these novel H7N9 HPAIV have caused human infection in three provinces and poultry farms outbreaks in eight provinces. We analyze these molecular features and compared the relative characteristics of one H7N9 LPAIV and two H7N9 HPAIVs isolated from chickens and two human-origin H7N9 HPAIVs in chicken and mice models. Our finding that all HPAIVs are both highly pathogenic and valid transmissibility in chickens. Strikingly, the human-origin viruses were more highly pathogenic than avian-origin viruses in mice, dynamic mutation were confirmed by NGS and Sanger sequencing. Our findings offer important insight into the origin, adaptation, pathogenicity, transmissibility to both poultry and mammals.

  • Viperin targets flavivirus virulence by inducing assembly of non-infectious capsid particles [PublishAheadOfPrint]

  • Efficient antiviral immunity requires interference with virus replication at multiple layers targeting diverse steps in the viral life cycle. Here we describe a novel flavivirus inhibition mechanism that results in interferon-mediated obstruction of tick-borne encephalitis virus particle assembly, and involves release of malfunctional membrane associated capsid (C) particles. This mechanism is controlled by the activity of the interferon-induced protein viperin, a broad spectrum antiviral interferon stimulated gene. Through analysis of the viperin-interactome, we identified the Golgi Brefeldin A resistant guanine nucleotide exchange factor 1 (GBF1), as the cellular protein targeted by viperin. Viperin-induced antiviral activity as well as C-particle release was stimulated by GBF1 inhibition and knock down, and reduced by elevated levels of GBF1. Our results suggest that viperin targets flavivirus virulence by inducing the secretion of unproductive non-infectious virus particles, by a GBF1-dependent mechanism. This yet undescribed antiviral mechanism allows potential therapeutic intervention.

    Importance The interferon response can target viral infection on almost every level, however, very little is known about interference of flavivirus assembly. Here we show that interferon, through the action of viperin, can disturb assembly of tick-borne encephalitis virus. The viperin protein is highly induced after viral infection and exhibit broad-spectrum antiviral activity. However, the mechanism of action is still elusive and appear to vary between the different viruses, indicating that cellular targets utilized by several viruses might be involved. In this study we show that viperin induce capsid particle release by interacting and inhibiting the function of the cellular protein Golgi Brefeldin A resistant guanine nucleotide exchange factor 1 (GBF1). GBF1 is a key protein in the cellular secretory pathway and essential in the life cycle of many viruses, also targeted by viperin, implicating GBF1 as a novel putative drug target.

  • Innate sensing of influenza A virus hemagglutinin glycoproteins by the host endoplasmic reticulum (ER) stress pathway triggers a potent antiviral response via ER-associated protein degradation [PublishAheadOfPrint]

  • Innate immunity provides an immediate defense against infection after host cells sense "danger" signals from microbes. Endoplasmic reticulum (ER) stress arises from accumulation of misfolded/unfolded proteins when protein load overwhelms the ER folding capacity, which activates the unfolded protein response (UPR) to restore the ER homeostasis. Here, we show that a mechanism for antiviral innate immunity is triggered after the ER stress pathway senses viral glycoproteins. When hemagglutinin (HA) glycoproteins from influenza A virus (IAV) are expressed in cells, ER stress is induced, resulting in rapid HA degradation via proteasomes. The ER-associated protein degradation (ERAD) pathway, an important UPR function for destruction of aberrant proteins, mediates HA degradation. Three class I aalpha;-mannosidases were identified to play a critical role in the degradation process, including EDEM1, EDEM2, and ERManI. HA degradation requires either ERManI enzymatic activity or EDEM1/EDEM2 enzymatic activity, when ERManI is not expressed, indicating that demannosylation is a critical step for HA degradation. Silencing of EDEM1, EDEM2, and ERManI strongly increases HA expression and promotes IAV replication. Thus, the ER stress pathway senses influenza HA as "non-self" or misfolded protein, and sorts HA to ERAD for degradation, resulting in inhibition of IAV replication.

    IMPORTANCE Viral nucleic acids are recognized as important inducers of innate antiviral immune responses that are sensed by multiple classes of sensors, but other inducers and sensors of viral innate immunity need to be identified and characterized. Here, we used influenza A virus (IAV) to investigate how host innate immunity is activated. We found that IAV hemagglutinin (HA) glycoproteins induce ER stress, resulting in HA degradation via ERAD and consequent inhibition of IAV replication. In addition, we have identified three class I aalpha;-mannosidases, EDEM1, EDEM2, and ERManI, which play a critical role in initiating HA degradation. Knockdown of these proteins substantially increases HA expression and IAV replication. The enzymatic activities and joint actions of these mannosidases are required for this antiviral activity. Our results suggest that viral glycoproteins induce a strong innate antiviral response through activating the ER stress pathway during viral infection.

  • gga-miR-130b suppresses infectious bursal disease virus replication via targeting the viral genome and cellular SOCS5 [PublishAheadOfPrint]

  • MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression post transcriptionally through silencing or degrading its targets, playing important roles in immune response. However, the role of miRNAs in host response against infectious bursal disease virus (IBDV) infection is not clear. In this study, we show that the expression of a series of miRNAs was significantly altered in DF-1 cells after infectious bursal disease virus (IBDV) infection. We found that gga-miR-130b inhibited IBDV replication via targeting the specific sequence of IBDV segment A and enhanced the expression of IFN-bbeta; by targeting Suppressors of Cytokine Signaling 5(SOCS5) in host cells. These findings indicate that gga-miR-130b-3p plays a crucial role in host defense against IBDV infection.

    Importance This work shows that gga-miR-130b suppresses IBDV replication via directly targeting the viral genome and cellular SOCS5, the negative regulator for type I interferon expression, revealing the mechanism underlying gga-miR-130-induced inhibition of IBDV replication. This information would be of great help to the understandings of how host cells combat pathogenic infection by self-encoded small RNA and further our knowledge of the role of MicroRNAs in cell response against viral infection.

  • Foamy Virus Vector Carries a Strong Insulator in Its Long Terminal Repeat Which Reduces Its Genotoxic Potential [PublishAheadOfPrint]

  • Strong viral enhancers in -retrovirus vectors have caused cellular proto-oncogene activation and leukemia, necessitating use of cellular promoters in llsquo;enhancer-lessrrsquo; self-inactivating integrating vectors. However, cellular promoters result in relatively low transgene expression, often leading to inadequate disease correction. Vectors derived from foamy virus, a nonpathogenic retrovirus, show higher preference for non-genic integrations than -retroviruses/lentiviruses and preferential integration near transcriptional start sites, like -retroviruses. We found that strong viral enhancer/promoters placed in foamy viral vectors caused extremely low immortalization of primary mouse hematopoietic stem/progenitor cells compared to analogous -retrovirus/lentivirus vectors carrying the same enhancer/promoters; an effect not explained solely by foamy virus' modest insertional site preference for non-genic regions, compared to -retrovirus/lentivirus vectors. Using CRISPR/Cas9-mediated targeted insertion of analogous proviral sequences into the LMO2 gene and then measuring LMO2 expression, we demonstrated a sequence specific effect of foamy virus, independent of insertional bias, contributing to reduced genotoxicity. We show that this effect is mediated by a 36-bp insulator located in the foamy virus LTR that has high affinity binding for the CCCTC-binding factor. Using our LMO2 activation assay, LMO2 expression was significantly increased when this insulator was removed from foamy virus, and significantly reduced when this insulator was inserted into the lentiviral LTR. Our results elucidate a mechanism underlying the low genotoxicity of foamy virus, identify a novel insulator, and support the use of foamy virus as a vector for gene therapy, especially when strong enhancer/promoters are required.

    IMPORTANCE Understanding the genotoxic potential of viral vectors is important in designing safe and efficacious vectors for gene therapy. Self-inactivating vectors, devoid of viral long-terminal-repeat enhancers, have proven safe; however, transgene expression from cellular promoters is often insufficient for full phenotypic correction. Foamy virus is an attractive vector for gene therapy. We found foamy virus vectors to be remarkably less genotoxic; well below what is expected from their integration site preferences. We demonstrate that the foamy virus long-terminal-repeats contain an insulator element that binds CCCTC-binding factor and reduce its insertional genotoxicity. Our study elucidates a mechanism behind the low genotoxic potential of foamy virus, identifies a unique insulator, and supports use of foamy virus as a vector for gene therapy.

  • Distinct roles of cellular ESCRT-I and ESCRT-III proteins in efficient entry and egress of budded virions of Autographa californica multiple nucleopolyhedrovirus [PublishAheadOfPrint]

  • The endosomal sorting complex required for transport (ESCRT) machinery is necessary for budding by many enveloped viruses. Recently, it was demonstrated that Vps4, the key regulator for recycling of the ESCRT-III complex, is required for efficient infection of the baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV). However, ESCRT assembly, regulation and function are complex and little is known regarding details of participation of specific ESCRT complexes in AcMNPV infection. In this study, the core components of ESCRT-I (Tsg101 and Vps28) and ESCRT-III (Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60) were cloned from Spodoptera frugiperda. Using a viral complementation system and RNAi assays, we found that ESCRT-I and ESCRT-III complexes are required for efficient entry of AcMNPV into insect cells. In cells knocking down or overexpressing dominant-negative (DN) forms of the components of ESCRT-I and ESCRT-III complexes, entering virions were partially trapped within the cytosol. To examine only egress, cells were transfected with the dsRNA targeting an individual ESCRT-I or ESCRT-III gene and viral bacmid DNA or viral bacmid DNA that expressed DN forms of ESCRT-I and ESCRT-III components. We found that ESCRT-III components (but not ESCRT-I components) are required for efficient nuclear egress of progeny nucleocapsids. In addition, we found that several baculovirus core or conserved proteins (Ac11, Ac76, Ac78, GP41, Ac93, Ac103, Ac142, and Ac146) interact with Vps4 and components of ESCRT-III. We propose that these viral proteins may form an "egress complex" that is involved in recruiting ESCRT-III components to a virus egress domain on the nuclear membrane.

    IMPORTANCE The ESCRT system is hijacked by many enveloped viruses, to mediate budding and release. Recently, it was found that Vps4, the key regulator of cellular ESCRT machinery, is necessary for efficient entry and egress of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). However, little is known about the roles of specific ESCRT complexes in AcMNPV infection. In this study, we demonstrated that ESCRT-I and ESCRT-III complexes are required for efficient entry of AcMNPV into insect cells. The components of ESCRT-III (but not ESCRT-I) are also necessary for efficient nuclear egress of progeny nucleocapsids. Several baculovirus core or conserved proteins were found to interact with Vps4 and components of ESCRT-III, and these interactions may suggest the formation of an "egress complex" involved in nuclear release or transport of viral nucleocapsids.

  • In vitro neutralization is not predictive of prophylactic efficacy of broadly neutralizing monoclonal antibodies CR6261 and CR9114 against lethal H2 influenza virus challenge in mice [PublishAheadOfPrint]

  • Influenza viruses of the H1N1, H2N2, and H3N2 subtypes have caused previous pandemics. H2 influenza viruses represent a pandemic threat due to continued circulation in wild birds and limited immunity in the human population. In the event of a pandemic, anti-viral agents are the mainstay for treatment, but broadly neutralizing antibodies (bNAbs) may be a viable alternative for short-term prophylaxis or treatment. The hemagglutinin stem binding bNAbs CR6261 and CR9114 have been shown to protect mice from severe disease following challenge with H1N1 and H5N1, and H1N1, H3N2, and influenza B viruses, respectively. Early studies with CR6261 and CR9114 showed weak in vitro activity against human H2 influenza viruses, but the in vivo efficacy against H2 viruses is unknown. Therefore, we evaluated these antibodies against human and animal origin H2 viruses: A/Ann Arbor/6/1960 (H2N2) [AA60] and A/swine/MO/4296424/06 (H2N3) [Sw06]. In vitro, CR6261 neutralized both H2 viruses, while CR9114 only neutralized Sw06. To evaluate prophylactic efficacy, mice were given CR6261 or CR9114 and intranasally challenged 24 hours later with lethal doses of AA60 or Sw06. Both antibodies reduced mortality, weight loss, airway inflammation, and pulmonary viral load. Using engineered bNAb variants, ADCC reporter assays, and Fc receptor deficient (Fcer1g-/-) mice, we show that the in vivo efficacy of CR9114 against AA60 is mediated by Fc receptor-dependent mechanisms. Collectively, these findings demonstrate the in vivo efficacy of CR6261 and CR9114 against H2 viruses, and emphasize the need for in vivo evaluation of bNAbs.

    IMPORTANCE: Broadly neutralizing antibodies (bNAbs) represent a strategy to prevent or treat infection by a wide range of influenza viruses. The evaluation of these antibodies against H2 viruses is important because H2 viruses caused a pandemic in 1957 and could cross into humans again. We demonstrate that CR6261 and CR9114 are effective against infection with H2 viruses of both human and animal origin in mice, despite the finding that CR9114 did not display in vitro neutralizing activity against the human H2 virus. These findings emphasize the importance of in vivo evaluation and testing of bNAbs.

  • Screening for Neuraminidase Inhibitor Resistance Markers among Avian Influenza Viruses of the N4, N5, N6 and N8 Neuraminidase Subtypes [PublishAheadOfPrint]

  • Several subtypes of avian influenza viruses (AIVs) are emerging as novel human pathogens, and the frequency of related infections has increased in recent years. Although neuraminidase (NA) inhibitors (NAIs) are the only class of antiviral drugs available for therapeutic intervention for AIV-infected patients, studies on NAI resistance among AIVs have been limited and markers of resistance are poorly understood. Previously, we identified unique NAI-resistance substitutions in AIVs of N3, N7, and N9 NA subtypes. Here, we report profiles of NA substitutions that confer NAI resistance in the AIVs of N4, N5, N6, and N8 NA subtypes using Gene-fragmented Random Mutagenesis. We generated libraries of mutant influenza viruses using reverse genetics (RG), and selected resistant variants in the presence of the NAIs oseltamivir carboxylate and zanamivir in MDCK cells. In addition, two substitutions, H274Y or R292K (N2 numbering) were introduced into each NA gene for comparison. We identified 37 amino acid substitutions within the NA gene, 16 of which (N4 = 4, N5 = 4, N6 = 4, and N8 = 4) conferred resistance to NAIs (oseltamivir carboxylate, zanamivir, or peramivir) as determined using a fluorescence-based NA inhibition assay. Substitutions conferring NAI resistance were mainly categorized as either novel NA subtype-specific (G/N147V/I, A246V and I427L) or previously reported in other subtypes (E119A/D/G/V, Q136K, E276D, R292K, and R371K). Our results demonstrate that each NA subtype possesses unique NAI resistance markers and knowledge of these substitutions in AIVs is important in facilitating antiviral susceptibility monitoring of NAI resistance in AIVs.

    IMPORTANCE The frequency of human infections with avian influenza viruses (AIVs) has increased in recent years. Despite the availability of vaccines, neuraminidase inhibitors (NAIs)mmdash;as the only available class of drugs for AIVs in humansmmdash;have been constantly used for treatment leading to the inevitable emergence of drug-resistant variants. To screen for substitutions conferring NAI resistance in AIV of N4, N5, N6, and N8 NA subtypes, random mutations within the target gene were generated and resistant viruses were selected from mutant libraries in the presence of individual drugs. We identified 16 NA substitutions conferring NAI resistance in the tested AIV subtypes, some are novel and subtype-specific, and others have been previously reported in other subtypes. Our findings will contribute to an increased and more comprehensive understanding of the mechanisms of NAI-induced inhibition of influenza and help lead to the development of drugs that bind to alternative interaction motifs.

  • Murine hepatitis virus nsp14 exoribonuclease activity is required for resistance to innate immunity [PublishAheadOfPrint]

  • Coronaviruses (CoVs) are positive-sense RNA viruses that infect numerous mammalian and avian species and are capable of causing severe and lethal disease in humans. CoVs encode several innate immune antagonists that counteract the host innate immune response to facilitate efficient viral replication. CoV non-structural protein 14 (nsp14) encodes 3' -to-5' exoribonuclease activity (ExoN), which performs a proofreading function and is required for high-fidelity replication. Outside of the order Nidovirales, arenaviruses are the only RNA viruses that encode an ExoN, which functions to degrade dsRNA replication intermediates. In this study, we tested the hypothesis that CoV ExoN may also function to antagonize the innate immune response. We demonstrate that viruses lacking ExoN activity [ExoN(-)] are sensitive to cellular pretreatment with interferon beta (IFN-bbeta;) in a dose-dependent manner. In addition, ExoN(-) virus replication was attenuated in wild-type bone marrow-derived macrophages (BMMs) and partially restored in interferon alpha/beta receptor deficient (IFNAR-/-) BMMs. ExoN(-) virus replication did not result in IFN-bbeta; gene expression, and in the presence of an IFN-bbeta;-mediated antiviral state, ExoN(-) viral RNA levels were not substantially reduced relative to untreated. However, ExoN(-) virus generated from IFN-bbeta; pretreated cells had reduced specific infectivity and decreased relative fitness, suggesting that ExoN(-) virus generated during an antiviral state is less viable to establish a subsequent infection. Overall, our data suggest murine hepatitis virus (MHV) ExoN activity is required for resistance to the innate immune response and antiviral mechanisms affecting the viral RNA sequence and/or an RNA modification act on viruses lacking ExoN activity.

    IMPORTANCE CoVs encode multiple antagonists that prevent or disrupt an efficient innate immune response. Additionally, no specific antiviral therapies or vaccines currently exist for human CoV infections. Therefore, the study of CoV innate immune antagonists is essential for understanding how CoVs overcome host defenses and to maximize potential therapeutic interventions. Here, we sought to determine the contributions of nsp14 ExoN activity in the induction of and resistance to the innate immune response. We show that viruses lacking nsp14 ExoN activity are more sensitive than WT-MHV to restriction by exogenous IFN-bbeta; and that viruses produced in the presence of an antiviral state are less capable of establishing a subsequent viral infection. Our results support the hypothesis that murine hepatitis virus ExoN activity is required for resistance to the innate immune response.

  • HIV-1 infection of primary CD4+ T cells regulates the expression of specific HERV-K (HML-2) elements. [PublishAheadOfPrint]

  • Endogenous retroviruses (ERVs) occupy extensive regions of the human genome. Although many of these retroviral elements have lost their ability to replicate, those whose insertion took place more recently, such as the HML-2 group of HERV-K elements, still retain intact open reading frames and the capacity to produce certain viral RNA and/or proteins. Transcription of these ERVs is, however, tightly regulated by dedicated epigenetic control mechanisms. Nonetheless, it has been reported that some pathologic states, such as viral infections and certain cancers, coincide with ERV expression suggesting transcriptional reawakening is possible. HML-2 elements are reportedly induced during HIV-1 infection, but the conserved nature of these elements has, until recently, rendered their expression profiling problematic.

    Here, we provide comprehensive HERV-K HML-2 expression profiles specific for productively HIV-1 infected primary human CD4+ T cells. We combined enrichment of HIV-1 infected cells using a reporter virus expressing a surface reporter for gentle and efficient purification with long-read Single Molecule Real-Time sequencing. We show that three HML-2 proviruses, 6q25.1, 8q24.3, and 19q13.42 are up-regulated on average between 3- and 5-fold in HIV-1 infected CD4+ T cells. One provirus, HML-2 12q24.33, in contrast, was repressed in the presence of active HIV replication.

    In conclusion, this report identifies the HERV-K HML-2 loci whose expression profiles differ upon HIV-1 infection in primary human CD4+ T cells. These data will help pave the way for further studies on the influence of endogenous retroviruses on HIV-1 replication.

    Importance Endogenous retroviruses inhabit big portions of our genome. And although they are mainly inert some of the evolutionarily younger members maintain the ability to express both RNA as well as proteins. We have developed an approach using long-read SMRT sequencing that produces long reads, that provides us with ability to obtain detailed and accurate HERV-K HML-2 expression profiles. We have now applied this approach to study HERV-K expression in the presence and absence of productive HIV-1 infection of primary human CD4+ T cells. In addition to using SMRT sequencing, our strategy also includes the magnetic selection of the infected cells so that levels of background expression due to uninfected cells are kept at a minimum. The results in this manuscript provide the blueprint for in-depth studies of the interactions of the authentic upregulated HERV-K HML-2 elements and HIV-1.

  • Flow cytometric detection of PrPSc in neurons and glial cells from prion-infected mouse brains [PublishAheadOfPrint]

  • In prion diseases, an abnormal isoform of prion protein (PrPSc) accumulates in neurons, astrocytes, and microglia in the brains of animals affected with prions. Detailed analyses of PrPSc-positive neurons and glial cells are required to clarify their pathophysiological roles in the disease. Here we report a novel method for the detection of PrPSc in neurons and glial cells from the brains of prion-infected mice by flow cytometry using PrPSc-specific staining with mAb 132. The combination of PrPSc staining and immunolabeling of neural cell markers clearly distinguished neurons, astrocytes, and microglia that were positive for PrPSc from those that were PrPSc negative. The flow cytometric analysis of PrPSc revealed the appearance of PrPSc-positive neurons, astrocytes, and microglia at 60 days after intracerebral prion inoculation, suggesting the presence of PrPSc in the glial cells, as well as in neurons, from an early stage of infection. Moreover, the kinetic analysis of PrPSc revealed a continuous increase in the proportion of PrPSc-positive cells for all cell types with disease progression. Finally, we applied this method to isolate neurons, astrocytes, and microglia positive for PrPSc from a prion-infected mouse brain by florescence-activated cell sorting. The method described here enables comprehensive analyses specific to PrPSc-positive neurons, astrocytes, and microglia that will contribute to the understanding of the pathophysiological roles of neurons and glial cells in PrPSc-associated pathogenesis.

    IMPORTANCE Although formation of PrPSc in neurons is associated closely with neurodegeneration in prion diseases, the mechanism of neurodegeneration is not understood completely. On the other hand, recent studies proposed the important roles of glial cells in PrPSc-associated pathogenesis, such as the intracerebral spread of PrPSc and clearance of PrPSc from the brain. Despite the great need for detailed analyses of PrPSc-positive neurons and glial cells, methods available for cell-type specific analysis of PrPSc have been limited thus far to microscopic observations. Here, we have established a novel high-throughput method for flow cytometric detection of PrPSc in cells with more accurate quantitative performance. By applying this method, we succeeded in isolating PrPSc-positive cells from the prion-infected mouse brains via fluorescence-activated cell sorting. This allows us to perform further detailed analysis specific to PrPSc-positive neurons and glial cells for the clarification of pathological changes in neurons and pathophysiological roles of glial cells.

  • Rice Stripe Tenuivirus Has a Greater Tendency To Use the Prime-and-Realign Mechanism in Transcription of Genomic than in Transcription of Antigenomic Template RNAs [PublishAheadOfPrint]

  • Most segmented negative sense RNA viruses employ a process termed cap-snatching during which they snatch capped-RNA leaders from host cellular mRNAs and use the snatched leaders as primers for transcription, leading to the synthesis of viral mRNAs with 5' heterogeneous sequences (HSs). With traditional methods, only a few HSs can be determined and identification of their donors is difficult. Here, the mRNA 5' -ends of Rice stripe tenuivirus (RSV) and Rice grassy stunt tenuivirus (RGSV) and those of their host rice were determined by high throughput sequencing. Millions of tenuiviral HSs were obtained and a large number of them mapped to the 5' -ends of corresponding host cellular mRNAs. Repeats of the dinucleotide AC, which are complementary to the U1G2 of the tenuiviral template 3' -U1G2U3G4UUUCG-, were found prevalent at the 3' termini of tenuiviral HSs. Most of these ACs did not match host cellular mRNAs, supporting the idea that tenuiviruses use the prime-and-realign mechanism during cap-snatching. We previously reported a greater tendency of RSV to use the prime-and-realign mechanism than RGSV in transcription with leaders cap-snatched from a co-infecting reovirus. Besides confirming this observation in natural tenuiviral infections, the data here additionally revealed that RSV has a greater tendency to use this mechanism in transcribing genomic- than in transcribing anigenomic templates. The data also suggested that tenuiviruses cap-snatch host cellular mRNAs from translation and photosynthesis related genes and capped-RNA leaders snatched by tenuiviruses base-pair with U1/U3 or G2/G4 of viral templates. These results provided unprecedented insights into the cap-snatching process of tenuiviruses.

    IMPORTANCE Many segmented negative sense RNA viruses (segmented NSVs) are medically or agriculturally important pathogens. The cap-snatching process is promising target for the development of antiviral strategies against this group of viruses. However, many details of this process remain poorly characterized. Tenuiviruses constitute a genus of agriculturally important segmented NSVs with several members of which being major viral pathogens of rice. Here, we for the first time adopted a high throughput sequencing strategy to determine the 5' heterogeneous sequences (HSs) of tenuiviruses and map them to host cellular mRNAs. Besides providing deep insights into the cap-snatching of tenuiviruses, the data obtained here provided clear evidence to support several previously proposed models regarding cap-snatching. Curiously and importantly, the data here revealed that not only different tenuiviruses but also the same tenuivirus in synthesizing different mRNAs uses the prime-and-realign mechanism with different tendencies during their cap snatching.

  • Relative resistance of MHC-B to Nef-mediated downregulation is conserved among primate lentiviruses and influences antiviral T cell responses in HIV-1-infected individuals [PublishAheadOfPrint]

  • Patient-derived HIV-1 subtype B Nef clones downregulate HLA-A more efficiently than HLA-B. However, it remains unknown whether this property is common to Nef proteins across primate lentiviruses, and how antiviral immune responses may be affected. We examined 263 Nef clones from diverse primate lentiviruses including different pandemic HIV-1 group M subtypes for their ability to downregulate MHC-A and MHC-B from the cell surface. Though lentiviral Nef proteins differed markedly in their absolute MHC-A and MHC-B downregulation abilities, all lentiviral Nef lineages downregulated MHC-A on average 11-32% more efficiently than MHC-B. Nef genotype/phenotype analyses in a cohort of HIV-1 subtype C-infected patients (N=168), together with site-directed mutagenesis, revealed Nef position 9 as a subtype-specific determinant of differential HLA-A vs. HLA-B downregulation activity. Nef clones harboring non-consensus variants at codon 9 downregulated HLA-B (though not HLA-A) significantly better than those harboring consensus at this site, resulting in reduced recognition of infected target cells by HIV-1-specific CD8+ effector cells in vitro. Among persons expressing protective HLA class I alleles, carriage of Nef codon 9 variants was also associated with reduced ex vivo HIV-specific T-cell responses. Our results demonstrate that Nef's inferior ability to downregulate MHC-B compared to MHC-A is conserved across primate lentiviruses, and suggest that this property influences antiviral cellular immune responses.

    IMPORTANCE Primate lentiviruses encode the Nef protein that plays an essential role in establishing persistent infection in their respective host species. Nef interacts with the cytoplasmic region of MHC-A and MHC-B molecules and downregulates them from the infected cell surface to escape recognition by host cellular immunity. Using a panel of Nef alleles isolated from diverse primate lentiviruses including pandemic HIV-1 group M subtypes, we demonstrate that Nef proteins across all lentiviral lineages downregulate MHC-A approximately 20% more effectively than MHC-B. We further identify a naturally polymorphic site at Nef position 9 that contributes to MHC-B downregulation function in HIV-1 subtype C, and show that carriage of Nef variants with enhanced MHC-B downregulation ability is associated with reduced breadth and magnitude of MHC-B-restricted cellular immune responses in HIV-infected individuals. Our study underscores an evolutionally conserved interaction between lentiviruses and primate immune systems that may contribute to pathogenesis.

  • Distribution of hepatitis B virus nuclear DNA [PublishAheadOfPrint]

  • Chronic hepatitis B affects over 300 million people who are at risk of developing liver cancer. The basis for the persistence of hepatitis B virus (HBV) in hepatocytes, even in the presence of available antiviral therapies, lies in the accumulation of covalently closed circular (ccc) DNA in nuclei of infected cells. While methods for cccDNA quantification from liver biopsies and cell lines expressing the virus are known, information about cccDNA formation, stability and turnover are lacking. In particular, little is known about the fate of cccDNA during cell division. To fill gaps in knowledge concerning cccDNA biology, we have developed a fluorescence imaging in situ hybridization (FISH)-based assay for the detection of duck hepatitis B virus (DHBV) cccDNA and HBV nuclear DNA in established cell lines. Using FISH, we determined the distribution of cccDNA under conditions mimicking chronic infections with and without antiviral therapy, which prevents de novo viral replication. Our results showed that the copy numbers of viral nuclear DNA can vary by as much as 1.8 orders of magnitude among individual cells, and that antiviral therapy leads to a reduction in nuclear DNA in a manner consistent with symmetrical distribution of viral DNA to daughter cells.

    Importance A mechanistic understanding of the stability of HBV cccDNA in the presence of antiviral therapy and during cell division induced by immune mediated lysis of infected hepatocytes, will be critical for the future design of curative antiviral therapies against chronic hepatitis B. To date, knowledge about cccDNA stability was largely derived from quantitative analyses of cccDNA levels present in liver samples, and little was known about the fate of cccDNA in individual cells. The development of a FISH-based assay for cccDNA tracking provided the first insights into the fate of DHBV cccDNA and nuclear HBV DNA during conditions mimicking antiviral therapy.

  • Prion Protein Devoid of the Octapeptide Repeat Region Delays BSE Pathogenesis in Mice [PublishAheadOfPrint]

  • Conformational conversion of the cellular isoform of prion protein PrPC, into the abnormally folded, amyloidogenic isoform, PrPSc, is a key pathogenic event in prion diseases including Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy (BSE) in animals. We previously reported that the octapeptide repeat (OR) region could be dispensable for converting PrPC into PrPSc after infection with RML prions. We demonstrated that mice transgenically expressing mouse PrP with deletion of the OR region on the PrP-knockout background, designated Tg(PrPOR)/Prnp0/0 mice, did not reduce susceptibility to RML scrapie prions, with abundant accumulation of PrPScOR in their brains. We show here that Tg(PrPOR)/Prnp0/0 mice were highly resistant to BSE prions, developing the disease with markedly elongated incubation times after infection with BSE prions. The conversion of PrPOR into PrPScOR was markedly delayed in their brains. These results suggest that the OR region may have a crucial role in the conversion of PrPC into PrPSc after infection with BSE prions. However, Tg(PrPOR)/Prnp0/0 mice remained susceptible to RML and 22L scrapie prions, developing the disease without elongated incubation times after infection with RML and 22L prions. PrPScOR accumulated only slightly less in the brains of RML- or 22L-infected Tg(PrPOR)/Prnp0/0 mice than PrPSc in control wild-type mice. Taken together, these results indicate that the OR region of PrPC could play a differential role in the pathogenesis of BSE prions and RML or 22L scrapie prions.

    IMPORTANCE Structure-function relationship studies of PrPC conformational conversion into PrPSc are worthwhile to understand the mechanism of the conversion of PrPC into PrPSc. We show here that, by inoculating the three different prion strains RML, 22L and BSE prions, into Tg(PrPOR)/Prnp0/0 mice, the OR region could play a differential role in the conversion of PrPC into PrPSc after infection with RML or 22L scrapie prions and BSE prions. PrPOR was efficiently converted into PrPScOR after infection with RML and 22L prions. However, the conversion of PrPOR into PrPScOR was markedly delayed after infection with BSE prions. Further investigation into the role of the OR region in the conversion of PrPC into PrPSc after infection with BSE prions might be helpful for understanding the pathogenesis of BSE prions.

  • Extended synaptotagmin 1 interacts with the Herpes simplex virus type 1 glycoprotein M and negatively modulates virus-induced membrane fusion [PublishAheadOfPrint]

  • Enveloped viruses typically encode their own fusion machinery to enter cells. Herpesviruses are particular as they fuse with a number of cellular compartments throughout their life cycle. As the uncontrolled mixing of the host membranes should be avoided in these events, the tight regulation of the viral fusion machinery is critical. While studying the herpes simplex virus type 1 glycoprotein gM, we identified the cellular protein E-Syt1 (Extended synaptotagmin-1) as an interaction partner. This took place in both infected and transfected cells, suggesting other viral proteins were not required for this interaction. Most interestingly, E-Syt1 is a member of the synaptotagmin family of membrane fusion regulators. However, this protein is rather known to promote the tethering of the endoplasmic reticulum (ER) with the plasma membrane. We now show that E-Syt1, along with the related E-Syt3, negatively modulates viral release into the extracellular milieu, cell-to-cell viral spread and viral entry, all processes that implicate membrane fusion events. Similarly, these E-Syt proteins impacted the formation of viral-induced syncytia. Altogether, these findings hint at the modulation of the viral fusion machinery by the E-Syt family of proteins.

    IMPORTANCE Viruses typically encode their own fusion apparatus to enable them to enter cells. For many viruses, this means a single fusogenic protein. However, herpesviruses are large entities that express several accessory viral proteins to regulate their fusogenic activity. The present study hints at the additional participation of cellular proteins in this process, suggesting the host can also modulate, to some extent, viral fusion. Hence the E-Syt proteins 1 and 3 seem to negatively modulate the different viral fusion events that take place during the HSV-1 viral life cycle. This could represent yet another innate immunity response to the virus.

  • Mechanisms of HERV-K (HML-2) transcription during human mammary epithelial cell transformation [PublishAheadOfPrint]

  • Increasing evidence suggests that repetitive elements may play a role in host gene regulation, particularly through the donation of alternative promoters, enhancers, splice sites, and termination signals. Elevated transcript expression of the endogenous retrovirus group HERV-K (HML-2) is seen in many human cancers, although the identity of the individual proviral loci contributing to this expression as well as their mechanism of activation has been unclear. Using high-throughput next-generation sequencing techniques optimized for the capture of HML-2 expression, we characterized the HML-2 transcriptome and means of activation in an in vitro model of human mammary epithelial cell transformation. Our analysis showed significant expression originating from fifteen HML-2 full-length proviruses, through four modes of transcription. The majority of expression was in antisense orientation and from proviruses integrated within introns. We found two instances of LTR-driven provirus transcription but no evidence to suggest that these active 5' LTRs were influencing nearby host gene expression. Importantly, LTR-driven transcription was restricted to tumorigenic cells, suggesting that LTR promoter activity is dependent upon the transcriptional environment of a malignant cell.

    IMPORTANCE Here, we use an in vitro model of human mammary epithelial cell transformation to assess how malignancy-associated shifts in the transcriptional milieu of a cell may impact HML-2 activity. We found fifteen proviruses to be significantly expressed through four different mechanisms, with the majority of transcripts being antisense copies of proviruses located within introns. We saw active 5' LTR use in tumorigenic cells only, suggesting that the cellular environment of a cancer cell is a critical component for induction of LTR promoter activity. These findings have implications for future studies investigating HML-2 as a target for immunotherapy or as a biomarker for disease.

  • Structure-function dissection of the Pseudorabies virus glycoprotein B fusion loops [PublishAheadOfPrint]

  • Conserved across the Herpesviridae family, glycoprotein B (gB) is responsible for driving fusion of the viral envelope with the host cell membrane for entry upon receptor binding and activation by the viral gH/gL complex. Although crystal structures of the gB ectodomain of several herpesviruses have been reported, the membrane fusion mechanism has remained elusive. Here, we report the X-ray structure of the Pseudorabies virus (PrV) gB ectodomain, revealing a typical class III post-fusion trimer that binds membranes via its fusion loops (FLs) in a cholesterol-dependent manner. Mutagenesis of FL residues allowed us to dissect those interacting with distinct sub-regions of the lipid bilayer and their role for membrane interactions. We tested 15 gB variants for their ability to bind to liposomes, and further investigated a subset of them in functional assays. We found that PrV gB FL residues Trp187, Tyr192, Phe275 and Tyr276, which were essential for liposome binding and for fusion in a cellular and viral context, form a continuous hydrophobic patch at the gB trimer surface. Together with reported results from other alpha-herpesvirus gBs, our data suggest a model in which Phe275 from the tip of FL2 protrudes deeper into the hydrocarbon core of the lipid bilayer, while the side chains of Trp187, Tyr192 and Tyr276 form a rim that inserts into the more superficial, interfacial region of the membrane to catalyze the fusion process. Comparative analysis with gB from beta- and gamma-herpesviruses suggest that this membrane-interaction mode is valid for gB from all herpesviruses.

    IMPORTANCE Herpesviruses are common human and animal pathogens, which infect cells by entering via fusion of viral and cellular membranes and which cause life-long and incurable infections. Central to the membrane fusion event for entry is glycoprotein B (gB), which is the most conserved envelope protein across the herpesvirus family. Like other viral fusion proteins, gB anchors itself into the target membrane via two polypeptide segments called fusion loops (FL). The molecular details of how gB FLs insert into the lipid bilayer have not been described. We provide here structural and functional data regarding key FL residues of gB from Pseudorabies virus, a porcine herpesvirus of veterinary concern, which allows us to propose, for the first time, a molecular model to understand how the initial interactions by gB from all herpesviruses with target membranes are established.

  • Hepatitis C virus replication depends on endosomal cholesterol homeostasis [PublishAheadOfPrint]

  • Similar to other positive-strand RNA viruses, hepatitis C virus (HCV) causes massive rearrangements of intracellular membranes, resulting in a membranous web (MW) composed of predominantly double membrane vesicles (DMVs), the presumed sites of RNA replication. DMVs are enriched for cholesterol, but mechanistic details on the source and recruitment of cholesterol to the viral replication organelle are only partially known. Here we focused on selected lipid transfer proteins implicated in direct lipid transfer at various endoplasmic reticulum (ER) - membrane contact sites. RNAi-mediated knockdown identified several hitherto unknown HCV dependency factors such as STARD3 (steroidogenic acute regulatory protein (StAR)-related lipid transfer domain protein 3), OSBPL1A and B (Oxysterol-binding protein-related protein 1A and B) and NPC1 (Niemann-Pick- type C1), all residing at late endosome and lysosome membranes and required for efficient HCV RNA replication, but not for replication of the closely related Dengue virus. Focussing on NPC1 we found that knockdown or pharmacological inhibition caused cholesterol entrapment in lysosomal vesicles concomitant with decreased cholesterol abundance at sites containing the viral replicase factor NS5A. In untreated HCV-infected cells unesterified cholesterol accumulated at the perinuclear region partially co-localizing with NS5A at DMVs, arguing for NPC1-mediated endosomal cholesterol transport to the viral replication organelle. Consistent with cholesterol being an important structural component of DMVs, reducing NPC1-dependent endosomal cholesterol transport impaired MW integrity. This suggests that HCV usurps lipid transfer proteins such as NPC1 at ER -- late endosome/lysosome membrane contact sites to recruit cholesterol to the viral replication organelle where it contributes to MW functionality.

    IMPORTANCE A key feature of the replication of positive-strand RNA viruses is the rearrangement of the host cell endomembrane system to produce a membranous replication organelle (RO). The underlying mechanisms are far from being fully elucidated. In this report we provide evidence that HCV RNA replication depends on a functional lipid transport along the endosomal - lysosomal pathway that is mediated by several lipid transfer proteins such as the Niemann-Pick Type C1 (NPC1) protein. Pharmacological inhibition of NPC1 function reduced viral replication, impaired the transport of cholesterol to the viral replication organelle and altered organelle morphology. Besides NPC1, our study reports the importance of additional endosomal and lysosomal lipid transfer proteins required for viral replication, thus contributing to our understanding of how HCV manipulates their function in order to generate a membranous replication organelle. These results might have implications for the biogenesis of replication organelles of other positive strand RNA viruses.

  • A Functional Carboxy-Terminal Fluorescent Protein Fusion to Pseudorabies Virus Small Capsid Protein VP26 [PublishAheadOfPrint]

  • Fluorescent protein fusions to herpesvirus capsids have proven to be a valuable method to study virus particle transport in living cells. Fluorescent protein fusions to the amino terminus of small capsid protein VP26 are the most widely-used method to visualize Pseudorabies Virus (PRV) and Herpes Simplex Virus (HSV) particles in living cells. However, these fusion proteins do not incorporate to full occupancy, and have modest effects on virus replication and pathogenesis. Recent cryo electron microscopy studies have revealed that herpesvirus small capsid proteins bind to capsids via their amino terminus, whereas the carboxy terminus is unstructured and may therefore better tolerate fluorescent protein fusions. Here we describe a new recombinant PRV expressing a carboxy-terminal VP26-mCherry fusion. Compared to previously characterized viruses expressing amino-terminal fusions, this virus expresses more VP26 fusion protein in infected cells, incorporates more VP26 fusion protein into virus particles, and individual virus particles exhibit brighter red fluorescence. We performed single particle tracking of fluorescent virus particles in primary neurons to measure anterograde and retrograde axonal transport, demonstrating the usefulness of this novel VP26-mCherry fusion for the study of viral intracellular transport.

    Importance Alpha herpesviruses are among the very few viruses that are adapted to invade the mammalian nervous system. Intracellular transport of virus particles in neurons is important, as this process underlies both mild peripheral nervous system infection and severe spread to the central nervous system. VP26, the small capsid protein of HSV and PRV, was one of the first herpesvirus proteins to be fused to a fluorescent protein. Since then, these capsid-tagged virus mutants have become a powerful tool to visualize and track individual virus particles. Improved capsid tags will facilitate fluorescence microscopy studies of virus particle intracellular transport, as a brighter particle will improve localization accuracy of individual particles and allow for shorter exposure times, reducing photo-toxicity and improving the time-resolution of particle tracking in live cells.

  • Deep sequencing of foot-and-mouth disease virus reveals RNA sequences involved in genome packaging [PublishAheadOfPrint]

  • Non-enveloped viruses protect their genomes by packaging them into an outer shell or capsid of virus-encoded proteins. Packaging and capsid assembly in RNA viruses can involve interactions between capsid proteins and secondary structures in the viral genome as exemplified by the RNA bacteriophage MS2 and as proposed for other RNA viruses of plants, animals and human. In the picornavirus family of non-enveloped RNA viruses, the requirements for genome packaging remain poorly understood. Here we show a novel and simple approach to identify predicted RNA secondary structures involved in genome packaging in the picornavirus foot-and-mouth disease virus (FMDV). By interrogating deep sequencing data generated from both packaged and unpackaged populations of RNA we have determined multiple regions of the genome with constrained variation in the packaged population. Predicted secondary structures of these regions revealed stem loops with conservation of structure and a common motif at the loop. Disruption of these features resulted in attenuation of virus growth in cell culture due to a reduction in assembly of mature virions. This study provides evidence for the involvement of predicted RNA structures in picornavirus packaging and offers a readily transferable methodology for identifying packaging requirements in many other viruses.

    Importance In order to transmit their genetic material to a new host, non-enveloped viruses must protect their genomes by packaging them into an outer shell or capsid of virus-encoded proteins. For many non-enveloped RNA viruses the requirements for this critical part of the viral life cycle remain poorly understood. We have identified RNA sequences involved in genome packaging of the picornavirus foot-and-mouth disease virus. This virus causes an economically devastating disease of livestock affecting both the developed and developing world. The experimental methods developed to carry out this work are novel, simple and transferable to the study of packaging signals in other RNA viruses. Improved understanding of RNA packaging may lead to novel vaccine approaches or targets for antiviral drugs with broad spectrum activity.

  • Myeloid dendritic cells repress human cytomegalovirus gene expression and spread by releasing interferon-unrelated soluble antiviral factors [PublishAheadOfPrint]

  • Cytomegalovirus (CMV) is a beta-herpesvirus that latently infects most adult humans worldwide and is a major cause of morbidity and mortality in immunocompromised hosts. Latent human CMV (HCMV) is believed to reside in precursors of myeloid-lineage, leukocytes and monocytes, which give raise to macrophages and dendritic cells. We report here that human monocyte derived DCs (mo-DC) suppress HCMV infection in coculture with infected fibroblasts target cells in an effector-to-target-ratio dependent manner. Intriguingly, optimal activation of mo-DC was achieved in coculture conditions, not by their direct infection with HCMV, implying that mo-DC may recognize unique molecular patterns on, or within, infected fibroblasts. We show that HCMV is controlled by secreted factors that act by priming defenses in target cells rather than by direct viral neutralization, but we excluded a role for IFNs in this control. The expression of lytic viral genes in infected cells and the progression of infection were significantly slowed down, but this effect was reversible, indicating that the control of infection depended on the transient induction of antiviral effector molecules in target cells. Using immediate-early or late-phase reporter HCMVs, we show that soluble factors secreted in the cocultures suppress HCMV replication at both stages of the infection and that their antiviral effect is robust and comparable in numerous batches of mo-DCs as well as in primary fibroblasts and stromal cells.

    Importance Human cytomegalovirus is a widespread opportunistic pathogen that can cause severe disease and complications in vulnerable individuals. This includes newborn children, HIV AIDS patients or transplant recipients. Although the majority of healthy humans carry this virus throughout their lives without symptoms, it is not exactly clear which tissues in the body are the main reservoirs of latent virus infection, or how the delicate balance between the virus and the immune system is maintained over the individual's lifetime. Here for the first time, we provide evidence for a novel mechanism of direct virus control by a subset of human innate immune cells called Dendritic Cells, which are regarded as a major site of virus latency and reactivation. Our findings may have important implications in HCMV disease prevention as well as development of novel therapeutic approaches.

  • Human embryonic stem cell derived neurons are highly permissive for varicella-zoster virus lytic infection [PublishAheadOfPrint]

  • Varicella-zoster virus (VZV) is highly cell-associated when grown in culture and has a much higher (4,000 to 20,000-fold increased) particle-to-plaque forming unit (PFU) ratio in vitro than herpes simplex virus (HSV). In contrast, VZV is highly infectious in vivo by airborne transmission. Neurons are major targets for VZV in vivo, in which the virus can establish latency and reactivate to produce infectious virus. Using neurons derived from human embryonic stem cells (hESC) and cell-free wild-type (WT) VZV, we demonstrate that neurons are nearly 100 times more permissive for WT VZV infection than very early passage human embryonic lung cells or MRC-5 diploid human fibroblasts, the cells used for vaccine production or virus isolation. The peak titers achieved after infection were ~10-fold higher in human neurons than MRC-5 cells and the viral genome copy number-to-PFU ratio for VZV in human neurons was 500 compared with 50,000 for MRC-5 cells. Thus, VZV may not necessarily have a higher particle-to-PFU ratio than other herpesviruses; instead, the cells previously used to propagate virus in vitro may have been suboptimal. Furthermore, based on electron microscopy, neurons infected with VZV produced fewer defective or incomplete viral particles than MRC-5 cells. Our data suggest that neurons derived from hESC may have advantages compared to other cells for studies of VZV pathogenesis, for obtaining stocks of virus with high titers, and for isolating VZV from clinical specimens.

    IMPORTANCE Varicella-zoster virus (VZV) causes chickenpox and shingles. Cell-free VZV has been difficult to obtain, both for in vitro studies and for vaccine production. While numerous cells lines have been tested for their ability to produce high titers of VZV, the number of total virus particles relative to the number of viral particles that can form plaques in culture has been reported to be extremely high relative to other viruses. We show that VZV grows to much higher titers in human neurons than in other cell types in vitro and that the number of total virus genomes relative to the number of viral particles that can form plaques in culture is much lower in human neurons than other cultured cells. These findings indicate that human neurons may be useful for studying VZV in vitro, for growing preparations of virus with high titers, and for isolating the virus from human samples.

  • The Impacts of Genome-wide Analyses on our Understanding of Human Herpesvirus Diversity and Evolution [PublishAheadOfPrint]

  • Until fairly recently, genome-wide evolutionary dynamics and within-host diversity were more commonly examined in the context of small viruses, than large double-stranded DNA viruses such as herpesviruses. The high mutation rates and more compact genomes of RNA viruses have inspired the investigation of population dynamics for these species, and recent data now suggest that herpesviruses might also be considered candidates for population modeling. High-throughput sequencing (HTS) and bioinformatics have expanded our understanding of herpesviruses through genome-wide comparisons of sequence diversity, recombination, allele frequency, and selective pressures. Here we discuss recent data on the mechanisms that generate herpesvirus genomic diversity and underlie the evolution of these virus families. We focus on human herpesviruses, with key insights drawn from veterinary herpesviruses and other large DNA virus families. We consider the impacts of cell culture on herpesvirus genomes, and how to accurately describe the viral populations under study. The need for a strong foundation of high quality genomes is also discussed, since it underlies all secondary genomic analyses such as RNA-Seq transcriptional profiling, chromatin binding, and ribosome footprinting. Areas where we foresee future progress are highlighted as well, such as the linking of viral genetic differences to phenotypic or clinical outcomes.

  • A tyrosine-based trafficking motif of the tegument protein pUL71 is crucial for human cytomegalovirus secondary envelopment [PublishAheadOfPrint]

  • The human cytomegalovirus (HCMV) tegument protein pUL71 is required for efficient secondary envelopment and accumulates at the Golgi-derived viral assembly complex (vAC) during infection. Analysis of various C-terminally truncated pUL71 fused to eGFP identified amino acids 23-34 as important determinants for its Golgi localization. Sequence analysis and mutational verification revealed the presence of an N-terminal tyrosine-based trafficking motif (Yxx) in pUL71. This led us to hypothesize a requirement of the Yxx motif for the function of pUL71 in infection. Mutation of both, the tyrosine residue and the entire Yxx motif, resulted in an altered distribution of mutant pUL71 at the plasma membrane and the cytoplasm during infection. Both Yxx mutant viruses exhibited similarly decreased focal growth and reduced virus yields in supernatants. Ultrastructurally, mutant virus infected cells exhibited an impaired secondary envelopment manifested by accumulations of capsids undergoing an envelopment process. Additionally, clusters of capsid accumulations surrounding the vAC were observed similarly to the ultrastructural phenotype of a UL71-deficient mutant. The importance of endocytosis and thus the Yxx motif for targeting pUL71 to the Golgi was further demonstrated when clathrin-mediated endocytosis was inhibited either by co-expression of AP180-C or by treatment with methyl-beta-cyclodextrin. Both conditions resulted in a plasma membrane accumulation of pUL71. Altogether, these data reveal the presence of a functional N-terminal endocytosis motif that is an important determinant for intracellular localization of pUL71 and furthermore required for the function of pUL71 during secondary envelopment of HCMV capsids at the vAC.

    IMPORTANCE Human cytomegalovirus (HCMV) is the leading cause of birth defects among congenital virus infections and can lead to live-threatening infections in immunocompromised hosts. Current antiviral treatments target viral genome replication and are increasingly overcome by viral mutations. Therefore, identifying new targets for antiviral therapy is important for future development of novel treatment options. A detailed molecular understanding of the complex virus morphogenesis will identify potential viral as well as cellular targets for antiviral intervention. Secondary envelopment is an important viral process through which infectious virus particles are generated and which involves the action of several viral proteins such as tegument protein pUL71. Targeting of pUL71 to the site of secondary envelopment appears to be crucial for its function during this process and regulated by utilizing host trafficking mechanisms that are commonly exploited by viral glycoproteins. Thus, intracellular trafficking, if targeted, might present a novel target for antiviral therapy.

  • Mutations in the Influenza A virus M1 protein enhance virus budding to complement lethal mutations in the M2 cytoplasmic tail [PublishAheadOfPrint]

  • The influenza A virus M1 and M2 proteins play important roles in virus assembly and in the morphology of virus particles. Mutations in the distal cytoplasmic tail region of M2, and in particular a tyrosine to alanine substitution at residue 76 (Y76A) was essential for infectious virus production and filament formation while having limited effects on total virus particle budding. Using a novel selection method, mutations at seven different M1 amino acids (residue 73, 94, 135, 136, or 138 or a double mutation 93/244) that are not found in circulating influenza virus strains or been previously identified to play a role in influenza A virus assembly were found to complement the lethal M2Y76A mutation. These M1 suppressor mutations restored infectious virus production in the presence of M2Y76A and mediated increased budding and filament formation even in the absence of M2. However, the efficiency of infectious virus replication was still dependent on the presence of the distal region of the M2 cytoplasmic tail. The data suggest influenza A virus budding and genome incorporation can occur independently, and provide further support for complementary roles of the M1 and M2 proteins in virus assembly.

    Importance Influenza virus particle assembly involves the careful coordination of various viral and host factors to optimally produce infectious virus particles. We have previously identified a mutation at position 76 of the influenza A virus M2 protein that drastically reduces infectious virus production and filament formation with minimal effects on virus budding. In this manuscript, we identified suppressor mutations in the M1 protein which complement this lethal M2 mutation by increasing the efficiency in which virus particles bud from infected cells and restoring filament formation at the infected cell surface. M2 distal cytoplasmic domain sequences were still required for optimal infectivity. This indicates that M1 and M2 can functionally replace each other in some, but not all aspects of virus particle assembly.

  • Varicella Zoster virus expresses multiple small non-coding RNAs [PublishAheadOfPrint]

  • Many herpesviruses express small non coding RNAs (sncRNA), including microRNAs (miR), that may play roles in regulating lytic and latent infections. None have yet been reported in varicella zoster virus (VZV, HHV-3). Here we analyzed next generation sequencing (NGS) data for small RNAs in VZV-infected fibroblasts and human embryonic stem cell-derived (hESC) neurons. Two independent bioinformatics analyses identified more than 20 VZV-encoded 20-24 nucleotide RNAs, some of which are predicted to have stem-loop precursors potentially representing miRs. These sequences are perfectly conserved between viruses from three clades of VZV. One NGS-identified sequence common to both bioinformatics analyses mapped to the repeat regions of the VZV genome, upstream of the predicted promoter of the immediate-early gene ORF63. This miR candidate was detected in each of 3 independent biological repetitions of NGS of RNA from fibroblasts and neurons productively infected with VZV using Taqman qPCR. Importantly, transfected synthetic RNA oligonucleotides antagonistic to the miR candidate significantly enhanced VZV plaque growth rates. The presence of 6 additional small non-coding RNAs was also verified by Taqman qPCR in productively infected fibroblasts and ARPE19 cells. Our results show that VZV, like other human herpesviruses, encodes several sncRNAs and miR, and some may regulate infection of host cells.

    Importance Varicella zoster virus is an important human pathogen, with herpes zoster being a major health issue in the aging and immune compromised populations. Small non coding RNAs (sncRNA) are recognized as important actors in modulating gene expression, and this study demonstrates the first reported VZV encoded sncRNAs. Many are clustered to a small genomic region, as seen in other human herpesviruses. At least one VZV sncRNA was expressed in productive infection of neurons and fibroblasts that is likely to reduce viral replication. Since sncRNAs have been suggested to be potential targets for antiviral therapies, identification of these molecules in VZV may provide a new direction for development of treatments for painful herpes zoster.

  • Human Parvovirus Infection of Human Airway Epithelia Induces Pyroptotic Cell Death via Inhibiting Apoptosis [PublishAheadOfPrint]

  • Human bocavirus 1 (HBoV1) is a human parvovirus that causes acute respiratory tract infections in young children. In this study, we confirmed that, when polarized/well-differentiated human airway epithelia are infected with HBoV1 in vitro, they develop damage characterized by barrier function disruption and cell hypotrophy. Cell death mechanism analyses indicated that the infection induced pyroptotic cell death characterized by caspase-1 activation. Unlike infections with other parvoviruses, HBoV1 infection did not activate the apoptotic or necroptotic cell death pathway. When the NLRP3-ASC-caspase-1 inflammasome-induced pathway was inhibited by shRNA, the HBoV1-induced cell death dropped significantly; thus, NLRP3 mediated by ASC appears to be the pattern recognition receptor driving HBoV1 infection-induced pyroptosis. HBoV1 infection induced steady increases in the expression of IL-1aalpha; and IL-18. HBoV1 infection also associated with the marked expression of the anti-apoptotic genes BIRC5 and IFI6. When the expression of BIRC5 and/or IFI6 was inhibited by shRNA, the infected cells underwent apoptosis rather than pyroptosis, as indicated by increased cleaved caspase-3 levels and the absence of caspase-1. BIRC5 and/or IFI6 gene inhibition also significantly reduced HBoV1 replication. Thus, HBoV1 infection of human airway epithelial cells activates anti-apoptotic proteins that suppress apoptosis and promote pyroptosis. This response may have evolved to confer a replicative advantage, thus allowing HBoV1 to establish a persistent airway epithelia infection. This is the first report of pyroptosis in airway epithelia infected by a respiratory virus.

    IMPORTANCE Microbial infection of immune cells often induces pyroptosis, which is mediated by a cytosolic protein complex called the inflammasome that senses microbial pathogens and then activates the proinflammatory cytokines interleukin (IL)-1 and IL-18. While virus-infected airway epithelia often activate NLRP3 inflammasomes, studies to date suggest that these viruses kill the airway epithelial cells via the apoptotic or necrotic pathways: involvement of the pyroptosis pathway has not been reported previously. Here, we show for the first time that virus infection of human airway epithelia can also induce pyroptosis. Human bocavirus 1 (HBoV1), a human parvovirus, causes lower respiratory tract infections in young children. This study indicates that HBoV1 kills airway epithelial cells by activating genes that suppress apoptosis and thereby promote pyroptosis. This strategy appears to promote HBoV1 replication and may have evolved to allow HBoV1 to establish a persistent infection of human airway epithelia.

  • Modelling of anti-latency treatment in HIV; What is the optimal duration of anti-retroviral-free HIV remission? [PublishAheadOfPrint]

  • A number of treatment strategies are currently being developed to promote anti-retroviral free HIV cure or remission. While complete elimination of the HIV reservoir would prevent recurrence of infection, it is not clear how different remission lengths would affect viral rebound and transmission. In this work we use a stochastic model to show that a treatment that achieves a one-year average time to viral remission will still lead to nearly a quarter of subjects experiencing viral rebound within the first three months. Given quarterly viral testing intervals, this leads to an expected 39 (95%UI 22-69) heterosexual transmissions and up to 262 (95%UI 107-534) homosexual transmissions per 1,000 treated subjects over a 10-year period. Thus, a balance between high initial treatment levels, risk of recrudescence, and risk of transmission should be considered when assessing the llsquo;usefulrrsquo; or optimal length of anti-retroviral-free HIV remission to be targeted. We also investigate the trade-off between increasing the average duration of remission, versus the risk of treatment failure (viral recrudescence) and the need for re-treatment. To minimise drug exposure, we find that the optimal target of anti-latency interventions is a 1700-fold reduction in the size of the reservoir, which would lead to an average time to recrudescence of 30 years. Interestingly, this is a significantly lower level of reduction than that required for complete elimination of the viral reservoir. Additionally we show that when shorter periods are targeted, there is a real probability of viral transmission occurring in between testing for viral rebound.

    Importance Current treatment of HIV involves patients taking anti-retroviral therapy to ensure that the level of virus remains at very low, or undetectable levels. Continuous therapy is required, as the virus persists in a latent state within cells, and when therapy is stopped the virus rebounds, usually within a couple of weeks. A major question is how to reduce the amount of persistent virus, and therefore allow a delay or remission until the virus returns after ceasing therapy. In this work we consider the probability that HIV will still rebound even after this reduction, and ask what the likelihood of viral transmission would be in this case.

  • Quiescence promotes latent HIV infection and resistance to reactivation from latency with histone deacetylase inhibitors [PublishAheadOfPrint]

  • Human immunodeficiency virus type-1 (HIV-1) establishes transcriptionally silent latent infections in many cell types, including resting memory T cells and hematopoietic stem and progenitor cells (HSPCs), which allow the virus to persist in infected individuals despite antiretroviral therapy. Developing in vitro models of HIV-1 latency that recapitulate the characteristics of latently-infected cells in vivo is crucial to identifying and developing effective latency-reversing therapies. HSPCs exist in a quiescent state in vivo, and quiescence is correlated with latent infections in T cells. However, current models for culturing HSPCs and for infecting T cells in vitro require that they be maintained in an actively proliferating state. Here, we describe a novel culture system in which primary human HSPCs cultured under hypothermic conditions are maintained in a quiescent state. We show that these quiescent HSPCs are susceptible to predominantly latent infection with HIV-1, while actively-proliferating and differentiating HSPCs obtain predominantly active infections. Furthermore, we demonstrate that the most primitive quiescent HSPCs are more resistant to spontaneous reactivation from latency than more differentiated HSPCs, and that quiescent HSPCs are resistant to reactivation by histone deacetylase inhibitors or P-TEFb activation, but are susceptible to reactivation by PKC agonists. We also demonstrate that inhibition of HSP90, a known regulator of HIV transcription, recapitulates the quiescence and latency phenotypes of hypothermia, suggesting that hypothermia and HSP90 inhibition may regulate these processes by similar mechanisms. In summary, these studies describe a novel model for studying HIV-1 latency in human primary cells maintained in a quiescent state.

    IMPORTANCE Human immunodeficiency virus type-1 (HIV-1) establishes a persistent infection for which there remains no feasible cure. Current approaches are unable to clear the virus despite decades of therapy due to the existence of latent reservoirs of integrated HIV-1, which can reactivate and contribute to viral rebound following treatment interruption. Previous clinical attempts to reactivate the latent reservoirs in an individual so that they can be eliminated by the immune response or viral cytopathic effect have failed, indicating a need for a better understanding of the processes regulating HIV-1 latency. Here we characterize a novel in vitro model of HIV-1 latency in primary hematopoietic stem and progenitor cells isolated from human cord blood that may better recapitulate the behavior of latently-infected cells in vivo. This model can be used to study mechanisms regulating latency and potential therapeutic approaches to reactivate latent infections in quiescent cells.

  • HIV-1 gp120 Protein and MVAgp140 Boost Immunogens Increase immunogenicity of a DNA/MVA HIV-1 Vaccine [PublishAheadOfPrint]

  • An important goal of human immunodeficiency virus (HIV) vaccine design is identification of strategies that elicit effective antiviral humoral immunity. One novel approach comprises priming with DNA and boosting with modified vaccinia Ankara (MVA) expressing HIV-1 Env on virus like particles. Here we evaluated whether the addition of a gp120 protein in alum or MVA expressed secreted gp140 (MVAgp140) could improve immunogenicity of a DNA prime - MVA boost vaccine. Five rhesus macaques per group received two DNA primes at weeks 0 and 8 followed by three MVA boosts (with or without additional protein or MVAgp140) at weeks 18, 26 and 40. Both boost immunogens enhanced the breadth of HIV-1 gp120 and V1V2 responses, antibody-dependent cellular cytotoxicity (ADCC), and low titer tier 1B and Tier 2 neutralizing antibody responses. However, there were differences in antibody kinetics, linear epitope specificity and CD4 T cell responses between the groups. The gp120 protein boost elicited earlier and higher peak responses; whereas, the MVAgp140 boost resulted in improved antibody durability and comparable peak responses after the final immunization. Linear V3 specific IgG responses were particularly enhanced by the gp120 boost whereas the MVAgp140 boost also enhanced responses to linear C5 and C2.2 epitopes. Interestingly, gp120, but not the MVAgp140 boost, increased peak CD4+ T cell responses. Thus, both gp120 and MVAgp140 can augment potential protection of a DNA/MVA vaccine by enhancing gp120 and V1/V2 antibody responses, whereas potential protection by gp120, but not MVAgp140 boosts, may be further impacted by increased CD4+ T cell responses.

    IMPORTANCE Prior immune correlates analyses in humans and nonhuman primates revealed the importance of antibody responses in preventing HIV-1 infection. A DNA prime - modified vaccinia Ankara (MVA) boost vaccine has proven to be potent in eliciting antibody responses. Here we explore the ability of boosts with recombinant gp120 protein or MVA-expressed gp140 to enhance antibody responses elicited by the GOVX-B11 DNA prime - MVA boost vaccine. We found that both types of immunogen boosts enhanced potentially protective antibody responses, whereas the gp120 protein boosts also increased CD4+ T cell responses. Our data provide important information for HIV vaccine designs that aim for effective and balanced humoral and T cell responses.

  • HIV exposed infants vaccinated with a MF59/rgp120 vaccine have higher magnitude anti-V1V2 IgG responses than adults immunized with the same vaccine [PublishAheadOfPrint]

  • In the RV144 vaccine trial, IgG responses against the HIV envelope variable loops 1 and 2 (V1V2) were associated with decreased HIV acquisition risk. We previously reported that infants immunized with a MF59 adjuvanted rgp120 vaccine developed higher magnitude anti-V1V2 IgG responses than adult RV144 vaccinees. To determine if the robust antibody response in infants is due to differences in vaccine regimens or to inherent differences between the adult and infant immune systems, we compared Env-specific IgG responses in adults and infants immunized with the same MF59 and Alum adjuvanted HIV envelope vaccines. At peak immunogenicity, the magnitude of gp120 and V1V2-specific IgG responses was comparable between adults and infants immunized with the Alum/MNrg120 vaccine (gp120 median MFI infant: 7,118; adult: 11,510; p=0.070; V1V2 median MFI infant: 512; adult: 804;; p=0.50), whereas infants immunized with the MF59/SF-2 rgp120 vaccine had higher magnitude antibody levels than adults (gp120 median MFI infant: 15,509; adult: 2,290; pllt;0.001; V1V2 median MFI infant: 23,926; adult: 1538; pllt;0.001). Six months after peak immunogenicity, infants maintained higher levels Env-specific IgG than adults. Anti-V1V2 IgG3 antibodies that were associated with decreased HIV-1 risk in RV144 vaccinees were present in 43% of MF59/rgp120 vaccinated infants but only in 12% of the vaccinated adults (p=0.0018). Finally, in contrast to rare vaccine-elicited Env-specific IgA in infants, rg120 vaccination elicited Env-specific IgA were frequently detected in adults. Our results suggest that vaccine adjuvants differently modulate gp120-specific antibody responses in adults and infants, and that infants can robustly respond to HIV Env immunization.

    IMPORTANCE More than 150,000 pediatric HIV infections continue to occur yearly, despite the availability of antiretroviral prophylaxis. A pediatric HIV vaccine could reduce the number of these ongoing infant infections, and also prime for long term immunity prior to sexual debut. We previously reported that immunization of infants with a MF59 adjuvanted recombinant gp120 vaccine induced higher magnitude potentially protective anti-V1V2 IgG responses than in adult vaccinees receiving the moderately effective RV144 vaccine. In the present study, we demonstrate that the robust response observed in infants is not due to differences in vaccine regimen or vaccine dose between adults and infants. Our results suggest that HIV vaccine adjuvants may differentially modulate immune responses in adults and infants, highlighting the need to conduct vaccine trials in pediatric populations.

  • RNASeq analysis of differentiated keratinocytes reveals a massive response to late events during human papillomavirus type 16 infection, including loss of epithelial barrier function. [PublishAheadOfPrint]

  • The human papillomavirus (HPV) replication cycle is tightly linked to epithelial cell differentiation. To examine HPV-associated changes in the keratinocyte transcriptome, RNAs isolated from undifferentiated and differentiated cell populations of normal, spontaneously immortalised, keratinocytes (NIKS), and NIKS stably transfected with HPV16 episomal genomes (NIKS16), were compared using RNASeq. HPV16 infection altered expression of 2862 cellular genes. Next, to elucidate the role of keratinocyte gene expression in late events during the viral life cycle, RNASeq was carried out on triplicate differentiated populations of NIKS (uninfected) and NIKS16 (infected). Of the top 966 genes altered (ggt;log2 = 1.8, 3.5-fold change) 670 genes were downregulated and 296 genes were up-regulated. HPV down-regulated many genes involved in epithelial barrier function that involves structural resistance to the environment and immunity to infectious agents. For example, HPV infection repressed expression of the differentiated keratinocyte-specific pattern recognition receptor TLR7, the Langerhans cell chemoattractant, CCL20, and proinflammatory cytokines, IL1A and IL1B. However, IRF1, IFN and viral restriction factors (IFIT1, 2, 3, 5, OASL, CD74, RTP4) were up-regulated. HPV infection abrogated gene expression associated with the physical epithelial barrier, including keratinocyte cytoskeleton, intercellular junctions and cell adhesion. qPCR and western blotting confirmed changes in expression of seven of the most significantly altered mRNAs. Expression of three genes showed statistically significant changes during cervical disease progression in clinical samples. Taken together, the data indicate that HPV infection manipulates the differentiating keratinocyte transcriptome to create an environment conducive to productive viral replication and egress.

    IMPORTANCE Human papillomavirus (HPV) genome amplification and capsid formation takes place in differentiated keratinocytes. The viral life cycle is intimately associated with host cell differentiation. Deep sequencing (RNASeq) of RNA from undifferentiated and differentiated uninfected and HPV16-positive keratinocytes showed that almost 3000 genes were differentially expressed in keratinocyte due to HPV16 infection. Strikingly, the epithelial barrier function of differentiated keratinocytes, comprising keratinocyte immune function and cellular structure, was found to be disrupted. These data provide new insights into virus-host interaction crucial for production of infectious virus and reveal that HPV infection remodels keratinocytes for completion of the virus replication cycle.

  • Characterization of the African swine fever virus decapping enzyme during infection [PublishAheadOfPrint]

  • African swine fever virus (ASFV) infection is characterized by a progressive decrease in cellular protein synthesis with a concomitant increase in viral protein synthesis, though the mechanism by which the virus achieves this phenomenon is still unknown. Decrease of cellular mRNA is observed during ASFV infection, suggesting that inhibition of cellular proteins is due to an active mRNA degradation process. ASFV carries a gene (Ba71V D250R/Malawi g5R) that encodes a decapping protein (ASFV-DP) which has a Nudix hydrolase motif and decapping activity in vitro. Here we show that ASFV-DP was expressed from early times and accumulated throughout the infection with a subcellular localization typical of the endoplasmic reticulum, co-localizing with the cap structure and interacting with the ribosomal protein L23a. ASFV-DP was capable of interaction with poly(A) RNA in cultured cells, primarily mediated by the N-terminal region of the protein. ASFV-DP also interacted with viral and cellular RNA in the context of infection, and its overexpression in infected cells resulted in decreased levels of both types of transcripts. This study points to ASFV-DP as a viral decapping enzyme involved in both the degradation of cellular mRNA and the regulation of viral transcripts.

    IMPORTANCE Virulent ASFV strains cause a highly infectious and lethal disease in domestic pigs for which there is no vaccine. Since 2007, an outbreak in the Caucasus region has spread to Russia, jeopardizing the European pig population and making it essential to deepen knowledge about this virus. Here we demonstrate that ASFV-DP is a novel RNA-binding protein implicated in the regulation of mRNA metabolism during infection, making it a good target for vaccine development.

  • HCMV utilizes a non-traditional STAT1 activation cascade via signaling through EGFR and integrins to efficiently promote the motility, differentiation, and polarization of infected monocytes [PublishAheadOfPrint]

  • Human cytomegalovirus infects peripheral blood monocytes and triggers biological changes that promote viral dissemination and persistence. We have shown that HCMV induces a pro-inflammatory state in infected monocytes, resulting in enhanced monocyte motility and transendothelial migration, prolonged monocyte survival, and differentiation toward a long-lived M1-like macrophage phenotype. Our data indicate that HCMV triggers these changes, in the absence of de novo viral gene expression and replication, through engagement and activation of EGFR and integrins on the surface of monocytes. We previously identified that HCMV induces the upregulation of multiple pro-inflammatory gene ontologies, with the interferon-associated gene ontology exhibiting the highest percentage of upregulated genes. However, the function of the HCMV-induced ISGs in infected monocytes remained unclear. We now show that HCMV induces the enhanced expression and activation of a key ISG transcriptional regulator, STAT1, via an IFN-independent, but EGFR- and integrin-dependent signaling pathway. Furthermore, we identified a biphasic activation of STAT1 that likely promotes two distinct phases of STAT1-mediated transcriptional activity. Moreover, our data show that STAT1 is required for efficient early HCMV-induced enhanced monocyte motility and later for HCMV-induced monocyte-to-macrophage differentiation and for the regulation of macrophage polarization, suggesting that STAT1 may serve as a molecular convergence point linking the biological changes that occur at early and later times post infection. Taken together, our results suggest that HCMV re-routes the biphasic activation of a traditionally anti-viral gene product through an EGFR- and integrin-dependent pathway in order to help promote the pro-viral activation and polarization of infected monocytes.

    IMPORTANCE HCMV promotes multiple functional changes in infected monocytes that are required for viral spread and persistence, including their enhanced motility and differentiation/polarization towards a pro-inflammatory M1 macrophage. We now show that HCMV utilizes the traditionally IFN-associated gene product, STAT1, to promote these changes. Our data suggest that HCMV utilizes EGFR- and integrin-dependent (but IFN-independent) signaling pathways to induce STAT1 activation, which may allow the virus to specifically dictate the biological activity of STAT1 during infection. Our data indicate that HCMV utilizes two phases of STAT1 activation, which we argue molecularly links the biological changes that occur following initial binding to those that continue to occur days to weeks following infection. Furthermore, our findings may highlight a unique mechanism for how HCMV avoids the anti-viral response during infection by hijacking the function of a critical component of the IFN response pathway.

  • Suppression of microRNA 424 levels by human papillomavirus proteins is necessary for differentiation-dependent genome amplification [PublishAheadOfPrint]

  • High-risk human papillomaviruses (HPVs) link their life cycle to epithelial differentiation and require activation of DNA damage pathways for efficient replication. HPVs modulate the expression of cellular transcription factors as well as cellular miRNAs to control these activities. One miRNA that has been reported to be repressed in HPV positive cancers of the cervix as well as oropharynx is miR-424. Our studies show that miR-424 levels are suppressed in cell lines that stably maintain HPV 31 or 16 episomes as well as cervical cancer lines that contain integrated genomes such as SiHa. Introduction of expression vectors for miR-424 reduced levels of HPV genomes in undifferentiated cells as well as amplification upon differentiation. Our studies show that the levels of two putative targets of miR-424 that function in DNA damage repair, CHK1 and Wee1, are suppressed in HPV positive cells providing an explanation for why this microRNA is targeted in HPV positive cells.

    IMPORTANCE This manuscript describes for the first time a critical role for miR-424 in the regulation of HPV replication. HPV E6 and E7 proteins suppress the levels of miR-424 and this is important for controlling the levels of CHK1 that plays a central role in viral replication. These findings should be of interest to the broad readership of Journal of Virology.

  • Induction of DNA damages upon Marek's disease virus infection: implication in viral replication and pathogenesis [PublishAheadOfPrint]

  • Marek's disease virus (MDV) is a highly contagious alphaherpesvirus that infects chickens and causes a deadly neoplastic disease. We previously demonstrated that MDV infection arrests cells in S-phase and that the tegument protein VP22 plays a major role in this process. In addition, expression of VP22 induces double strand breaks (DSB) in the cellular DNA, suggesting that DNA damage and the associated cellular response might be favorable for the MDV lifecycle. Here, we addressed the role of DNA damage in MDV replication and pathogenesis. We demonstrated that MDV induces DSB during lytic infection in vitro and in the PBMCs of infected animals. Intriguingly, we did not observe DNA damage in latently infected MDV-induced lymphoblastoid cells, while MDV reactivation resulted in the onset of DNA lesions, suggesting that DNA damage and/or the resulting DNA damage response might be required for efficient MDV replication and reactivation. In addition, reactivation was significantly enhanced by the induction of DNA damage using a number of chemicals. Finally, we used recombinant viruses to show that VP22 is required for the induction of DNA damage in vivo and that this likely contributes to viral oncogenesis.

    IMPORTANCE Marek's Disease Virus is an oncogenic alphaherpesvirus that causes fatal T-cell lymphomas in chickens. MDV causes substantial losses in poultry industry and is also used as a small-animal model for virus-induced tumor formation. DNA damage is not only implicated in tumor development but also aids in the life cycle of several viruses, however its role in MDV replication, latency and reactivation remains elusive. Here, we demonstrated that MDV induces DNA lesions during lytic replication in vitro and in vivo. DNA damage was not observed in latently infected cells, however is reinitiated during reactivation. Reactivation was significantly enhanced by the induction of DNA damage. Recombinant viruses that lacked the ability to induced DNA damage were defective in the induction of tumors, suggesting that DNA damage might also contribute to cellular transformation processes leading to MDV-lymphomagenesis.

  • COBRA HA elicits hemagglutination-inhibition antibodies against a panel of H3N2 influenza virus co-circulating variants. [PublishAheadOfPrint]

  • Each influenza season, a set of wild-type viruses, representing one H1N1, one H3N2, 1-2 influenza B isolates, are selected for inclusion in the annual seasonal influenza vaccine. In order to develop broadly reactive subtype specific influenza vaccines, a methodology called computationally optimized broadly reactive antigens (COBRA) was used to design novel hemagglutinin (HA) vaccine immunogens. COBRA technology was effectively used to design HA immunogens that elicited antibodies that neutralized H5N1 and H1N1 isolates. In this report, the development and characterization of seventeen prototype H3N2 COBRA HA proteins were screened in mice and ferrets for the elicitation of antibodies with HAI activity against human seasonal H3N2 viruses that were isolated over the last 48 years. The most effective COBRA HA vaccine regimens elicited antibodies with broader HAI activity against a panel of H3N2 viruses compared to wild-type H3 HA vaccines. The top leading COBRA HA candidates were tested against co-circulating variants. These variants were not efficiently detected by antibodies elicited by the wild-type HA from viruses selected as the vaccine candidates. The T-11 COBRA HA vaccine elicited antibodies with HAI and neutralization activity against all co-circulating variants from 2004-2007. This is the first report demonstrating broader breadth of vaccine induced antibodies against co-circulating H3N2 strains compared to the wild-type HA antigens that were represented in commercial influenza vaccines.

    IMPORTANCE There is a need for an improved influenza vaccine that elicits immune responses that recognize a broader number of influenza virus strains to prevent infection and transmission. Using the COBRA approach, a set of vaccines against influenza viruses in the H3N2 subtype were tested for the ability to elicit antibodies that neutralize virus infection against not only historical vaccine strains of H3N2, but also a set of co-circulating variants that circulated between 2004-2007. Three of the H3N2 COBRA vaccines recognized all the co-circulating strains during this era, but the chosen wild-type vaccine strains were not able to elicit antibodies with HAI activity against these co-ciruclating strains. Therefore, the COBRA vaccines have the ability to not only elicit protective antibodies against the dominant vaccine strains, but also minor circulating strains that can evolve into the dominant vaccine strains in the future.

  • An in vitro approach to identify the key amino acids in the low susceptibility of rabbit PrP to misfolding [PublishAheadOfPrint]

  • Prion diseases or transmissible spongiform encephalopathies (TSEs) are a group of rare progressive neurodegenerative disorders caused by an abnormally folded prion protein (PrPSc). This is capable of transforming the normal cellular prion protein (PrPC) in to new infectious PrPSc. Interspecies prion transmissibility studies performed by experimental challenge and the outbreak of bovine spongiform encephalopathy that occurred in the late 1980s-1990's showed that while some species were readily susceptible to TSEs (sheep, mice and cats), others were apparently resistant (rabbits, dogs and horses) when exposed to the same agent.

    To study the mechanisms of low susceptibility to TSEs of certain species, the mouse-rabbit transmission barrier was used as a model. To identify which specific amino acid residues determine high or low susceptibility to PrPSc propagation, Protein Misfolding Cyclic Amplification (PMCA), which mimics PrPC to PrPSc conversion with accelerated kinetics, was used. This allowed amino acid substitutions in rabbit PrP and accurate analysis of misfolding propensities. Wild-type rabbit recombinant PrP could not be misfolded into a protease-resistant self-propagating isoform in vitro despite seeding with at least twelve different infectious prions from diverse origins. Therefore, rabbit recombinant PrP mutants were designed containing every single amino acid substitution that distinguishes rabbit recombinant PrP from mouse recombinant PrP. Key amino acid residue substitutions were identified that make rabbit recombinant PrP susceptible to misfolding, and using these, protease-resistant misfolded recombinant rabbit PrP was generated. Additional studies characterized the mechanisms by which these critical amino acid residue substitutions increased the misfolding susceptibility of rabbit PrP.

    IMPORTANCE Prion disorders are invariably fatal, untreatable diseases typically associated with long incubation periods and characteristic spongiform changes associated with neuronal loss in the brain. Development of any treatment or preventative measure is dependent upon a detailed understanding of the pathogenesis and understanding the mechanism by which certain species appear resistant to TSEs is critical. Rabbits are highly resistant to naturally acquired TSEs and even under experimental conditions induction of clinical disease is not easy. Using recombinant rabbit PrP as a model, this study describes critical molecular determinants that confer this high resistance to transmissible spongiform encephalopathies.

  • Full-length isoforms of KSHV LANA accumulate in the cytoplasm of cells undergoing the lytic cycle of replication [PublishAheadOfPrint]

  • The latency-associated nuclear antigen (LANA) of the Kaposi sarcoma herpesvirus (KSHV) performs a variety of functions to establish and maintain KSHV latency. During latency, LANA localizes to discrete punctate spots in the nucleus where it tethers viral episomes to cellular chromatin and interacts with nuclear components to regulate cellular and viral gene expression. Using highly sensitive tyramide signal amplification (TSA), we determined that LANA localizes to the cytoplasm in different cell types undergoing the lytic cycle of replication after de novo primary infection and after spontaneous, TPA-, or ORF50/RTA-induced activation. We confirmed the presence of cytoplasmic LANA in a subset of cells in lytically-active multicentric Castleman disease lesions. The induction of cellular migration by scratch-wounding confluent cell cultures, culturing under subconfluent conditions or induction of cell differentiation in primary cultures upregulated the number of cells permissive for primary lytic KSHV infection. The induction of lytic replication was characterized by high level expression of cytoplasmic LANA and nuclear ORF59, a marker of lytic replication. Subcellular fractionation studies revealed the presence of multiple isoforms of LANA in the cytoplasm of ORF50/RTA-activated Vero cells undergoing primary infection. Mass spectrometry analysis demonstrated that cytoplasmic LANA isoforms were full-length, containing the N-terminal nuclear localization signal. These results suggest that trafficking of LANA to different subcellular locations is a regulated phenomenon, which allows LANA to interact with cellular components in different compartments during both the latent and replicative stages of the KSHV lifecycle.

    IMPORTANCE Kaposi sarcoma herpesvirus (KSHV) causes AIDS-related malignancies, including lymphomas and Kaposi sarcoma. KSHV establishes life-long infections using its latency-associated nuclear antigen (LANA). During latency, LANA localizes to the nucleus where it connects viral and cellular DNA complexes and regulates gene expression allowing the virus to maintain long-term infections. Our research shows that intact LANA traffics to the cytoplasm of cells undergoing permissive lytic infections and latently-infected cells in which the virus is induced to replicate. This suggests that LANA plays important roles in the cytoplasm and nuclear compartments of the cell during different stages of the KSHV life cycle. Determining cytoplasmic function and mechanism for regulation of the nuclear localization of LANA will enhance our understanding of the biology of this virus, leading to therapeutic approaches to eliminate infection and block its pathological effects.

  • Prior Dengue virus exposure shapes T cell immunity to Zika virus in humans [PublishAheadOfPrint]

  • While progress has been made in characterizing humoral immunity to Zika virus (ZIKV) in humans, little is known regarding the corresponding T cell responses to ZIKV. Here we investigate the kinetics and viral epitopes targeted by T cells responding to ZIKV and address the critical question of whether pre-existing dengue virus (DENV) T cell immunity modulates these responses. We find that memory T cell responses elicited by prior infection with DENV or vaccination with Tetravalent Dengue Attenuated Vaccines (TDLAV) recognize ZIKV-derived peptides. This cross-reactivity is explained by the sequence similarity of the two viruses, as the ZIKV peptides recognized by DENV-elicited memory T cells are identical or highly conserved in DENV and ZIKV. DENV exposure prior to ZIKV infection also influences the timing and magnitude of the T cell response. ZIKV-reactive T cells in the acute phase of infection are detected earlier and in greater magnitude in DENV-immune patients. Conversely, the frequency of ZIKV-reactive T cells continues to rise in the convalescent phase in DENV-naive donors, but declines in DENV pre-exposed donors, compatible with more efficient control of ZIKV replication and/or clearance of ZIKV antigen. The quality of responses is also influenced by previous DENV exposure, and ZIKV-specific CD8 T cells form DENV pre-exposed donors selectively up-regulated granzyme B and PD1, as compared to DENV-naïve donors. Finally, we discovered that ZIKV structural proteins (E, prM and C) are major targets of both the CD4 and CD8 T cell responses, whereas DENV T cell epitopes are found primarily in nonstructural proteins.

    IMPORTANCE The issue of potential ZIKV and DENV cross-reactivity and how pre-existing DENV T cell immunity modulates ZIKA T cell responses is of great relevance as the two viruses often co-circulate and ZIKA virus has been spreading in geographical regions where DENV is endemic or hyper-endemic. Our data show that memory T cell responses elicited by prior infection with DENV recognize ZIKV-derived peptides and that DENV exposure prior to ZIKV infection influences the timing, magnitude and quality of the T cell response. Additionally we show that ZIKV-specific responses target different proteins than DENV-specific responses, pointing towards important implications for vaccine design against this global threat.

  • Deletion of the African swine fever virus gene DP148R does not reduce virus replication in culture but reduces virus virulence in pigs and induces high levels of protection against challenge [PublishAheadOfPrint]

  • Many of the approximately 165 proteins encoded by African swine fever virus do not have significant similarity to known proteins and have not been studied experimentally. One such protein is DP148R. We showed that the DP148R gene is transcribed at early times post-infection. Deletion of this gene did not reduce virus replication in macrophages showing that is not essential for replication in these cells. However deleting this gene from a virulent isolate, Benin 97/1, dramatically reduced the virulence of the virus in vivo. All pigs infected with the BeninDP148R virus survived infection showing only transient mild clinical signs soon after immunisation. Following challenge with the parental virulent virus all pigs immunised by the intramuscular route (11/11) and all except one immunised by the intranasal route (5/6) survived. Mild or no clinical signs were observed after challenge. As expected control non-immune pigs developed signs of acute ASF. Virus genome and infectious virus were observed soon after immunisation coincident with the onset of clinical signs (~106 genome copies or TCID50/ml). Levels of virus genome declined over an extended period of up to 60 days post-immunisation. In contrast infectious virus was no longer detectable by days 30 to 35. IFN- was detected in serum between days 4 and 7 post-immunisation, and IFN- producing cells were detected in all pigs analysed following stimulation of immune lymphocytes with whole virus. ASFV specific antibodies were first detected from day 10 post-immunisation.

    IMPORTANCE African swine fever (ASF) is endemic in Africa, parts of the Trans Caucasus, Russian Federation and several European countries. The lack of a vaccine hinders control. Many of the ASF virus genes lack similarity to known genes and have not been characterised. We have shown that one of these, DP148R, is transcribed early during virus replication in cells and can be deleted from the virus genome without reducing virus replication. The gene deleted virus, BeninDP148R caused mild clinical signs in pigs and induced high levels of protection against challenge with parental virulent virus. Therefore deletion of this gene can provide a target for rational development of vaccines.

  • Coat protein mutations that alter the flux of morphogenetic intermediates through the {varphi}X174 early assembly pathway [PublishAheadOfPrint]

  • Two scaffolding proteins orchestrate X174 morphogenesis. The internal scaffolding protein B mediates the formation of pentameric assembly intermediates, whereas the external scaffolding protein D organizes 12 of these intermediates into procapsids. Aromatic amino acid side chains mediate most coat-internal scaffolding protein interactions. One residue in the internal scaffolding protein and three in the coat protein constitute the core of the B protein binding cleft. The three coat gene codons were randomized separately to ascertain the chemical requirements of the encoded amino acids and the morphogenetic consequences of mutation. The resulting mutants exhibited a wide range of recessive phenotypes, which could generally be explained within a structural context. Mutants with phenylalanine, tyrosine and methionine substitutions were phenotypically indistinguishable from wild-type. However, tryptophan substitutions were detrimental at two sites. Charged residues were poorly tolerated, conferring extreme temperature sensitive and lethal phenotypes. Eighteen lethal and conditional lethal mutants were genetically and biochemically characterized. The primary defect associated with the missense substitutions ranged from inefficient internal scaffolding protein B binding to faulty procapsid elongation reactions mediated by external scaffolding protein D. Elevating B protein concentrations above wild-type levels via exogenous, cloned gene expression, compensated for inefficient B protein binding as did suppressing mutations within gene B. Similarly, elevating D protein concentrations above wild-type levels or compensatory mutations within gene D suppressed faulty elongation. Some of the parental mutations were pleiotropic, affecting multiple morphogenetic reactions. This progressively reduced the flux of intermediates through the pathway. Accordingly, multiple mechanisms, which may be unrelated, could restore viability.

    IMPORTANCE Genetic analyses have been instrumental in deciphering the temporal events of many biochemical pathways. However, pleiotropic effects can complicate analyses. Vis-aagrave;-vis virion morphogenesis, an improper protein-protein interaction within an early assembly intermediate can influence the efficiency of all subsequent reactions. Consequently, the flux of assembly intermediates cumulatively decreases as the pathway progresses. During morphogenesis, X174 coat protein participates in at least four well-defined reactions, each one characterized by an interaction with a scaffolding or structural protein. In this study, genetic analyses, biochemical characterizations and physiological assays, i.e. elevating the protein levels with which the coat protein interacts, are used to elucidate pleiotropic effects that may alter the flux of intermediates through a morphogenetic pathway.

  • Coxsackievirus B escapes the infected cell in ejected mitophagosomes [PublishAheadOfPrint]

  • Coxsackievirus B (CVB) is a common enterovirus that can cause various systemic inflammatory diseases. Because CVB lacks an envelope, it has been thought to be inherently cytolytic, wherein CVB can only escape from the infected host cell by causing it to rupture. In recent years, however, we and others have observed that various naked viruses such as CVB can trigger the release of infectious extracellular microvesicles (EMVs) that contain viral material. This mode of cellular escape has been suggested to allow the virus to be masked from the adaptive immune system. Additionally, we have previously reported that these viral EMVs have LC3, suggesting they originated from autophagosomes. We now report that CVB-infected cells trigger DRP1-mediated fragmentation of mitochondria, which is a precursor to autophagic mitochondrial elimination (mitophagy). However, rather than being degraded by lysosomes, mitochondria-containing autophagosomes are released from the cell. We believe that CVB localizes to mitochondria, induces mitophagy, and subsequently disseminates from the cell in an autophagosome-bound mitochondria-virus complex. Suppressing the mitophagy pathway in HL-1 cardiomyocytes either with siRNA or Mdivi-1 caused marked reduction in virus production. The findings in this study suggest that CVB subverts mitophagy machinery to support viral dissemination in released EMVs.

    IMPORTANCE- Coxsackievirus B (CVB) can cause a number of life-threatening inflammatory diseases. Though CVB is well known to disseminate via cytolysis, recent reports have revealed a second pathway in which CVB can become encapsulated in host membrane components to escape the cell in an exosome-like particle. In this study, we report that these membrane-bound structures derive from mitophagosomes. Blocking various steps in the mitophagy pathway reduced levels of intracellular and extracellular virus. Not only does this study reveal a novel mechanism of picornaviral dissemination, but it also sheds light on new therapeutic targets to treat CVB and potentially other picornaviral infections.

  • Elicitation of protective antibodies against a broad panel of H1N1 viruses in ferrets pre-immune to historical H1N1 influenza viruses [PublishAheadOfPrint]

  • Most pre-clinical animal studies test influenza vaccines in immunologically naïve animal models, even though the results of vaccination may not accurately reflect the effectiveness of vaccine candidates in humans that have pre-existing immunity to influenza. In this study, novel, broadly-reactive influenza vaccine candidates were assessed in pre-immune ferrets. These animals were infected with different H1N1 isolates before being vaccinated or infected with another influenza virus. Previously, our group has described the design and characterization of computationally optimized broadly reactive HA antigens (COBRA) for H1N1 isolates. Vaccinating ferrets with virus-like particle (VLP) vaccines expressing COBRA HA proteins elicited antibodies with HAI activity against more H1N1 viruses in the panel than VLP vaccines expressing wild-type HA proteins. Specifically, ferrets infected with the 1986 virus and vaccinated with a single dose of the COBRA HA VLP vaccines elicited antibodies with HAI activity against 11-14 of the 15 H1N1 viruses isolated between 1934 and 2013. A subset of ferrets was infected with influenza viruses expressing the COBRA HA antigens. These COBRA pre-immune ferrets had superior breadth of hemagglutination-inhibition (HAI) activity following vaccination with COBRA HA VLP vaccines than COBRA pre-immune ferrets vaccinated with VLP vaccines expressing wild-type HA proteins. Overall, priming naïve ferrets with COBRA HA based viruses or using COBRA HA based vaccines to boost pre-existing antibodies induced by wild-type H1N1 viruses, COBRA HA antigens elicited sera with the broadest HAI reactivity against multiple antigenic H1N1 viral variants. This is the first report demonstrating the effectiveness of a broadly-reactive or universal influenza vaccine in a pre-immune ferret model.

    IMPORTANCE: Currently, many groups are testing influenza vaccine candidates to meet the challenge of developing a vaccine that elicits broadly reactive and long-lasting protective immune responses. The goal of these vaccines is to stimulate immune responses that react against most, if not all, circulating influenza strains, over a long period of time in all populations of people. Commonly, these experimental vaccines are tested in naïve animal models that do not have anti-influenza Immune responses, however, humans have pre-existing immunity to influenza viral antigens, particularly antibodies to the HA and NA glycoproteins. Therefore, this study investigated how pre-existing antibodies to historical influenza viruses influenced HAI-specific antibodies and protective efficacy using a broadly protective vaccine candidate.

  • HUMAN METAPNEUMOVIRUS INDUCES FORMATION OF INCLUSION BODIES FOR EFFICIENT GENOME REPLICATION AND TRANSCRIPTION [PublishAheadOfPrint]

  • Human metapneumovirus (HMPV) causes significant upper and lower respiratory disease to all age groups worldwide. The virus possesses a negative-sense single-stranded RNA genome of approximately 13.3 Kb encapsidated by multiple copies of the nucleoprotein (N), giving rise to helical nucleocapsids. In addition, copies of the phosphoprotein (P) and the large RNA polymerase (L) decorate the viral nucleocapsids. After viral attachment, endocytosis, and fusion mediated by the viral glycoproteins, HMPV nucleocapsids are released into the cell cytoplasm. To visualize the subsequent steps of genome transcription and replication, a fluorescence in situ hybridization (FISH) protocol was established to detect different viral RNA subpopulations in infected cells. The FISH probes were specific for detection of HMPV positive-sense RNA (+RNA) and genomic RNA (vRNA). Time-course analysis of human bronchial epithelial BEAS-2B cells infected with HMPV revealed the formation of inclusion bodies (IBs) from early times post-infection. HMPV IBs were shown to be cytoplasmic sites of active transcription and replication, with translation of viral proteins closely associated. Inclusion body formation was consistent with an actin-dependent coalescence of multiple early replicative sites. Time-course RT-qPCR analysis suggested that coalescence of inclusion bodies is a strategy to efficiently replicate and transcribe the viral genome. These results provide a better understanding of the steps following HMPV entry and have important clinical implications.

    IMPORTANCE Human metapneumovirus (HMPV) is a recently discovered pathogen that affects human populations of all ages worldwide. Reinfections are common throughout life, but no vaccines or antiviral treatments are currently available. In this work, a spatio-temporal analysis of HMPV replication and transcription in bronchial epithelial-derived immortal cells was performed. HMPV was shown to induce formation of large cytoplasmic granules, named inclusion bodies, for genome replication and transcription. Unlike other cytoplasmic structures such as stress granules and P-bodies, inclusion bodies are exclusively present in infected cells and contain HMPV RNA and proteins to more efficiently transcribe and replicate the viral genome. Though nuanced, inclusion body formation corresponds to a more generalized strategy used by different viruses, including filoviruses and rhabdoviruses, for genome transcription and replication. Thus, understanding inclusion body formation is crucial for the discovery of innovative therapeutic targets.

  • T cell-macrophage fusion triggers multinucleated giant cell formation for HIV-1 spreading [PublishAheadOfPrint]

  • HIV-1-infected macrophages participate in virus dissemination and establishment of virus reservoirs in host tissues, but the mechanisms for virus cell-to-cell transfer to macrophages remain unknown. Here, we reveal the mechanisms for cell-to-cell transfer from infected T cells to macrophages and virus spreading between macrophages. We show that contacts between infected T lymphocytes and macrophages lead to cell fusion for fast and massive transfer of CCR5-tropic viruses to macrophages. Through the merge of viral material between T cells and macrophages, these newly formed lymphocyte/macrophage fused cells acquire the ability to fuse with neighboring non-infected macrophages. Together, these two-step envelope-dependent cell fusion processes lead to the formation of highly virus-productive multinucleated giant cells reminiscent of the infected multinucleated giant macrophages detected in HIV-1-infected patients and SIV-infected macaques. These mechanisms represent an original mode of virus transmission for viral spreading and a new model for the formation of macrophage virus reservoirs during infection.

    IMPORTANCE We reveal a very efficient mechanism involved in cell-to-cell transfer from infected T cells to macrophages and subsequent virus spreading between macrophages by a two-step cell fusion process. Infected T cells first establish contacts and fuse with macrophage targets. The newly formed lymphocyte/macrophage fused cells then acquire the ability to fuse with surrounding uninfected macrophages leading to the formation of infected multinucleated giant cells that can survive for a long time as evidenced in vivo in lymphoid organs and the central nervous system. This route of infection may be a major determinant for virus dissemination and the formation of macrophage virus reservoirs in host tissues during HIV-1 infection.

  • Functional Stability of HIV-1 Envelope Trimer Affects Accessibility to Broadly Neutralizing Antibodies at its Apex [PublishAheadOfPrint]

  • The trimeric envelope glycoprotein spike (Env) of HIV-1 is the target of vaccine development to elicit broadly neutralizing antibodies (bnAbs). Env trimer instability and heterogeneity in principle make subunit interfaces inconsistent targets for the immune response. Here, we investigate how functional stability of Env relates to neutralization sensitivity to V2 bnAbs and V3 crown antibodies that engage subunit interfaces upon binding to unliganded Env. Env heterogeneity was inferred when antibodies neutralized a mutant Env with a plateau of less than 100% percentage neutralization. A statistically significant correlation was found between the stability of mutant Envs and the MPN of V2 bnAb, PG9, as well as an inverse correlation between stability of Env and neutralization by V3 crown antibody, 447-52D. A number of Env-stabilizing mutations and V2 bnAb-enhancing mutations were identified in Env, but did not always overlap indicating distinct requirements of functional stabilization versus antibody recognition. Blocking complex glycosylation of Env affected V2 bnAb recognition, as previously described, but also notably increased functional stability of Env. This study shows how instability and heterogeneity affect antibody sensitivity of HIV-1 Env, which is relevant to vaccine design involving its dynamic apex.

    IMPORTANCE The Env trimer is the only viral protein on the surface of HIV-1 and is the target of neutralizing antibodies that reduce viral infectivity. Quaternary epitopes at the apex of the spike are recognized by some of the most potent and broadly neutralizing antibodies to date. Being that their glycan-protein hybrid epitopes are at subunit interfaces, the resulting heterogeneity can lead to partial neutralization. Here, we screened for mutations in Env that allowed for complete neutralization by the bnAbs. We found that when mutations outside of V2 increased V2 bnAb recognition they often also increased Env stability-of-function and decreased binding by narrowly neutralizing antibodies to the V3 crown. Three mutations together increased neutralization by V2 bnAb and eliminated binding by V3 crown antibodies. These results may aid the design of immunogens that elicit antibodies to the trimer apex.

  • Processing of Potato Spindle Tuber Viroid (PSTVd) RNAs in Yeast, a Nonconventional Host [PublishAheadOfPrint]

  • Potato spindle tuber viroid (PSTVd) is a circular, single-stranded, noncoding RNA plant pathogen that is a useful model for the processing of noncoding RNA in eukaryotes. Infective PSTVd circles are replicated via an asymmetric rolling circle mechanism to form linear multimeric RNAs. An endonuclease cleaves these into monomers; a ligase seals these into mature circles. All eukaryotes may have such enzymes for processing noncoding RNA. As a test, we investigated the processing of three PSTVd RNA constructs in the yeast Saccharomyces cerevisiae. Of these, only one form, the construct that adopts a previously described tetraloop-containing conformation (TL), produces circles.TL has 16 nucleotides of the 3' end duplicated at the 5' end and a 3' end produced by self-cleavage of a delta ribozyme. The other two constructs, an exact monomer flanked by ribozymes, and a trihelix-forming RNA with requisite 5' and 3' duplications do not produce circles. The TL circles contain non-native nucleotides resulting from the 3' -end created by the ribozyme and the 5' -end from an endolytic cleavage by yeast at a site distinct from where potato enzymes cut these RNAs. RNAs from all three transcripts are cleaved in places not on path for circle formation, and are likely RNA decay. We propose that these constructs fold into distinct RNA structures that interact differently with host cell RNA metabolism enzymes, resulting in varying susceptibility to degradation versus processing. We conclude that PSTVd RNA is opportunistic and may use different processing pathways in different hosts.

    IMPORTANCE In higher eukaryotes, the majority of transcribed RNA does not encode proteins. These noncoding RNA are responsible for messenger RNA regulation, control of the expression of regulatory microRNAs, sensing changes in the environment using riboswitches (RNAs that change shape in response to environmental signals), catalysis, and more roles that are still being uncovered. Some of these functions may be remnants from the RNA world, and as such would be part of the evolutionary past of all forms of modern life. Viroids are noncoding RNAs that can cause disease in plants. Since they encode no proteins, they depend on their own RNA and host proteins for replication and pathogenicity. It is likely that viroids hijack critical host RNA pathways for processing the host's own noncoding RNA. These pathways are still unknown. Elucidating these pathways should reveal new biological functions of noncoding RNA.

  • Atomic Structures of Minor Proteins VI and VII in the Human Adenovirus [PublishAheadOfPrint]

  • Human adenoviruses (Ad) are dsDNA viruses associated with infectious diseases, yet better known as tools for gene delivery and oncolytic anti-cancer therapy. Atomic structures of Ad provide the basis for the development of antivirals and for engineering efforts towards more effective applications. Since 2010, atomic models of human Ad5 have been independently derived from photographic film cryoEM and X-ray crystallography, but discrepancies exist concerning the assignment of cement proteins IIIa, VIII and IX. To clarify these discrepancies, here we have employed the technology of direct electron-counting to obtain a cryoEM structure of human Ad5 at 3.2 AAring; resolution. Our improved structure unambiguously confirmed our previous cryoEM models of proteins IIIa, VIII and IX and explained the likely cause of conflict in the crystallography models. The improved structure also allows the identification of three new components in the cavities of hexons nndash; the cleaved N-terminus of precursor protein VI (pVIn), the cleaved N-terminus of precursor protein VII (pVIIn2), and mature protein VI. The binding of pVIIn2mmdash;-by extension that of genome-condensing pVIImmdash;-to hexons is consistent with the previously proposed dsDNA genome-capsid co-assembly for adenoviruses, which resembles that of ssRNA viruses but differs from the well-established mechanism of pumping dsDNA into a preformed protein capsid, as exemplified by tailed bacteriophages and herpesviruses.

    IMPORTANCE Adenovirus is a double-edged sword to humans nndash; as a widespread pathogen and a bioengineering tool for anti-cancer and gene therapy. Atomic structure of the virus provides the basis for antiviral and application developments, but conflicting atomic models from conventional/film cryoEM and X-ray crystallography for important cement proteins IIIa, VIII, and IX have caused confusion. Using the cutting-edge cryoEM technology with electron counting, we improved the structure of human adenovirus type 5 and confirmed our previous models of cement proteins IIIa, VIII, and IX, thus clarifying the inconsistent structures. The improved structure also reveals atomic details of membrane-lytic protein VI and genome-condensing protein VII and supports the previously proposed genome-capsid co-assembly mechanism for adenoviruses.

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