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

Table of Contents for this page:

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

    JVI Current Issue

  • Editorial Board [Masthead]

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

  • Coinfections: Another Variable in the Herpesvirus Latency-Reactivation Dynamic [Gems]

  • Chronic viruses, such as herpesviruses, shape host physiology. These viruses modulate the inflammatory state of the immune system and have evolved to harness inflammation as a mechanism to regulate viral latency and reactivation. In this review, I examine some of the recent work demonstrating the important role of inflammation in the regulation of the herpesvirus life cycle and discuss recent work that implicates coinfection in the regulation of herpesvirus latency.

  • Commandeering the Ribosome: Lessons Learned from Dicistroviruses about Translation [Gems]

  • To replicate, all viruses depend entirely on the enslavement of host cell ribosomes for their own advantage. To this end, viruses have evolved a multitude of translational strategies to usurp the ribosome. RNA-based structures known as internal ribosome entry sites (IRESs) are among the most notable mechanisms employed by viruses to seize host ribosomes. In this article, we spotlight the intergenic region IRES from the Dicistroviridae family of viruses and its importance as a model for IRES-dependent translation and in understanding fundamental properties of translation.

  • Vaccination with Live Attenuated Simian Immunodeficiency Virus (SIV) Protects from Mucosal, but Not Necessarily Intravenous, Challenge with a Minimally Heterologous SIV [Pathogenesis and Immunity]

  • Few studies have evaluated the impact of the viral challenge route on protection against a heterologous simian immunodeficiency virus (SIV) challenge. We vaccinated seven macaques with a live attenuated SIV that differed from SIVmac239nef by 24 amino acids, called m3KOnef. All animals were protected from an intrarectal SIVmac239 challenge, whereas only four animals were protected from subsequent intravenous SIVmac239 challenge. These data suggest that immune responses elicited by vaccination with live attenuated SIV in an individual animal can confer protection from intrarectal challenge while remaining insufficient for protection from intravenous challenge.

    IMPORTANCE Our study is important because we show that vaccinated animals can be protected from a mucosal challenge with a heterologous SIV, but the same animals are not necessarily protected from intravenous challenge with the same virus. This is unique because in most studies, either vaccinated animals are challenged multiple times by the same route or only a single challenge is performed. An individually vaccinated animal is rarely challenged multiple times by different routes, so protection from different challenge routes cannot be measured in the same animal. Our data imply that vaccine-elicited responses in an individual animal may be insufficient for protection from intravenous challenge but may be suitable for protection from a mucosal challenge that better approximates human immunodeficiency virus (HIV) exposure.

  • Lipopolysaccharide and Tumor Necrosis Factor Alpha Inhibit Interferon Signaling in Hepatocytes by Increasing Ubiquitin-Like Protease 18 (USP18) Expression [Pathogenesis and Immunity]

  • Inflammation may be maladaptive to the control of viral infection when it impairs interferon (IFN) responses, enhancing viral replication and spread. Dysregulated immunity as a result of inappropriate innate inflammatory responses is a hallmark of chronic viral infections such as, hepatitis B virus and hepatitis C virus (HCV). Previous studies from our laboratory have shown that expression of an IFN-stimulated gene (ISG), ubiquitin-like protease (USP)18 is upregulated in chronic HCV infection, leading to impaired hepatocyte responses to IFN-aalpha;. We examined the ability of inflammatory stimuli, including tumor necrosis factor alpha (TNF-aalpha;), lipopolysaccharide (LPS), interleukin-6 (IL-6) and IL-10 to upregulate hepatocyte USP18 expression and blunt the IFN-aalpha; response. Human hepatoma cells and primary murine hepatocytes were treated with TNF-aalpha;/LPS/IL-6/IL-10 and USP18, phosphorylated (p)-STAT1 and myxovirus (influenza virus) resistance 1 (Mx1) expression was determined. Treatment of Huh7.5 cells and primary murine hepatocytes with LPS and TNF-aalpha;, but not IL-6 or IL-10, led to upregulated USP18 expression and induced an IFN-aalpha; refractory state, which was reversed by USP18 knockdown. Liver inflammation was induced in vivo using a murine model of hepatic ischemia/reperfusion injury. Hepatic ischemia/reperfusion injury led to an induction of USP18 expression in liver tissue and promotion of lymphocytic choriomeningitis replication. These data demonstrate that certain inflammatory stimuli (TNF-aalpha; and LPS) but not others (IL-6 and IL-10) target USP18 expression and thus inhibit IFN signaling. These findings represent a new paradigm for how inflammation alters hepatic innate immune responses, with USP18 representing a potential target for intervention in various inflammatory states.

    IMPORTANCE Inflammation may prevent the control of viral infection when it impairs the innate immune response, enhancing viral replication and spread. Blunted immunity as a result of inappropriate innate inflammatory responses is a common characteristic of chronic viral infections. Previous studies have shown that expression of certain interferon-stimulated genes is upregulated in chronic HCV infection, leading to impaired hepatocyte responses. In this study, we show that multiple inflammatory stimuli can modulate interferon stimulated gene expression and thus inhibit hepatocyte interferon signaling via USP18 induction. These findings represent a new paradigm for how inflammation alters hepatic innate immune responses, with the induction of USP18 representing a potential target for intervention in various inflammatory states.

  • Two Outbreak Sources of Influenza A (H7N9) Viruses Have Been Established in China [Genetic Diversity and Evolution]

  • Due to enzootic infections in poultry and persistent human infections in China, influenza A (H7N9) virus has remained a public health threat. The Yangtze River Delta region, which is located in eastern China, is well recognized as the original source for H7N9 outbreaks. Based on the evolutionary analysis of H7N9 viruses from all three outbreak waves since 2013, we identified the Pearl River Delta region as an additional H7N9 outbreak source. H7N9 viruses are repeatedly introduced from these two sources to the other areas, and the persistent circulation of H7N9 viruses occurs in poultry, causing continuous outbreak waves. Poultry movements may contribute to the geographic expansion of the virus. In addition, the AnH1 genotype, which was predominant during wave 1, was replaced by JS537, JS18828, and AnH1887 genotypes during waves 2 and 3. The establishment of a new source and the continuous evolution of the virus hamper the elimination of H7N9 viruses, thus posing a long-term threat of H7N9 infection in humans. Therefore, both surveillance of H7N9 viruses in humans and poultry and supervision of poultry movements should be strengthened.

    IMPORTANCE Since its occurrence in humans in eastern China in spring 2013, the avian H7N9 viruses have been demonstrating the continuing pandemic threat posed by the current influenza ecosystem in China. As the viruses are silently circulated in poultry, with potentially severe outcomes in humans, H7N9 virus activity in humans in China is very important to understand. In this study, we identified a newly emerged H7N9 outbreak source in the Pearl River Delta region. Both sources in the Yangtze River Delta region and the Pearl River Delta region have been established and found to be responsible for the H7N9 outbreaks in mainland China.

  • B Cell Responses during Secondary Dengue Virus Infection Are Dominated by Highly Cross-Reactive, Memory-Derived Plasmablasts [Cellular Response to Infection]

  • Dengue virus (DENV) infection results in the production of both type-specific and cross-neutralizing antibodies. While immunity to the infecting serotype is long-lived, heterotypic immunity wanes a few months after infection. Epidemiological studies link secondary heterotypic infections with more severe symptoms, and cross-reactive, poorly neutralizing antibodies have been implicated in this increased disease severity. To understand the cellular and functional properties of the acute dengue virus B cell response and its role in protection and immunopathology, we characterized the plasmablast response in four secondary DENV type 2 (DENV2) patients. Dengue plasmablasts had high degrees of somatic hypermutation, with a clear preference for replacement mutations. Clonal expansions were also present in each donor, strongly supporting a memory origin for these acutely induced cells. We generated 53 monoclonal antibodies (MAbs) from sorted patient plasmablasts and found that DENV-reactive MAbs were largely envelope specific and cross neutralizing. Many more MAbs neutralized DENV than reacted to envelope protein, emphasizing the significance of virion-dependent B cell epitopes and the limitations of envelope protein-based antibody screening. A majority of DENV-reactive MAbs, irrespective of neutralization potency, enhanced infection by antibody-dependent enhancement (ADE). Interestingly, even though DENV2 was the infecting serotype in all four patients, several MAbs from two patients neutralized DENV1 more potently than DENV2. Further, half of all type-specific neutralizing MAbs were also DENV1 biased in binding. Taken together, these findings are reminiscent of original antigenic sin (OAS), given that the patients had prior dengue virus exposures. These data describe the ongoing B cell response in secondary patients and may further our understanding of the impact of antibodies in dengue virus pathogenesis.

    IMPORTANCE In addition to their role in protection, antibody responses have been hypothesized to contribute to the pathology of dengue. Recent studies characterizing memory B cell (MBC)-derived MAbs have provided valuable insight into the targets and functions of B cell responses generated after DENV exposure. However, in the case of secondary infections, such MBC-based approaches fail to distinguish acutely induced cells from the preexisting MBC pool. Our characterization of plasmablasts and plasmablast-derived MAbs provides a focused analysis of B cell responses activated during ongoing infection. Additionally, our studies provide evidence of OAS in the acute-phase dengue virus immune response, providing a basis for future work examining the impact of OAS phenotype antibodies on protective immunity and disease severity in secondary infections.

  • Identification of the Fusion Peptide-Containing Region in Betacoronavirus Spike Glycoproteins [Virus-Cell Interactions]

  • The fusion peptides (FP) play an essential role in fusion of viral envelope with cellular membranes. The location and properties of the FPs in the spike (S) glycoproteins of different coronaviruses (CoV) have not yet been determined. Through amino acid sequence analysis of S proteins of representative CoVs, we identified a common region as a possible FP (pFP) that shares the characteristics of FPs of class I viral fusion proteins, including high Ala/Gly content, intermediate hydrophobicity, and few charged residues. To test the hypothesis that this region contains the CoV FP, we systemically mutated every residue in the pFP of Middle East respiratory syndrome betacoronavirus (MERS-CoV) and found that 11 of the 22 residues in the pFP (from G953 to L964, except for A956) were essential for S protein-mediated cell-cell fusion and virus entry. The synthetic MERS-CoV pFP core peptide (955IAGVGWTAGL964) induced extensive fusion of liposome membranes, while mutant peptide failed to induce any lipid mixing. We also selectively mutated residues in pFPs of two other bbeta;-CoVs, severe acute respiratory syndrome coronavirus (SARS-CoV) and mouse hepatitis virus (MHV). Although the amino acid sequences of these two pFPs differed significantly from that of MERS-CoV and each other, most of the pFP mutants of SARS-CoV and MHV also failed to mediate membrane fusion, suggesting that these pFPs are also the functional FPs. Thus, the FPs of 3 different lineages of bbeta;-CoVs are conserved in location within the S glycoproteins and in their functions, although their amino acid sequences have diverged significantly during CoV evolution.

    IMPORTANCE Within the class I viral fusion proteins of many enveloped viruses, the FP is the critical mediator of fusion of the viral envelope with host cell membranes leading to virus infection. FPs from within a virus family, like influenza viruses or human immunodeficiency viruses (HIV), tend to share high amino acid sequence identity. In this study, we determined the location and amino acid sequences of the FPs of S glycoproteins of 3 bbeta;-CoVs, MERS-CoV, SARS-CoV, and MHV, and demonstrated that they were essential for mediating cell-cell fusion and virus entry. Interestingly, in marked contrast to the FPs of influenza and HIV, the primary amino acid sequences of the FPs of bbeta;-CoVs in 3 different lineages differed significantly. Thus, during evolution the FPs of bbeta;-CoVs have diverged significantly in their primary sequences while maintaining the same essential biological functions. Our findings identify a potential new target for development of drugs against CoVs.

  • The Coxsackievirus and Adenovirus Receptor: Glycosylation and the Extracellular D2 Domain Are Not Required for Coxsackievirus B3 Infection [Virus-Cell Interactions]

  • The coxsackievirus and adenovirus receptor (CAR) is a member of the immunoglobulin superfamily (IgSF) and functions as a receptor for coxsackie B viruses (CVBs). The extracellular portion of CAR comprises two glycosylated immunoglobulin-like domains, D1 and D2. CAR-D1 binds to the virus and is essential for virus infection; however, it is not known whether D2 is also important for infection, and the role of glycosylation has not been explored. To understand the function of these structural components in CAR-mediated CVB3 infection, we generated a panel of human (h) CAR deletion and substitution mutants and analyzed their functionality as CVB receptors, examining both virus binding and replication. Lack of glycosylation of the CAR-D1 or -D2 domains did not adversely affect CVB3 binding or infection, indicating that the glycosylation of CAR is not required for its receptor functions. Deletion of the D2 domain reduced CVB3 binding, with a proportionate reduction in the efficiency of virus infection. Replacement of D2 with the homologous D2 domain from chicken CAR, or with the heterologous type C2 immunoglobulin-like domain from IgSF11, another IgSF member, fully restored receptor function; however, replacement of CAR-D2 with domains from CD155 or CD80 restored function only in part. These data indicate that glycosylation of the extracellular domain of hCAR plays no role in CVB3 receptor function and that CAR-D2 is not specifically required. The D2 domain may function largely as a spacer permitting virus access to D1; however, the data may also suggest that D2 affects virus binding by influencing the conformation of D1.

    IMPORTANCE An important step in virus infection is the initial interaction of the virus with its cellular receptor. Although the role in infection of the extracellular CAR-D1, cytoplasmic, and transmembrane domains have been analyzed extensively, nothing is known about the function of CAR-D2 and the extracellular glycosylation of CAR. Our data indicate that glycosylation of the extracellular CAR domain has only minor importance for the function of CAR as CVB3 receptor and that the D2 domain is not essential per se but contributes to receptor function by promoting the exposure of the D1 domain on the cell surface. These results contribute to our understanding of the coxsackievirus-receptor interactions.

  • The Human Papillomavirus 16 E7 Oncoprotein Attenuates AKT Signaling To Promote Internal Ribosome Entry Site-Dependent Translation and Expression of c-MYC [Transformation and Oncogenesis]

  • While the role of high-risk human papillomavirus (HPV) oncoproteins E6 and E7 in targeting p53 and retinoblastoma (Rb) has been intensively studied, how E6 and E7 manipulate cellular signaling cascades to promote the viral life cycle and cancer development is less understood. Keratinocytes containing the episomal HPV-16 genome had decreased activation of AKT, which was phenocopied by HPV-16 E7 expression alone. Attenuation of phosphorylated AKT (pAKT) by E7 was independent of the Rb degradation function of E7 but could be ablated by a missense mutation in the E7 carboxy terminus, H73E, thereby defining a novel structure-function phenotype for E7. Downstream of AKT, reduced phosphorylation of p70 S6K and 4E-BP1 was also observed in E7-expressing keratinocytes, which coincided with an increase in internal ribosomal entry site (IRES)-dependent translation that enhanced the expression of several cellular proteins, including MYC, Bax, and the insulin receptor. The decrease in pAKT mediated by E7 is in contrast to the widely observed increase of pAKT in invasive cervical cancers, suggesting that the activation of AKT signaling could be acquired during the progression from initial productive infections to invasive carcinomas.

    IMPORTANCE HPV causes invasive cervical cancers through the dysregulation of the cell cycle regulators p53 and Rb, which are degraded by the viral oncoproteins E6 and E7, respectively. Signaling cascades contribute to cancer progression and cellular differentiation, and how E6 and E7 manipulate those pathways remains unclear. The phosphoinositol 3-kinase (PI3K)/AKT pathway regulates cellular processes, including proliferation, cell survival, and cell differentiation. Surprisingly, we found that HPV-16 decreased the phosphorylation of AKT (pAKT) and that this is a function of E7 that is independent of the Rb degradation function. This is in contrast to the observed increase in AKT signaling in nearly 80% of cervical cancers, which typically show an acquired mutation within the PI3K/AKT cascade leading to constitutive activation of the pathway. Our observations suggest that multiple changes in the activation and effects of AKT signaling occur in the progression from productive HPV infections to invasive cervical cancers.

  • Roles of Us8A and Its Phosphorylation Mediated by Us3 in Herpes Simplex Virus 1 Pathogenesis [Pathogenesis and Immunity]

  • The herpes simplex virus 1 (HSV-1) Us8A gene overlaps the gene that encodes glycoprotein E (gE). Previous studies have investigated the roles of Us8A in HSV-1 infection using null mutations in Us8A and gE; therefore, the role of Us8A remains to be elucidated. In this study, we investigated the function of Us8A and its phosphorylation at serine 61 (Ser-61), which we recently identified as a phosphorylation site by mass spectrometry-based phosphoproteomic analysis of HSV-1-infected cells, in HSV-1 pathogenesis. We observed that (i) the phosphorylation of Us8A Ser-61 in infected cells was dependent on the activity of the virus-encoded Us3 protein kinase; (ii) the Us8A null mutant virus exhibited a 10-fold increase in the 50% lethal dose for virulence in the central nervous system (CNS) of mice following intracranial infection compared with a repaired virus; (iii) replacement of Ser-61 with alanine (S61A) in Us8A had little effect on virulence in the CNS of mice following intracranial infection, whereas it significantly reduced the mortality of mice following ocular infection to levels similar to the Us8A null mutant virus; (iv) the Us8A S61A mutation also significantly reduced viral yields in mice following ocular infection, mainly in the trigeminal ganglia and brains; and (v) a phosphomimetic mutation at Us8A Ser-61 restored wild-type viral yields and virulence. Collectively, these results indicate that Us8A is a novel HSV-1 virulence factor and suggest that the Us3-mediated phosphorylation of Us8A Ser-61 regulates Us8A function for viral invasion into the CNS from peripheral sites.

    IMPORTANCE The DNA genomes of viruses within the subfamily Alphaherpesvirinae are divided into unique long (UL) and unique short (Us) regions. Us regions contain alphaherpesvirus-specific genes. Recently, high-throughput sequencing of ocular isolates of HSV-1 showed that Us8A was the most highly conserved of 13 herpes simplex virus 1 (HSV-1) genes mapped to the Us region, suggesting Us8A may have an important role in the HSV-1 life cycle. However, the specific role of Us8A in HSV-1 infection remains to be elucidated. Here, we show that Us8A is a virulence factor for HSV-1 infection in mice, and the function of Us8A for viral invasion into the central nervous system from peripheral sites is regulated by Us3-mediated phosphorylation of the protein at Ser-61. This is the first study to report the significance of Us8A and its regulation in HSV-1 infection.

  • Pathogenesis of Influenza D Virus in Cattle [Pathogenesis and Immunity]

  • Cattle have been proposed as the natural reservoir of a novel member of the virus family Orthomyxoviridae, which has been tentatively classified as influenza D virus (IDV). Although isolated from sick animals, it is unclear whether IDV causes any clinical disease in cattle. To address this aspect of Koch's postulates, three dairy calves (treatment animals) held in individual pens were inoculated intranasally with IDV strain D/bovine/Mississippi/C00046N/2014. At 1 day postinoculation, a seronegative calf (contact animal) was added to each of the treatment animal pens. The cattle in both treatment and contact groups seroconverted, and virus was detected in their respiratory tracts. Histologically, there was a significant increase in neutrophil tracking in tracheal epithelia of the treatment calves compared to control animals. While infected and contact animals demonstrated various symptoms of respiratory tract infection, they were mild, and the calves in the treatment group did not differ from the controls in terms of heart rate, respiratory rate, or rectal temperature. To mimic zoonotic transmission, two ferrets were exposed to a plastic toy fomite soaked with infected nasal discharge from the treatment calves. These ferrets did not shed the virus or seroconvert. In summary, this study demonstrates that IDV causes a mild respiratory disease upon experimental infection of cattle and can be transmitted effectively among cattle by in-pen contact, but not from cattle to ferrets through fomite exposure. These findings support the hypothesis that cattle are a natural reservoir for the virus.

    IMPORTANCE A novel influenza virus, tentatively classified as influenza D virus (IDV), was identified in swine, cattle, sheep, and goats. Among these hosts, cattle have been proposed as the natural reservoir. In this study, we show that cattle experimentally infected with IDV can shed virus and transmit it to other cattle through direct contact, but not to ferrets through fomite routes. IDV caused minor clinical signs in the infected cattle, fulfilling another of Koch's postulates for this novel agent, although other objective clinical endpoints were not different from those of control animals. Although the disease observed was mild, IDV induced neutrophil tracking and epithelial attenuation in cattle trachea, which could facilitate coinfection with other pathogens, and in doing so, predispose animals to bovine respiratory disease.

  • Quantitation of Productively Infected Monocytes and Macrophages of Simian Immunodeficiency Virus-Infected Macaques [Virus-Cell Interactions]

  • Despite the success of combined antiretroviral therapy (ART), human immunodeficiency virus (HIV) infection remains a lifelong infection because of latent viral reservoirs in infected patients. The contribution of CD4+ T cells to infection and disease progression has been extensively studied. However, during early HIV infection, macrophages in brain and other tissues are infected and contribute to tissue-specific diseases, such as encephalitis and dementia in brain and pneumonia in lung. The extent of infection of monocytes and macrophages has not been rigorously assessed with assays comparable to those used to study infection of CD4+ T cells and to evaluate the number of CD4+ T cells that harbor infectious viral genomes. To assess the contribution of productively infected monocytes and macrophages to HIV- and simian immunodeficiency virus (SIV)-infected cells in vivo, we developed a quantitative virus outgrowth assay (QVOA) based on similar assays used to quantitate CD4+ T cell latent reservoirs in HIV- and SIV-infected individuals in whom the infection is suppressed by ART. Myeloid cells expressing CD11b were serially diluted and cocultured with susceptible cells to amplify virus. T cell receptor bbeta; RNA was measured as a control to assess the potential contribution of CD4+ T cells in the assay. Virus production in the supernatant was quantitated by quantitative reverse transcription-PCR. Productively infected myeloid cells were detected in blood, bronchoalveolar lavage fluid, lungs, spleen, and brain, demonstrating that these cells persist throughout SIV infection and have the potential to contribute to the viral reservoir during ART.

    IMPORTANCE Infection of CD4+ T cells and their role as latent reservoirs have been rigorously assessed; however, the frequency of productively infected monocytes and macrophages in vivo has not been similarly studied. Myeloid cells, unlike lymphocytes, are resistant to the cytopathic effects of HIV. Moreover, tissue-resident macrophages have the ability to self-renew and persist in the body for months to years. Thus, tissue macrophages, once infected, have the characteristics of a potentially stable viral reservoir. A better understanding of the number of productively infected macrophages is crucial to further evaluate the role of infected myeloid cells as a potential viral reservoir. In the study described here we compared the frequency of productively infected CD4+ T cells and macrophages in an SIV-infected macaque model. We developed a critical assay that will allow us to quantitate myeloid cells containing viral genomes that lead to productive infection in SIV-infected macaques and assess the role of macrophages as potential reservoirs.

  • Trimer Enhancement Mutation Effects on HIV-1 Matrix Protein Binding Activities [Structure and Assembly]

  • The HIV-1 matrix (MA) protein is the amino-terminal domain of the HIV-1 precursor Gag (Pr55Gag) protein. MA binds to membranes and RNAs, helps transport Pr55Gag proteins to virus assembly sites at the plasma membranes of infected cells, and facilitates the incorporation of HIV-1 envelope (Env) proteins into virions by virtue of an interaction with the Env protein cytoplasmic tails (CTs). MA has been shown to crystallize as a trimer and to organize on membranes in hexamer lattices. MA mutations that localize to residues near the ends of trimer spokes have been observed to impair Env protein assembly into virus particles, and several of these are suppressed by the 62QR mutation at the hubs of trimer interfaces. We have examined the binding activities of wild-type (WT) MA and 62QR MA variants and found that the 62QR mutation stabilized MA trimers but did not alter the way MA proteins organized on membranes. Relative to WT MA, the 62QR protein showed small effects on membrane and RNA binding. However, 62QR proteins bound significantly better to Env CTs than their WT counterparts, and CT binding efficiencies correlated with trimerization efficiencies. Our data suggest a model in which multivalent binding of trimeric HIV-1 Env proteins to MA trimers contributes to the process of Env virion incorporation.

    IMPORTANCE The HIV-1 Env proteins assemble as trimers, and incorporation of the proteins into virus particles requires an interaction of Env CT domains with the MA domains of the viral precursor Gag proteins. Despite this knowledge, little is known about the mechanisms by which MA facilitates the virion incorporation of Env proteins. To help elucidate this process, we examined the binding activities of an MA mutant that stabilizes MA trimers. We found that the mutant proteins organized similarly to WT proteins on membranes, and that mutant and WT proteins revealed only slight differences in their binding to RNAs or lipids. However, the mutant proteins showed better binding to Env CTs than the WT proteins, and CT binding correlated with MA trimerization. Our results suggest that multivalent binding of trimeric HIV-1 Env proteins to MA trimers contributes to the process of Env virion incorporation.

  • Short Intracellular HIV-1 Transcripts as Biomarkers of Residual Immune Activation in Patients on Antiretroviral Therapy [Genome Replication and Regulation of Viral Gene Expression]

  • HIV-1 patients continue to remain at an abnormal immune status despite prolonged combination antiretroviral therapy (cART), which results in an increased risk of non-AIDS-related diseases. Given the growing recognition of the importance of understanding and controlling the residual virus in patients, additional virological markers to monitor infected cells are required. However, viral replication in circulating cells is much poorer than that in lymph nodes, which results in the absence of markers to distinguish these cells from uninfected cells in the blood. In this study, we identified prematurely terminated short HIV-1 transcripts (STs) in peripheral blood mononuclear cells (PBMCs) as an efficient intracellular biomarker to monitor viral activation and immune status in patients with cART-mediated full viral suppression in plasma. STs were detected in PBMCs obtained from both treated and untreated patients. ST levels in untreated patients generally increased with disease progression and decreased after treatment initiation. However, some patients exhibited sustained high levels of ST and low CD4+ cell counts despite full viral suppression by treatment. The levels of STs strongly reflected chronic immune activation defined by coexpression of HLA-DR and CD38 on CD8+ T cells, rather than circulating proviral load. These observations represent evidence for a relationship between viral persistence and host immune activation, which in turn results in the suboptimal increase in CD4+ cells despite suppressive antiretroviral therapy. This cell-based measurement of viral persistence contributes to an improved understanding of the dynamics of viral persistence in cART patients and will guide therapeutic approaches targeting viral reservoirs.

    IMPORTANCE Combination antiretroviral therapy (cART) suppresses HIV-1 load to below the detectable limit in plasma. However, the virus persists, and patients remain at an abnormal immune status, which results in an increased risk of non-AIDS-related complications. To achieve a functional cure for HIV-1 infection, activities of viral reservoirs must be quantified and monitored. However, latently infected cells are difficult to be monitored. Here, we identified prematurely terminated short HIV-1 transcripts (STs) as an efficient biomarker for monitoring viral activation and immune status in patients with cART-mediated full viral suppression in plasma. This cell-based measurement of viral persistence will contribute to our understanding of the impact of residual virus on chronic immune activation in HIV-1 patients during cART.

  • Differential Inductions of RNA Silencing among Encapsidated Double-Stranded RNA Mycoviruses in the White Root Rot Fungus Rosellinia necatrix [Pathogenesis and Immunity]

  • RNA silencing acts as a defense mechanism against virus infection in a wide variety of organisms. Here, we investigated inductions of RNA silencing against encapsidated double-stranded RNA (dsRNA) fungal viruses (mycoviruses), including a partitivirus (RnPV1), a quadrivirus (RnQV1), a victorivirus (RnVV1), a mycoreovirus (RnMyRV3), and a megabirnavirus (RnMBV1) in the phytopathogenic fungus Rosellinia necatrix. Expression profiling of RNA silencing-related genes revealed that a dicer-like gene, an Argonaute-like gene, and two RNA-dependent RNA polymerase genes were upregulated by RnMyRV3 or RnMBV1 infection but not by other virus infections or by constitutive expression of dsRNA in R. necatrix. Massive analysis of viral small RNAs (vsRNAs) from the five mycoviruses showed that 19- to 22-nucleotide (nt) vsRNAs were predominant; however, their ability to form duplexes with 3' overhangs and the 5' nucleotide preferences of vsRNAs differed among the five mycoviruses. The abundances of 19- to 22-nt vsRNAs from RnPV1, RnQV1, RnVV1, RnMyRV3, and RnMBV1 were 6.8%, 1.2%, 0.3%, 13.0%, and 24.9%, respectively. Importantly, the vsRNA abundances and accumulation levels of viral RNA were not always correlated, and the origins of the vsRNAs were distinguishable among the five mycoviruses. These data corroborated diverse interactions between encapsidated dsRNA mycoviruses and RNA silencing. Moreover, a green fluorescent protein (GFP)-based sensor assay in R. necatrix revealed that RnMBV1 infection induced silencing of the target sensor gene (GFP gene and the partial RnMBV1 sequence), suggesting that vsRNAs from RnMBV1 activated the RNA-induced silencing complex. Overall, this study provides insights into RNA silencing against encapsidated dsRNA mycoviruses.

    IMPORTANCE Encapsidated dsRNA fungal viruses (mycoviruses) are believed to replicate inside their virions; therefore, there is a question of whether they induce RNA silencing. Here, we investigated inductions of RNA silencing against encapsidated dsRNA mycoviruses (a partitivirus, a quadrivirus, a victorivirus, a mycoreovirus, and a megabirnavirus) in Rosellinia necatrix. We revealed upregulation of RNA silencing-related genes in R. necatrix infected with a mycoreovirus or a megabirnavirus but not with other viruses, which was consistent with the relatively high abundances of vsRNAs from the two mycoviruses. We also showed common and different molecular features and origins of the vsRNAs from the five mycoviruses. Furthermore, we demonstrated the activation of RNA-induced silencing complex by mycoviruses in R. necatrix. Taken together, our data provide insights into an RNA silencing pathway against encapsidated dsRNA mycoviruses which is differentially induced among encapsidated dsRNA mycoviruses; that is, diverse replication strategies exist among encapsidated dsRNA mycoviruses.

  • Life Cycle Characterization of Sulfolobus Monocaudavirus 1, an Extremophilic Spindle-Shaped Virus with Extracellular Tail Development [Virus-Cell Interactions]

  • We provide here, for the first time, insights into the initial infection stages of a large spindle-shaped archaeal virus and explore the following life cycle events. Our observations suggest that Sulfolobus monocaudavirus 1 (SMV1) exhibits a high adsorption rate and that virions adsorb to the host cells via three distinct attachment modes: nosecone association, body association, and body/tail association. In the body/tail association mode, the entire virion, including the tail(s), aligns to the host cell surface and the main body is greatly flattened, suggesting a possible fusion entry mechanism. Upon infection, the intracellular replication cycle lasts about 8 h, at which point the virions are released as spindle-shaped tailless particles. Replication of the virus retarded host growth but did not cause lysis of the host cells. Once released from the host and at temperatures resembling that of its natural habitat, SMV1 starts developing one or two tails. This exceptional property of undergoing a major morphological development outside, and independently of, the host cell has been reported only once before for the related Acidianus two-tailed virus. Here, we show that SMV1 can develop tails of more than 900 nm in length, more than quadrupling the total virion length.

    IMPORTANCE Very little is known about the initial life cycle stages of viruses infecting hosts of the third domain of life, Archaea. This work describes the first example of an archaeal virus employing three distinct association modes. The virus under study, Sulfolobus monocaudavirus 1, is a representative of the large spindle-shaped viruses that are frequently found in acidic hot springs. The results described here will add valuable knowledge about Archaea, the least studied domain in the virology field.

  • Contributions of Charged Residues in Structurally Dynamic Capsid Surface Loops to Rous Sarcoma Virus Assembly [Structure and Assembly]

  • Extensive studies of orthoretroviral capsids have shown that many regions of the CA protein play unique roles at different points in the virus life cycle. The N-terminal domain (NTD) flexible-loop (FL) region is one such example: exposed on the outer capsid surface, it has been implicated in Gag-mediated particle assembly, capsid maturation, and early replication events. We have now defined the contributions of charged residues in the FL region of the Rous sarcoma virus (RSV) CA to particle assembly. Effects of mutations on assembly were assessed in vivo and in vitro and analyzed in light of new RSV Gag lattice models. Virus replication was strongly dependent on the preservation of charge at a few critical positions in Gag-Gag interfaces. In particular, a cluster of charges at the beginning of FL contributes to an extensive electrostatic network that is important for robust Gag assembly and subsequent capsid maturation. Second-site suppressor analysis suggests that one of these charged residues, D87, has distal influence on interhexamer interactions involving helix aalpha;7. Overall, the tolerance of FL to most mutations is consistent with current models of Gag lattice structures. However, the results support the interpretation that virus evolution has achieved a charge distribution across the capsid surface that (i) permits the packing of NTD domains in the outer layer of the Gag shell, (ii) directs the maturational rearrangements of the NTDs that yield a functional core structure, and (iii) supports capsid function during the early stages of virus infection.

    IMPORTANCE The production of infectious retrovirus particles is a complex process, a choreography of protein and nucleic acid that occurs in two distinct stages: formation and release from the cell of an immature particle followed by an extracellular maturation phase during which the virion proteins and nucleic acids undergo major rearrangements that activate the infectious potential of the virion. This study examines the contributions of charged amino acids on the surface of the Rous sarcoma virus capsid protein in the assembly of appropriately formed immature particles and the maturational transitions that create a functional virion. The results provide important biological evidence in support of recent structural models of the RSV immature virions and further suggest that immature particle assembly and virion maturation are controlled by an extensive network of electrostatic interactions and long-range communication across the capsid surface.

  • Incongruity between Prion Conversion and Incubation Period following Coinfection [Prions]

  • When multiple prion strains are inoculated into the same host, they can interfere with each other. Strains with long incubation periods can suppress conversion of strains with short incubation periods; however, nothing is known about the conversion of the long-incubation-period strain during strain interference. To investigate this, we inoculated hamsters in the sciatic nerve with long-incubation-period strain 139H prior to superinfection with the short-incubation-period hyper (HY) strain of transmissible mink encephalopathy (TME). First, we found that 139H is transported along the same neuroanatomical tracks as HY TME, adding to the growing body of evidence indicating that PrPSc favors retrograde transneuronal transport. In contrast to a previous report, we found that 139H interferes with HY TME infection, which is likely due to both strains targeting the same population of neurons following sciatic nerve inoculation. Under conditions where 139H blocked HY TME from causing disease, the strain-specific properties of PrPSc corresponded with the strain that caused disease, consistent with our previous findings. In the groups of animals where incubation periods were not altered, we found that the animals contained a mixture of 139H and HY TME PrPSc. This finding expands the definition of strain interference to include conditions where PrPSc formation is altered yet disease outcome is unaltered. Overall, these results contradict the premise that prion strains are static entities and instead suggest that strain mixtures are dynamic regardless of incubation period or clinical outcome of disease.

    IMPORTANCE Prions can exist as a mixture of strains in naturally infected animals, where they are able to interfere with the conversion of each other and to extend incubation periods. Little is known, however, about the dynamics of strain conversion under conditions where incubation periods are not affected. We found that inoculation of the same animal with two strains can result in the alteration of conversion of both strains under conditions where the resulting disease was consistent with infection with only a single strain. These data challenge the idea that prion strains are static and suggests that strain mixtures are more dynamic than previously appreciated. This observation has significant implications for prion adaptation.

  • Antibody Responses with Fc-Mediated Functions after Vaccination of HIV-Infected Subjects with Trivalent Influenza Vaccine [Vaccines and Antiviral Agents]

  • This study seeks to assess the ability of seasonal trivalent inactivated influenza vaccine (TIV) to induce nonneutralizing antibodies (Abs) with Fc-mediated functions in HIV-uninfected and HIV-infected subjects. Functional influenza-specific Ab responses were studied in 30 HIV-negative and 27 HIV-positive subjects immunized against seasonal influenza. All 57 subjects received the 2015 TIV. Fc-mediated antihemagglutinin (anti-HA) Ab activity was measured in plasma before and 4 weeks after vaccination using Fc-receptor-binding assays, NK cell activation assays, and phagocytosis assays. At baseline, the HIV-positive group had detectable but reduced functional Ab responses to both vaccine and nonvaccine influenza antigens. TIV enhanced Fc-mediated Ab responses in both HIV-positive and HIV-negative groups. A larger rise was generally observed in the HIV-positive group, such that there was no difference in functional Ab responses between the two groups after vaccination. The 2015 TIV enhanced functional influenza-specific Ab responses in both HIV-negative and HIV-positive subjects to a range of influenza HA proteins. The increase in functional Ab responses in the HIV-positive group supports recommendations to immunize this at-risk group.

    IMPORTANCE Infection with HIV is associated with increasing disease severity following influenza infections, and annual influenza vaccinations are recommended for this target group. However, HIV-infected individuals respond relatively poorly to vaccination compared to healthy individuals, particularly if immunodeficient. There is therefore a need to increase our understanding of immunity to influenza in the context of underlying HIV infection. While antibodies can mediate direct virus neutralization, interactions with cellular Fc receptors may be important for anti-influenza immunity in vivo by facilitating antibody-dependent cellular cytotoxicity (ADCC) and/or antibody-dependent phagocytosis (ADP). The ability of seasonal influenza vaccines to induce antibody responses with potent Fc-mediated antiviral activity is currently unclear. Probing the ADCC and ADP responses to influenza vaccination has provided important new information in the quest to improve immunity to influenza.

  • Transmembrane Domains of NS2B Contribute to both Viral RNA Replication and Particle Formation in Japanese Encephalitis Virus [Genome Replication and Regulation of Viral Gene Expression]

  • Flavivirus nonstructural protein 2B (NS2B) is a transmembrane protein that functions as a cofactor for viral NS3 protease. The cytoplasmic region (amino acids 51 to 95) alone of NS2B is sufficient for NS3 protease activity, whereas the role of transmembrane domains (TMDs) remains obscure. Here, we demonstrate for the first time that flavivirus NS2B plays a critical role in virion assembly. Using Japanese encephalitis virus (JEV) as a model, we performed a systematic mutagenesis at the flavivirus conserved residues within the TMDs of NS2B. As expected, some mutations severely attenuated (L38A and R101A) or completely destroyed (G12L) viral RNA synthesis. Interestingly, two mutations (G37L and P112A) reduced viral RNA synthesis and blocked virion assembly. None of the mutations affected NS2B-NS3 protease activity. Because mutations G37L and P112A affected virion assembly, we selected revertant viruses for these two mutants. For mutant G37L, replacement with G37F, G37H, G37T, or G37S restored virion assembly. For mutant P112A, insertion of K at position K127 (leading to K127KK) of NS2B rescued virion assembly. A biomolecular fluorescent complementation (BiFC) analysis demonstrated that (i) mutation P112A selectively weakened NS2B-NS2A interaction and (ii) the adaptive mutation K127KK restored NS2B-NS2A interaction. Collectively, our results demonstrate that, in addition to being a cofactor for NS3 protease, flavivirus NS2B also functions in viral RNA replication, as well as virion assembly.

    IMPORTANCE Many flaviviruses are important human pathogens. Understanding the molecular mechanisms of the viral infection cycle is essential for vaccine and antiviral development. In this study, we demonstrate that the TMDs of JEV NS2B participate in both viral RNA replication and virion assembly. A viral genetic study and a BiFC assay demonstrated that interaction between NS2B and NS2A may participate in modulating viral assembly in the flavivirus life cycle. Compensatory-mutation analysis confirmed that there was a correlation between viral assembly and NS2B-NS2A interaction. TMDs of NS2B may serve as novel antiviral targets to prevent flavivirus infection, and the structure determination of NS2B will help us to understand the functional mechanism of NS2B in viral RNA replication and assembly. The results have uncovered a new function of flavivirus NS2B in virion assembly, possibly through interaction with the NS2A protein.

  • Interleukin-2 Therapy Induces CD4 Downregulation, Which Decreases Circulating CD4 T Cell Counts, in African Green Monkeys [Pathogenesis and Immunity]

  • African green monkeys (AGMs) are natural hosts of simian immunodeficiency virus (SIVAGM). Because these animals do not develop simian AIDS despite maintaining high viral loads, there is considerable interest in determining how these animals have evolved to avoid SIV disease progression. Unlike nonnatural hosts of SIV, adult AGMs maintain low levels of CD4+ T cells at steady states and also have a large population of virus-resistant CD8aalpha;aalpha; T cells that lack CD4 expression despite maintaining T helper cell functionalities. In recent work, we have shown that homeostatic cytokines can induce CD4 downregulation in AGM T cells in vitro. Through administering therapeutic doses of recombinant human interleukin-2 (IL-2) to AGMs, we show here that this mechanism is operative in vivo. IL-2 therapy induced transient yet robust proliferation in all major T cell subsets. Within the CD4+ T cell population, those that were induced into cycle by IL-2 exhibited characteristics of CD4-to-CD8aalpha;aalpha; conversion. In all animals receiving IL-2, circulating CD4+ T cell counts and proportions tended to be lower and CD4nndash; CD8aalpha;aalpha;+ T cell counts tended to be higher. Despite reductions in circulating target cells, the viral load was unaffected over the course of study.

    IMPORTANCE The data in this study identify that homeostatic cytokines can downregulate CD4 in vivo and, when given therapeutically, can induce AGMs to sustain very low levels of circulating CD4+ T cells without showing signs of immunodeficiency.

  • Novel Structure and Unexpected RNA-Binding Ability of the C-Terminal Domain of Herpes Simplex Virus 1 Tegument Protein UL21 [Structure and Assembly]

  • Proteins forming the tegument layers of herpesviral virions mediate many essential processes in the viral replication cycle, yet few have been characterized in detail. UL21 is one such multifunctional tegument protein and is conserved among alphaherpesviruses. While UL21 has been implicated in many processes in viral replication, ranging from nuclear egress to virion morphogenesis to cell-cell spread, its precise roles remain unclear. Here we report the 2.7-AAring; crystal structure of the C-terminal domain of herpes simplex virus 1 (HSV-1) UL21 (UL21C), which has a unique aalpha;-helical fold resembling a dragonfly. Analysis of evolutionary conservation patterns and surface electrostatics pinpointed four regions of potential functional importance on the surface of UL21C to be pursued by mutagenesis. In combination with the previously determined structure of the N-terminal domain of UL21, the structure of UL21C provides a 3-dimensional framework for targeted exploration of the multiple roles of UL21 in the replication and pathogenesis of alphaherpesviruses. Additionally, we describe an unanticipated ability of UL21 to bind RNA, which may hint at a yet unexplored function.

    IMPORTANCE Due to the limited genomic coding capacity of viruses, viral proteins are often multifunctional, which makes them attractive antiviral targets. Such multifunctionality, however, complicates their study, which often involves constructing and characterizing null mutant viruses. Systematic exploration of these multifunctional proteins requires detailed road maps in the form of 3-dimensional structures. In this work, we determined the crystal structure of the C-terminal domain of UL21, a multifunctional tegument protein that is conserved among alphaherpesviruses. Structural analysis pinpointed surface areas of potential functional importance that provide a starting point for mutagenesis. In addition, the unexpected RNA-binding ability of UL21 may expand its functional repertoire. The structure of UL21C and the observation of its RNA-binding ability are the latest additions to the navigational chart that can guide the exploration of the multiple functions of UL21.

  • Molecular Characterizations of Surface Proteins Hemagglutinin and Neuraminidase from Recent H5Nx Avian Influenza Viruses [Structure and Assembly]

  • During 2014, a subclade highly pathogenic avian influenza (HPAI) A(H5N8) virus caused poultry outbreaks around the world. In late 2014/early 2015, the virus was detected in wild birds in Canada and the United States, and these viruses also gave rise to reassortant progeny, composed of viral RNA segments (vRNAs) from both Eurasian and North American lineages. In particular, viruses were found with N1, N2, and N8 neuraminidase vRNAs, and these are collectively referred to as H5Nx viruses. In the United States, more than 48 million domestic birds have been affected. Here we present a detailed structural and biochemical analysis of the surface antigens of H5N1, H5N2, and H5N8 viruses in addition to those of a recent human H5N6 virus. Our results with recombinant hemagglutinin reveal that these viruses have a strict avian receptor binding preference, while recombinantly expressed neuraminidases are sensitive to FDA-approved and investigational antivirals. Although H5Nx viruses currently pose a low risk to humans, it is important to maintain surveillance of these circulating viruses and to continually assess future changes that may increase their pandemic potential.

    IMPORTANCE The H5Nx viruses emerging in North America, Europe, and Asia pose a great public health concern. Here we report a molecular and structural study of the major surface proteins of several H5Nx influenza viruses. Our results improve the understanding of these new viruses and provide important information on their receptor preferences and susceptibilities to antivirals, which are central to pandemic risk assessment.

  • Entry Sites of Venezuelan and Western Equine Encephalitis Viruses in the Mouse Central Nervous System following Peripheral Infection [Pathogenesis and Immunity]

  • Venezuelan and western equine encephalitis viruses (VEEV and WEEV; Alphavirus; Togaviridae) are mosquito-borne pathogens causing central nervous system (CNS) disease in humans and equids. Adult CD-1 mice also develop CNS disease after infection with VEEV and WEEV. Adult CD-1 mice infected by the intranasal (i.n.) route, showed that VEEV and WEEV enter the brain through olfactory sensory neurons (OSNs). In this study, we injected the mouse footpad with recombinant WEEV (McMillan) or VEEV (subtype IC strain 3908) expressing firefly luciferase (fLUC) to simulate mosquito infection and examined alphavirus entry in the CNS. Luciferase expression served as a marker of infection detected as bioluminescence (BLM) by in vivo and ex vivo imaging. BLM imaging detected WEEV and VEEV at 12 h postinoculation (hpi) at the injection site (footpad) and as early as 72 hpi in the brain. BLM from WEEV.McM-fLUC and VEEV.3908-fLUC injections was initially detected in the brain's circumventricular organs (CVOs). No BLM activity was detected in the olfactory neuroepithelium or OSNs. Mice were also injected in the footpad with WEEV.McM expressing DsRed (Discosoma sp.) and imaged by confocal fluorescence microscopy. DsRed imaging supported our BLM findings by detecting WEEV in the CVOs prior to spreading along the neuronal axis to other brain regions. Taken together, these findings support our hypothesis that peripherally injected alphaviruses enter the CNS by hematogenous seeding of the CVOs followed by centripetal spread along the neuronal axis.

    IMPORTANCE VEEV and WEEV are mosquito-borne viruses causing sporadic epidemics in the Americas. Both viruses are associated with CNS disease in horses, humans, and mouse infection models. In this study, we injected VEEV or WEEV, engineered to express bioluminescent or fluorescent reporters (fLUC and DsRed, respectively), into the footpads of outbred CD-1 mice to simulate transmission by a mosquito. Reporter expression serves as detectable bioluminescent and fluorescent markers of VEEV and WEEV replication and infection. Bioluminescence imaging, histological examination, and confocal fluorescence microscopy were used to identify early entry sites of these alphaviruses in the CNS. We observed that specific areas of the brain (circumventricular organs [CVOs]) consistently showed the earliest signs of infection with VEEV and WEEV. Histological examination supported VEEV and WEEV entering the brain of mice at specific sites where the blood-brain barrier is naturally absent.

  • Dynamic Nucleolar Targeting of Dengue Virus Polymerase NS5 in Response to Extracellular pH [Virus-Cell Interactions]

  • The nucleolar subcompartment of the nucleus is increasingly recognized as an important target of RNA viruses. Here we document for the first time the ability of dengue virus (DENV) polymerase, nonstructural protein 5 (NS5), to accumulate within the nucleolus of infected cells and to target green fluorescent protein (GFP) to the nucleolus of live transfected cells. Intriguingly, NS5 exchange between the nucleus and nucleolus is dynamically modulated by extracellular pH, responding rapidly and reversibly to pH change, in contrast to GFP alone or other nucleolar and non-nucleolar targeted protein controls. The minimal pH-sensitive nucleolar targeting region (pHNTR), sufficient to target GFP to the nucleolus in a pH-sensitive fashion, was mapped to NS5 residues 1 to 244, with mutation of key hydrophobic residues, Leu-165, Leu-167, and Val-168, abolishing pHNTR function in NS5-transfected cells, and severely attenuating DENV growth in infected cells. This is the first report of a viral protein whose nucleolar targeting ability is rapidly modulated by extracellular stimuli, suggesting that DENV has the ability to detect and respond dynamically to the extracellular environment.

    IMPORTANCE Infections by dengue virus (DENV) threaten 40% of the world's population yet there is no approved vaccine or antiviral therapeutic to treat infections. Understanding the molecular details that govern effective viral replication is key for the development of novel antiviral strategies. Here, we describe for the first time dynamic trafficking of DENV nonstructural protein 5 (NS5) to the subnuclear compartment, the nucleolus. We demonstrate that NS5's targeting to the nucleolus occurs in response to acidic pH, identify the key amino acid residues within NS5 that are responsible, and demonstrate that their mutation severely impairs production of infectious DENV. Overall, this study identifies a unique subcellular trafficking event and suggests that DENV is able to detect and respond dynamically to environmental changes.

  • Roles of Capsid-Interacting Host Factors in Multimodal Inhibition of HIV-1 by PF74 [Virus-Cell Interactions]

  • The viral capsid of HIV-1 interacts with a number of host factors to orchestrate uncoating and regulate downstream events, such as reverse transcription, nuclear entry, and integration site targeting. PF-3450074 (PF74), an HIV-1 capsid-targeting low-molecular-weight antiviral compound, directly binds to the capsid (CA) protein at a site also utilized by host cell proteins CPSF6 and NUP153. Here, we found that the dose-response curve of PF74 is triphasic, consisting of a plateau and two inhibitory phases of different slope values, consistent with a bimodal mechanism of drug action. High PF74 concentrations yielded a steep curve with the highest slope value among different classes of known antiretrovirals, suggesting a dose-dependent, cooperative mechanism of action. CA interactions with both CPSF6 and cyclophilin A (CypA) were essential for the unique dose-response curve. A shift of the steep curve at lower drug concentrations upon blocking the CA-CypA interaction suggests a protective role for CypA against high concentrations of PF74. These findings, highlighting the unique characteristics of PF74, provide a model in which its multimodal mechanism of action of both noncooperative and cooperative inhibition by PF74 is regulated by interactions of cellular proteins with incoming viral capsids.

    IMPORTANCE PF74, a novel capsid-targeting antiviral against HIV-1, shares its binding site in the viral capsid protein (CA) with the host factors CPSF6 and NUP153. This work reveals that the dose-response curve of PF74 consists of two distinct inhibitory phases that are differentially regulated by CA-interacting host proteins. PF74's potency depended on these CA-binding factors at low doses. In contrast, the antiviral activity of high PF74 concentrations was attenuated by cyclophilin A. These observations provide novel insights into both the mechanism of action of PF74 and the roles of host factors during the early steps of HIV-1 infection.

  • Herpes Simplex Virus 1 Serine Protease VP24 Blocks the DNA-Sensing Signal Pathway by Abrogating Activation of Interferon Regulatory Factor 3 [Virus-Cell Interactions]

  • The interferon (IFN)-mediated antiviral response is a central aspect of host defense; however, viruses have evolved multiple strategies to counteract IFN-mediated responses in order to successfully infect the host. Herpes simplex virus 1 (HSV-1), a typical human-restricted DNA virus, is capable of counteracting host immune responses via several distinct viral proteins, thus establishing a lifelong latent infection. In this study, we demonstrate that the VP24 protein, a serine protease of HSV-1 essential for the formation and maturation of capsids, is a novel antagonist of the beta interferon (IFN-bbeta;) pathway. Here, VP24 was shown for the first time to dampen interferon stimulatory DNA (ISD)-triggered IFN-bbeta; production and inhibit IFN-bbeta; promoter activation induced by cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) and by STING, respectively. Further study demonstrated that ectopic expression of VP24 selectively blocked IFN regulatory factor 3 (IRF3) but not NF-B promoter activation. In addition, VP24 was demonstrated to downregulate ISD-induced phosphorylation and dimerization of IRF3 during HSV-1 infection with a VP24 stable knockdown human foreskin fibroblast cell line. The underlying molecular mechanism is that VP24 abrogates the interaction between TANK-binding kinase 1 (TBK1) and IRF3, hence impairing IRF3 activation. These results illustrate that VP24 is able to block the production of IFN-bbeta; by inhibiting IRF3 activation, which may represent a critical adaptation to enable viral effective replication within the host.

    IMPORTANCE This study demonstrated that HSV-1 protein VP24 could inhibit IFN-bbeta; production and promoter activation triggered by ISD, cGAS and STING and by STING, respectively. VP24 selectively blocked IRF3 promoter activation and ISD-induced phosphorylation and dimerization of IRF3 without affecting the NF-B promoter activation during viral infection. VP24 also inhibited IRF3 activation by impeding the interaction between TBK1 and IRF3 during viral infection. This study provides new insights into the immune evasion mediated by HSV-1 and identifies VP24 as a crucial effector for HSV-1 to evade the host DNA-sensing signal pathway.

  • Cell-Free Hepatitis B Virus Capsid Assembly Dependent on the Core Protein C-Terminal Domain and Regulated by Phosphorylation [Structure and Assembly]

  • Multiple subunits of the hepatitis B virus (HBV) core protein (HBc) assemble into an icosahedral capsid that packages the viral pregenomic RNA (pgRNA). The N-terminal domain (NTD) of HBc is sufficient for capsid assembly, in the absence of pgRNA or any other viral or host factors, under conditions of high HBc and/or salt concentrations. The C-terminal domain (CTD) is deemed dispensable for capsid assembly although it is essential for pgRNA packaging. We report here that HBc expressed in a mammalian cell lysate, rabbit reticulocyte lysate (RRL), was able to assemble into capsids when (low-nanomolar) HBc concentrations mimicked those achieved under conditions of viral replication in vivo and were far below those used previously for capsid assembly in vitro. Furthermore, at physiologically low HBc concentrations in RRL, the NTD was insufficient for capsid assembly and the CTD was also required. The CTD likely facilitated assembly under these conditions via RNA binding and protein-protein interactions. Moreover, the CTD underwent phosphorylation and dephosphorylation events in RRL similar to those seen in vivo which regulated capsid assembly. Importantly, the NTD alone also failed to accumulate in mammalian cells, likely resulting from its failure to assemble efficiently. Coexpression of the full-length HBc rescued NTD assembly in RRL as well as NTD expression and assembly in mammalian cells, resulting in the formation of mosaic capsids containing both full-length HBc and the NTD. These results have important implications for HBV assembly during replication and provide a facile cell-free system to study capsid assembly under physiologically relevant conditions, including its modulation by host factors.

    IMPORTANCE Hepatitis B virus (HBV) is an important global human pathogen and the main cause of liver cancer worldwide. An essential component of HBV is the spherical capsid composed of multiple copies of a single protein, the core protein (HBc). We have developed a mammalian cell-free system in which HBc is expressed at physiological (low) concentrations and assembles into capsids under near-physiological conditions. In this cell-free system, as in mammalian cells, capsid assembly depends on the C-terminal domain (CTD) of HBc, in contrast to other assembly systems in which HBc assembles into capsids independently of the CTD under conditions of nonphysiological protein and salt concentrations. Furthermore, the phosphorylation state of the CTD regulates capsid assembly and RNA encapsidation in the cell-free system in a manner similar to that seen in mammalian cells. This system will facilitate detailed studies on capsid assembly and RNA encapsidation under physiological conditions and identification of antiviral agents that target HBc.

  • Correction for Shin et al., Histone Deacetylase Classes I and II Regulate Kaposi's Sarcoma-Associated Herpesvirus Reactivation [Author Corrections]

  • Correction for Colman et al., Genome-Wide Analysis of Host mRNA Translation during Hepatitis C Virus Infection [Author Corrections]

  • Correction for Soh and Whelan, Tracking the Fate of Genetically Distinct Vesicular Stomatitis Virus Matrix Proteins Highlights the Role for Late Domains in Assembly [Author Corrections]

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    JVI Accepts: Articles Published Ahead of Print

  • Tegument protein ORF45 plays an essential role in virion morphogenesis of murine gammaherpesvirus-68 [PublishAheadOfPrint]

  • Tegument proteins play critical roles in herpesvirus morphogenesis. ORF45 is a conserved tegument protein of gammaherpesviruses, however, its role in virion morphogenesis is largely unknown. In this work, we determined the ultrastructural localization of murine gammaherpesvirus-68 (MHV-68) ORF45 and found that it was incorporated into virions around the site of host-derived vesicles. Notably, absence of ORF45 inhibited nucleocapsid egress and blocked cytoplasmic virion maturation, demonstrating that ORF45 is essential for MHV-68 virion morphogenesis.

  • Critical role of the PA-X C-terminal domain of influenza A virus on its subcellular localization and shutoff activity [PublishAheadOfPrint]

  • PA-X is a recently identified influenza virus protein, which is composed of PA N-terminal 191 amino acids and unique C-terminal 41 or 61 residues. We and others showed that PA-X has a strong ability to suppress host protein synthesis via host mRNA decay, which is mediated by endonuclease activity in its N-terminal domain. However, the mechanism of host mRNA degradation, especially where and how PA-X targets mRNAs has not been analyzed. In this study, we determined the localization of PA-X and the role of the C-terminal unique region on shutoff activity. Quantitative subcellular localization analysis revealed that PA-X was located equally in both cytoplasm and nucleus. By characterizing a series of PA-X C-terminal deletion mutants, we found that the first 9 amino acids were sufficient for nuclear localization, but an additional 6 residues were required to induce the maximum shutoff activity observed with intact PA-X. Importantly, forced nuclear localization of the PA-X C-terminal deletion mutant enhanced shutoff activity, highlighting the ability of nuclear PA-X to degrade host mRNAs more efficiently. However, PA-X also inhibited luciferase expression from transfected mRNAs synthesized in vitro, suggesting that PA-X also degrades mRNAs in the cytoplasm. Among the basic amino acids in the PA-X C-terminal region, 3 residues, 195K, 198K, and 199R were identified as key residues for inducing host shutoff and nuclear localization. Overall, our data indicate a critical role for the 15 residues in the PA-X C-terminal domain in degrading mRNAs in both the cytoplasm and nucleus.

    IMPORTANCE Influenza A viruses express PA-X proteins to suppress global host gene expression, including host antiviral genes to allow efficient viral replication in infected cells. However, little is known about how PA-X induces host shutoff. In this study, we showed that PA-X localized equally in both the cytoplasm and nucleus of the cells, but the nuclear localization of PA-X mediated by its C-terminal region has a significant impact on shutoff activity. Three basic residues at the C-terminal region play a critical role in nuclear localization, but additional basic residues were required for maximum shutoff activity. Our findings indicate that PA-X targets and degrades mRNAs in both the nucleus and cytoplasm, and that the first 15 residues of the PA-X unique C-terminal region play a critical role in shutoff activity.

  • Release of infectious hepatitis C virus from Huh7 cells occurs via a trans-Golgi network to endosome pathway independent of VLDL [PublishAheadOfPrint]

  • The release of infectious hepatitis C virus (HCV) particles from infected cells remains poorly characterized. We previously demonstrated that virus release is dependent on the endosome sorting complex required for transport (ESCRT). Here, we show a critical role of TGN-endosome trafficking during the assembly, but principally the secretion of infectious virus. This was demonstrated by both siRNA mediated silencing of TGN-associated adaptor proteins, and a panel of dominant negative (DN) Rab GTPases involved in TGN-endosome trafficking steps. Importantly, interfering with factors critical for HCV release did not have a concomitant effect on secretion of either triglyceride, ApoB or ApoE, indicating that particles are likely released from Huh7 cells via pathways distinct to that of VLDL. Finally we show that HCV NS2 perturbs trans-Golgi network (TGN) architecture, redistributing TGN membranes to closely associate with HCV core residing on lipid droplets. These findings support the notion that HCV hijacks TGN-endosome trafficking to facilitate particle assembly and release. Moreover, whist essential for assembly and infectivity, the trafficking of mature virions is seemingly independent of host lipoproteins.

    Importance. The mechanisms by which infectious hepatitis C virus particles are assembled and released from the cell are poorly understood. We show that the virus subverts host cell trafficking pathways to effect the release of virus particles and disrupts the structure of the Golgi apparatus, a key cellular organelle involved in secretion. In addition we demonstrate that the mechanisms used by the virus to exit the cell are distinct from those used by the cell to release lipoproteins, suggesting that the virus effects an unique modification to cellular trafficking pathways.

  • HIV-1-Specific Chimeric Antigen Receptors Based on Broadly-Neutralizing Antibodies [PublishAheadOfPrint]

  • Although the use chimeric antigen receptors (CARs) based on single chain antibodies for gene immunotherapy of cancers is increasing due to promising recent results, the earliest CAR therapeutic trials were for HIV-1 infection in the late 1990s. This approach utilized a CAR based on human CD4 as a binding domain, and was abandoned for lack of efficacy. The growing number of HIV-1 broadly neutralizing antibodies (bnAbs) offers the opportunity to generate novel CARs that may be more active and revisit this modality for HIV-1 immunotherapy. We used sequences from seven well-defined bnAbs varying in binding sites and generated single chain antibody-based CARs. These included 10E8, 3BNC117, PG9, PGT126, PGT128, VRC01, and X5. Each novel CAR exhibited conformationally relevant expression on the surface of transduced cells, mediated specific proliferation and killing in response to HIV-1-infected cells, and conferred potent antiviral activity (reduction of viral replication in log10 units) to transduced CD8+ T lymphocytes. Their antiviral activity was reproducible but varied according to the strain of virus. These findings indicated that bnAbs are excellent candidates for developing novel CARs to consider in the immunotherapeutic treatment of HIV-1.

    IMPORTANCE While chimeric antigen receptors (CARs) using single chain antibodies as binding domains are growing in popularity for gene immunotherapy of cancers, the earliest human trials of CARs were for HIV-1 infection. However, those trials failed and the approach was abandoned for HIV-1. The only tested CAR against HIV-1 was based on using CD4 as the binding domain. The growing availability of HIV-1 broadly neutralizing antibodies (bnAbs) affords the opportunity to revisit gene immunotherapy for HIV-1 using novel CARs based on single chain antibodies. Here we construct and test a panel of seven novel CARs based on diverse bnAb types, and show that all are functional against HIV-1.

  • Human Cytomegalovirus pUL93 Links Nucleocapsid Maturation and Nuclear Egress [PublishAheadOfPrint]

  • Human cytomegalovirus (HCMV) pUL93 and pUL77 are both essential for virus growth but their functions in the virus life cycle remain mostly unresolved. Homologs of pUL93 and pUL77 in herpes simplex virus (HSV-1) and pseudorabies virus (PRV) are known to interact to form a complex at capsid vertices known as the capsid vertex-specific component (CVSC), which likely stabilizes nucleocapsids during virus maturation and also aids in nuclear egress. In herpesviruses, nucleocapsids assemble and partially mature in nuclear replication compartments and then travel to the inner nuclear membrane (INM) for nuclear egress. The factors governing the recruitment of nucleocapsids to the INM are not known. Kinetic analysis of pUL93 demonstrates that this protein is expressed late during infection and localizes primarily to the nucleus of infected cells. pUL93 associates with both virions and capsids, and interacts with the components of the nuclear egress complex (NEC), namely pUL50, pUL53, and pUL97, during infection. Also, multiple regions in pUL93 can independently interact with pUL77, which has been shown to help retain viral DNA during capsid assembly. These studies, combined with our earlier report of an essential role of pUL93 in viral DNA packaging, indicate that pUL93 may serve as an important link between nucleocapsid maturation and nuclear egress.

    IMPORTANCE Human cytomegalovirus (HCMV) causes life-threatening disease and disability in immunocompromised patients and congenitally infected newborns. In this study, we investigated the functions of HCMV essential tegument protein pUL93 and determined that it interacts with the components of the nuclear egress complex, namely pUL50, pUL53, and pUL97. We also found that pUL93 specifically interacts with pUL77, which helps retain viral DNA during capsid assembly. Together, our data point towards an important role of pUL93 in linking virus maturation to nuclear egress. In addition to expanding our knowledge of the process of HCMV maturation, information from these studies will also be utilized to develop new antiviral therapies.

  • MAVS expressed by hematopoietic cells is critical for control of West Nile virus infection and pathogenesis [PublishAheadOfPrint]

  • West Nile virus (WNV) is the most important cause of epidemic encephalitis in North America. Innate immune responses, which are critical for control of WNV infection, are initiated by signaling through pathogen recognition receptors, RIG-I and MDA5, and their downstream adaptor molecule, MAVS. Here we show that a deficiency of MAVS in hematopoietic cells resulted in increased mortality and delayed WNV clearance from the brain. In Mavs-/- mice, a dysregulated immune response was detected, characterized by a massive influx of macrophages and virus-specific T cells into the infected brain. These T cells were polyfunctional and lysed peptide-pulsed target cells in vitro. However, virus-specific T cells in the brains of infected Mavs-/- mice exhibited lower functional avidity than those in wild-type animals, and even virus-specific memory T cells generated by prior immunization could not protect Mavs-/- mice from WNV-induced lethal disease. Concomitant with ineffective virus clearance, macrophage numbers were increased in the Mavs-/- brain and both macrophages and microglia exhibited an activated phenotype. Microarray analyses of leukocytes in the infected Mavs-/- brain showed a preferential expression of genes associated with activation and inflammation. Together, these results demonstrate a critical role for MAVS in hematopoietic cells in augmenting the kinetics of WNV clearance and thereby preventing a dysregulated and pathogenic immune response.

    IMPORTANCE West Nile virus (WNV) is the most important cause of mosquito-transmitted encephalitis in the United States. The innate immune response is known to be critical for protection in infected mice. Here, we show that expression of MAVS, a key adaptor molecule in the RIG-I-like receptor RNA sensing pathway, in hematopoietic cells is critical for protection from lethal WNV infection. In the absence of MAVS, there is a massive infiltration of myeloid cells and virus-specific T cells into the brain and over-exuberant production of pro-inflammatory cytokines. These results demonstrate the important role that MAVS expression in hematopoietic cells has in regulating the inflammatory response in the WNV-infected brain.

  • Viral evasion and manipulation of host RNA quality control pathways [PublishAheadOfPrint]

  • Viruses have evolved diverse strategies to maximize the functional and coding capacities of their genetic material. Individual viral RNAs are often used as substrates for both replication and translation and can contain multiple, sometimes overlapping, open reading frames. Further, viral RNAs engage in a wide variety of interactions with both host and viral proteins to modify the activities of important cellular factors and direct their own trafficking, packaging, localization, stability, and translation. However, adaptations increasing the information density of small viral genomes can have unintended consequences. In particular, viral RNAs have developed features that mark them as potential targets of host RNA quality control pathways. This review focuses on ways in which viral RNAs run afoul of cellular the mRNA quality control and decay machinery, as well as strategies developed by viruses to circumvent or exploit cellular mRNA surveillance.

  • Glutathione depletion is linked with Th2 polarization in mice with a retrovirus-induced immunodeficiency syndrome, MAIDS: role of pro-glutathione molecules as immunotherapeutics [PublishAheadOfPrint]

  • LP-BM5 murine leukemia virus injection causes murine AIDS, a disease characterized by many dysfunctions of immunocompetent cells. To establish whether the disease is characterized by glutathione imbalance, reduced glutathione (GSH) and cysteine were quantified in different organs. A marked redox imbalance, consisting in GSH and/or cysteine depletion, was found in the lymphoid organs, such as the spleen and lymph nodes. Moreover, a significant decrease in cysteine and GSH levels was measured in pancreas and brain respectively at 5 weeks post infection. Th2 immune response was predominant at all the times investigated as revealed by the expression of Th1/Th2 cytokines. Furthermore, an investigation of the activation status of peritoneal macrophages showed that the expression of genetic markers of alternative activation, namely, Fizz1, Ym1, and Arginase1, was induced. Conversely, expression of iNOS, a marker of classical activation of macrophages, was detected only when Th1 cytokines were more expressed. In vitro studies revealed that during the very early phases of infection, GSH depletion and down-regulation of interleukin-12 p40 mRNA were correlated with the level of the infecting dose of LP-BM5 used to infect macrophages. Treatment of LP-BM5-infected mice with I-152, a N-acetyl-cysteine supplier, restored GSH/cysteine levels in the organs, reduced the expression of alternatively activated macrophage markers, and increased IFN- production while decreased Th2 cytokines, such as IL-4 and IL-5. Our findings thus establish a link between GSH deficiency and Th1/Th2 disequilibrium in LP-BM5 infection, and I-152 can be used to restore the GSH level and a balanced Th1/Th2 response in infected mice.

    IMPORTANCE This study represents the first report of association between Th2 polarization and alteration of the redox state in LP-BM5 infection. Moreover, it is shown the evidence that LP-BM5 infection causes a decrease in thiol content of peritoneal macrophages which can influence IL-12 production.

    The restoration of GSH levels by GSH-replenishing molecules can represent a new therapeutic avenue to fight this retroviral infection re-establishing the Th1/Th2 balance. The immunotherapy based on the use of pro-GSH molecules would permit to more effectively combat LP-BM5 infection and probably all those viral infections characterized by GSH deficiency and Th1/Th2 imbalance.

  • Generating bona fide mammalian prions with internal deletions. [PublishAheadOfPrint]

  • Mammalian prions are PrP proteins with altered structures causing transmissible fatal neurodegenerative diseases. They are self-perpetuating through formation of beta-sheet-rich assemblies that seed conformational change of cellular PrP. Pathological PrP usually forms an insoluble protease-resistant core exhibiting beta-sheet structures but no more alpha helical content, loosing the three alpha helices contained in the correctly folded PrP. The lack of a high-resolution prion structure makes it difficult to understand the dynamics of conversion and to identify elements of the protein involved in this process. To determine whether completeness of residues within the protease-resistant domain is required for prions, we performed serial deletions in the helix H2 C-terminus of ovine PrP, as this region has previously shown some tolerance to sequence changes without preventing prion replication. Deletions of either four or five residues essentially preserved the overall PrP structure and mutant PrP expressed in RK13 cells were efficiently converted into bona fide prions upon challenge by three different prion strains. Remarkably, deletions in PrP facilitated the replication of two strains that otherwise do not replicate in this cellular context. Prions with internal deletion were self-propagating and de novo infectious for naïve homologous and wild-type PrP expressing cells. Even more, they caused transmissible spongiform encephalopathies in mice, with similar biochemical signatures and neuropathologies than the original strains. Prion convertibility and transfer of strain-specific information are thus preserved despite shortening of an alpha helix in PrP and removal of residues within prions. These findings provide new insights into sequence/structure/infectivity relationship for prions.

    IMPORTANCE Prions are misfolded PrP proteins that convert the normal protein into a replicate of their own abnormal form. They are responsible for invariably fatal neurodegenerative disorders. Other aggregation-prone proteins appear to have a prion-like mode of expansion in brains, such as in Alzheimer's or Parkinson's diseases. To date resolution of prion structure remains elusive. Thus to genetically define the landscape of regions critical for prion conversion, we tested the effect of short deletions. We found that, surprisingly, removal of a portion of PrP, the C-terminus of alpha-helix H2, did not hamper prion formation, but generated infectious agents with an internal deletion that showed characteristics essentially similar to those of original infecting strains. Thus we demonstrate that completeness of the residues inside prions is not necessary for maintaining infectivity and the main strain-specific information, while reporting one of the few if not the only bona fide prions with an internal deletion.

  • A Genome-Wide RNAi Screen Identifies a Role for Wnt/Beta-Catenin Signaling During Rift Valley Fever Virus Infection [PublishAheadOfPrint]

  • Rift Valley fever virus (RVFV) is an arbovirus within the Bunyaviridae family capable of causing serious morbidity and mortality in humans and livestock. To identify host factors involved in bunyavirus replication, we employed genome-wide RNA interference (RNAi) screening and identified 381 genes whose knockdown reduced infection. The Wnt pathway was the most represented pathway when gene hits were functionally clustered. With further investigation, we found that RVFV infection activated Wnt signaling, was enhanced when Wnt signaling was pre-activated, was reduced with knockdown of bbeta;-catenin, and was blocked using Wnt signaling inhibitors. Similar results were found using distantly related bunyaviruses La Crosse virus and California encephalitis virus, suggesting a conserved role for Wnt signaling in bunyaviral infection. We propose a model where bunyaviruses activate Wnt responsive genes to regulate optimal cell cycle conditions needed to promote efficient viral replication. The findings in this study should aid in the design of efficacious host-directed anti-viral therapeutics.

    IMPORTANCE: RVFV is a mosquito-borne bunyavirus that is endemic to Africa but has demonstrated a capacity for emergence in new territories (e.g. Arabian Peninsula). As a zoonotic pathogen that primarily affects livestock, RVFV can also cause lethal hemorrhagic fever and encephalitis in humans. Currently, there are no treatments or fully-licensed vaccines for this virus. Using high-throughput RNAi screening, we identified canonical Wnt signaling as an important host pathway regulating RVFV infection. The beneficial role of Wnt signaling was observed for RVFV, along with other disparate bunyaviruses, indicating a conserved bunyaviral replication mechanism involving Wnt signaling. These studies supplement our knowledge of the fundamental mechanisms of bunyavirus infection and provide new avenues for countermeasure development against pathogenic bunyaviruses.

  • Deep Sequencing of the HIV-1 env Gene Reveals Discrete X4 Lineages and Linkage Disequilibrium Between X4 and R5 Viruses In the V1/V2 and V3 Variable Regions [PublishAheadOfPrint]

  • HIV-1 requires the CD4 receptor and a co-receptor (CCR5 (R5 phenotype] or CXCR4 [X4 phenotype]) to enter cells. Co-receptor tropism can be assessed by either phenotypic or genotypic analysis, the latter using bioinformatics algorithms to predict tropism based on the env V3 sequence. We used the Primer ID sequencing strategy with the MiSeq sequencing platform to reveal the structure of viral populations in the V1/V2 and C2/V3 regions of the HIV-1 env gene in 30 late stage and 6 early stage subjects. We also used end-point dilution PCR followed by cloning of env genes to create pseudotyped virus to explore the link between genotypic predictions and phenotypic assessment of coreceptor usage. We found out that the most stringently sequence-based calls of X4 variants (Geno2Pheno false positive rate [FPR] lle; 2%) formed distinct lineages within the viral population, and these were detected in 24 of 30 late-stage samples (80%), significantly higher than has been seen previously using other approaches. Non-X4 lineages were not skewed toward lower FPR scores in X4-containing populations. Phenotypic assays showed that variants with intermediate FPR (2nndash;20%) could be either X4/dual or R5 variants, although the X4 variants made up only about 25% of the lineages with an FPR of less than 10%, and these carried a distinctive sequence change. Phylogenetic analysis of both V1/V2 and C2/V3 regions showed evidence of recombination within but very little between the X4 and R5 lineages, suggesting these populations are genetically isolated.

    IMPORTANCE Primer ID sequencing provides a novel approach to study genetic structures of viral populations. X4 variants may be more prevalent than previously reported when assessed using next generation sequencing (NGS) and with greater depth of sampling than SGA. Phylogenetic analysis to identify lineages of sequences with intermediate FPR values may provide additional information for accurately predicting X4 variants using V3 sequences. Limited recombination occurs between X4 and R5 lineages, suggesting X4 and R5 variants are genetically isolated and may be replicating in different cell types, or that X4/R5 recombinants have reduced fitness.

  • A role of influenza exposure history in determining pandemic susceptibility and CD8+ T cell responses [PublishAheadOfPrint]

  • Novel influenza viruses often cause differential infection patterns across different age groups, defined as heterogeneous demographic susceptibility. This occurred during the A/H2N2 pandemic, when children experienced higher influenza attack rates than adults. As the recognition of conserved epitopes across influenza subtypes by CD8+ cytotoxic T lymphocytes (CTLs) limit influenza disease, we hypothesised that conservation of CTL antigenic peptides (Ag-p) in viruses circulating before the pH2N2-1957 may have resulted in differential CTL immunity. We compared viruses isolated in the years preceding the pandemic (1941nndash;1957) to which children and adults were exposed, with viruses circulating decades earlier (1918nndash;1940), which could infect adults only. Consistent with phylogenetic models, influenza viruses circulating 1941-1957, which infected children, shared with pH2N2 the majority (~89%) of CTL-peptides within the most immunogenic nucleoprotein, matrix-1 and polymerase basic-1, thus providing evidence for minimal pH2N2 CTL escape in children. Our study, however, identified potential CTL immune evasion from pH2N2 irrespective of age, within HLA-A*03:01+ individuals for PB1471-L473V/N476I variants and HLA-B*15:01+ population for NP404-414-V408I mutant. Further experiments using the murine model of B-cell-deficient mice showed that multiple influenza infections resulted in superior protection from influenza-induced morbidity, coinciding with accumulation of tissue resident memory CD8+ T-cells in the lung. Our study suggests that protection against H2N2-1957 pandemic influenza was most likely linked to the number of influenza virus infections prior to the pandemic challenge rather than differential pre-existing CTL immunity. Thus, the regimen of a CTL-based vaccine/vaccine-component may benefit from periodic boosting to achieve fully protective, asymptomatic influenza infection.

    IMPORTANCE Due to a lack of cross-reactive neutralizing antibodies, children are particularly susceptible to influenza infections caused by novel viral strains. Pre-existing T cell immunity directed at conserved viral regions, however, can provide protection against influenza viruses, promote rapid recovery and better clinical outcomes. When we asked whether high susceptibility of children (as compared to adults) to the pandemic H2N2 influenza strain was associated with immune evasion from T-cell immunity, we found high conservation within T-cell antigenic regions in pandemic H2N2. However, the number of influenza infections prior to the challenge was linked to protective, asymptomatic infections and establishment of tissue resident memory T cells. Our study supports development of vaccines that prime and boost T cells to elicit cross-strain protective T cells, especially tissue resident memory T cells, for life-long immunity against distinct influenza viruses.

  • MF59 adjuvant effects on inducing isotype-switched IgG antibodies and protection after immunization with T-dependent influenza virus vaccine in the absence of CD4+ T cells [PublishAheadOfPrint]

  • CD4+ T cells play a central role in orchestrating adaptive immunity. To better understand the roles of CD4+ T cells in the adjuvant effects, we investigated the efficacy of T-dependent influenza virus split vaccine with MF59rreg; or alum in CD4-knockout (CD4KO) and wild-type (WT) mice. CD4+ T cells were required for the induction of IgG antibody responses to split vaccine and alum adjuvant effects. In contrast, MF59rreg; was found to be highly effective in raising isotype-switched IgG antibodies to T-dependent influenza split vaccine in CD4KO mice or CD4-depleted WT mice, equivalent to those in intact WT mice, thus overcoming the deficiency of CD4+ T cells in helping B cells and inducing immunity against influenza virus. MF59-adjuvanted influenza split vaccination was able to induce protective CD8+ T cells and long-lived antibody-secreting cells in CD4KO mice. MF59 adjuvant effects in CD4KO mice might be associated with uric acid, inflammatory cytokines, and recruitment of multiple immune cells at the injection site, but their cellularity and phenotypes were different from those in WT mice. These findings suggest a new paradigm of CD4-independent adjuvant mechanisms, providing the rationales to improve vaccine efficacy in infants, elderly, and immune-compromised patients as well as in healthy adults.

    IMPORTANCE MF59-adjuvanted influenza vaccines were licensed for human vaccination, but the detailed mechanisms are not fully elucidated. CD4+ T cells are required to induce antibody isotype switching and long-term memory responses. In contrast, we discovered that MF59 was highly effective in inducing isotype-switched IgG antibodies and long-term protective immune responses to T-dependent influenza vaccine independent of CD4+ T cells. These findings are highly significant (1). MF59 can overcome a defect of CD4+ T cells in inducing protective immunity to T-dependent influenza split vaccination (2). A CD4-independent pathway can be an alternative mechanism for certain adjuvants such as MF59 (3). This study has significant implications for improving vaccine efficacies in young children, elderly, and immune-compromised populations.

  • Notwithstanding circumstantial alibis, cytotoxic T cells can be major killers of HIV-1 infected cells. [PublishAheadOfPrint]

  • Several experiments suggest that in the chronic phase of HIV-1 infection CD8+cytotoxic T lymphocytes (CTL) contribute very little to the death of productively infected cells. First, the expected life span of productively infected cells is fairly long, i.e., about one day. Second, this life span is hardly affected by the depletion of CD8+T cells. Third, the rate at which mutants escaping a CTL response take over the viral population tends to be slow. Our main result is that all these observations are perfectly compatible with killing rates that are much faster than one per day once we invoke the fact that infected cells proceed through an eclipse phase of about one day before they start producing virus. Assuming that the major protective effect of CTL is cytolytic, we demonstrate that mathematical models with an eclipse phase account for the data when the killing is fast, and when it varies over the life cycle of infected cells. Considering the steady state corresponding to the chronic phase of the infection, we find that the rate of immune escape, and the rate at which the viral load increases following CD8+T cell depletion, should reflect the viral replication rate, . A meta analysis of previous data shows that viral replication rates during chronic infection vary between 0.5lle;lle;1 day-1. Balancing such fast viral replication requires killing rates that are several times larger than , implying that most productively infected cells would die by cytolytic effects.

    Importance Most current data suggest that Cytotoxic T cells (CTL) mediate their control of HIV-1 infection by non-lytic mechanisms, i.e., data suggest that CTL hardly kill. This interpretation of these data has been based upon the general mathematical model for HIV infection. Because this model ignores the eclipse phase between the infection of a target cell and the start of viral production by that cell, we re-analyze the same data sets with novel models that do account for the eclipse phase. We find that the data are perfectly consistent with lytic control by CTL, and predict that most productively infected cells are killed by CTL. Because that the killing rate should balance the viral replication rate, we estimate both parameters from a large set of published experiments depleting CD8+T cells in SIV infected monkeys. This confirms that the killing rate can be much faster than is currently appreciated.

  • Visualization and sequencing of membrane remodeling leading to influenza virus fusion [PublishAheadOfPrint]

  • Protein-mediated membrane fusion is an essential step in many fundamental biological events including enveloped virus infection. The nature of protein and membrane intermediates and the sequence of membrane remodeling during these essential processes remain poorly understood. Here we used cryo-electron tomography (cryo-ET) to image the interplay between influenza virus and vesicles with a range of lipid compositions. By following the population kinetics of membrane fusion intermediates imaged by cryo-ET, we found that membrane remodeling commenced with the hemagglutinin fusion protein spikes grappling to the target membrane, followed by localized target membrane dimpling as local clusters of hemagglutinin started to undergo conformational refolding. The local dimples then transitioned to extended, tightly apposed contact zones where the two proximal membrane leaflets were in most cases indistinguishable from each other, suggesting significant dehydration and possible intermingling of the lipid head groups. Increasing the content of fusion-enhancing cholesterol or bis-monoacylglycerophosphate in the target membrane, led to an increase in extended contact zone formation. Interestingly, hemifused intermediates were found to be extremely rare in the influenza virus fusion system studied here, most likely reflecting the instability of this state and its rapid conversion to postfusion complexes, which increased in population over time. By tracking the populations of fusion complexes over time, the architecture and sequence of membrane reorganization leading to efficient enveloped virus fusion were thus resolved.

    IMPORTANCE Enveloped viruses employ specialized surface proteins to mediate fusion of cell and viral membranes resulting in the formation of pores through which the viral genetic material is transferred into the cell. In influenza virus, the trimeric hemagglutinin (HA) glycoprotein spike mediates host cell attachment and membrane fusion. While structures of a subset of conformations and parts of the fusion machinery have been characterized, the nature and sequence of membrane deformations during fusion have largely eluded characterization. Building upon studies that focused on early stages of HA-mediated membrane remodeling, here cryo-electron tomography (cryo-ET) was used to image the 3-dimensional organization of intact influenza virions at different stages of fusion with liposomes, leading all the way to completion of the fusion reaction. By monitoring the evolution of fusion intermediate populations over the course of acid-induced fusion, we identified the progression of membrane reorganization that leads to efficient fusion by an enveloped virus.

  • Targeted mutagenesis of guinea pig cytomegalovirus using CRISPR/Cas9-mediated gene editing [PublishAheadOfPrint]

  • The cytomegaloviruses are among the most genetically complex mammalian viruses, with viral genomes that often exceed 230 kilobase pairs. Manipulation of cytomegalovirus genomes is largely performed using infectious bacterial artificial chromosomes, which necessitates the maintenance of the viral genome in E. coli and successful reconstitution of virus from permissive cells after transfection of the BAC. Here we describe an alternative strategy for the mutagenesis of guinea pig cytomegalovirus that utilizes CRISPR/Cas9-mediated genome editing to introduce targeted mutations to the viral genome. Transient transfection and drug selection was used to restrict lytic replication of guinea pig cytomegalovirus to cells that express Cas9 and virus-specific guide RNA. The result was highly efficient editing of the viral genome that introduced targeted insertion or deletion mutations to nonessential viral genes. Cotransfection of multiple virus-specific guide RNAs or a homology repair template was used for targeted, markerless deletions of viral sequence or to introduce exogenous sequence by homology driven repair. As CRISPR-Cas9 mutagenesis occurs directly in infected cells, this methodology avoids selective pressures that may occur during propagation of the viral genome in bacteria and may facilitate the genetic manipulation of low-passage or clinical CMV isolates.

    IMPORTANCE The cytomegalovirus genome is complex and viral adaptations to cell culture have complicated the study of infection in vivo. Recombineering of viral bacterial artificial chromosomes enabled the study of recombinant cytomegaloviruses. Here we report the development of an alternative approach using CRISPR/Cas9-based mutagenesis in guinea pig cytomegalovirus, a small animal model of congenital cytomegalovirus disease. CRISPR/Cas9-mutagenesis can introduce the same types of mutations to the viral genome as bacterial artificial chromosome recombineering but does so directly in virally infected cells. CRISPR/Cas9 mutagenesis is not dependent on a bacterial intermediate, and defined viral mutants can be recovered after a limited number of viral genome replications, minimizing the risk of spontaneous mutation.

  • Dynein Regulators Are Important For Ecotropic Murine Leukemia Virus Infection [PublishAheadOfPrint]

  • During the early steps of infection, retroviruses must direct the movement of the viral genome into the nucleus to complete their replication cycle. This process is mediated by cellular proteins that interact first with the reverse transcription complex, and later with the preintegration complex (PIC), allowing it to reach and enter the nucleus. For simple retroviruses such as murine leukemia virus (MLV), the identity of the cellular proteins involved in trafficking of the PIC in infection is unknown. To identify cellular proteins that interact with MLV PIC, we developed a replication-competent MLV virus in which the integrase protein was tagged with a FLAG epitope. Using a combination of immunoprecipitation and mass spectrometry, we established that microtubule motor dynein regulator DCTN2/p50/dynamitin interacts with the MLV preintegration complex early in infection, suggesting a direct interaction between the incoming viral particles and the dynein complex regulators. Further experiments showed that RNAi mediated silencing of either DCTN2/p50/dynamitin or another dynein regulator, NudEL, profoundly reduced the efficiency of infection by ecotropic but not amphotropic MLV reporter viruses. We propose that the cytoplasmic dynein regulators are a critical component of the host machinery needed for infection by those retroviruses entering the cell via the ecotropic envelop pathway.

    IMPORTANCE Retroviruses must access the chromatin of the host cells to integrate the viral DNA, but before this crucial event, they must reach the nucleus. The movement through the cytoplasm -a crowded environment where diffusion is slow- is thought to utilize retrograde transport along the microtubule network by the dynein complex. Different viruses use different components of this multisubunit complex. We find that the preintegration complex of the murine leukemia virus (MLV) interacts with the dynein complex and that regulators of this complex are essential for infection. Our study provides the first insight into the requirements for retrograde transport of MLV preintegration complex.

  • Mosaic H5 hemagglutinin provides broad humoral and cellular immune responses against influenza viruses [PublishAheadOfPrint]

  • The most effective way to prevent influenza infection is via vaccination. However, the constant mutation of influenza viruses due to antigenic drift and shift compromises vaccine efficacy. This represents a major challenge to the development of a cross-protective vaccine that can protect against circulating viral antigenic diversity. Using the modified vaccinia Ankara (MVA) virus, we had previously generated a recombinant vaccine against highly pathogenic avian influenza (H5N1) based on an in silico mosaic approach. This MVA-H5M construct protected mice against multiple clades of H5N1 and H1N1 viruses. We have now further characterized the immune responses using immune-depletion of T cells and passive serum transfer, and these studies indicate that antibodies are the main contributors in homosubtypic protection (H5N1 clades). When compared with a MVA construct expressing HA from influenza A/VN/1203/04, MVA-HA), the MVA-H5M vaccine markedly increased and broadened B cell and T cell responses against H5N1 virus. The MVA-H5M also provided effective protection with no morbidity against H5N1 challenge; whereas, MVA-HA vaccinated mice showed clinical signs and experienced significant weight loss. In addition, MVA-H5M induced CD8+ T cell responses that play a major role in heterosubtypic protection (H1N1). Finally, expression of the H5M gene as either a DNA vaccine or subunit protein protected mice against H5N1 challenge, indicating the effectiveness of the mosaic sequence without viral vectors for the development of a universal influenza vaccine.

    IMPORTANCE Influenza viruses infect up to one billion people around the globe each year and are responsible for 300,000-500,000 deaths annually. Vaccines are still the main intervention to prevent infection but they fail to provide effective protection against heterologous strains of viruses. We developed broadly reactive H5N1 vaccine based on an in silico mosaic approach and previously demonstrated that modified vaccinia Ankara expressing an H5 mosaic hemagglutinin prevented infection with multiple clades of H5N1 and limited severe disease after H1N1 infection. Further characterization revealed that antibody responses and T cells are main contributors to protection against H5N1 and H1N1 viruses, respectively. The vaccine also broadens both T cell and B cell responses compared to native H5 vaccine from influenza A/Vietnam/1203/04. Finally, delivering the H5 mosaic as a DNA vaccine or as a purified protein demonstrated effective protection similar to the viral vector approach.

  • Measurements of intrahost viral diversity are extremely sensitive to systematic errors in variant calling [PublishAheadOfPrint]

  • With next generation sequencing technologies, it is now feasible to efficiently sequence patient-derived virus populations at a depth of coverage sufficient to detect rare variants. However, each sequencing platform has characteristic error profiles, and sample collection, target amplification, and library preparation are additional processes whereby errors are introduced and propagated. Many studies account for these errors by using ad hoc quality thresholds and/or previously published statistical algorithms. Despite common usage, the majority of these approaches have not been validated under conditions that characterize many studies of intrahost diversity. Here we use defined populations of influenza virus to mimic the diversity and titer typically found in patient-derived samples. We identified single nucleotide variants using two commonly used variant callers, DeepSNV and LoFreq. We found that the accuracy of these variant callers was lower than expected and exquisitely sensitive to input titer. Small reductions in specificity had a significant impact on the number of minority variants identified and subsequent measures of diversity. We were able to increase the specificity of DeepSNV to ggt;99.95% by applying an empirically validated set of quality thresholds. When applied to a set of influenza samples from a household-based cohort study, these changes resulted in a 10-fold reduction in measurements of viral diversity. We have made our sequence data and analysis code available so that others may improve on our work and use our dataset to benchmark their own bioinformatic pipelines. Our work demonstrates that inadequate quality control and validation can lead to significant overestimation of intrahost diversity.

    Importance Advances in sequencing technology have made it feasible to sequence patient-derived viral samples at a level sufficient for detection of rare mutations. These high-throughput, cost-effective methods are revolutionizing the study of within-host viral diversity. However, these techniques are error prone, and the methods commonly used to control for these errors have not been validated under the conditions that characterize patient-derived samples. Here we show that these conditions affect measurements of viral diversity. We found that the accuracy of previously benchmarked analysis pipelines were greatly reduced under patient-derived conditions. By carefully validating our sequencing analysis using known control samples, we were able to identify biases in our method and improve our accuracy to acceptable levels. Application of our modified pipeline to a set of influenza samples from a cohort study provide a realistic picture of intrahost diversity and suggest the need for rigorous quality control in such studies.

  • CD47 plays a role as a negative regulator in inducing protective immune responses to vaccination against influenza virus [PublishAheadOfPrint]

  • An integrin-associated protein CD47, which is a ligand for the inhibitory receptor signal regulatory protein aalpha;, is expressed on B and T cells as well as on most innate immune cells. However, the roles of CD47 in the immune responses to viral infection or vaccination remain unknown. We have investigated the role of CD47 in inducing humoral immune responses after intranasal infection with virus or immunization with influenza virus-like particles (VLP). Virus infection or vaccination with VLP containing hemagglutinin (HA) from A/PR8/34 influenza virus induced higher levels of antigen-specific IgG2c isotype dominant antibodies in CD47-deficient (CD47KO) mice than those in wild type (WT) mice. CD47KO mice with vaccination showed higher protective efficacy against lethal challenge, as evidenced by no loss in body weight and reduced lung viral titers compared to WT mice. In addition, inflammatory responses which include cytokine production, leukocyte infiltrates, and interferon gamma (IFN-)-producing CD4+ T cells as well as anti-inflammatory cytokine (IL-10) were reduced in the lung of vaccinated CD47KO mice after challenge with influenza virus. Analysis of lymphocytes indicated that GL7+ germinal center B cells were induced at higher levels in the draining lymph nodes of CD47KO mice as compared with those in WT mice. Notably, CD47KO mice exhibited significant increases in the numbers of antigen-specific memory B cells in spleens and plasma cells in bone marrow despite their lower levels of background IgG antibodies. Thus, these results suggest that CD47 plays a role as a negative regulator in inducing protective immune responses to influenza vaccination.

    IMPORTANCE Molecular mechanisms that control B cell activation to produce protective antibodies upon viral vaccination remain poorly understood. The CD47 molecule is known to be a ligand for the inhibitory receptor signal regulatory protein aalpha; and expressed on the surfaces of most immune cell types. CD47 was previously demonstrated to play an important role in modulating the migration of monocytes, neutrophils, polymorphonuclear neutrophils, and dendritic cells into the inflamed tissues. Results in this study demonstrate new roles of CD47 in negatively regulating the induction of protective IgG antibodies, germinal center B cells, and plasma cells secreting antigen-specific antibodies as well as macrophages upon influenza vaccination and challenge. As consequences, vaccinated CD47 deficient mice resulted in better control of influenza viral infection and enhanced protection. This study provides insights into understanding the regulatory functions of CD47 in inducing adaptive immunity to vaccination.

  • Small-molecule fusion inhibitors bind the pH-sensing SSP-GP2 subunit interface of the Lassa virus envelope glycoprotein [PublishAheadOfPrint]

  • Arenavirus species are responsible for severe life-threatening hemorrhagic fevers in western Africa and South America. Without effective antiviral therapies or vaccines, these viruses pose serious public health and biodefense concerns. Chemically distinct small-molecule inhibitors of arenavirus entry have recently been identified and shown to act on the arenavirus envelope glycoprotein (GPC) to prevent membrane fusion. In the tripartite GPC complex, pH-dependent membrane fusion is triggered through a poorly understood interaction between the stable signal peptide (SSP) and the transmembrane fusion subunit GP2, and our genetic studies have suggested that these small-molecule inhibitors act at this interface to antagonize fusion activation. Here, we have designed and synthesized photoaffinity derivatives of the 4-acyl-1,6-dialkylpiperazin-2-one class of fusion inhibitors and demonstrate specific labeling of both the SSP and GP2 subunits in a native-like Lassa virus (LASV) GPC trimer expressed in insect cells. Photoaddition is competed by the parental inhibitor and other chemically distinct compounds active against LASV, but not those specific to New World arenaviruses. These studies provide direct physical evidence that these inhibitors bind at the SSP-GP2 interface. We also find that GPC containing the uncleaved GP1-GP2 precursor is not susceptible to photocrosslinking, suggesting that proteolytic maturation is accompanied by conformational changes at this site. Detailed mapping of residues modified by the photoaffinity adducts may provide insight to guide the further development of these promising lead compounds as potential therapeutic agents to treat Lassa hemorrhagic fever.

    IMPORTANCE Hemorrhagic fever arenaviruses cause lethal infections in humans and, in the absence of licensed vaccines or specific antiviral therapies, are recognized to pose significant threats to public health and biodefense. Lead small-molecule inhibitors that target the arenavirus envelope glycoprotein (GPC) have recently been identified and shown to block GPC-mediated fusion of the viral and cellular endosomal membranes, thereby preventing virus entry into the host cell. Genetic studies suggest that these inhibitors act through a unique pH-sensing intersubunit interface in GPC, but atomic-level structural information is unavailable. In this report, we utilize novel photoreactive fusion inhibitors and photoaffinity labeling to obtain direct physical evidence for inhibitor binding at this critical interface in Lassa virus GPC. Future identification of modified residues at the inhibitor-binding site will help elucidate the molecular basis for fusion activation and its inhibition, and guide the development of effective therapies to treat arenaviral hemorrhagic fevers.

  • Both Cis- and Trans- Activities of the Foot-and-Mouth Disease Virus 3D Polymerase are Essential for Viral RNA Replication. [PublishAheadOfPrint]

  • The Picornaviridae is a large family of positive-sense RNA viruses that contains numerous human and animal pathogens, including foot-and-mouth disease virus (FMDV). The picornavirus replication complex comprises a co-ordinated network of protein-protein and protein-RNA interactions involving multiple viral and host-cellular factors. Many of the proteins within the complex possess multiple roles in viral RNA replication, some of which can be provided in trans (i.e. via expression from a separate RNA molecule), whilst other are required in cis (i.e. expressed from the template RNA molecule). In vitro studies have suggested that multiple copies of the RNA-dependent RNA-polymerase (RdRp), 3D, are involved in the viral replication complex. However, it is not clear whether all these molecules are catalytically active or what other function(s) they provide. In this study, we aimed to distinguish between catalytically-active 3D molecules and those which build a replication complex. We report a novel non-enzymatic cis-acting function of 3D that is essential for viral genome replication. Using a FMDV replicon in complementation experiments, our data demonstrate that this cis-acting role of 3D is distinct from the catalytic activity, which is predominantly trans-acting. Immunofluorescence studies suggest that both cis- and trans-acting 3D molecules localise to the same cellular compartment. However, our genetic and structural data suggest that 3D interacts in cis with RNA stem-loops that are essential for viral RNA replication. Together, this study identifies a previously undescribed aspect of picornavirus replication complex structure-function and an important methodology for probing such interactions further.

    IMPORTANCE Foot-and-mouth disease virus (FMDV) is an important animal pathogen responsible for foot-and-mouth disease. The disease is endemic in many parts of the world with outbreaks within livestock resulting in major economic losses. Propagation of the viral genome occurs within replication complexes and understanding this process can facilitate the development of novel therapeutic strategies. Many of the non-structural proteins involved in replication possess multiple functions in the viral life-cycle, some of which can be supplied to the replication complex from a separate genome (i.e. in trans) whilst other must originate from the template (i.e. in cis). Here, we present an analysis of cis- and trans-activities of the RNA-dependent RNA-polymerase, 3D. We demonstrate a novel cis-acting role of 3D in replication. Our data suggest that this role is distinct from its enzymatic functions and requires interaction with the viral genome. Our data furthers the understanding of genome replication of this important pathogen.

  • Transformation by Polyomavirus Middle T Antigen Involves A Unique Bimodal Interaction with the Hippo Effector YAP [PublishAheadOfPrint]

  • Murine polyomavirus has repeatedly provided insights into tumorigenesis, revealing such key control mechanisms as tyrosine phosphorylation and PI3K signaling. We recently demonstrated that polyomavirus ST antigen binds YAP, a major effector of Hippo signaling, to regulate differentiation. Here we characterize YAP as a target of MT antigen important for transformation. Through a surface including residues R103 and D182, wild-type MT binds to the YAP WW domains. Mutation of either R103 or D182 of MT abrogates YAP binding without affecting binding to other signaling molecules or the strength of PI3K or Ras signaling. Either genetic abrogation of YAP binding to MT or silencing of YAP via shRNA reduced MT transformation, suggesting that YAP makes a positive contribution to the transformed phenotype. MT targets YAP both by activating signaling pathways that affect it and by binding to it. MT signaling, whether from wild-type or YAP-binding mutant MT, promoted YAP phosphorylation at S127 and S381/397(YAP2/YAP1). Consistent with the known functions of these phosphorylated serines, MT signaling leads to loss of YAP from the nucleus and degradation. Binding of YAP to MT brings it together with PP2A leading to YAP's dephosphorylation in the MT complex. It also leads to YAP's enrichment in membranes. Taken together, these results indicate that YAP promotes MT transformation via mechanisms that may depart from YAP's canonical oncogenic transcriptional activation functions.

    IMPORTANCE The highly conserved Hippo/YAP pathway is important for tissue development and homeostasis. Increasingly, changes in this pathway are being associated with cancer. Middle T (MT) is the primary polyomavirus oncogene responsible for tumor formation. In this study we show that MT signaling promotes YAP phosphorylation, loss from the nucleus and increased turn-over. Notably MT genetics demonstrate that YAP binding to MT is important for transformation. Because MT also binds PP2A, the YAP bound to MT is dephosphorylated, stabilized and localized to membranes. Taken together, these results indicate that YAP promotes MT transformation via mechanisms that depart from YAP's canonical oncogenic transcriptional activation functions.

  • Identification of diverse mycoviruses through metatranscriptomics characterization of the viromes of five major fungal plant pathogens [PublishAheadOfPrint]

  • Mycoviruses can have a marked effect on natural fungal communities and influence plant health and productivity. However, a comprehensive picture of mycoviral diversity is still lacking. To characterize the viromes of five widely dispersed plant pathogenic fungi, Colletotrichum truncatum, Macrophomina phaseolina, Diaporthe longicolla, Rhizoctonia solani, and Sclerotinia sclerotiorum, a high-throughput sequencing-based metatranscriptomic approach was used to detect viral sequences. Total RNA and double-stranded RNA (dsRNA) from mycelia and RNA from samples enriched for virus particles were sequenced. Sequence data were assembled de novo, and contigs with predicted amino acid sequence similarities to viruses in the non-redundant protein database were selected. The analysis identified 72 partial or complete genome segments representing 66 previously undescribed mycoviruses. Using primers specific for each viral contig, at least one fungal isolate was identified that contained each virus. The novel mycoviruses showed affinity with 15 distinct lineages: Barnaviridae, Benyviridae, Chrysoviridae, Endornaviridae, Fusariviridae, Hypoviridae, Mononegavirales, Narnaviridae, Ophioviridae, Ourmiavirus, Partitiviridae, Tombusviridae, Totiviridae, Tymoviridae, and Virgaviridae. More than half of the viral sequences were predicted to be members of the Mitovirus genus in the family Narnaviridae, which replicate within mitochondria. Five viral sequences showed strong affinity with three families (Benyviridae, Ophioviridae, and Virgaviridae) that previously contained no mycovirus species. The genomic information provides insight into the diversity and taxonomy of mycoviruses and coevolution of mycoviruses and their fungal hosts.

    IMPORTANCE Plant pathogenic fungi reduce crop yields, which affects food security worldwide. Plant-host resistance is considered a sustainable disease management option, but may often be incomplete or lacking for some crops to certain fungal pathogens or strains. In addition, the rising issues of fungicide resistance demand alternative strategies to reduce the negative impacts of fungal pathogens. Those fungus-infecting viruses (mycoviruses) that attenuate fungal virulence maybe welcome additions for mitigating plant diseases. By high throughput sequencing of the RNAs from 275 isolates of five fungal plant pathogens, 66 previously undescribed mycoviruses were identified. In addition to identifying new potential biological control agents, these results expand the grand view of the diversity of mycoviruses and provide possible insights into the importance of intracellular and extracellular transmission in fungi-virus coevolution.

  • ISG12a restricts hepatitis C virus infection through ubiquitination-dependent degradation pathway [PublishAheadOfPrint]

  • Interferons (IFNs) restrict various kinds of viral infection via inducing hundreds of IFN-stimulated genes (ISGs), while the functions of the majority of ISGs are broadly unclear. Here, we show that a high IFN-inducible gene ISG12a exhibits non-apoptotic antiviral effect to HCV infection. Viral NS5A protein is targeted specifically by ISG12a that mediates NS5A degradation via ubiquitination-dependent proteasomal pathway. K374R mutation in NS5A domain III abrogates ISG12a-induced ubiquitination and degradation of NS5A. SKP2 is identified as an ubiquitin E3 ligase for NS5A. ISG12a functions as a crucial adaptor that promotes SKP2 to interact with and degrade viral protein. Moreover, the antiviral effect of ISG12a is dependent on the E3 ligase activity of SKP2. These findings uncover an intriguing mechanism by which ISG12a restricts viral infection and provide clues for understanding the actions of innate immunity.

    IMPORTANCE Upon virus invasion, IFNs induce numerous ISGs to control viral spread, while the functions of the majority of ISGs are broadly unclear. The study showed a novel antiviral mechanism of ISGs and elucidated that ISG12a recruits an E3 ligase SKP2 for ubiquitination and degradation of viral protein and restricts viral infection. These findings provide important insights into exploring the working principles of innate immunity.

  • A novel open and infectious form of Echovirus 1 [PublishAheadOfPrint]

  • One of the hallmarks of enterovirus genome delivery is the formation of an uncoating intermediate particle. Based on earlier studies of mostly heated picornavirus particles, intermediate particles were shown to have externalized the innermost capsid protein (VP4) and exposed the N-terminus of VP1 and to have reduced infectivity. Here, in addition to the native and intact particle type, we have identified another type of infectious Echovirus 1 (E1) particle population during infection. Our results show that E1 is slightly altered during entry, which leads to the broadening of the major virion peak in the sucrose gradient. In contrast, CsCl gradient separation revealed that in addition to the light intact and empty particles, a dense particle peak appeared during infection in cells. When the broad peak from the sucrose gradient was subjected to CsCl gradient, it revealed light and dense particles, further suggesting that the shoulder represents the dense particle. The dense particle was permeable to SYBR Green II, it still contained most of its VP4, and it was able to bind to its receptor aalpha;2bbeta;1 integrin and showed high infectivity. A thermal assay further showed that the aalpha;2bbeta;1 integrin binding domain, I-domain, stabilized the virus particle. Finally, heating E1 particles to super-physiological temperatures produced more fragile particles with aberrant ultrastructural appearances, suggesting that they are distinct from the dense E1 particles. These results describe a more open and highly infectious E1 particle that is naturally produced during infection and may represent a novel form of an uncoating intermediate.

    IMPORTANCE In this paper, we have characterized a possible uncoating intermediate particle of E1 that is produced in cells during infection. Before releasing their genome into the host cytosol, enteroviruses go through structural changes in their capsid, forming an uncoating intermediate particle. It has been shown earlier that structural changes can be induced by receptor interactions and, in addition, by heating the native virion to super-physiological temperatures. Here, we demonstrate that an altered, still infectious E1 particle is found during infection. This particle has a more open structure and it cannot be formed by heating. It still contains VP4 protein and is able to bind to its receptor and cause infection. Moreover, we show that in contrast to some other enteroviruses, the receptor-virion interaction has a stabilizing effect on E1. This paper highlights the differences between enterovirus species and further increases our understanding of various uncoating forms of enteroviruses.

  • Functional incompatibility between the generic NF-{kappa}B motif and a subtype-specific Sp1III element drives the formation of HIV-1 subtype C viral promoter [PublishAheadOfPrint]

  • Of the various genetic subtypes of HIV-1, HIV-2 and SIV, only in subtype C of HIV-1, a genetically variant NF-B binding site is found at the core of the viral promoter in association with a subtype-specific Sp1III motif. How the subtype-associated variations in the core transcription factor binding sites (TFBS) influence gene expression from the viral promoter has not been examined previously. Using panels of infectious viral molecular clones, we demonstrate that subtype-specific NF-B and Sp1III motifs have evolved for optimal gene expression, and neither of the motifs can be substituted by a corresponding TFBS variant.The variant NF-B motif binds NF-B with an affinity two-fold higher than that of the generic NF-B site. Importantly, in the context of an infectious virus, the subtype-specific Sp1III motif demonstrates a profound loss of function in association with the generic NF-B motif. An additional substitution of the Sp1III motif fully restores viral replication suggesting that the subtype C specific Sp1III has evolved to function with the variant, but not generic, NF-B motif. A change of only two base pairs in the central NF-B motif completely suppresses viral transcription from the provirus and converts the promoter into heterochromatin refractory to TNF-aalpha; induction. The present work represents the first demonstration of functional incompatibility between an otherwise functional NF-B motif and a unique Sp1 site in the context of HIV-1 promoter. Our work provides important leads as per the evolution of HIV-1 subtype C viral promoter with relevance for gene expression regulation and viral latency.

    Importance: Subtype-specific genetic variations provide a powerful tool to examine how these variations offer a replication advantage to specific viral subtypes if any. Only in subtype C of HIV-1, two genetically distinct transcription factor binding sites are positioned at the most critical location of the viral promoter. Since a single promoter regulates viral gene expression, the promoter variations can play a critical role in determining the replication fitness of the viral strains. Our work for the first time provides a scientific explanation for the presence of a unique NF-B binding motif in subtype C, a major HIV-1 genetic family responsible for half of the global HIV-1 infections. The results offer compelling evidence that subtype C viral promoter is not only stronger but also is endowed with a qualitative-gain-of-function advantage. The genetically variant NF-B, and the Sp1III motifs may be responsive to specific cell signal-pathways differentially, and these mechanisms must be examined.

  • CD8+ T cell breadth and ex vivo virus inhibition capacity distinguish between viremic controllers with and without protective HLA class I alleles [PublishAheadOfPrint]

  • The mechanisms of viral control and loss of viral control in chronically infected individuals with or without protective HLA class I alleles are not fully understood. We therefore characterized longitudinally the immunological and virological features that may explain divergence in disease outcome in 70 HIV-1 C-clade infected antiretroviral therapy (ART)-naïve South African adults, 35 of whom possessed protective HLA class I alleles. We demonstrate that, over five years of longitudinal study, 35% of individuals with protective HLA class I alleles lost viral control compared to none of individuals without protective HLA class I alleles (p=0.06). Sustained HIV-1 control in patients with protective HLA class I alleles was characteristically related to the breadth of HIV-1 CD8+ T-cell responses against Gag and enhanced ability of CD8+ T cells to suppress viral replication ex vivo. In some cases loss of virological control was associated with reduction in the total breadth of CD8+ T cell responses in the absence of differences in HIV-1-specific CD8+ T cell polyfunctionality or proliferation. In contrast, viremic controllers without protective HLA class I alleles possessed low breadth of HIV-1-specific CD8+ T cell responses characterized by reduced ability to suppress viral replication ex vivo. These data suggest that the control of HIV-1 in individuals with protective HLA class I alleles may be driven by broad CD8+ T cell responses with potent viral inhibitory capacity while control among individuals without protective HLA class I alleles may be more durable and mediated by CD8+ T cell independent mechanisms.

    Importance Host mechanisms of natural HIV-1 control are not fully understood. In a longitudinal study of antiretroviral therapy (ART)-naïve individuals, we show that those with protective HLA-I alleles subsequently experienced virologic failure compared to those without protective alleles. Among individuals with protective HLA-I alleles, viremic control was associated with broad CD8+ T cells that targeted the Gag protein, and CD8+ T cells from these individuals exhibited superior virus inhibition capacity. In individuals without protective HLA-I alleles, HIV-1-specific CD8+ T cell responses were narrow and poorly inhibited virus replication. These results suggest that broad, highly functional cytotoxic T cells (CTL) against the HIV-1 Gag protein are associated with control among those with protective HLA-I alleles and that loss of these responses eventually leads to viremia. A subset of individuals appears to have alternative, non-CTL mechanisms of viral control. These controllers may hold the key to an effective HIV vaccine.

  • Cell-to-Cell Contact and Nectin-4 Govern Spread of Measles Virus from Primary Human Myeloid Cells to Primary Human Airway Epithelial Cells [PublishAheadOfPrint]

  • Measles is a highly contagious, acute viral illness. Immune cells within the airways are likely first targets of infection and these cells traffic measles virus (MeV) to lymph nodes for amplification and subsequent systemic dissemination. Infected immune cells are thought to return MeV back to the airways; however, the mechanisms responsible for virus transfer to pulmonary epithelial cells are poorly understood. To investigate this process, we collected blood from human donors and generated primary myeloid cells, specifically monocyte-derived macrophages (MDMs) and dendritic cells (DCs). MDMs and DCs were infected with MeV and then applied to primary cultures of well-differentiated airway epithelial cells from human donors (HAE). Consistent with previous results using free virus, infected MDMs or DCs were incapable of transferring MeV to HAE when applied to the apical surface. Likewise, infected MDMs or DCs applied to the basolateral surface of HAE grown on small pore (0.4 mmu;m) support membranes did not transfer virus. In contrast, infected MDMs and DCs applied to the basolateral surface of HAE grown on large pore (3.0 mmu;m) membranes successfully transferred MeV. Confocal microscopy demonstrated that MDMs and DCs are capable of penetrating large pore membranes but not small pore membranes. Further, by using a nectin-4 blocking antibody or recombinant MeV unable to enter cells through nectin-4, we demonstrated formally that transfer from immune cells to HAE occurs in a nectin-4 dependent manner. Thus both infected MDMs and DCs rely on cell-to-cell contacts and nectin-4 to efficiently deliver MeV to the basolateral surface of HAE.

    IMPORTANCE Measles virus spreads rapidly and efficiently in human airway epithelial cells. This rapid spread is based on cell-cell contact rather than on particle release and re-entry. Here we posit that MeV transfer from infected immune cells to epithelial cells also occurs by cell-cell contact rather than through cell-free particles. In addition, we seek to determine which immune cells transfer MeV infectivity to the human airway epithelium. Our studies are based on two types of human primary cells: 1) myeloid cells generated from donated blood and 2) well-differentiated airway epithelial cells derived from donor lungs. We show that different types of myeloid cells, i.e. monocyte-derived macrophages and dendritic cells, transfer infection to airway epithelial cells. Furthermore, cell-to-cell contact is an important component of successful MeV transfer. Our studies elucidate a mechanism by which the most contagious human respiratory virus is delivered to the airway epithelium.

  • HIV frameshift site RNA stability correlates with frameshift efficiency and decreased viral infectivity [PublishAheadOfPrint]

  • HIV replication is strongly dependent upon a programmed ribosomal frameshift. Here we investigate the relationship between the thermodynamic stability of the HIV-1 RNA frameshift site stem-loop, frameshift efficiency and infectivity using pseudotyped HIV-1 and HEK293T cells. The data reveal a strong correlation between frameshift efficiency and local, but not overall, RNA thermodynamic stability. Mutations that modestly increase the local stability of the frameshift site RNA stem-loop structure increase frameshift efficiency 2-3 fold in cells. Thus, frameshift efficiency is determined by the strength of the thermodynamic barrier encountered by the ribosome. These data agree with previous in vitro measurements, suggesting there are no viral or host-specific factors that modulate frameshifting. The data also indicate that there are no sequence-specific requirements for the frameshift site stem-loop. A linear correlation between Gag-Pol levels in cells and in virions support a stochastic virion assembly mechanism. We further demonstrate that the surrounding genomic RNA secondary structure influences frameshift efficiency, and that a mutation that commonly arises in response to protease inhibitor therapy creates a functional, but inefficient secondary slippery site. Finally, HIV-1 mutants with enhanced frameshift efficiencies are significantly less infectious, suggesting that compounds that increase frameshift efficiency by as little as 2-fold may be effective at suppressing HIV-1 replication.

    IMPORTANCE HIV, like many retroviruses, utilizes a -1 programmed ribosomal frameshift to generate viral enzymes in the form of a Gag-Pol polyprotein precursor. Thus, frameshifting is essential for viral replication. Here, we utilize a panel of mutant HIV to demonstrate that in cells, frameshifting efficiency is correlated with the stability of the local thermodynamic barrier to ribosomal translocation. Increasing the stability of the frameshift site RNA increases the frameshift efficiency 2-3 fold. Mutant viruses with increased frameshift efficiencies have significantly reduced infectivity. These data suggest this effect might be exploited in the development of novel anti-viral strategies.

  • Uukuniemi virus as a tick-borne virus model [PublishAheadOfPrint]

  • In the last decade, novel tick-borne pathogenic phleboviruses in the family Bunyaviridae, all closely related to Uukuniemi virus (UUKV), have emerged in different continents. To reproduce the tick-mammal switch in vitro, we first established a reverse genetics system to rescue UUKV with a genome the closest to that of the authentic virus isolated from the Ixodes ricinus tick reservoir. The IRE/CTVM19 and IRE/CTVM20 cell lines, both derived from I. ricinus, were susceptible to the virus rescued from plasmid DNAs and supported production of the virus over many weeks, indicating that infection is persistent. The glycoprotein GC was mainly highly-mannosylated on tick cell-derived viral progeny. The second envelope viral protein, GN, carried mostly N-glycans not recognized by the classical glycosidases PNGase F and Endo H. Treatment with bbeta;-mercaptoethanol did not impact the apparent molecular weight of GN. On viruses originating from mammalian BHK-21 cells, GN glycosylations were exclusively sensitive to PNGase F and the electrophoresis mobility of the protein was substantially slower after the reduction of disulfide bonds. Furthermore, the amount of viral nucleoprotein per foci forming units differed markedly whether viruses were produced in tick or BHK-21 cells, suggesting a higher infectivity for tick cell-derived viruses. Together, our results indicate that UUKV particles derived from vector tick cells have glycosylation and structural specificities that may influence the initial infection in mammalian hosts. This study also highlights the importance of working with viruses originating from arthropod vector cells when investigating the cell biology of arbovirus transmission and entry into mammalian hosts.

    Importance Tick-borne phleboviruses represent a growing threat to humans globally. Although ticks are important vectors of infectious emerging diseases, previous studies have mainly involved virus stocks produced in mammalian cells. This limitation tends to minimize the importance of host alternation in virus transmission to humans and initial infection at the molecular level. With this study, we have developed an in vitro tick cell-based model that allows production of the tick-borne Uukuniemi virus to high titers. Using this system, we found that virions derived from tick cells have specific structural properties and N-glycans that may enhance virus infectivity for mammalian cells. By shedding light on molecular aspects of tick-derived viral particles, our data illustrate the importance of considering the host switch in studying early virus-mammalian receptor/cell interactions. The information gained here lays the basis for future research, not only on tick-borne phleboviruses, but on all viruses and other pathogens transmitted by ticks.

  • Bat SARS-like coronavirus WIV1 encodes an extra accessory protein ORFX involved in modulation of host immune response [PublishAheadOfPrint]

  • Bats harbor severe acute respiratory syndrome-like coronaviruses (SL-CoVs) from which the causative agent of the 2002-3 SARS pandemic is thought to have originated. However, despite a large number of genetically diverse SL-CoV sequences detected in bats, only two strains (named WIV1 and WIV16) have been successfully cultured in vitro. These two strains differ from SARS-CoV only in containing an extra ORF (named ORFX) between ORF6 and ORF7 with no homology to any known protein sequences. In this study, we constructed a full-length cDNA clone of SL-CoV WIV1 (rWIV1), an ORFX deletion mutant (rWIV1-X), and a GFP-expressing mutant (rWIV1-GFP-X). Northern blot and fluorescent microscopy indicate that ORFX was expressed during WIV1 infection. Virus infection assay showed that rWIV1-X replicated as efficiently as rWIV1 in Vero E6, Calu-3, and HeLa-hACE2 cells. Further study showed that ORFX could inhibit interferon production and activates NF-B. Our results demonstrate for the first time that the unique ORFX in the WIV1 strain is a functional gene involving modulation of host immune response, but not essential for in vitro viral replication.

    Importance Bats harbor genetically diverse SARS-like coronaviruses (SL-CoVs) and some of them have the potential of interspecies transmission. A unique open reading frame (ORFX) was identified in the genome of two recently isolated bat SL-CoV strains (WIV1 aamp; 16). It will therefore be critical to clarify whether and how this protein would contribute to virulence during viral infection. Here we revealed that the unique ORFX is a functional gene involving in the modulation of host immune response, but not essential for in vitro viral replication. Our results provide important information for further exploration of the ORFX function in the future. Moreover, the reverse genetics system we constructed will be helpful for pathogenesis study of this group of viruses and develop therapeutics for future control of emerging SARS-like infections.

  • p53 is a Host Cell Regulator during Herpes Simplex Encephalitis [PublishAheadOfPrint]

  • p53 is a critical host cell factor in the cellular response to a broad range of stress factors. We recently reported that p53 is required for efficient herpes simplex virus 1 (HSV-1) replication in cell culture. However, a defined role for p53 in HSV-1 replication and pathogenesis in vivo remains elusive. In this study, we examined the effects of p53 on HSV-1 infection in vivo using p53-deficient mice. Following intracranial inoculation, p53 knock-out reduced viral replication in the brains of mice and led to significantly reduced HSV-1 mortality due to encephalitis. These results suggest that p53 is an important host cell regulator for HSV-1 replication and pathogenesis in the central nervous system (CNS).

    IMPORTANCE HSV-1 causes sporadic encephalitis, which, even with antiviral therapy, can result in severe neurological defects and even death. Many host cell factors involved in the regulation of CNS HSV-1 infection have been investigated using genetically modified mice. However, most of these factors are immunological regulators and act via immunological pathways in order to restrict CNS HSV-1 infection. They therefore provide limited information on intrinsic host cell regulators that may be involved in the facilitation of CNS HSV-1 infection. Here we demonstrate that a host cell protein, p53, which has generally been considered to be a host cell restriction factor for various viral infections, is required for efficient HSV-1 replication and pathogenesis in the CNS of mice. This is the first report showing that p53 positively regulates viral replication and pathogenesis in vivo and provides insights into its molecular mechanism that may suggest novel clinical treatment options for HSV-1 encephalitis.

  • MxA mediates SUMO-induced resistance to Vesicular Stomatitis Virus [PublishAheadOfPrint]

  • Multiple cellular pathways are regulated by small ubiquitin like modifier (SUMO) modification, including ubiquitin-mediated proteolysis, signal transduction, innate immunity and antiviral defense. In this report, we investigated the effects of SUMO on the replication of two members of the Rhabdoviridae family, Vesicular stomatitis virus (VSV) and rabies virus (RABV). We show that stable expression of SUMO in human cells confers resistance to VSV infection in an interferon-independent manner. We demonstrate that SUMO expression did not alter VSV entry but blocked primary mRNA synthesis leading to a reduction of viral protein synthesis and viral production, thus protecting cells from VSV-induced cell lysis. MxA is known to inhibit VSV primary transcription. Interestingly, we found that the MxA protein was highly stabilized in SUMO-expressing cells. Furthermore, extracts from cells stably expressing SUMO exhibited an increase in MxA oligomers suggesting that SUMO plays a role in protecting MxA from degradation, thus providing a stable intracellular pool of MxA available to combat invading viruses. Importantly, MxA depletion in SUMO-expressing cells abrogated the anti-VSV effect of SUMO. Furthermore, SUMO expression results in IRF3 SUMOylation, subsequently decreasing the RABV-induced IRF3 phosphorylation and interferon synthesis. As expected, this renders SUMO-expressing cells more sensitive to RABV infection even though MxA was stabilized in SUMO-expressing cells, since its expression did not confer resistance to RABV. Our findings demonstrate opposing effects of SUMO expression on two viruses of the same family, intrinsically inhibiting VSV infection through MxA stabilization while enhancing RABV infection by decreasing IFN induction.

    IMPORTANCE We report that SUMO expression reduces interferon synthesis upon RABV or VSV infection. Therefore, SUMO renders cells more sensitive to RABV but unexpectedly renders cells resistant to VSV by blocking primary mRNA synthesis. Unlike the interferon-mediated innate immune response, intrinsic antiviral resistance is mediated by constitutively expressed restriction factors. Among the various anti-VSV restriction factors, only MxA is known to inhibit VSV primary transcription and we show here that its expression does not alter RABV infection. Interestingly, MxA depletion abolished the inhibition of VSV by SUMO, demonstrating that MxA mediates SUMO-induced intrinsic VSV resistance. Furthermore, MxA oligomerization is known to be critical for its protein stability and we show that higher levels of oligomers were formed in cells expressing SUMO compared to wild-type cells, suggesting that SUMO may play a role in protecting MxA from degradation, providing a stable intracellular pool of MxA able to protect cells from viral infection.

  • Identification and Characterization of a Ribose 2-O-Methyltransferase Encoded by the Ronivirus Branch of Nidovirales [PublishAheadOfPrint]

  • The order Nidovirales currently comprises four virus families: Arteriviridae, Coronaviridae (divided into the subfamilies Coronavirinae and Torovirinae), Roniviridae and the recently recognized Mesoniviridae. RNA cap formation and methylation have been best studied for coronaviruses, with emphasis on the identification and characterization of two virus-encoded methyltransferases (MTases) involved in RNA capping, a guanine-N7-MTase and a ribose-2rrsquo; -O-MTase. Although bioinformatic analyses suggest that these MTases may also be encoded by other nidoviruses with large genomes, such as toroviruses and roniviruses, no experimental evidence has been reported thus far. In this study, we show that a ronivirus, gill-associated nidovirus (GAV), encodes the 2rrsquo; -O-MTase activity, although we could not detect 2rrsquo; -O-MTase activity for the homologous protein of a torovirus, equine torovirus, which is more closely related to coronaviruses. Like the coronavirus 2rrsquo; -O-MTase, the roniviral 2rrsquo; -O-MTase harbors a catalytic K-D-K-E tetrad that is conserved among 2rrsquo; -O-MTases and can only target the N7-methylated cap structure of adenylate-primed RNA substrates. However, in contrast with coronavirus, roniviral 2rrsquo; -O-MTase does not require a protein cofactor for stimulation of its activity, and differs in its preference for several biochemical parameters such as reaction temperature and pH. Furthermore, the ronivirus 2rrsquo; -O-MTase can be targeted by MTase inhibitors. These results extend our current understanding of nidovirus RNA cap formation and methylation beyond the coronavirus family.

    IMPORTANCE: Methylation of the 5rrsquo; -cap structure of viral RNAs plays important roles in genome replication and evasion of innate recognition of viral RNAs by cellular sensors. It is known that coronavirus nsp14 acts as an N7-(guanine)-methyltransferase (MTase) and nsp16 as 2rrsquo; -O-MTase, which are involved in the modification of RNA cap structure. However, these enzymatic activities have not been shown for any other nidoviruses beyond coronaviruses in the order Nidovirales. In this study, we identified a 2rrsquo; -O-methyltransferase encoded by ronivirus and it shows common and unique features in comparison with that of coronaviruses. Ronivirus 2rrsquo; -O-MTase does not need a protein co-factor for MTase activity whereas coronavirus nsp16 needs the stimulating factor nsp10 for its full activity. The conserved K-D-K-E catalytic tetrad is identified in ronivirus 2rrsquo; -O-MTase. These results extend our understanding of nidovirus RNA capping and methylation beyond coronaviruses and also strengthen the evolutionary and functional link between roniviruses and coronaviruses.

  • Complete unique genome sequence, expression profile and salivary gland tissue tropism of the herpesvirus-7 homolog in pigtailed macaques [PublishAheadOfPrint]

  • Human herpesviruses HHV-6A, 6B and 7 are classified as roseoloviruses, and are highly prevalent in the human population. Roseolovirus reactivation in an immunocompromised host can cause severe pathologies. While the pathogenic potential of HHV-7 is unclear, it can reactivate HHV-6 from latency and thus contributes to severe pathological conditions associated with HHV-6. Because of the ubiquitous nature of roseoloviruses, their roles in such interactions and resulting pathological consequences have been difficult to study. Furthermore, the lack of a relevant animal model for HHV-7 infection has hindered a better understanding of its contribution to roseolovirus associated diseases.

    Using Next-Gen sequence analysis, we have characterized the unique genome of an uncultured novel pig-tailed macaque roseolovirus. Detailed genomic analysis revealed the presence of gene homologs to all 84 known HHV-7 ORFs. Phylogenetic analysis confirmed that the virus is a macaque homolog of HHV-7, which we have provisionally named MneHV7.

    Using high-throughput RNA sequencing, we observed that the salivary gland tissue samples from nine different macaques had distinct MneHV7 gene expression patterns and that the overall number of viral transcripts correlated with viral loads in parotide tissue and saliva. Immunohistochemistry staining confirmed that like HHV-7, MneHV7 exhibits natural tropism for salivary gland ductal cells. We also observed staining for MneHV7 in peripheral nerve ganglia present in salivary gland tissues suggesting that HHV-7 may also have a tropism for the peripheral nervous system.

    Our data demonstrate that MneHV7-infected macaques represent a relevant animal model that may help clarify the causality between roseolovirus reactivation and diseases.

    Importance Human herpesviruses HHV-6A, 6B and 7 are classified as Roseoloviruses. We have recently discovered that pigtailed macaques are naturally infected with viral homologs of HHV-6 and HHV-7 which we provisionally named MneHV6 and MneHV7, respectively. In this study we confirm that MneHV7 is genetically and biologically similar to its human counterpart HHV-7. We determine the complete unique MneHV7 genome sequence and provide a comprehensive annotation of all genes. We also characterize viral transcription profiles in salivary glands from naturally infected macaques. We show that broad transcriptional activity across most of the viral genome is associated with high viral loads in infected parotids and that late viral protein expression is detected in salivary duct cells and peripheral nerve ganglia. Our study provides new insights into the natural behavior of an extremely prevalent virus and establishes a basis for subsequent investigations of the mechanisms that cause HHV-7 reactivation and associated disease.

  • Requirement of HSP90 chaperoning in addition to phosphoprotein for folding but not for supporting enzymatic activities of measles and Nipah virus L polymerases [PublishAheadOfPrint]

  • Non-segmented negative-stranded RNA viruses, or Mononegavirales, share a conserved gene order and the use of elaborate transcription and replication machinery made up of at least four molecular partners. These partners have co-evolved with the acquisition of the permanent encapsidation of the entire genome by the nucleoprotein (N) and the use of this N-RNA complex as a template for the viral polymerase composed of the phosphoprotein (P) and the large enzymatic protein (L). Not only is P required for polymerase function, but it also stabilizes L protein, through an unknown underlying molecular mechanism. By using NVP-AUY922 and/or 17-DMAG as specific inhibitors of the cellular heat shock protein 90 (HSP90), we report that the efficient chaperoning of L by HSP90 requires P in the case of measles, Nipah and vesicular stomatitis viruses. While the production of P remains unchanged in the presence of HSP90 inhibitors, the production of soluble and functional L requires both P and HSP90 activity. Measles virus P can bind the N-terminus of L in the absence of HSP90 activity. Both HSP90 and P are required for the folding of L as evidenced by a luciferase reporting insert fused within measles virus L. HSP90 acts as a true chaperon: its activity is transient and dispensable for the activity of measles and Nipah polymerases of virion origin. That the cellular chaperoning of a viral polymerase into a soluble functional enzyme requires the assistance of another viral protein constitutes a new paradigm that seems to be conserved within the Mononegavirales order.

    IMPORTANCE Viruses are obligate intracellular parasites that require a cellular environment for their replication. Some viruses particularly depend on the cellular chaperoning apparatus. We report here that for measles virus the successful chaperoning of the viral L polymerase mediated by heat shock protein 90 (HSP90) requires the presence of the viral phosphoprotein (P). Indeed, whilst P protein binds to the N-terminus of L independently of HSP90 activity, both HSP90 and P are required to produce stable, soluble, folded and functional L proteins. Once formed, the mature P+L complex no longer requires HSP90 to exert its polymerase functions. Such a new paradigm for the maturation of a viral polymerase appears to be conserved for several members of the Mononegavirales order including Nipah and vesicular stomatitis virus.

  • HIV-1 Gag, Envelope, and extracellular determinants cooperate to regulate the stability and turnover of virological synapses [PublishAheadOfPrint]

  • Retroviruses spread more efficiently when infected and uninfected cells form tight, physical interfaces known as virological synapses (VSs). VS formation is initiated by adhesive interactions between viral Envelope (Env) glycoproteins on the infected cell and CD4 receptor molecules on the uninfected cell. How high avidity Env-CD4 linkages are resolved over time is unknown. Here, we describe a tractable two-color, long-term (ggt;24 hr) live cell imaging strategy to study VS turnover in the context of a large cell population, quantitatively. We show that Env's conserved cytoplasmic tail (CT) can potently signal the recruitment of Gag capsid proteins to the VS, a process also dependent on residues within Gag's N-terminal matrix (MA) domain. Additionally, we demonstrate that Env's CT and Gag's MA domain both regulate the duration of interactions between viral donor and target cells, as well as the stability of this interaction over time (i.e., its capacity to resolve or form a syncytium). Finally, we report the unexpected finding that modulating extracellular fluid viscosity markedly impacts target T cell trafficking and thus affects the duration, stability, and turnover of virus-induced cell-cell contacts. Combined, these results suggest a stepwise model for viral cell-to-cell transmission wherein 1) Env-receptor interactions anchor target cells to infected cells, 2) Env signals Gag's recruitment to the cell-cell contact dependent on an intact Env CT and Gag MA, and 3) Env CT and Gag MA, in conjunction with extracellular forces, combine to regulate VS stability and infectious outcomes.

    IMPORTANCE HIV-1 spreads efficiently at physical, cell-cell interfaces known as virological synapses. The VS provides for spatiotemporal coupling of virus assembly and entry into new host cells and may transmit signals relevant to pathogenesis. Disrupting this mode of transmission may be critical to the goal of abolishing viral persistence in infected individuals. Here we describe a long-term live cell imaging strategy for studying virus-induced effects on cell behavior in the context of a large cell population. We demonstrate cooperative roles for viral Gag capsid proteins and Envelope glycoproteins in regulating VS formation and turnover. We also show that modulating fluid viscosity markedly affects T cell trafficking and VS stability. Thus, extracellular factors also play an important role in modulating the nature of infectious cell-cell interactions. In sum, our study provides new tools and insights relevant to exposing vulnerabilities in how HIV-1 and other viruses spread infection among cells, tissues, and people.

  • Arteriviruses, pegiviruses, and lentiviruses are common among wild African monkeys [PublishAheadOfPrint]

  • Non-human primates (NHPs) are a historically important source of zoonotic viruses and are a gold-standard model for research on many human pathogens. However, with the exception of simian immunodeficiency virus (SIV, family Retroviridae), the blood-borne viruses harbored by these animals in the wild remain incompletely characterized. Here, we report the discovery and characterization of two novel simian pegiviruses (family Flaviviridae) and two novel simian arteriviruses (family Arteriviridae) in wild African green monkeys from Zambia (malbroucks, Chlorocebus cynosuros) and South Africa (vervet monkeys, Chlorocebus pygerythrus). We examine several aspects of infection including viral load, genetic diversity, evolution, geographic distribution, as well as host factors such as age, sex, and plasma cytokines. In combination with previous efforts to characterize blood-borne RNA viruses in wild primates across sub-Saharan Africa, these discoveries demonstrate that in addition to SIV, simian pegiviruses and simian arteriviruses are widespread and prevalent among many African cercopithecoid (i.e., Old World) monkeys.

    IMPORTANCE Primates are an important source of viruses that infect humans and serve as an important laboratory model of human virus infection. Here, we discover two new viruses in African green monkeys from Zambia and South Africa. In combination with previous virus discovery efforts, this finding suggests that these virus types are widespread among African monkeys. Our analysis suggests that one of these virus types, the simian arteriviruses, may have the potential to jump between different primate species and cause disease. In contrast, the other virus type, the pegiviruses, are thought to reduce the disease caused by HIV in humans. However, we did not observe a similar protective effect in SIV-infected African monkeys co-infected with pegiviruses, possibly because SIV causes little-to-no disease in these hosts.

  • Both CD8+ and CD4+ T-Cells Contribute to Corneal Clouding and Viral Clearance Following Vaccinia Virus Infection in C57BL/6 Mice [PublishAheadOfPrint]

  • Vaccinia virus (VACV) keratitis is a serious complication following Smallpox vaccination and can lead to blindness. Pathologic mechanisms involved in ocular VACV infection are poorly understood. Previous studies have used rabbits, but the lack of immune reagents and transgenic or knockout animals makes them less suitable for mechanistic studies. We report that infection of C57BL/6 mice with 1x107 plaque forming units (PFU) of Vaccinia virus strain WR results in blepharitis, corneal neovascularization, and stromal keratitis. The DryVax strain of VACV was completely attenuated. Infection required corneal scarification and replication-competent virus and the severity of ocular disease was similar in 4- to 6-week-old and 1-year-old mice. Viral titers peaked on day 5 post-infection at approximately 1x106 PFU and virus had not cleared by day 13 post-infection. Neutrophils were found in the peripheral cornea on day 1 after infection and then declined, followed by infiltration of both CD4+ and CD8+ T-cells that remained peripheral throughout the infection. Blood vessel growth extended 2-5 mm into the cornea from the limbus. Infection of CD4-/- or CD8-/, or antibody depleted mice resulted in very similar disease severity and corneal clouding indicating that both T-cell subsets were involved in the immunopathological response. Depletion of both CD4+ and CD8+ T-cells resulted in significantly more severe disease and failure to clear virus. Based on our results, the pathology of VACV keratitis is significantly different from Herpes simplex virus keratitis. Further studies are likely to reveal novel information regarding virulence and immune responses to viral ocular infection.

    Importance Potentially blinding eye infections can occur after vaccination for Smallpox. Very little is known about the pathologic mechanisms that are involved and the information that is available was generated using rabbit models. The lack of immunological reagents for rabbits makes such studies difficult. We have characterized a mouse model of vaccinia virus ocular disease using C57BL/6 mice and strain WR and show that both CD4+ and CD8+ T-cell subsets play a role in the blinding eye disease and controlling virus replication. Based on these results vaccinia virus keratitis is significantly different from Herpes simplex virus keratitis and further studies using this model should generate novel insights into immunopathological responses to viral ocular infection.

  • HIV-1 Vpu Antagonizes CD317/tetherin by Adaptor protein-1-Mediated Exclusion from Virus Assembly Sites [PublishAheadOfPrint]

  • The host cell restriction factor CD317/tetherin traps virions at the surface of producer cells to prevent their release. The HIV-1 accessory protein Vpu antagonizes this restriction. Vpu reduces the cell surface density of the restriction factor and targets it for degradation; however, these activities are dispensable for enhancing particle release. Instead, Vpu has been suggested to antagonize CD317/tetherin by preventing recycling of internalized CD317/tetherin to the cell surface, blocking anterograde transport of newly synthesized CD317/tetherin, and/or displacing the restriction factor from virus assembly sites at the plasma membrane. On the molecular level, antagonism relies on the physical interaction of Vpu with CD317/tetherin. Recent findings suggested that phosphorylation of a di-serine motif enables Vpu to bind to adaptor protein 1 (AP-1) trafficking complexes via two independent interaction motifs and to couple CD317/tetherin to the endocytic machinery. Here we used a panel of Vpu proteins with specific mutations in individual interaction motifs to define which interactions are required for antagonism of CD317/tetherin. Impairing recycling or anterograde transport of CD317/tetherin to the plasma membrane was insufficient for antagonism. In contrast, excluding CD317/tetherin from HIV-1 assembly sites depended on Vpu motifs for interaction with AP-1 and CD317/tetherin and correlated with antagonism of the particle release restriction. Consistently, interference with AP-1 function or its expression blocked these Vpu activities. Our results define displacement from HIV-1 assembly sites as active principle of CD317/tetherin antagonism by Vpu and support a role of tripartite complexes between Vpu, AP-1 and CD317/tetherin in this process.

    Importance CD317/tetherin poses an intrinsic barrier to human immunodeficiency virus (HIV-1) replication in human cells by trapping virus particles at the surface of producer cells and thereby preventing their release. The viral protein Vpu antagonizes this restriction, and molecular interactions with the restriction factor and adaptor protein complex 1 (AP-1) were suggested to mediate this activity. Vpu modulates intracellular trafficking of CD317/tetherin and excludes the restriction factor from HIV-1 assembly sites at the plasma membrane, but the relative contribution of these effects to antagonism remain elusive. Using a panel of Vpu mutants as well as interference with AP-1 function and expression, we show here that Vpu antagonizes CD317/tetherin by blocking its recruitment to viral assembly sites in an AP-1-dependent manner. These results refine our understanding of the molecular mechanisms of CD317/tetherin antagonism and suggest complexes of Vpu with the restriction factor and AP-1 as targets for potential therapeutic intervention.

  • Initiation of antiretroviral therapy restores CD4+ TSCM homeostasis in SIV-infected macaques [PublishAheadOfPrint]

  • Treatment of human immunodeficiency virus (HIV) infection with antiretroviral therapy (ART) has significantly improved prognosis. Unfortunately, interruption of ART almost invariably results in viral rebound, attributed to a pool of long-lived, latently-infected cells. Based on their longevity and proliferative potential, CD4+ Tmemory stem cells (TSCM) have been proposed as an important site of HIV persistence. In a previous study, we found that, in simian immunodeficiency virus (SIV)-infected rhesus macaques (RM), CD4+ TSCM are preserved in number but show (i) a decrease in the frequency of CCR5+ cells, (ii) an expansion of the fraction of proliferating Ki-67+ cells, and (iii) high levels of SIV-DNA. To understand the impact of ART on both CD4+ TSCM homeostasis and virus persistence, we conducted a longitudinal analysis of these cells in the blood and lymph nodes of twenty-five SIV-infected RM. We found that ART induced a significant restoration of CD4+CCR5+ TSCM in both blood and lymph nodes, and a reduction in the fraction of proliferating CD4+Ki-67+ TSCM in blood (but not lymph nodes). Importantly, we found that the level of SIV-DNA in CD4+ transitional memory (TTM-) and effector memory (TEM-) T cells declined ~100-fold after ART in both blood and lymph nodes, while the level of SIV-DNA in CD4+ TSCM and central memory T cells (TCM-) did not significantly change. These data suggest that ART is effective at partially restoring CD4+ TSCM homeostasis and the observed stable level of virus in TSCM supports the hypothesis that these cells are a critical contributor to SIV persistence.

    IMPORTANCE: Understanding the role of various CD4+ T cell memory subsets in immune homeostasis and HIV/SIV persistence during antiretroviral therapy (ART) is critical to effectively treat and cure HIV infection. T memory stem cells (TSCM) are a unique memory T cell subset with enhanced self-renewal capacity and the ability to differentiate into other memory T cell subsets, such as central and transitional memory T cells (TCM and TTM, respectively). CD4+ TSCM are disrupted, but not depleted during pathogenic SIV infection. We find ART is partially effective at restoring CD4+ TSCM homeostasis and that SIV-DNA harbored within this subset contracts more slowly compared to virus harbored in shorter-lived subsets, such as TTM and effector memory (TEM). Because of their ability to persist long-term in an individual, understanding the dynamics of virally infected CD4+ TSCM during suppressive ART is important for future therapeutic interventions aimed at modulating immune activation and purging the HIV reservoir.

  • Identification of a functionally relevant AAV Rep68 oligomeric interface [PublishAheadOfPrint]

  • The life cycle of the human parvovirus adeno-associated virus (AAV) is orchestrated by four Rep proteins. The large Rep proteins Rep78 and Rep68 are remarkably multi-functional and display a range of biochemical activities, including DNA binding, nicking and unwinding. Functionally, Rep78 and Rep68 are involved in transcriptional regulation, DNA replication and genomic integration. Structurally, the Rep proteins share a AAA+ domain characteristic of the SF3 family of helicases, with the large Rep proteins additionally containing an N-terminal origin-binding domain (OBD) domain that specifically binds and nicks DNA. The combination of these domains, coupled with dynamic oligomerization properties, is the basis for the remarkable multifunctionality displayed by Rep68 and Rep78 during the AAV life cycle. In this report we describe an oligomeric interface formed by Rep68 and demonstrate how disrupting this interface has drastic effects both on the oligomerization and functionality of the Rep proteins. Our results support a role for the four-helical bundle in the helicase domain of Rep68 as a bona fide oligomerization domain (OD). We have identified key residues in the OD that are critical for the stabilization of the Rep68-Rep68 interface; mutation of these key residues disrupts the enzymatic activities of Rep68, including DNA binding and nicking, and compromises viral DNA replication and transcriptional regulation of the viral promoters. Taken together, our data contribute to our understanding of the dynamic and substrate-responsive Rep78/68 oligomerization that is instrumental in the regulation of the DNA transitions that take place during the AAV life cycle.

    Importance The limited genome size of small viruses has driven the evolution of highly multifunctional proteins that integrate different domains and enzymatic activities within a single polypeptide. The Rep68 protein from adeno-associated virus (AAV) combines a DNA binding and endonuclease domain with a helicase-ATPase domain, which together support DNA replication, transcriptional regulation and site-specific integration. The coordination of the enzymatic activities of Rep68 remains poorly understood, however Rep68 oligomerization and Rep68-DNA interactions have been suggested to play a crucial role. We investigated the determinants of Rep68 oligomerization and identified a hydrophobic interface necessary for Rep68 activity during the AAV life cycle. Our results provide new insights into the molecular mechanisms underlying the regulation of the versatile Rep proteins. Efficient production of AAV-based gene therapy vectors requires optimal Rep expression levels, and studies such as the one presented here could contribute to further optimization of AAV production schemes.

  • Bile Acids Act as Soluble Host Restriction Factors Limiting Cytomegalovirus Replication in Hepatocytes [PublishAheadOfPrint]

  • The liver constitutes a prime site of cytomegalovirus (CMV) replication and latency. Hepatocytes produce, secrete and recycle a chemically diverse set of bile acids with the result that interactions between bile acids and cytomegalovirus inevitably occur. Here we determined the impact of naturally occurring bile acids on mouse CMV (MCMV) replication. In primary mouse hepatocytes, physiological concentrations of taurochenodeoxycholic acid (TCDC) glycochenodeoxycholic acid and to a lesser extent taurocholic acid significantly reduced MCMV-induced gene expression and diminished the generation of virus progeny, while several other bile acids did not exert antiviral effects. The anti-cytomegalovirus activity required active import of bile acids via sodium-taurocholate cotransporting polypeptide (NTCP) and was consistently observed in hepatocytes but not in fibroblasts. Under conditions in which IFN-aalpha; lacks antiviral activity, physiological TCDC concentrations were similarly effective as interferon (IFN)-. A detailed investigation of distinct steps of the viral life cycle revealed that TCDC deregulates viral transcription and diminishes global translation in infected cells.

    Importance Cytomegaloviruses are members of bbeta;-herpesvirinae. Primary infection leads to latency from which cytomegaloviruses can reactivate under immunocompromised conditions and cause severe disease manifestations including hepatitis. The present study describes an unanticipated antiviral activity of conjugated bile acids on MCMV replication in hepatocytes. Bile acids negatively influence viral transcription and exhibit a global effect on translation. Our data identify bile acids as site-specific soluble host restriction factors against MCMV, which may allow rational design of anticytomegalovirus drugs using bile acids as lead compounds.

  • Structural and biochemical analyses of swine MHC class I complexes and prediction of the epitope map in important influenza A strains [PublishAheadOfPrint]

  • The lack of a peptide-swine leukocyte antigen class I (pSLA I) complex structure presents difficulties for the study of swine cytotoxic T lymphocyte (CTL) immunity and molecule vaccine development to eliminate important swine viral diseases, such as influenza A virus (IAV). Here, after cloning and comparing of 28 SLA I allelic genes from Chinese Heishan pigs, pSLA-3*hs0202 was crystalized and solved. The SLA-3*hs0202 binding with sbbeta;2m and a KMNTQFTAV (HA-KMN9) peptide from the 2009 pandemic swine H1N1 strain clearly displayed two distinct conformations with HA-KMN9 peptides in the structures, which are believed to be beneficial to stimulate a broad spectrum of CTL immune response. Notably, we found that different HA-KMN9 conformations are caused not only by the flexibility of the side chains of residues in the peptide-binding groove (PBG) but also by the skewing of aalpha;1 and aalpha;2 helixes forming the PBG. In addition, alanine scanning and CD spectra confirmed that the B, D and F pockets play critical biochemical roles in determining the peptide-binding motif of SLA-3*hs0202. Based on biochemical parameters and comparisons to similar pockets in other known major histocompatibility complex class I (MHC I) structures, the fundamental motif for SLA-3*hs0202 was determined as llsquo;X-(M/A/R)-(N/Q/R/F)-X-X-X-X-X-(V/I)rrsquo; by refolding in vitro and multiple mutant peptides. Finally, 28 SLA-3*hs0202-restricted epitope candidates were identified from important IAV strains, and two of them have been found in humans as HLA-A*0201-specific IAV epitopes. Structural and biochemical illumination of pSLA-3*hs0202 can benefit vaccine development to control IAV in swine.

    IMPORTANCE We crystalized and solved the first SLA-3 structure, SLA-3*hs0202, and found that it could present the same IAV peptide with two distinct conformations. Unlike previously findings showing that variable peptide conformations are only caused by the flexibility of the side chains in the groove, the skewing of the aalpha;1 and aalpha;2 helixes is important in the different peptide conformations in SLA-3*hs0202. We also determined the fundamental motif for SLA-3*hs0202 as llsquo;X-(M/A/R)-(N/Q/R/F)-X-X-X-X-X-(V/I)rrsquo; based on a series of structural and biochemical analyses, and 28 SLA-3*hs0202-restricted epitope candidates were identified from important IAV strains. We believe our structure and analyses of pSLA-3*hs0202 can benefit vaccine development to control IAV in swine.

  • Association between HA stem-reactive antibodies and influenza A/H1N1 infection during the 2009 pandemic [PublishAheadOfPrint]

  • The discovery of influenza broadly neutralizing antibodies prompted efforts to develop universal vaccines. Influenza virus stem-reactive (SR), broadly-neutralizing (BrN) antibodies have been detected by screening antibody phage display libraries. However, studies of SR-BrN antibodies in human serum, and their association with natural infection, are limited. To address this, pre- and post-pandemic sera from a prospective community cohort study in Viet Nam were assessed for antibodies that inhibit SR-BrN mAb (C179) binding to H1N1 pandemic 2009 virus (H1N1pdm09). Of 270 households, 33 with at least one confirmed H1N1pdm09 illness, or at least two seroconverters were included. The included households comprised 71 infected and 41 non-infected participants. Sera were tested as two-fold dilutions between 1:5 and 1:40. 50% C179 inhibition (IC50) titers did not exceed 10, although both IC50 titers, and % C179 inhibition by sera diluted 1:5 or 1:10, correlated with HI and MN titers (all p llt; 0.001). Thirteen (12%) participants had detectable pre-pandemic IC50 titers but only one reached a titer of 10. This proportion increased to 44% after the pandemic when 39 participants had a titer of 10, and 67% of infected compared to 44% of non-infected had detectable IC50 titers (p llt; 0.001). The low levels of SR-reactive antibodies in pre-pandemic sera were not associated with subsequent H1N1pdm09 infection (p = 0.241), and the higher levels induced by H1N1pdm09 infection returned to pre-pandemic levels within two years. The findings indicate that natural infection induces only low titers of SR antibodies that are not sustained.

    Importance Universal influenza vaccines could have substantial health and economic benefits. The focus of universal vaccine research has been to induce antibodies that prevent infection by diverse influenza strains. These so-called broadly-neutralizing antibodies are readily detected in mice and ferrets after infection with a series of distinct influenza strains. The 2009 H1N1 pandemic provided an opportunity to investigate whether infection with a novel strain induced broadly-neutralizing antibodies in humans. We found that broadly neutralizing antibodies were induced, but levels were low and poorly maintained. This could represent an obstacle for universal vaccine development and warrants further investigation.

  • Identification and validation of small molecules that enhance recombinant Adeno-associated virus transduction following high throughput screen [PublishAheadOfPrint]

  • While the recent success of AAV-mediated gene therapy in clinical trials is promising, challenges still face the widespread applicability of recombinant AAV(rAAV). A major goal is to enhance the transduction efficiency of vectors in order to achieve therapeutic levels of gene expression at a vector dose that is below the immunological response threshold. In an attempt to identify novel compounds that enhance rAAV transduction, we performed two high-throughput screens comprising 2396 compounds. We identified 13 compounds that were capable of enhancing transduction, 12 of which demonstrated vector-specific effects and one that could also enhance vector-independent transgene expression. Many of these compounds shared similar properties and could be categorized into five groups: Epipodophyllotoxins (group 1), inducers of DNA damage (group 2), effectors of epigenetic modification (group 3), anthracyclines (group 4), and proteasome inhibitors (group 5). We optimized dosing for the identified compounds in several immortalized human cell lines as well as normal diploid cells. We found that the group 1 epipodophyllotoxins (teniposide and etoposide) consistently produced the greatest transduction enhancement. We also explored transduction enhancement among single-stranded, self-complementary, and fragment vectors and found that the compounds could impact fragmented rAAV2 transduction to an even greater extent than single-stranded vectors. In vivo analysis of rAAV2 and all of the clinically-relevant compounds revealed that, consistent with our in vitro results, teniposide exhibited the greatest level of transduction enhancement. Finally, we explored the capability of teniposide to enhance transduction of fragment vectors in vivo using an AAV8 capsid, which is known to exhibit robust liver tropism. Consistent with our in vitro results, teniposide co-administration greatly enhanced fragmented rAAV8 transduction at 48h and 8 days. This study provides a foundation based on rAAV small molecule screen methodology which is ideally studied for more diverse libraries of compounds that can be tested for potentiating rAAV transduction.

    IMPORTANCE This study seeks to enhance the ability of adeno-associated viral vectors for therapeutic gene delivery applicable to the treatment of diverse diseases. To do this, a comprehensive panel of FDA-approved drugs was tested in human cells and in animal models to determine if they increased adeno-associated virus gene delivery. The results demonstrate that particular groups of drugs enhance adeno-associated virus gene delivery by unknown mechanisms. In particular, the enhancement of gene delivery was approximately 50-100 times better using teniposide, which is also used as chemotherapy for cancer. Collectively, these results highlight the potential for FDA-approved drug enhancement of adeno-associated virus gene therapy which could result in a safer and effective treatments for diverse acquired or genetic diseases.

  • In vivo analysis of infectivity, fusogenicity, and incorporation of a mutagenic viral glycoprotein library reveals determinants for viral incorporation. [PublishAheadOfPrint]

  • Enveloped viruses utilize transmembrane surface glycoproteins to gain entry into target cells. Glycoproteins from diverse viral families can be incorporated into non-native viral particles in a process termed pseudotyping, however, the molecular mechanisms governing acquisition of these glycoproteins are poorly understood. For Murine Leukemia Virus envelope (MLV Env) glycoprotein, incorporation into foreign viral particles has been shown to be an active process, but it does not appear to be caused by direct interactions among viral proteins. Here, we coupled in vivo selection systems with Illumina next-generation sequencing (NGS) to test hundreds-of-thousands of MLV Env mutants for their ability to be enriched in viral particles and to perform other glycoprotein functions. NGS analyses on a subset of these mutants predicted that the residues important for incorporation are in the MPER, particularly W127 and W137, and the residues in the membrane spanning domain (MSD) and also immediately flanking it (T140-L163). These predictions were validated by directly measuring the impact of mutations in these regions on fusogenicity, infectivity, and incorporation. We suggest that these two regions dictate pseudotyping through interactions with specific lipid environments formed during viral assembly.

    IMPORTANCE Researchers from numerous fields routinely exploit the ability to manipulate viral tropism by swapping viral surface proteins. However, this process, termed pseudotyping, is poorly understood at the molecular level. For Murine Leukemia Virus envelope (MLV Env) glycoprotein, incorporation into foreign viral particles is an active process, but it does not appear to occur through direct viral protein-protein interactions. In this study, we tested hundreds-of-thousands of MLV Env mutants for their ability to be enriched in viral particles as well as perform other glycoprotein functions. Our analyses on a subset of these mutants predict that the glycoprotein regions embedded in and immediately flanking the viral membrane dictate active incorporation into viral particles. We suggest pseudotyping occurs through specific lipid-protein interactions at the viral assembly site.

  • Functional Upregulation of the DNA Cytosine Deaminase APOBEC3B by Polyomaviruses [PublishAheadOfPrint]

  • The APOBEC3 family of DNA cytosine deaminases has important roles in innate immunity and cancer. It is unclear how DNA tumor viruses regulate these enzymes and how these interactions, in turn, impact the integrity of both the viral and cellular genomes. Polyomavirus (PyVs) are small DNA viral pathogens that contain oncogenic potentials. In this study we examine the effects of PyV infection on APOBEC3 expression and activity. We demonstrate that APOBEC3B is specifically upregulated by BK polyomavirus (BKPyV) infection in primary kidney cells and that the upregulated enzyme is active. We further show that the BKPyV large T antigen, as well as large T antigens from related polyomaviruses, is alone capable of upregulating APOBEC3B expression and activity. Furthermore, we assess the impact of A3B on productive BKPyV infection and viral genome evolution. Although the specific knockdown of APOBEC3B has little short-term effect on productive BKPyV infection, our informatics analyses indicate that the preferred target sequences of APOBEC3B are depleted in BKPyV genomes and that this motif underrepresentation is enriched on the non-transcribed stand of the viral genome, which is also the lagging strand during viral DNA replication. Our results suggest that PyV infection upregulates APOBEC3B activity to influence virus sequence composition over longer evolutionary periods. These findings also imply that the increased activity of APOBEC3B may contribute to PyV-mediated tumorigenesis.

    IMPORTANCE Polyomaviruses (PyVs) are a group of emerging pathogens that can cause severe diseases including cancers in immunosuppressed individuals. Here we describe the findings that PyV infection specifically induces the innate immune DNA cytosine deaminase, APOBEC3B. The induced APOBEC3B enzyme is fully functional and therefore may exert mutational effects on both viral and host cell DNA. Consistent with this idea, we provide bioinformatic evidence that BK polyomavirus genomes are depleted for APOBEC3B-preferred target motifs and enriched for the corresponding predicted reaction products. These data imply that the interplay between PyV infection and APOBEC proteins may have significant impact on both viral evolution and virus-induced tumorigenesis.

  • Feline Calicivirus infection disrupts the assembly of cytoplasmic stress granules and induces G3BP1 cleavage [PublishAheadOfPrint]

  • In response to stress such as virus infection, cells can stall translation by storing mRNAs away in cellular compartments called stress granules (SGs). This defence mechanism favours cell survival by limiting the use of energy and nutrients until the stress is resolved. In some cases it may also block viral propagation as viruses are dependent on the host cell resources to produce viral proteins. Human norovirus is a member of the Caliciviridae family responsible for gastroenteritis outbreaks worldwide. Previous studies on caliciviruses have identified mechanisms by which they can usurp the host translational machinery, using the viral protein genome-linked VPg, or regulate host protein synthesis through the MAPK pathway. Herein we examined the effect of feline calicivirus (FCV) infection on SGs accumulation. We show that FCV infection impairs the assembly of SGs despite an increased phosphorylation of eukaryotic initiation factor eIF2aalpha;, a hallmark of stress pathway activation. Furthermore SGs did not accumulate in FCV-infected cells that are stressed with arsenite or hydrogen peroxide. FCV infection resulted in the cleavage of the SG-nucleating protein Ras-GTPase activating SH3 domain-binding protein (G3BP1), which is mediated by the viral 3C-like proteinase NS6Pro. Using mutational analysis, we identified the FCV-induced cleavage site within G3BP1, which differs from the poliovirus 3C proteinase cleavage site previously identified. Finally, we showed that NS6Pro-mediated G3BP1 cleavage impairs SGs assembly. In contrast, murine norovirus (MNV) infection did not impact arsenite-induced SG assembly or G3BP1 integrity suggesting that related caliciviruses have distinct effects on the stress response pathway.

    IMPORTANCE Human noroviruses are a major cause of viral gastroenteritis, and it is important to understand how they interact with the infected host cell. Feline calicivirus (FCV) and murine norovirus (MNV) are used as models to understand norovirus biology. Recent studies have suggested that the assembly of stress granules is central in orchestrating stress and antiviral responses to restrict viral replication. Overall, our study provides the first insight on how caliciviruses impair stress granules assembly by targeting the nucleating factor G3BP1 via the viral proteinase NS6Pro. This work provides new insights into host-pathogen interactions that regulate stress pathways during FCV infection.

  • Suppression of Kaposi's sarcoma-associated herpesvirus infection and replication by 5 AMP-activated protein kinase [PublishAheadOfPrint]

  • The host intracellular antiviral restriction factors inhibit viral infection and replication. The 5rrsquo; AMP-activated protein kinase (AMPK) is a cellular energy sensor regulating metabolic homeostasis. Activated AMPK inhibits the replication of numerous RNA viruses but enhances the entry of vaccinia virus. However, the role of AMPK in herpesvirus infection is unclear. In this study, we showed that the constitutive AMPK activity restricted Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication in primary human umbilical vein endothelial cells while KSHV infection did not markedly affect the endogenous AMPK activity. Knockdown of the AMPKaalpha;1 considerably enhanced the expression of viral lytic genes and the production of infectious virions while overexpression of a constitutively active AMPK had the opposite effects. Accordingly, an AMPK inhibitor Compound C augmented viral lytic gene expressions and virion productions but an AMPK agonist AICAR suppressed both. Furthermore, a common diabetes drug metformin that carries an AMPK agonistic activity drastically inhibited the expression of viral lytic genes and the production of infectious virions, suggesting metformin as a therapeutic agent for KSHV infection and replication. Together, these results identify the host AMPK as a KSHV restriction factor that can serve as a potential therapeutic target.

    IMPORTANCE Host cells encode specific proteins to restrict viral infection and replication. Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus associated with several cancers. In this study, we have identified 5rrsquo; AMP-activated protein kinase (AMPK), a cellular energy sensor, as a restriction factor of KSHV lytic replication during primary infection. Activation of AMPK suppresses while inhibition of AMPK enhances KSHV lytic replication by regulating the expression of viral genes. AICAR and metformin, both of which are AMPK agonists currently used in clinics for the treatment of conditions associated with metabolic disorders, inhibit KSHV lytic replication. Thus, our works has identified AMPK as a potential therapeutic target, and AICAR and metformin as potential therapeutic agents for KSHV-associated cancers.

  • HCV-induced up-regulation of miR-146a-5p in hepatocytes promotes viral infection and deregulates metabolic pathways associated with liver disease pathogenesis [PublishAheadOfPrint]

  • Hepatitis C virus (HCV)-induced chronic liver disease is a leading cause of hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying HCC development following chronic HCV infection remain poorly understood. MicroRNAs (miRNAs) play an important role in homeostasis within the liver and deregulation of the miRNome has been associated with liver disease including HCC. While host miRNAs are essential for HCV replication, viral infection in turn appears to induce alterations of intrahepatic miRNA networks. Although the cross-talk between HCV and liver cell miRNAs most likely contributes to liver disease pathogenesis, the functional involvement of miRNAs in HCV-driven hepatocyte injury and HCC remains elusive. Here, we combined a hepatocyte-like based model system, high-throughput small RNA-sequencing, computational analysis and functional studies to investigate HCV-miRNA interactions that may contribute to liver disease and HCC. Profiling analyses indicated that HCV infection differentially regulated the expression of 72 miRNAs by at least two-fold including miRNAs that were previously described to target genes associated with inflammation, fibrosis and cancer development. Further investigation demonstrated that miR-146a-5p was consistently increased in HCV-infected hepatocyte-like cells and primary human hepatocytes as well as in liver tissue from HCV-infected patients. Genome-wide microarray and computational analyses indicated that miR-146a-5p over-expression modulates pathways that are related to liver disease and HCC development. Furthermore, we showed that miR-146a-5p positively impacts on late steps of the viral replication cycle thereby increasing HCV infection. Collectively, our data indicate that the HCV-induced increase in miR-146a-5p expression both promotes viral infection and is relevant for pathogenesis of liver disease.

    IMPORTANCE HCV is a leading cause of chronic liver disease and cancer. However, how HCV induces liver cancer remains poorly understood. There is accumulating evidence that viral cure does not eliminate the risk for HCC development. Thus, there is an unmet medical need to develop novel approaches to predict and prevent virus-induced HCC. miRNA expression is known to be deregulated in liver disease and cancer. Furthermore, miRNAs are essential for HCV replication and HCV infection alters miRNA expression. However, how miRNAs contribute to HCV-driven pathogenesis remains elusive. Here, we show that HCV induces miRNAs that may contribute to liver injury and carcinogenesis. miR-146a-5p was consistently increased in different cell-based models of HCV infection and HCV patient-derived liver tissue. Furthermore, miR-146a-5p increases HCV infection. Collectively, our data are relevant to understand viral pathogenesis and may open perspectives for novel biomarkers and prevention of virus-induced liver disease and HCC.

  • NF-{kappa}B Signaling Regulates Expression of Epstein-Barr Virus BART MicroRNAs and Long noncoding RNAs in Nasopharyngeal Carcinoma [PublishAheadOfPrint]

  • Epstein-Barr virus (EBV) expresses few viral proteins in nasopharyngeal carcinoma (NPC), but high levels of BamHI-A rightward transcripts (BARTs), which include noncoding RNAs (lncRNAs) and BART microRNAs (miRNAs). It is hypothesized that the mechanism for regulation of BARTs may relate to EBV pathogenesis in NPC. We showed that nuclear factor-B (NF-B) activates the BART promoters and modulates the expression of BARTs in EBV-infected NPC cells, but that introduction of mutations into the putative NF-B binding sites abolished activation of BART promoters by NF-B. Binding of p50 subunits to NF-B sites in the BART promoters was confirmed in electrophoretic mobility shift assays (EMSA) and further demonstrated in vivo using chromatin immunoprecipitation (ChIP) analysis. Expression of BART miRNAs and lncRNAs correlated with NF-B activity in EBV-infected epithelial cells, while treatment of EBV-harboring NPC C666-1 cells with aspirin (ASA) and the IB kinase inhibitor, PS-1145, inhibited NF-B activity, resulting in downregulation of BART expression. Expression of EBV LMP1 activates BART promoters, whereas an LMP1 mutant which cannot induce NF-B activation does not activate BART promoters, further supporting the idea that expression of BARTs is regulated by NF-B signaling. Expression of LMP1 is tightly regulated in NPC cells and this study confirmed that miR-BART5-5p downregulates LMP1 expression, suggesting a feedback loop between BART miRNA and LMP1 mediated NF-B activation in the NPC setting. These findings provide new insights into the mechanism underlying the deregulation of BARTs in NPC and identify a regulatory loop through which BARTs support EBV latency in NPC.

    IMPORTANCE Nasopharyngeal carcinoma (NPC) cells are ubiquitously infected with Epstein-Barr virus (EBV). Notably, EBV expresses very few viral proteins in NPC cells, presumably to avoid triggering an immune response, but high levels of EBV BART miRNAs and lncRNAs which exhibit complex functions associated with EBV pathogenesis. The mechanism for regulation of BARTs is critical for understanding NPC oncogenesis. This study provides multiple lines of evidence to show that expression of BARTs is subject to regulation by NF-B signaling. EBV LMP1 is a potent activator of NF-B signaling and we demonstrate that LMP1 can upregulate expression of BARTs through NF-B signaling, but that miR-BARTs are also able to downregulate LMP1 expression. It appears that aberrant NF-B signaling and expression of BARTs form an autoregulatory loop for maintaining EBV latency in NPC cells. Further exploration of how targeting NF-B signaling interrupts EBV latency in NPC cells may reveal new options for NPC treatment.

  • CD69 deficiency enhances the host response to Vaccinia virus infection through altered NK cell homeostasis [PublishAheadOfPrint]

  • During the host response to viral infection, the transmembrane CD69 protein is highly up-regulated in all immune cells. We have studied the role of CD69 in the murine immune response to Vaccinia Virus (VACV) infection and we report that the absence of CD69 enhances protection against VACV both at short and long times post- infection in immunocompetent and immunodeficient mice. NK cells were implicated in the increased infection control, since the differences were greatly diminished when they were depleted. This role of NK cells was not based on an altered NK cell reactivity, since CD69 did not affect NK cell activation threshold in response to MHC-I NK targets or PKC activation. Instead, NK cell numbers were increased in the spleen and peritoneum of CD69-deficient infected mice. That was not just secondary to a better infection control in CD69 deficient mice, since NK cell numbers were already augmented in the spleens and the blood of uninfected CD69-/- mice. CD69 deficient NK cells from infected mice not have altered proliferation capacity. However, a lower spontaneous cell death rate was observed in CD69-/- lymphocytes. Thus, our results suggest that CD69 limits the innate immune response to VACV virus infection at least in part through cell homeostatic survival.

    IMPORTANCE Herein, we show that increased natural killer (NK) cell numbers augmented the host response and survival after infection with Vaccinia virus. This phenotype is found in the absence of CD69 in immunocompetent and immunodeficient hosts. As part of the innate immune system, NK lymphocytes are activated and participate in the defense against infection. Several works have focused on the NK cells contribution to the protection to infection with Vaccinia virus. In this study it is demonstrated that the augmented early NK response in the absence of CD69 is responsible of the increased protection during infection with Vaccinia virus even at late times of infection. This work points to CD69 molecule may be a target of therapy to augment response to poxvirus infection.

  • Unexpected Inter-farm Transmission Dynamics During a Highly Pathogenic Avian Influenza Epidemic [PublishAheadOfPrint]

  • Next Generation Sequencing technology is now being increasingly applied to study the within and between host population dynamics of viruses. However, information on avian influenza virus evolution and transmission during a naturally occurring epidemic is still limited. Here, we use deep sequencing data obtained from clinical samples collected from five industrial holdings and a backyard farm infected during the 2013 highly pathogenic avian influenza (HPAI) H7N7 epidemic in Italy to unravel i) the epidemic virus population diversity, ii) the evolution of virus pathogenicity, and iii) the pathways of viral transmission between different holdings and sheds. We show a high level of genetic diversity of the HPAI H7N7 viruses within a single farm as a consequence of separate bottlenecks and founder effects. In particular, we identified the co-circulation in the index case of two viral strains showing a different insertion at the Hemagglutinin cleavage site, as well as nine nucleotide differences at the consensus level and 92 minority variants. To assess inter-farm transmission, we combined epidemiological and genetic data and identified the index case as the major source of the virus, suggesting the spread of different viral haplotypes from the index farm to the other industrial holdings, probably at different time points. Our results revealed inter-farm transmission dynamics that the epidemiological data alone could not unravel and demonstrated that delay in the disease detection and stamping out was the major cause of the emergence and the spread of the HPAI strain.

    IMPORTANCE The within and between host evolutionary dynamics of a highly pathogenic avian influenza (HPAI) strain during a naturally occurring epidemic is currently poorly understood. Here, we perform for the first time an in-depth sequence analysis of all the samples collected during a HPAI epidemic and demonstrate the importance to complement outbreak investigations with genetic data to reconstruct the transmission dynamics of the viruses and to evaluate the within and between farms genetic diversity of the viral population. We show that the evolutionary transition from the low pathogenic to the highly pathogenic form occurred within the first infected flock where we identified haplotypes with hemagglutinin cleavage site of different lengths. We also identify the index case as the major source of virus, indicating that prompt application of depopulation measures is essential to limit virus spread to other farms.

  • Broad cross protection is induced by a human papillomavirus vaccine composed of L1/L2 chimeric virus-like particles in preclinical models [PublishAheadOfPrint]

  • At least 15 high-risk human papillomavirus (HPV) types are linked to anogenital preneoplastic lesions and cancer. Currently, there are 3 licensed prophylactic HPV vaccines based on virus-like particles (VLPs) of L1 major-capsid proteins from 2, 4 or 9 HPV types, respectively, including the AS04-adjuvated HPV-16/18 vaccine. The L2 minor-capsid protein contains HPV-neutralizing epitopes that are well-conserved across numerous high-risk HPV types. Therefore the objective of our study was to assess the capacity to broaden vaccine-mediated protection using AS04-adjuvanted vaccines based on VLP chimeras of L1 and 2 L2 epitopes. Several chimeric VLPs were constructed by inserting L2 epitopes within the DE loop and/or C-terminus of L1. Based on the shape, yield, size and immunogenicity, 1 of 7 chimeras was selected for further evaluation in mouse and rabbit challenge models. The chimeric VLP consisted of HPV-18 L1 inserted with HPV-33 L2 (amino residues 17-36; L1 DE loop) and HPV-58 L2 (amino residues 56-75; L1 C-terminus). This chimeric L1/L2 VLP vaccine induced persistent immune responses and protected against all of the different HPV types evaluated (HPV-6, 11, 16, 31, 35, 39, 45, 58 and 59 as pseudovirions or quasivirions) in both mouse and rabbit challenge models. The degree and breadth of protection in the rabbit were further enhanced when the chimeric L1/L2 VLP was formulated with the L1 VLPs from the HPV-16/18 vaccine. Therefore the novel HPV-18 L1/L2 chimeric VLP (alone or in combination with HPV-16/18 VLPs) formulated with AS04 has the potential to provide a broad protective efficacy in human subjects.

    Importance From evaluations in human papillomavirus (HPV)-protection models in rabbits and mice; our study has identified a prophylactic vaccine with the potential to target a wide range of HPV types linked to anogenital cancer. The three currently licensed vaccines contain virus-like particles (VLPs) of L1 major capsid proteins from 2, 4, or 9 HPV types. Rather than increasing the diversity of L1 VLPs, this vaccine contains VLPs based on recombinant chimera of 2 highly conserved neutralizing epitopes from the L2 capsid protein inserted into L1. Our study demonstrated that the chimeric L1/L2 VLP is an effective vehicle for displaying 2 different L2 epitopes and can be used at an equivalent quantity to what is used in the licensed vaccines. Hence using the chimeric L1/L2 VLP may be a more cost-effective approach for vaccine formulation than adding different VLPs for each HPV type.

  • The membrane anchors of the structural flavivirus proteins and their role in virus assembly [PublishAheadOfPrint]

  • The structural proteins of flaviviruses carry a unique set of transmembrane domains (TMDs) at their C-termini that are derived from the mode of viral polyprotein processing. They function as internal signal and stop-transfer sequences during protein translation, but possible additional roles in protein interactions required during assembly and maturation of viral particles are ill-defined. To shed light on the role of TMDs in these processes, we engineered a set of tick-borne encephalitis virus mutants in which these structural elements were replaced in different combinations by the homologous sequences of a distantly related flavivirus (Japanese encephalitis virus). The effects of these modifications were analyzed with respect to protein synthesis, viral particle secretion, specific infectivity, and acidic-pH-induced maturation processes. We provide evidence that interactions involving the double membrane anchor of the envelope protein E (a unique feature compared to other viral fusion proteins) contribute substantially to particle assembly, stability and maturation. Disturbances of the inter- and intra-TMD interactions of E resulted in the secretion of a higher proportion of capsid-less subviral particles at the expense of whole virions, suggesting a possible role in the still incompletely understood mechanism of capsid integration during virus budding. In contrast, the TMD initially anchoring the C protein to the endoplasmic reticulum membrane does not appear to take part in envelope protein interactions. We also show that E TMDs are involved in the envelope protein rearrangements that are triggered by acidic pH in the Trans-Golgi-Network and represent a hallmark of virus maturation.

    Importance The assembly of flaviviruses occurs in the endoplasmic reticulum and leads to the formation of immature, non-infectious particles composed of an RNA-containing capsid surrounded by a lipid membrane with the two integrated envelope proteins prM and E arranged in an icosahedral lattice. By which mechanism the capsid is formed and integrated into the budding viral envelope is currently unknown. We provide evidence that the transmembrane domains (TMDs) of E are essential for the formation of capsid-containing particles and that disturbances of these interactions lead to the preferential formation of capsid-less subviral particles at the expense of whole virions. E TMD interactions also appear to be essential for the envelope protein rearrangements required for virus maturation and the generation of infectious virions. Our data thus provide new insights into the biological functions of E TMDs and extend their role during viral polyprotein processing to additional functions in particle assembly and maturation.

  • Identification of respiratory syncytial virus nonstructural protein 2 residues essential for exploitation of the host ubiquitin system and inhibition innate immune responses. [PublishAheadOfPrint]

  • Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in young children worldwide. The RSV nonstructural protein 2 (NS2) is a multifunctional protein that primarily acts to antagonize the innate immune system by targeting STAT2 for proteasomal degradation. We investigated the structural determinants of NS2 important for interaction with the host ubiquitin system to degrade STAT2 during infection. We found that NS2 expression enhances ubiquitination of host proteins. Bioinformatics analysis provided a platform for identification of specific residues that limit NS2-induced ubiquitination. Combinations of multiple mutations displayed an additive effect on reducing NS2-induced ubiquitination. Using a reverse genetics system, we generated recombinant RSV (rRSV) containing NS2 ubiquitin mutations, which maintained their effect on ubiquitin expression during infection. Interestingly, STAT2 degradation activity was ablated in the NS2 ubiquitin mutant rRSV. In addition, NS2 ubiquitin mutations decreased rRSV replication, indicating a correlation between NS2's ubiquitin function and antagonism of innate immune signaling to enhance viral replication. Our approach at targeting NS2 residues required for NS2 inhibition of immune responses provides a mechanism for attenuating RSV for vaccine development.

    IMPORTANCE RSV has been circulating globally for more than 60 years, causing severe respiratory disease in pediatric, elderly and immunocompromised populations. Production of a safe, effective vaccine against RSV is a public health priority. The NS2 protein is an effective target for prevention and treatment of RSV due to its antagonistic activity against the innate immune system. However, NS2-deleted RSV vaccine candidates rendered RSV over-attenuated or poorly immunogenic. Alternatively, we can modify essential NS2 structural features to marginally limit viral growth while maintaining immune responses, providing the necessary balance between antigenicity and safety required for an effective vaccine. We coupled bioinformatics analysis with reverse genetics to introduce mutations into RSV's negative-sense genome. In this way we constructed rRSV NS2 ubiquitin mutants that limited NS2's ability to antagonize the innate immune system, thereby attenuating rRSV growth and increasing innate immune responses.

  • The foot-and-mouth disease carrier state divergence in cattle [PublishAheadOfPrint]

  • The pathogenesis of persistent foot-and-mouth disease virus (FMDV) infection was investigated in 46 cattle that were either naïve or vaccinated using a recombinant, adenovirus-vectored vaccine two weeks before challenge. The prevalence of FMDV persistence was similar in both groups (62% vaccinated; 67% non-vaccinated) despite vaccinated cattle having been protected from clinical disease. Antemortem infection dynamics demonstrated that the subclinical divergence between FMDV carriers and animals that cleared the infection had occurred by 10 days post infection (dpi) in vaccinated cattle, and by 21 dpi in non-vaccinated animals. Anatomic distribution of virus in subclinically infected, vaccinated cattle was restricted to the pharynx throughout both early and persistent phases of infection. In non-vaccinated cattle, systemically disseminated virus was cleared from peripheral sites by 10 dpi, while selectively persisting within the nasopharynx of a subset of animals. The quantities of viral RNA shed in oropharyngeal fluid during FMDV persistence were similar in vaccinated and non-vaccinated cattle. FMDV structural and non-structural proteins were localized to follicle-associated epithelium of the dorsal soft palate and dorsal nasopharynx in persistently infected cattle. Host transcriptome analysis of tissue samples processed by laser capture microdissection indicated suppression of antiviral host factors (IRF-7, CXCL10, IFN-, IFN-, and IL10) associated with persistent FMDV. Contrastingly, during the transitional phase of infection, expression of IFN- mRNA was higher in follicle-associated epithelium of animals that had cleared infection. This work provides novel insights in the intricate mechanisms of FMDV persistence and contributes to further understanding of this critical aspect of FMDV pathogenesis.

    IMPORTANCE The existence of a prolonged, asymptomatic carrier state is a political impediment for control and potential eradication of foot-and-mouth disease (FMD). When FMD outbreaks occur, they are often extinguished by massive depopulation of livestock due to fear of undiagnosed subclinically infected animals despite uncertainty of the biological relevance of FMD virus (FMDV) persistence. The work described herein elucidates aspects of the FMDV carrier state in cattle which may facilitate identification and/or abrogation of asymptomatic FMDV infection. The divergence between animals that clear infection and those that develop persistent infection was demonstrated to occur earlier than previously established. The host anti-viral response was down-regulated in tissues maintaining persistent FMDV, whereas up-regulation of IFN- mRNA was found in epithelium of cattle that had recently cleared infection. This suggests that clearing FMDV infection is associated with enhanced mucosal anti-viral response whereas FMDV persistence is associated with a suppression of the host anti-viral response.

  • Lassa virus cell entry via dystroglycan involves an unusual pathway of macropinocytosis [PublishAheadOfPrint]

  • The pathogenic Old World arenavirus Lassa virus (LASV) causes a severe hemorrhagic fever with high mortality in humans. Several LASV receptors, including dystroglycan (DG), TAM receptor tyrosine kinases, and C-type lectins have been identified, suggesting complex receptor use. Upon receptor binding, LASV enters the host cell via an unknown clathrin- and dynamin-independent pathway that delivers the virus to late endosomes, where fusion occurs. Here we investigated the mechanisms underlying LASV endocytosis in human cells in the context of productive arenavirus infection, using recombinant lymphocytic choriomeningitis virus (LCMV) expressing LASV glycoprotein (rLCMV-LASVGP). We found that rLCMV-LASVGP entered human epithelial cells via DG using a macropinocytosis-related pathway independently of alternative receptors. Dystroglycan-mediated entry of rLCMV-LASVGP required sodium hydrogen exchangers, actin, the GTPase Cdc42, and its downstream targets PAK-1 and N-Wasp. Unlike other viruses that enter cells via macropinocytosis, rLCMV-LASVGP entry did not induce overt changes in cellular morphology and hardly affected actin dynamics or fluid uptake. Screening of kinase inhibitors identified PKC, PI3K, and the receptor tyrosine kinase HGFR as regulators of rLCMV-LASVGP entry. The HGFR inhibitor EMD 1214063, a candidate anti-cancer drug, showed anti-viral activity against rLCMV-LASVGP at the level of entry. When combined with ribavirin, which is currently used to treat human arenavirus infection, EMD 1214063 showed additive anti-viral effects. In sum, our study reveals that DG can link LASV to an unusual pathway of macropinocytosis that causes only minimal perturbation of the host cell and identifies cellular kinases a possible novel targets for therapeutic intervention.

    IMPORTANCE Lassa virus (LASV) causes several hundred thousand infections per year in Western Africa with high mortality among hospitalized patients. The current lack of a vaccine and the limited therapeutic options at hand make the development of new drugs against LASV a high priority. In the present study, we uncover that LASV entry into human cells via its major receptor dystroglycan involves an unusual pathway of macropinocytosis and define a set of cellular factors implicated in the regulation of LASV entry. A screen of kinase inhibitors revealed HGFR as a possible candidate target for anti-viral drugs against LASV. An HGFR candidate inhibitor currently evaluated for cancer treatment showed potent anti-viral activity and additive drug effects with ribavirin, which is used in the clinic to treat human LASV infection. In sum, our study reveals novel fundamental aspects of LASV-host cell interaction and highlights a possible candidate drug target for therapeutic intervention.

  • The Nipah virus C and W proteins contribute to respiratory disease in ferrets [PublishAheadOfPrint]

  • Nipah virus (NiV) is a highly lethal paramyxovirus that recently emerged as a causative agent of febrile encephalitis and severe respiratory disease in humans. The ferret model has emerged as the preferred small animal model to study NiV disease, but much is still unknown about the viral determinants of pathogenesis for NiV including the contribution of the C protein in ferrets. Additionally, studies have yet to examine the synergistic effects of the various P gene products on pathogenesis in animal models. Using recombinant NiVs (rNiVs), we examine the sole contribution of the NiV C protein and the combined C and W protein contributions in the ferret model of NiV pathogenesis. We show that a rNiV void of C expression resulted in 100% mortality albeit with less respiratory disease, similar to our previously reported rNiV void of W expression; this is in stark contrast to the attenuated phenotype observed in previous hamster studies utilizing rNiVs void of C expression. We also observed that a rNiV void of both C and W expression resulted in limited respiratory disease; however, there was severe neurological disease leading to 60% mortality and the surviving ferrets demonstrating similar sequelae to human survivors of NiV encephalitis.

    IMPORTANCE Nipah virus (NiV) is a human pathogen capable of causing lethal respiratory and neurological disease. Many human survivors have long-lasting neurological impairment. Using a ferret model, this study demonstrated the roles of the NiV C and W proteins in pathogenesis, where the lack of either the C or W protein independently decreased clinical respiratory disease, but did not decrease lethality. Abolishing both C and W expression, however, severely decreased respiratory disease and destruction of splenic germinal centers. These ferrets still suffered severe neurological disease with 60% succumbing to disease, with the survivors experiencing long-term neurological impairment as seen in human survivors. This new ferret NiV C and W knock-out model may allow, for the first time, the examination of interventions to prevent or mitigate the neurological damage and sequelae experienced by human survivors.

  • Human cytomegalovirus induces an atypical activation of Akt to stimulate the survival of short-lived monocytes [PublishAheadOfPrint]

  • Human cytomegalovirus (HCMV) is a pervasive herpesvirus responsible for significant morbidity and mortality among immunodeficient/naive hosts. Following a primary HCMV infection, circulating blood monocytes mediate the systemic spread of the virus. Extending the short 48-hour lifespan of monocytes is critical to the viral dissemination process as these blood-borne cells are non-permissive for virus replication until fully differentiated into macrophages. Here, we show that HCMV glycoprotein gB binding to cellular epidermal growth factor receptor (EGFR) during HCMV entry initiated a rapid activation of the apoptosis suppressor Akt within 15 minutes, which was maintained through 72 hours. The virus-induced activation of Akt was more robust than with the normal myeloid growth factor M-CSF and was essential for infected monocytes to bypass the 48-hour viability checkpoint. Activation of PI3K following EGFR engagement by HCMV mediated the phosphorylation of Akt. Moreover, HCMV entry drove a switch away from the PI3K p110 isoform, which was required for the viability of uninfected monocytes, to the p110bbeta; isoform in order to facilitate the Akt-dependent prosurvival state within infected cells. Simultaneously, in contrast to M-CSF, HCMV promoted a rapid increase in SHIP1 expression leading to signaling through a noncanonical Akt activation pathway. To ensure maximum Akt activity, HCMV also induced an early phosphorylation-dependent inactivation of the negative regulator PTEN. Overall, our data indicate that HCMV hijacks the upstream Akt signaling network to induce a non-traditional activation of Akt and subsequently a prosurvival decision at the 48-hour cell fate checkpoint, a vital step for HCMV's dissemination and persistence strategy.

    IMPORTANCE HCMV is found throughout the world with a prevalence of 55-100% within the human population. HCMV infection is generally asymptomatic in immunocompetent or naive individuals, but is a significant cause of morbidity and mortality among the immunocompromised. Widespread organ inflammation is associated with symptomatic infections, which is a direct consequence of the viral dissemination strategy. Inflammatory peripheral blood monocytes facilitate spread of HCMV. However, HCMV must subvert the naturally short lifespan of monocytes. In this work, we demonstrate that HCMV induces the activation of Akt, an antiapoptotic protein, in distinct manner from that of normal myeloid growth factors. Moreover, we decipher how HCMV dysregulates the upstream Akt signaling network during viral entry to promote an Akt-dependent prosurvival state following infection. Delineating the virus-specific mechanisms that regulate cellular prosurvival pathways in order to drive the survival of HCMV-infected monocytes is important to identifying new anti-HCMV therapeutic targets.

  • The Emerging Duck Flavivirus is not Pathogenic for Primates and is Highly Sensitive to Mammal Interferon Signaling [PublishAheadOfPrint]

  • Flaviviruses pose a significant threat to both animals and humans. Recently, a novel flavivirus, duck Tembusu virus (DTMUV), was identified as the causative agent of a serious duck viral disease in Asia. Its rapid spread, expanding host ranges and uncertain transmission routes have raised substantial concerns regarding its potential threats to non-avian hosts, including humans. Here, we demonstrate that DTMUV is not pathogenic for non-human primates and is highly sensitive to mammal type I interferon signaling. In vitro assays demonstrated that DTMUV infected and replicated efficiently in various mammalian cell lines. Further tests in mice demonstrated high neurovirulence and the age-dependent neuroinvasiveness of the virus. In particular, the inoculation of DTMUV in rhesus monkeys did not result in either viremia or apparent clinical symptoms, although DTMUV-specific humoral immune responses were detected. Furthermore, we revealed that although avian IFN failed to inhibit DTMUV in avian cells, DTMUV were more sensitive to the antiviral effects of type I interferon than other known human pathogenic flaviviruses. Knockout of the type I IFN receptor caused apparent viremia, viscerotropic disease and mortality in mice, indicating a vital role of IFN signaling in protection against DTMUV infection. Collectively, we provide direct experimental evidence that this novel avian-origin DTMUV possesses a limited capability to establish infection in immunocompetent primates due to its decreased antagonistic activity in the mammal IFN system. Furthermore, our findings highlight the potential risk of DTMUV infection in immunocompromised individuals and warrant studies on the cross-species transmission and pathogenesis of this novel flavivirus.

    IMPORTANCE Mosquito-borne flaviviruses comprise a large group of pathogenic and nonpathogenic members. The pathogenic flaviviruses include dengue, West Nile and Japanese encephalitis viruses, and the nonpathogenic flaviviruses normally persist in a natural cycle and rarely cause disease in humans. A novel flavivirus DTMUV (also known as duck egg-drop syndrome flavivirus, DEDSV) was identified in 2012 in ducks and then rapidly spread to several Asian countries. This new flavivirus was then shown to infect multiple avian species, resulting in neurological symptoms with unknown routes of transmission. There is public concern regarding its potential transmission from birds to humans and other non-avian hosts. Our present study shows that the mammalian IFN system can efficiently eliminate DTMUV infection and that the emergence of severe DTMUV-associated disease in mammals, especially humans, is unlikely. Currently, DTMUV infection mostly affects avian species.

  • Functional regulation of AcMNPV-miR-1 in baculovirus replication [PublishAheadOfPrint]

  • AcMNPV-miR-1, an Autographa californica nucleopolyhedrovirus-encoded microRNA (miRNA), down-regulates the ac94 gene, reducing the production of infectious budded virions and accelerating the formation of occlusion-derived virions. In the current study, four viruses that constitutively over-express AcMNPV-miR-1 were constructed to further explore the function of the miRNA. In addition to the ac94 gene, two new viral gene targets (ac18 and ac95) of AcMNPV-miR-1 were identified, and the possible interacting proteins were verified and tested. In the context of AcMNPV-miR-1 over-expression, ac18 was slightly up-regulated, and ac95 was down-regulated. Several interacting proteins were identified, and a functional pathway for AcMNPV-miR-1 was deduced. AcMNPV-miR-1 over-expression decreased budded virus infectivity, reduced viral DNA replication, accelerated polyhedra formation, and promoted viral infection efficiency in Trichoplusia ni larvae, suggesting that AcMNPV-miR-1 restrains virus infection of cells but facilitates virus infection of larvae.

    IMPORTANCE Recently, microRNAs (miRNAs) have been widely reported as moderators or regulators of mammalian cellular processes, especially disease-related pathways in humans. However, the roles played by miRNAs encoded by baculoviruses, which infect numerous beneficial insects and agricultural pests, have rarely been described. To explore the actions of virus-encoded miRNAs, we investigated a miRNA encoded by Autographa californica nucleopolyhedrovirus (AcMNPV-miR-1). We previously identified this miRNA through the exogenous addition of AcMNPV-miR-1 mimics. In the current study, we constitutively over-expressed AcMNPV-miR-1 and analyzed the resultant effects to more comprehensively assess what is indeed the function of this miRNA during viral infection. In addition, we widely explored the target genes for the miRNA in the viral and host genome and proposed a possible functional network for AcMNPV-miR-1, which provides a better general understanding of virus-encoded miRNAs. In brief, our study implied that AcMNPV-miR-1 constrains viral replication and cellular infection but enhances larval infection.

  • Tetraspanin CD151 promotes initial events in human cytomegalovirus infection [PublishAheadOfPrint]

  • Human cytomegalovirus (HCMV), a beta-herpesvirus, can cause life-threatening disease in immunocompromised individuals. Viral envelope glycoproteins mediating binding to and penetration into target cells have been identified previously. In contrast, cellular proteins supporting HCMV during entry are largely unknown.

    In order to systematically identify host genes affecting initial steps of HCMV infection, a targeted RNA interference screen of 96 cellular genes was performed in endothelial cells with a virus strain expressing the full set of known glycoprotein H and L (gH/gL) complexes. The approach yielded five pro-viral host factors from different protein families and eight anti-viral host factors, mostly growth factor receptors. The tetraspanin CD151 was uncovered as a novel pro-viral host factor and analyzed further. Like endothelial cells, fibroblasts were also less susceptible to HCMV infection after CD151 depletion. Virus strains with different sets of gH/gL complexes conferring either a broad or a narrow cell tropism were equally impaired. Infection of CD151 depleted cells by a fluorescent virus with differentially labelled capsid and envelope revealed a role of CD151 in viral penetration but not in adsorption to the cell.

    In conclusion, the tetraspanin CD151 emerges as a novel host factor in HCMV entry and as a putative antiviral target.

    IMPORTANCE At present, the events at the virus-cell interface and the cellular proteins involved during the HCMV entry steps are scarcely understood. In this study, several host factors with a putative role in this process were identified. The tetraspanin CD151 is discovered as a previously unrecognized pro-viral host factor for HCMV and found to support viral penetration into the target cells. The findings shed light on the cellular contribution during the initial steps of infection and open a new direction in HCMV research.

  • Calcium-dependent Rubella Virus Fusion Occurs in Early Endosomes [PublishAheadOfPrint]

  • The E1 membrane protein of rubella virus (RuV) is a class II membrane fusion protein structurally related to the fusion proteins of the alphaviruses, flaviviruses and phleboviruses. Virus entry is mediated by a low pH-dependent fusion reaction through E1's insertion into the cell membrane and refolding to a stable homotrimer. Unlike the other described class II proteins, RuV E1 contains 2 fusion loops, which complex a metal ion between them by interactions with residues N88 and D136. Insertion of the E1 protein into the target membrane, fusion and infection require calcium and are blocked by alanine substitution of N88 or D136. Here we addressed the requirements of E1 for calcium binding and the intracellular location of the calcium requirement during virus entry. Our results demonstrated that N88 and D136 are optimally configured to support RuV fusion and are strongly selected for during the virus lifecycle. While E1 has some similarities with cellular proteins that bind calcium and anionic lipids, RuV membrane binding was independent of anionic lipids. Virus fusion occurred within early endosomes, and chelation of intracellular calcium showed that calcium within the early endosome was required for virus fusion and infection. Calcium triggered the reversible insertion of E1 into the target membrane at neutral pH, but E1 homotrimer formation and fusion required low pH. Thus RuV E1, unlike other known class II fusion proteins, has distinct triggers for membrane insertion and fusion protein refolding mediated respectively by endosomal calcium and low pH.

    IMPORTANCE Rubella virus causes a mild disease of childhood but infection of pregnant women frequently results in miscarriage or severe birth defects. In spite of an effective vaccine, RuV disease remains a serious problem in many developing countries. RuV infection of host cells involves endocytic uptake and low pH-triggered membrane fusion, and is unusual in its requirement for calcium binding by the membrane fusion protein. Here we addressed the mechanism of the calcium requirement and the required location of calcium during virus entry. Calcium and low pH were both essential during the virus fusion reaction, which was shown to occur in the early endosome compartment.

  • Biphasic CD8+ T-cell defense in simian immunodeficiency virus control by acute-phase passive neutralizing antibody immunization [PublishAheadOfPrint]

  • Identifying human immunodeficiency virus type 1 (HIV-1) control mechanisms by neutralizing antibodies (NAbs) is critical for anti-HIV-1 strategies. Recent in vivo studies on animals infected with simian immunodeficiency virus (SIV) and related viruses have shown the efficacy of post-infection NAb passive immunization for viremia reduction, and one suggested mechanism is its occurrence through modulation of cellular immune responses. Here we describe SIV control in macaques showing biphasic CD8+ cytotoxic T lymphocyte (CTL) responses following acute-phase NAb passive immunization. Analysis of four SIVmac239-infected rhesus macaque pairs matched with major histocompatibility complex class I haplotypes, respectively, found that counterparts receiving day 7 anti-SIV polyclonal NAb infusion all suppressed viremia for up to 2 years without accumulating viral CTL escape mutations. In the first phase of primary viremia control attainment, CD8+ cells had high capacities to suppress SIVs carrying CTL escape mutations. Conversely, in the second sustained phase of SIV control, CTL responses converged to a pattern of immunodominant CTL preservation. During this sustained phase of viral control, SIV epitope-specific CTLs showed retention of phosphorylated extracellular signal-related kinase (ERK)hi/phosphorylated AMP-activated protein kinase (AMPK)lo subpopulations, implying their correlation with SIV control. Results suggest that virus-specific CTLs functionally boosted by acute-phase NAbs may drive robust AIDS virus control.

    IMPORTANCE In early HIV infection, NAb responses are lacking and CTL responses are insufficient, which leads to viral persistence. Hence it is important to identify immune responses that can successfully control such HIV replication. Here, we show that monkeys receiving NAb passive immunization in early SIV infection strictly control viral replication for years. Passive infusion of NAbs, with CTL cross-priming capacity, resulted in induction of functionally boosted early CTL responses showing enhanced suppression of CTL escape mutant virus replication. Accordingly, the NAb-infused animals did not show accumulation of viral CTL escape mutations during sustained SIV control and immunodominant CTL responses were preserved. This early functional augmentation of CTLs by NAbs provides key insights into the design of lasting and viral escape mutation-free protective immunity against HIV-1 infection.

  • Robust Lys63-linked Ubiquitination of RIG-I Promotes Cytokine Eruption in Early Influenza B Virus Infection [PublishAheadOfPrint]

  • Influenza A and B virus infections both cause a host innate immunity response. Here, we report that the robust production of type I and III interferons (IFNs), IFN-stimulated genes, and pre-inflammatory factors can be induced by influenza B virus infection rather than influenza A virus in alveolar epithelial cells (A549) during early infection. This response is mainly dependent on the RIG-I-mediated signaling pathway. The infection by influenza B virus promotes intense Lys63-linked ubiquitination of RIG-I, resulting in cytokine eruption. It is known that the influenza A virus NS1 protein (NS1-A) interacts with RIG-I and TRIM25 to suppress the activation of RIG-I-mediated signaling. However, the present results indicate that the influenza B virus NS1 protein (NS1-B) is unable to interact with RIG-I but engages in the formation of a RIG-I/TRIM25/NS1-B ternary complex. Furthermore, we demonstrate that the N-terminal RNA-binding domain (RBD) of NS1-B is responsible for interaction with TRIM25, and this interaction blocks the inhibitory effect on RIG-I ubiquitination from the NS1-B C-terminus. Our findings reveal a novel mechanism for the host cytokine response to influenza B virus infection through regulatory interplay between host and viral proteins.

    IMPORTANCE Influenza B virus generally causes local mild epidemics but is occasionally lethal to individuals. Existing studies describe the broad characteristics of influenza B virus epidemiology and pathology. However, to develop better prevention and treatments for the disease, determining the concrete molecular mechanisms of pathogenesis becomes pivotal to understand how the host reacts to the challenge of influenza B virus. Thus, we aimed to characterize the host innate immune response against influenza B virus infection. Here, we show that vigorous Lys63-linked ubiquitination of RIG-I and cytokine eruption dependent on RIG-I-mediated signal transduction are induced by virus infection. Additionally, TRIM25 positively regulates RIG-I-mediated signaling by ablating the inhibitory function of NS1-B on RIG-I ubiquitination.

  • Migration of Nucleocapsids in Vesicular Stomatitis Virus-Infected Cells is Dependent on Both Microtubules and Actin Filaments [PublishAheadOfPrint]

  • The distribution of vesicular stomatitis virus (VSV) nucleocapsids in the cytoplasm of infected cells was analyzed by scanning confocal fluorescence microscopy using a newly developed quantitative approach called the border-to-border distribution method. Nucleocapsids were located near the cell nucleus at early times postinfection (2 hr), but were redistributed during infection toward the edges of the cell. This redistribution was inhibited by treatment with nocodazole, colcemid, or cytochalasin D indicating it is dependent on both microtubules and actin filaments. The role of actin filaments in nucleocapsid mobility was also confirmed by live cell imaging of fluorescent nucleocapsids of a virus containing P protein fused to enhanced green fluorescent protein. However, in contrast to the overall redistribution in the cytoplasm, the incorporation of nucleocapsids into virions as determined in pulse-chase experiments was dependent on the activity of actin filaments with little if any effect of inhibition of microtubule function. These results indicate that the mechanisms by which nucleocapsids are transported to the farthest reaches of the cell differ from those required for incorporation into virions. This is likely due to the ability of nucleocapsids to follow shorter paths to the plasma membrane mediated by actin filaments.

    IMPORTANCE Nucleocapsids of nonsegmented negative strand viruses like VSV are assembled in the cytoplasm during genome RNA replication and must migrate to the plasma membrane for assembly into virions. Nucleocapsids are too large to diffuse in the cytoplasm in the time required for virus assembly, and must be transported by cytoskeletal elements. Previous results suggested that microtubules were responsible for migration of VSV nucleocapsids to the plasma membrane for virus assembly. Data presented here show that both microtubules and actin filaments are responsible for mobility of nucleocapsids in the cytoplasm, but that actin filaments play a larger role than microtubules in incorporation of nucleocapsids into virions.

  • Long-term Spontaneous control of HIV-1 relates to low frequency of infected cells and inefficient viral reactivation [PublishAheadOfPrint]

  • HIV establishes reservoirs of infected cells that persist despite effective antiretroviral therapy (ART). In most patients, the virus begins to replicate soon after treatment interruption. However, a low frequency of infected cells at the time of treatment interruption has been associated with delayed viral rebound. Likewise, individuals who control the infection spontaneously, so-called "HIV-1 controllers" (HICs) carry particularly low levels of infected cells. It is unclear, however, whether and how this small number of infected cells contributes to durable viral control. Here we compared 38 HICs with 12 patients on effective combined ART ("cART patients") and found that the low frequency of infected cells in the former subjects was associated both with less efficient viral reactivation in resting CD4+ T cells and with less efficient virion production ex vivo. We also found that a potent HIV-specific CD8+ T cell response was only present in those HICs whose CD4+ T cells produced virus ex vivo. Long-term spontaneous control of HIV infection in HICs thus appears to be sustained on the inefficient reactivation of viruses from a limited number of infected cells, and the capacity of HICs to activate a potent HIV-specific CD8+ T cell response to counteract efficient viral reactivation events.

    IMPORTANCE There is a strong scientific interest in developing strategies to eradicate the HIV-1 reservoir. Very rare HIV-1 infected patients are able to spontaneously control viremia for long periods of time (HIV-1 controllers, HIC) and are put forward as a model of HIV-1 remission. Here, we show that the low viral reservoirs found in HIC are a critical part of the mechanisms underlying viral control and traduces in a lower probability of HIV-1 reactivation events resulting in limited HIV-1 release and spread. We found that those HIC in whom viral reactivation and spread in vitro from CD4+ T cells was most difficult were those with shrunk CD8+ T cell responses. These results suggest that low HIV-1 reservoirs, in some settings, decisively contribute to at least temporary control of infection without antiretroviral therapy. We believe that this work provides information of relevance in the context of the search for HIV-1 remission.

  • Rearrangement of influenza virus spliced segments for the development of live attenuated vaccines [PublishAheadOfPrint]

  • Influenza viral infections represent a serious public health problem, causing a contagious respiratory disease, which is most effectively prevented through vaccination. Segments seven (M) and eight (NS) of influenza virus encode mRNA transcripts that are alternatively spliced to express two different viral proteins. This study describes the generation, using reverse genetics, of three different recombinant influenza A/Puerto Rico/8/1934 (PR8) H1N1 viruses containing individual or combined modified M and/or NS viral segments in which the overlapping open reading frames of M1/M2 and/or NS1/NEP, respectively, have been split by using the porcine teschovirus-1 (PTV-1) 2A autoproteolytic cleavage site. Viruses with an M split segment were impaired in replication at non-permissive high temperatures, whereas high virus titers could be obtained at permissive low temperatures (33ddeg;C). Furthermore, viruses containing the M split segment were highly attenuated in vivo, while retaining immunogenicity and protection against a lethal challenge with wild-type PR8. These results indicate that influenza viruses can be effectively attenuated by the rearrangement of spliced segments and represents an excellent option as safe, immunogenic and protective live-attenuated vaccines. Moreover, this is the first time in which an influenza virus containing a restructured M segment has been described. Reorganization of M segment to encode M1 and M2 from two separate, non-overlapping, independent open reading frames represents a useful tool to independently study mutations in the M1 and M2 viral proteins without affecting the other viral M product.

    IMPORTANCE Vaccination represents our best therapeutic option against influenza viral infections. However, the efficacy of current influenza vaccines is suboptimal, and novel approaches are necessary for the prevention of disease caused by this important human respiratory pathogen. In this work, we describe a novel approach to generate safer and more efficient live attenuated influenza vaccines (LAIV) based on recombinant viruses encoding non-overlapping and independent M1/M2 (Ms) or both M1/M2 and NS1/NEP (Ms/NSs) open reading frames. Viruses containing a modified M segment were highly attenuated in mice, but able to confer, upon a single intranasal immunization, complete protection against a lethal homologous challenge with wild-type virus. Notably, protection efficacy conferred by our split M viruses was better than that obtained with the current temperature sensitive influenza LAIV. Altogether, these results open a new avenue for the development of safer and more protective LAIV based on genome reorganization of spliced viral RNA segments.

  • Comparison of Antibody-Dependent Cell-Mediated Cytotoxicity and Virus Neutralization by HIV-1 Env-specific Monoclonal Antibodies [PublishAheadOfPrint]

  • Although antibodies to the HIV-1 envelope glycoprotein have been studied extensively for their ability to block viral infectivity, little data is currently available on non-neutralizing functions of these antibodies, such as their ability to eliminate virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 Env-specific antibodies of diverse specificities, including potent broadly neutralizing and non-neutralizing antibodies, were therefore tested for ADCC against cells infected with a lab-adapted HIV-1 isolate (HIV-1NL4-3), a primary HIV-1 isolate (HIV-1JR-FL), and a simian-human immunodeficiency virus adapted for pathogenic infection of rhesus macaques (SHIVAD8-EO). In accordance with the sensitivity of these viruses to neutralization, HIV-1NL4-3-infected cells were considerably more sensitive to ADCC, both in terms of the number of antibodies and magnitude of responses, than cells infected with HIV-1JR-FL or SHIVAD8-EO. ADCC activity generally correlated with antibody binding to Env on the surface of virus-infected cells and with viral neutralization; however, neutralization was not always predictive of ADCC, as instances of ADCC in the absence of detectable neutralization, and vice versa, were observed. These results reveal incomplete overlap in the specificities of antibodies that mediate these antiviral activities, and provide insights into the relationship between ADCC and neutralization important for the development of antibody-based vaccines and therapies for combating HIV-1 infection.

    IMPORTANCE This study provides fundamental insights into the relationship between antibody-dependent cell-mediated cytotoxicity (ADCC) and virus neutralization that may help to guide the development of antibody-based vaccines and immunotherapies for the prevention and treatment of HIV-1 infection.

  • Human norovirus interactions with histo-blood group antigens and human milk oligosaccharides [PublishAheadOfPrint]

  • Human noroviruses interact with both human histo-blood group antigens (HBGAs) and human milk oligosaccharides (HMOs). The former is believed to be important for a virus infection, while the latter might act as a natural decoy in the host during an infection. However, certain noroviruses are known to bind poorly to HBGAs, yet still cause infections; some interact with numerous HBGA types, but are non-prevalent; and yet others bind HBGAs and seem to be increasing in prevalence. HBGAs and HMOs can be found as soluble antigens in humans, both can be structurally alike, and both can interact with equivalent residues at an identical binding pocket on the capsid. In this Gem, we discuss HBGA and HMO binding studies for human noroviruses, concentrating on the clinically important genogroup II noroviruses. In short, the roles of HBGA and HMO interactions are still an unclear in a norovirus infection.

  • CSF-1R antagonists sensitize HIV-1-infected macrophages to TRAIL-mediated killing [PublishAheadOfPrint]

  • Strategies aimed at eliminating persistent viral reservoirs from HIV-1-infected individuals have focused on CD4+ T-cell reservoirs. However, very little attention has been given to approaches that could promote elimination of tissue macrophage reservoirs. HIV-1 infection of macrophages induces phosphorylation of colony-stimulating factor 1 receptor (CSF-1R), which confers resistance to apoptotic pathways driven by TNF-related apoptosis-inducing ligand (TRAIL), thereby promoting viral persistence. In this study we assessed whether CSF-1R antagonists (PLX647, PLX3397 and PLX5622) restored apoptotic sensitivity of HIV-1-infected macrophages in vitro. PLX647, PLX3397 and PLX5622, at clinically relevant concentrations, blocked the activation of CSF-1R and reduced viability of infected macrophages as well as the extent of viral replication. Our data show that strategies targeting MCSF signaling could be used to promote elimination of HIV-1-infected myeloid cells and contribute to the elimination of persistent viral reservoirs.

    IMPORTANCE As the HIV/AIDS research field explores approaches to eliminate HIV-1 in individuals on suppressive antiviral therapy, those approaches will need to eliminate both CD4+ T-cell and myeloid cell reservoirs. Most of the attention has focused on CD4+ T-cell reservoirs and scant attention has been paid to myeloid cell reservoirs. The distinct nature of the infection in myeloid cells versus CD4+ T-cells will likely dictate different approaches in order to achieve their elimination. For CD4+ T-cells, most strategies focus on promoting virus reactivation to promote immune-mediated clearance and/or elimination by viral cytopathicity. Macrophages resist viral cytopathic effects and CD8+ T-cell killing. Therefore, we have explored clearance strategies that render macrophages sensitive to viral cytopathicity. This research helps inform the design of strategies to promote clearance of the macrophage reservoir in infected individuals on suppressive antiviral therapy.

  • Analysis of conformational stability of abnormal prion protein aggregates across the spectrum of Creutzfeldt-Jakob disease prions [PublishAheadOfPrint]

  • The wide phenotypic variability of prion diseases is thought to depend on the interaction of a host genotype with prion strains that have self-perpetuating biological properties enciphered in distinct conformations of the misfolded prion protein, PrPSc. The latter concept is largely based on indirect approaches studying the effect of proteases or denaturing agents on the physicochemical properties of PrPSc aggregates. Furthermore, most data come from studies on rodent-adapted prion strains, making current understanding of the molecular basis of strains and phenotypic variability in naturally occurring diseases, especially in humans, more limited. To fill this gap, we studied the effect of guanidine hydrochloride (GdnHCl) and heating on PrPSc aggregates extracted from 60 sporadic CJD and 6 variant CJD brains. While denaturation curves obtained after exposure of PrPSc to increasing GndHCl concentrations showed a similar profile among the 7 CJD types analysed, PrPSc exposure to increasing temperature revealed significantly different and type-specific responses. In particular, MM1 and VV2, the most prevalent and faster replicating CJD types, showed stable and highly resistant PrPSc aggregates, whereas VV1, a rare and slow propagating type, revealed unstable aggregates that easily dissolved at low temperature. Taken together our results indicate that the molecular interactions mediating the aggregation state of PrPSc, possibly enciphering strain diversity, are differently targeted by GdnHCl, temperature and proteases. Furthermore, the detected positive correlation between thermo-stability of PrPSc aggregates and disease transmission efficiency makes inconsistent the proposed hypothesis that a decrease in conformational stability of prions results in an increase in their replication efficiency.

    IMPORTANCE Prion strains are defined as infectious isolates propagating distinctive phenotypic traits after transmission to syngenic hosts. Although the molecular basis of prion strains is not fully understood, it is largely accepted that variations in prion protein conformation drive the molecular changes leading to the different phenotypes. In this study, we exposed abnormal prion protein aggregates, encompassing the spectrum of human prion strains, to both guanidine hydrochloride and thermal unfolding. Remarkably, while exposure to increasing temperature revealed significant strain-specific differences in the denaturation profile of the protein, treatment with guanidine hydrochloride did not. The findings suggest that thermal and chemical denaturation perturb the structure of prion protein aggregates differently. Moreover, since the most thermo-stable prion protein types were those associated with the most prevalent phenotypes and most rapidly and efficiently transmitting strains, the results suggest a direct correlation between the strain replication efficiency and the thermo-stability of prion protein aggregates.

  • Evolution of Neuroadaptation in the Periphery and Purifying Selection in the Brain Contribute to Compartmentalization of Simian Immunodeficiency Virus (SIV) in the Brain of Rhesus Macaques with SIV-Associated Encephalitis [PublishAheadOfPrint]

  • The emergence of a distinct subpopulation of human and simian immunodeficiency virus (HIV/SIV) sequences within the brain (compartmentalization) during infection is hypothesized to be linked with acquired immunodeficiency syndrome (AIDS)-related central nervous system (CNS) neuropathology. However, the exact evolutionary mechanism responsible for HIV/SIV brain compartmentalization has not been thoroughly investigated. Using extensive viral sampling from several different peripheral tissues and cell types as well as three distinct regions within the brain from two well-characterized rhesus macaque models of neuroAIDS, we have been able to perform in-depth evolutionary analyses that have been unattainable in HIV-infected subjects. The results indicate that, despite multiple introductions of virus into the brain over the course of infection, brain sequence compartmentalization in macaques with SIV-associated CNS neuropathology likely results from late viral entry of virus that has acquired through evolution in the periphery sufficient adaptation for the distinct microenvironment of the CNS.

    IMPORTANCE HIV-associated neurocognitive disorders remain prevalent among HIV type 1-infected individuals, whereas our understanding of the critical components of disease pathogenesis, such as virus evolution and adaptation, remains limited. Building upon earlier findings of specific viral subpopulations in the brain, we present novel yet fundamental results concerning the evolutionary patterns driving this phenomenon in two well-characterized animal models of neuroAIDS and provide insight into the timing of entry of virus into the brain and selective pressure associated with viral adaptation to this particular microenvironment. Such knowledge is invaluable for therapeutic strategies designed to slow or even prevent neurocognitive impairment associated with AIDS.

  • Highly pathogenic avian influenza H5N6 viruses exhibit enhanced affinity for human type sialic acid receptor and in-contact transmission in model ferrets [PublishAheadOfPrint]

  • Since May 2014, highly pathogenic avian influenza (HPAI) H5N6 virus has been reported to cause six severe human infections three of which were fatal. The biological properties of this subtype, in particular its relative pathogenicity and transmissibility in mammals are not known. We characterized the virus receptor binding affinity, pathogenicity and transmissibility in mice and ferrets of four H5N6 isolates derived from waterfowl in China from 2013-2014. All four H5N6 viruses have acquired binding affinity for human-like SAaalpha;2,6Gal linked receptor to be able to attach to human tracheal epithelial and alveolar cells. The emergent H5N6 viruses, which share high sequence similarity with the human isolate A/Guangzhou/39715/2014 (H5N6), were fully infective and highly transmissible by direct contact in ferrets but showed less severe pathogenicity in comparison with their parental H5N1 virus. The present results highlight the threat of emergent H5N6 viruses to poultry and human health and the need to closely track their continual adaptation in humans.

    IMPORTANCE Extended epizootics and panzootics of H5N1 viruses have led to the emergence of the novel clade of H5 virus subtypes including H5N2, H5N6 and H5N8 reassortants. Avian H5N6 viruses from this clade have caused three fatalities out of six severe human infections in China since the first case in 2014. However, the biological properties of this subtype, especially the pathogenicity and transmission in mammals are not known. Here, we found that natural avian H5N6 viruses have acquired high affinity for human-type virus receptor. In comparison with parental clade 2.3.4 H5N1 virus, emergent H5N6 isolates showed less severe pathogenicity in mice and ferrets, but acquired efficient in-contact transmission in ferrets. These findings suggest that the threat of avian H5N6 viruses to humans should not be ignored.

  • Monoclonal antibodies to different components of the human cytomegalovirus (HCMV) pentamer gH/gL/pUL128L and trimer gH/gL/gO as well as antibodies elicited during primary HCMV infection prevent epithelial cell syncytium formation [PublishAheadOfPrint]

  • Human cytomegalovirus (HCMV) may cause disseminated/end-organ disease in congenitally infected newborns and immunosuppressed transplant recipients. Two glycoprotein complexes gHgLgO and gH/gL/pUL128/pUL130/pUL131 (gH/gL/pUL128L, referred to as the pentamer) are required for HCMV entry into fibroblasts and endothelial/epithelial cells, respectively, in the presence of the viral fusion protein gB. In addition, gH/gL/gO has recently been reported to be required also for infection of endothelial/epithelial cells. Virus entry into human fibroblasts involves fusion of the virus envelope with the plasma membrane, whereas entry into endothelial/epithelial cells involves macropinocytosis or endocytosis and low-pH-dependent fusion with endosomes. A large set of neutralizing monoclonal antibodies (mAbs) directed to gH, gB, and multiple components of the pentamer was developed. In addition, novel anti-gO human monoclonal antibodies were recently isolated. It is known that epithelial cell infection with a HCMV wild strain at a high multiplicity of infection produces a large number of syncytia. Incubation of heavily HCMV VR1814-infected ARPE-19 epithelial cells with neutralizing mAbs to one, two or three components of the pUL128L portion of the pentamer blocked syncytium formation at an antibody concentration of 10mmu;g/ml, whereas only a partial inhibitory effect was displayed by mAbs to gO, gH, or gB at the same concentration. A blocking effect was also exhibited by convalescent-phase sera from primary HCMV infection. These findings indicate that the pentamer is required for syncytium formation in epithelial cells.

    IMPORTANCE Human cytomegalovirus (HCMV) mostly infects epithelial and endothelial cells in vivo. Recently, the pentamer protein complex gH/gL/pUL128L has been identified as required for infection of these cells, in association with the other protein complex gH/gL/gO. In primary infections, HCMV migrates to endothelial cells and then to leukocytes, which disseminate the infection throughout the body. Then, virus spreads to organs and tissues, mostly yielding either single or multinucleated epithelial giant cells (syncytia), depending on the viral load. Potent neutralizing human mAbs directed to distinct binding sites of the pUL128L portion of the pentamer were shown in the past to block virus dissemination. Here, mAbs to pUL128L are shown to block syncytium formation, with higher potency than mAbs to gO, gH or gB, thus suggesting their role in limiting virus dissemination. This finding provides additional information useful for the development of anti-HCMV therapeutic antibodies and subunit vaccines.

  • Characterization of human and murine T-cell immunoglobulin mucin domain 4 (TIM-4) IgV domain residues critical for Ebola virus entry [PublishAheadOfPrint]

  • Phosphatidylserine (PtdSer) receptors that are responsible for the clearance of dying cells have recently been appreciated to mediate enveloped virus entry. Ebola virus (EBOV), a member of the Filoviridae family of viruses, utilizes PtdSer receptors for entry into target cells. The PtdSer receptors, human and murine T-cell immunoglobulin mucin domain proteins, TIM-1 and TIM-4, mediate filovirus entry by binding to PtdSer on the surface of virions via a conserved PtdSer binding pocket within the amino terminal IgV domain. While the residues within the TIM-1 IgV domain that are important for EBOV entry are characterized, the molecular details of virion/TIM-4 interactions have yet to be investigated. As sequences and structural alignments of the TIM proteins suggest distinct differences in the TIM-1 and TIM-4 IgV domain structures, we sought to characterize TIM-4 IgV domain residues required for EBOV entry. Using vesicular stomatitis virus pseudovirions bearing EBOV glycoprotein (EBOV GP/VSVG), we evaluated virus binding and entry into cells expressing TIM-4 molecules mutated within the IgV domain, allowing us to identify residues important for entry. Similar to TIM-1, residues in the PtdSer binding pocket of murine and human TIM-4 (mTIM-4 and hTIM-4) were found to be important for EBOV entry. However, additional TIM-4-specific residues were also identified to impact EBOV entry, with a total of eight mTIM-4 and fourteen hTIM-4 IgV domain residues being critical for virion binding and internalization. Together these findings provide a greater understanding of the interaction of TIM-4 with EBOV virions.

    IMPORTANCE With more than 28,000 cases and over 11,000 deaths during the largest and most recent Ebola virus (EBOV) outbreak, there has been increased emphasis on the development of therapeutics against filoviruses. Many therapies under investigation target EBOV cell entry. T-cell immunoglobulin mucin domain (TIM) proteins are cell surface factors important for entry of many enveloped viruses, including EBOV. TIM family member, TIM-4, is expressed on macrophages and dendritic cells, which are early cellular targets during EBOV infection. Here, we performed a mutagenesis screen of the IgV domain of murine and human TIM-4 to identify residues that are critical for EBOV entry. Surprisingly, we identified more human TIM-4 IgV domain residues that are required for EBOV entry than murine TIM-4 residues. Defining TIM IgV residues needed for EBOV entry clarifies the virus/receptor interactions and paves the way for the development of novel therapeutics targeting virus binding to this cell surface receptor.

  • Furin Cleavage of L2 During Papillomavirus Infection: Minimal Dependence on Cyclophilins [PublishAheadOfPrint]

  • Despite an abundance of evidence supporting an important role for the cleavage of minor capsid protein L2 by cellular furin, direct cleavage of capsid-associated L2 during Human Papillomavirus Type 16 (HPV16) infection remains poorly characterized. The conserved cleavage site, close to the L2 N-terminus, confounds observation and quantification of the small cleavage product by SDS-PAGE. To overcome this difficulty, we increased the size shift by fusing a compact protein domain, the Propionibacterium shermanii transcarboxylase domain (PSTCD), to the N-terminus of L2. The infectious PSTCD-L2 virus displayed an appreciable L2 size shift during infection of HaCaT keratinocytes. Cleavage under standard cell culture conditions rarely exceeded 35% of total L2. Cleavage levels were enhanced by the addition of exogenous furin and the absolute levels of infection correlated to the level of L2 cleavage. Cleavage occurs on both the HaCaT cell surface and ECM. Contrary to current models, experiments on the involvement of cyclophilins revealed little if any role for these cellular enzymes in the modulation of furin cleavage. HPV16 L2 contains two consensus cleavage sites, Arg5 (2RHKR5) and Arg12 (9RTKR12). Mutant PSTCD-L2 viruses demonstrated that although furin can cleave either site, cleavage must occur at Arg12 as cleavage at Arg5 alone is insufficient for successful infection. Mutation of the conserved cysteine residues reveal that the Cys22-Cys28 disulfide bridge is not required for cleavage. The PSTCD-L2 virus or similar N-terminal fusions will be valuable tools to study additional cellular and viral determinants of furin cleavage.

    IMPORTANCE Furin cleavage of minor capsid protein L2 during papillomavirus infection has been difficult to directly visualize and quantify, confounding efforts to study this important step of HPV infection. Fusion of a small protein domain to the N-terminus greatly facilitates direct visualization of the cleavage product, revealing important characteristics of this critical process. Contrary to the current model, we find that cleavage is largely independent of cyclophilins, suggesting that cyclophilins either act in parallel or downstream of furin to trigger exposure of a conserved N-terminal L2 epitope (RG-1) during infection. Based on this finding, we strongly caution against using L2 RG-1 epitope exposure as a convenient but indirect proxy of furin cleavage.

  • NSs virulence factor of Rift Valley fever virus engages the F-box proteins FBXW11 and {beta}-TRCP1 to degrade the antiviral protein kinase PKR [PublishAheadOfPrint]

  • Rift Valley fever virus (RVFV, family Bunyaviridae, genus Phlebovirus) is a relevant pathogen of both humans and livestock in Africa. The non-structural protein NSs is a major virulence factor, known to suppress the type I interferon (IFN) response by inhibiting host cell transcription and by proteasomal degradation of a major antiviral IFN effector, the translation-inhibiting protein kinase PKR. Here, we identified components of the modular SCF (Skp1, Cul1, F-Box protein) - type E3-ubiquitin ligases as mediators of PKR destruction by NSs. siRNAs against the conserved SCF subunit Skp1 protected PKR from NSs-mediated degradation. Consequently, RVFV replication was severely reduced in Skp1-depleted cells when PKR was present. SCF complexes have a variable F-box protein subunit that determines the substrate specificity for ubiquitination. We performed an siRNA screen for all (about 70) human F-box proteins and identified FBXW11 to be involved in PKR degradation. The partial stabilization of PKR by FBXW11 depletion upregulated PKR autophosphorylation and phosphorylation of the PKR substrate eIF2aalpha;, and caused a shutoff of host cell protein synthesis in RVFV-infected cells. To maximally protect PKR from NSs action, knockdown of the structurally and functionally related FBXW1 (also known as bbeta;-TRCP1) in addition to FBXW11 deletion was necessary. Consequently, NSs was found to interact with both FBXW11 and bbeta;-TRCP1. Thus, NSs eliminates the antiviral kinase PKR by recruitment of SCF-type E3-ubiquinin ligases containing FBXW11 and bbeta;-TRCP1 as substrate recognition subunits. This antagonism of PKR by NSs is essential for efficient RVFV replication in mammalian cells.

    SIGNIFICANCE Rift Valley fever virus is a pathogen of humans and animals that has the potential to spread from Africa and the Arabian Peninsula to other continents. A major virulence mechanism is the proteasomal degradation of the antiviral kinase PKR by the viral protein NSs. Here, we demonstrate that NSs requires E3-ubiquitin ligase complexes of the SCF (Skp1, Cul1, F-Box protein) - type to destroy PKR. SCF-type complexes can engage variant ubiquitination substrate recognition subunits, and we found the F-box proteins FBXW11 and bbeta;-TRCP1 as being relevant for the NSs action against PKR. Thus, we identified the host cell factors that are critically needed by Rift Valley fever virus to uphold its replication against the potent antiviral kinase PKR.

  • NOD1 participates in the innate immune response triggered by the hepatitis C viral polymerase [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) triggers innate immunity signalling in the infected cell. Replication of the viral genome is dispensable for this phenotype and we and others have recently shown that NS5B, the viral RNA dependent RNA polymerase synthesizes double stranded RNA (dsRNA) from cellular templates thus eliciting an inflammatory response, notably via activation of type I interferon and lymphotoxin bbeta;. Here we investigated intracellular signal transduction pathways involved in this process. Using HepaRG cells, a model that largely recapitulates the in vivo complexities of the innate immunity receptors signaling, we have confirmed that NS5B triggered increased expression of the canonical pattern recognition receptors specific for dsRNA, namely RIG-I, MDA5 and TLR3. Unexpectedly, intracellular dsRNA also led to accumulation of NOD1, a receptor classically involved in recognition of bacterial peptidoglycans. NOD1 activation, confirmed by analysis of its downstream targets, was likely due to its interaction with dsRNA and was independent of RIG-I and MAVS signaling. It is likely to have a functional significance in the cellular response in the context of HCV infection since interference with NOD1 pathway severely reduced inflammatory response elicited by NS5B.

    IMPORTANCE In this study we show that NOD1, a PRR that normally senses bacterial peptidoglycans, is activated by HCV viral polymerase, probably through an interaction with dsRNA, suggesting that NOD1 acts as an RNA ligand recognition receptor. In consequence, interference with NOD1-mediated signaling significantly weakens the inflammatory response to dsRNA. These results add a new level of complexity to the understanding of the crosstalk between different classes of pattern recognition receptors and may be related to certain complications of chronic hepatitis C.

  • The Herpes Simplex Virus Virion Host Shutoff Protein Enhances Translation of Viral True Late mRNAs Independently of Suppressing Protein Kinase R and Stress Granule Formation [PublishAheadOfPrint]

  • The herpes simplex virus virion host shutoff (vhs) ribonuclease destabilizes cellular and viral mRNAs, suppresses host protein synthesis, dampens antiviral responses, and stimulates translation of viral mRNAs. Vhs mutants display a host range phenotype: translation of viral true late mRNAs is severely impaired and stress granules accumulate in HeLa cells, while translation proceeds normally in Vero cells. We found that vhs-deficient virus activates the double stranded RNA activated protein kinase PKR much more strongly than wild-type virus in HeLa cells, while PKR is not activated in Vero cells, raising the possibility that PKR might play roles in stress granule induction and/or inhibiting translation in restrictive cells. We tested this possibility by evaluating the effects of inactivating PKR. Eliminating PKR in HeLa cells abolished stress granule formation but had only minor effects on viral true late protein levels. These results document an essential role for PKR in stress granule formation by a nuclear DNA virus, indicate that induction of stress granules is the consequence rather than the cause of the translational defect, and are consistent with our previous suggestion that vhs promotes translation of viral true late mRNAs by preventing mRNA overload rather than by suppressing eIF2aalpha; phosphorylation.

    IMPORTANCE The herpes simplex virus vhs ribonuclease plays multiple roles during infection, including suppressing PKR activation, inhibiting the formation of stress granules, and promoting translation of viral late mRNAs. A key question is the extent to which these activities are mechanistically connected. Our results demonstrate that PKR is essential for stress granule formation in the absence of vhs, but plays at best a secondary role in suppressing translation of viral mRNAs. Thus, the ability of vhs to promote translation of viral mRNAs can be largely uncoupled from PKR suppression, demonstrating that this viral RNase modulates at least two distinct aspects of RNA metabolism.

  • Hepatitis B surface antigen activates myeloid dendritic cells via a soluble CD14-dependent mechanism. [PublishAheadOfPrint]

  • Hepatitis B virus (HBV) infection can cause chronic liver disease which is associated with increased risk of liver cirrhosis, liver failure and liver cancer. Clearance of HBV infection requires effective HBV-specific immunity, however the immunological mechanisms that determine the development of effective HBV-specific immunity are poorly understood. Dendritic cells (DC) play a pivotal role in the regulation of anti-viral immunity. Here, we investigated the interaction between HBV surface antigen (HBsAg), the main envelop glycoprotein of HBV, and BDCA1+ myeloid dendritic cells (mDC).

    Exposure of peripheral blood-derived BDCA1+ mDC to HBsAg resulted in strong DC maturation, cytokine production and enhanced capacity to activate antigen-specific CTL. By using neutralizing antibodies, crucial roles for CD14 and TLR4 in HBsAg-mediated BDCA1+ mDC maturation were identified. Concordantly, HBsAg-mediated DC maturation required FCS or human plasma, naturally containing soluble CD14 (sCD14). Intriguingly, HBsAg-induced DC maturation was significantly reduced in umbilical cord blood-plasma that contained less sCD14 than adult plasma, indicating that sCD14 is an important host factor for recognition of HBsAg by DC and subsequent DC activation. A direct interaction between sCD14 and HBsAg was demonstrated by using ELISA. Moreover, sCD14-HBsAg complexes were detected both in vitro and in serum of HBV-infected patients. Abundance of sCD14-HBsAg complexes varied between chronic HBV disease stages and correlated with activation of BDCA1+ mDC in vivo.

    We conclude that HBsAg activates BDCA1+ DC via a sCD14-dependent mechanism. These findings provide important novel insights into initiation of HBV-specific immunity and facilitate development of effective immunotherapeutic interventions for HBV.

    IMPORTANCE Hepatitis B virus (HBV) infection is a significant health problem as it causes progressive liver injury and liver cancer in patients with chronic HBV infection, which affects approximately 250 million individuals worldwide. Part of adults and the majority of neonates fail to mount an effective immune response and consequently develop chronic infection. The viral and host factors involved in the initiation of effective HBV-specific immune responses remain poorly understood. Here we identified CD14 and TLR4 as receptors for HBsAg, the main HBV envelop antigen. HBsAg induced strong maturation of dendritic cells (DC), which have a central role in regulation of virus-specific immunity. These results provide essential novel insights into the mechanisms underlying initiation of HBV-specific immunity. Intriguingly, since neonates have naturally low sCD14, the finding that serum-derived sCD14 is a crucial host factor for recognition of HBsAg by DC may have implications for immunity of neonates to HBV infection.

  • Rotavirus NSP1 associates with components of the cullin RING ligase family of E3 ubiquitin ligases [PublishAheadOfPrint]

  • The rotavirus nonstructural protein NSP1 acts as an antagonist of the host antiviral response by inducing degradation of key proteins required to activate interferon (IFN) production. Protein degradation induced by NSP1 is dependent on the proteasome, and the presence of a RING domain near the N-terminus has led to the hypothesis that NSP1 is an E3 ubiquitin ligase. To examine this hypothesis, pull-down assays were performed followed by mass spectrometry to identify components of the host ubiquitination machinery that associate with NSP1. Multiple components of cullin RING ligases (CRLs), which are essential multi-subunit ubiquitination complexes, were identified in association with NSP1. The mass spectrometry was validated in both transfected and infected cells to show that the NSP1 protein from different strains of rotavirus associated with key components of CRL complexes, most notably the cullin scaffolding proteins Cul3 and Cul1. In vitro binding assays using purified proteins confirmed that NSP1 specifically interacted with Cul3, and the N-terminal region of Cul3 was responsible for binding to NSP1. To test if NSP1 was using CRL3 to induce degradation of the target proteins IRF3 or bbeta;-TrCP, Cul3 levels were knocked-down using an siRNA approach. Unexpectedly, loss of Cul3 did not rescue IRF3 or bbeta;-TrCP from degradation in infected cells. The results indicate that rather than actively using CRL complexes to induce degradation of target proteins required for IFN production, NSP1 may be using cullin-containing complexes to prevent another cellular activity.

    IMPORTANCE The ubiquitin-proteasome pathway plays an important regulatory role in numerous cellular functions, and many viruses have evolved mechanisms to exploit or manipulate this pathway to enhance replication and spread. Rotavirus, a major cause of severe gastroenteritis in young children that causes approximately 420,000 deaths worldwide each year, utilizes the ubiquitin-proteasome system to subvert the host innate immune response by inducing the degradation of key components required for the production of IFN. Here we show that NSP1 from different rotavirus strains associate with the scaffolding proteins Cul1 and Cul3 of CRL ubiquitin ligase complexes. Nonetheless, knockdown of Cul1 and Cul3 suggest that NSP1 induces the degradation of some target proteins independently of its association with CRL complexes, stressing a need to further investigate the mechanistic details of how NSP1 subverts the host IFN response.

  • The autophagosomal SNARE protein syntaxin 17 is an essential factor for the hepatitis C virus life cycle [PublishAheadOfPrint]

  • Syntaxin 17 is an autophagosomal SNARE (Soluble N-ethyl maleimidesensitive factor attachment protein receptor) required for the fusion of autophagosomes with lysosomes to form autolysosomes and thereby to deliver the enclosed contents for degradation. Hepatitis C virus (HCV) induces autophagy. In light of the observation that much more viral particles are formed than infectious viral particles are finally released by HCV-infected cells, regulation of fusion between autophagosomes and lysosomes might fulfill a key function controlling the number of released virions.

    HCV-replicating cells possess a decreased amount of syntaxin 17 due to impaired expression and increased turnover of syntaxin 17. Overexpression of syntaxin 17 in HCV-replicating cells diminishes the number of released infectious viral particles and decreases the amount of intracellular retained viral particles by favoring the formation of autolysosomes, in which HCV particles are degraded. Inhibition of lysosomal acidification by bafilomycin rescues the decreased release of virions from syntaxin 17-overexpressing cells, while induction of autophagy by rapamycin enforces the impairment of release under these conditions. Vice versa inhibition of syntaxin 17 expression by specific siRNAs results in elevated amount of intracellular retained viral particles and facilitates the release of HCV virions by impairment of autophagosomal-lysosomal fusion. HCV genome replication, however, is not affected by modulation of syntaxin 17 expression.

    These data identify syntaxin 17 as a novel factor controlling the release of HCV. This is achieved by regulation of autophagosome-lysosome fusion that affects the equilibrium between release of infectious viral particles and lysosomal degradation of intracellular retained viral particles.

    IMPORTANCE Hepatitis C virus (HCV) induces autophagy. Syntaxin 17 is an autophagosomal SNARE required for the fusion of autophagosomes with lysosomes. In HCV-infected cells a major fraction of the de novo synthesized viral particles is not released but intracellularly degraded. In this context the effect of HCV on the amount and distribution of syntaxin 17 and the relevance of syntaxin 17 for the viral life cycle were investigated. This study demonstrates that in HCV-replicating cells the amount of syntaxin 17 decreased. In addition, syntaxin 17 is identified as a novel factor controlling the release of HCV and the relevance of autophagosome-lysosome fusion as a regulator for the amount of released viral particles is revealed. Taken together, syntaxin 17 is involved in the regulation of autophagosome-lysosome fusion and thereby affects the equilibrium between release of infectious viral particles and lysosomal degradation of intracellularly retained viral particles.

  • Discovery of a co-regulatory interaction between KSHV ORF45 and the viral protein kinase ORF36 [PublishAheadOfPrint]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of three human malignancies. KSHV ORF36 encodes a serine/threonine viral protein kinase, which is conserved throughout all herpesviruses. Although several studies have identified the viral and cellular substrates of conserved herpesvirus protein kinases (CHPKs), the precise functions of KSHV ORF36 during lytic replication remain elusive. Here, we report that ORF36 interacts with another lytic protein, ORF45, in a manner dependent on ORF36 kinase activity. We mapped the regions of ORF36 and ORF45 involved in the binding. Their association appears to be mediated by electrostatic interactions, since deletion of either the highly basic N-terminus of ORF36 or an acidic patch of ORF45 abolished the binding. Additionally, dephosphorylation of ORF45 protein dramatically reduced its association with ORF36. Importantly, ORF45 enhances both the stability and kinase activity of ORF36. Consistent with previous studies of CHPK homologs, we detected ORF36 protein in extracellular virions. To investigate the roles of ORF36 in the context of KSHV lytic replication, we employed BAC mutagenesis to engineer both ORF36-null and kinase-dead (KD) mutants. We found that ORF36-null/mutant virions are moderately defective in viral particle production and are further deficient in primary infection. In summary, our results uncover a functionally important interaction between ORF36 and ORF45, and indicate a significant role of ORF36 in the production of infectious progeny virions.

    IMPORTANCE STATEMENT Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus with a significant public health burden. KSHV ORF36 encodes a serine/threonine viral protein kinase, whose functions throughout the viral life cycle have not been elucidated. Here, we report that ORF36 interacts with another KSHV protein, ORF45. We mapped the regions of ORF36 and ORF45 involved in their association, and further characterized the consequences of this interaction. We engineered ORF36 mutant viruses in order to investigate the functional roles of ORF36 in the context of KSHV lytic replication, and confirmed that ORF36 is a component of KSHV virions. Moreover, we found that ORF36 mutants are defective in virion production and primary infection. In summary, we discovered and characterized a functionally important interaction between KSHV ORF36 and ORF45, and our results suggest a significant role of ORF36 in the production of infectious progeny virions, a process critical for KSHV pathogenesis.

  • Role of WDHD1 in human papillomavirus-mediated oncogenesis identified by transcriptional profiling of E7 expressing cells [PublishAheadOfPrint]

  • The E7 oncoprotein of the high-risk human papillomavirus (HPV) plays a major role in HPV-induced carcinogenesis. E7 abrogates the G1 cell cycle checkpoint and induces genomic instability but the mechanism is not fully understood. In this study, we performed RNA-seq to characterize the transcriptional profile of keratinocytes expressing HPV-16 E7. At the transcriptome level, two hundred thirty six genes were differentially expressed between E7 and vector control cells. A subset of the differentially expressed genes, most of them novel to E7 expressing cells, was further confirmed by real-time PCR. Of interest, the activities of multiple transcription factors were altered in E7 expressing cells. Through bioinformatics analysis, pathways altered in E7 expressing cells were investigated. The upregulated genes were enriched in cell cycle and DNA replication, as well as DNA metabolic process, transcription, DNA damage, DNA repair and nucleotide metabolism. Specifically, we focused our studies on WDHD1 (WD repeat and HMG-box DNA-binding protein), one of the genes that was upregulated in E7 expressing cells. WDHD1 is a component of the replisome that regulates DNA replication. Recent studies suggest that WDHD1 may also function as a DNA replication initiation factor as well as a G1 checkpoint regulator. We found that in E7 expressing cells, the steady-state level of WDHD1 protein was increased along with half-life. Moreover, downregulation of WDHD1 reduced E7-induced G1 checkpoint abrogation and re-replication, demonstrating a novel function for WDHD1. These studies shed light on mechanisms by which HPV induces genomic instability and have therapeutic implications.

    IMPORTANCE The high-risk HPV types induce cervical cancer and encode an E7 oncoprotein that plays a major role in HPV-induced carcinogenesis. However, the mechanism by which E7 induces carcinogenesis is not fully understood; specific anti-HPV agents are not available. In this study, we performed RNA-seq to characterize transcriptional profiling of keratinocytes expressing HPV-16 E7 and identified more than two hundred genes that were differentially expressed between E7 and vector control cells. Through bioinformatics analysis, pathways altered in E7 expressing cells were identified. Significantly, WDHD1, one of the genes that is upregulated in E7 expressing cells, was found to play an important role in E7-induced G1 checkpoint abrogation and re-replication. These studies shed light on mechanisms by which HPV induces genomic instability and have therapeutic implications.

  • HIV-1 Protease, Reverse Transcriptase, and Integrase Variation [PublishAheadOfPrint]

  • HIV-1 protease (PR), reverse transcriptase (RT), and integrase (IN) variability presents a challenge to laboratories performing genotypic resistance testing. This challenge will grow with increased sequencing of samples enriched for proviral DNA such as dried blood spots and increased use of next-generation sequencing (NGS) to detect low-abundance HIV-1 variants. We analyzed PR and RT sequences from ggt;100,000 individuals and IN sequences from ggt;10,000 individuals to characterize variation at each amino acid position, identify mutations indicating APOBEC-mediated G-to-A editing, and identify mutations resulting from selective drug pressure. Forty-seven percent of PR, 37% of RT and 34% of IN positions had one or more amino acid variants with a prevalence gge;1%. Seventy percent of PR, 60% of RT and 60% of IN positions had one or more variants with a prevalence gge;0.1%. Overall 201 PR, 636 RT and 346 IN variants had a prevalence gge;0.1%. The median inter-subtype prevalence-ratio was 2.9-, 2.1- and 1.9-fold for these PR, RT and IN variants, respectively. Only 5.0% of PR, 3.7% of RT and 2.0% of IN variants had a median inter-subtype prevalence-ratio gge;10-fold. Variants at lower prevalences were more likely to differ biochemically and to be part of an electrophoretic mixture compared to high prevalence variants. There were 209 mutations indicative of APOBEC-mediated G-to-A editing and 326 non-polymorphic treatment-selected mutations. Identifying viruses with a high number of APOBEC-associated mutations will facilitate the quality control of dried blood spot sequencing. Identifying sequences with a high proportion of rare mutations will facilitate the quality control of NGS.

    IMPORTANCE: Most antiretroviral drugs target three HIV-1 proteins: PR, RT, and IN. These proteins are highly variable: many different amino acids can be present at the same position in viruses from different individuals. Some of the amino acid variants cause drug resistance and occur mainly in individuals receiving antiretroviral drugs. Some variants result from a human cellular defense mechanism called APOBEC-mediated hypermutation. Many variants result from naturally occurring mutation. Some variants may represent technical artifacts. We studied PR and RT sequences from ggt;100,000 individuals and IN sequences from ggt;10,000 individuals to quantify variation at each amino acid position in these three HIV-1 proteins. We performed analyses to determine which amino acid variants resulted from antiretroviral drug selection pressure, APOBEC-mediated editing, and naturally occurring variation. Our results provide information essential to clinical, research, and public health laboratories performing genotypic resistance testing by sequencing HIV-1 PR, RT, and IN.

  • RNA-free and ribonucleoprotein-associated influenza virus polymerases directly bind the serine-5 phosphorylated carboxyl-terminal domain of host RNA polymerase II [PublishAheadOfPrint]

  • Influenza viruses subvert the transcriptional machinery of their hosts to synthesise their own viral mRNA. Ongoing transcription by cellular RNA polymerase II (Pol II) is required for viral mRNA synthesis. By a process known as cap-snatching, the virus steals short 5' capped RNA fragments from host capped RNAs and uses these to prime viral transcription. An interaction between the influenza A virus RNA polymerase and the C-terminal domain (CTD) of the large subunit of Pol II has been established, but the molecular details of this interaction remain unknown. We show here that influenza virus ribonucleoprotein (vRNP) complex binds to the CTD of transcriptionally engaged Pol II. Furthermore, we provide evidence that the viral polymerase binds directly to the serine-5 phosphorylated form of the Pol II CTD, both in the presence and absence of viral RNA, and show that this interaction is conserved in evolutionarily distant influenza viruses. We propose a model in which direct binding of the viral RNA polymerase in the context of vRNPs to Pol II early in infection facilitates cap-snatching, while we suggest that binding of free viral polymerase to Pol II late in infection may trigger Pol II degradation.

    IMPORTANCE Influenza viruses cause yearly epidemics and occasional pandemics that pose a threat to human health as well as represent a large economic burden to healthcare systems globally. Existing vaccines are not always effective as they may not exactly match the circulating viruses. Furthermore, there are a limited number of antivirals available, and development of resistance to these is a concern. New measures to combat influenza are needed, but before they can be developed it is necessary to better understand the molecular interactions between influenza viruses and their host cells. By providing further insights into the molecular details of how influenza viruses hijack the host transcriptional machinery, we aim to uncover novel targets for development of antivirals.

  • Influenza Hemagglutinin Glycoproteins with Different N-glycan Patterns Activate Dendritic Cells in Vitro [PublishAheadOfPrint]

  • Influenza HA N-glycans serve important regulatory roles in the control of virus virulence, antigenicity, receptor-binding specificity, and viral immune escape. Considered essential for controlling innate and adaptive immune responses against influenza virus infections, DCs trigger pro-inflammatory and adaptive immune responses in hosts. In this study, we engineered CHO cell lines expressing recombinant HA from pandemic H1, H5, and H7 influenza viruses. rH1HA, rH5HA and rH7HA proteins were obtained as wild type or in the presence of kifunensine (KIF), or further with Endo H treated (KIF+E) to generate single GlcNAc N-glycans, consisting of (i) terminally sialylated complex type N-glycans, (ii) high mannose-type N-glycans, and (iii) single-N-acetylglucosamine (GlcNAc)-type N-glycans. Our results show that high mannose-type and single GlcNAc-type N-glycans, but not complex-type N-glycans, are capable of inducing more active hIL12 p40, hIL12 p70 and hIL-10 production in human DCs. The significantly increased expression levels of the HLA-DR, CD40, CD83 and CD86, as well reduced endocytotic capacity in human DC were noted in the high mannose-type rH1HA and single GlcNAc-type rH1HA groups compared to the complex type N-glycans rH1HA group. Our data indicate that native avian rHA (H5N1 and H7N9) are more immunostimulatory than human rHA (pH1N1). The high-mannose type or single GlcNAc type N-glycans of both avian and human HA types are more stimulatory than the complex type N-glycans. The HA-stimulated DC activation was partially through mannose receptor(s). These results provide more understanding of the contribution of glycosylation of viral proteins to the immune responses and may have implications for vaccine development.

    IMPORTANCE Influenza viruses trigger seasonal epidemics or pandemics with mild-to-severe consequences for human and poultry populations. DCs are the most potent professional antigen-presenting cells, which play a crucial role for the link of innate and adaptive immunity. In this study, we obtained stable-expression CHO cells to produce rH1HA, rH5HA and rH7HA proteins containing distinct N-glycans patterns. These rHA proteins, each with a distinct N-glycan pattern, were used to investigate interactions with mouse and human DCs. Our data indicate that native avian rHA (H5N1 and H7N9) are more immunostimulatory than human rHA (pH1N1). High mannose type- and single GlcNAc-type N-glycans were more effective than complex-type N-glycans in triggering mouse and human DC activation and maturation. We believe these results provide some useful information for influenza vaccine development regarding how influenza HA proteins with different types of N-glycans activate DCs.

  • Interferon alpha Subtype-specific Suppression of HIV-1 Infection in vivo [PublishAheadOfPrint]

  • Although all twelve subtypes of human interferon alpha (IFNaalpha;) bind the same receptor, recent results demonstrate they elicit unique host responses and display distinct efficacies in the control of different viral infections. The IFNaalpha;2 subtype is currently in HIV-1 clinical trials but it has not consistently reduced viral loads in HIV-1 patients and is not the most effective subtype against HIV-1 in vitro. We now demonstrate in humanized mice that, when delivered at the same high clinical dose, the human IFNaalpha;14 subtype has very potent anti-HIV-1 activity whereas IFNaalpha;2 does not. In both post-exposure prophylaxis and treatment of acute infections, IFNaalpha;14 but not IFNaalpha;2 significantly suppressed HIV-1 replication and proviral loads. Furthermore, HIV-1-induced immune hyper-activation, which is a prognosticator of disease progression, was reduced by IFNaalpha;14 but not IFNaalpha;2. Whereas ineffective IFNaalpha;2 therapy was associated with CD8+ T cell activation, successful IFNaalpha;14 therapy was associated with increased intrinsic and innate immunity including significantly higher induction of tetherin and MX2, increased APOBEC3G signature mutations in HIV-1 proviral DNA, and higher frequencies of TRAIL+ NK cells. These results identify IFNaalpha;14 as a potent new therapeutic that operates via mechanisms distinct from antiretroviral drugs. The ability of IFNaalpha;14 to reduce both viremia and proviral loads in vivo suggests that it has strong potential as a component of a cure strategy for HIV-1 infections. The broad implication of these results is that the antiviral efficacy of each individual IFNaalpha; subtype should be evaluated against the specific virus being treated.

    IMPORTANCE The naturally occurring antiviral protein, IFNaalpha;2, is used to treat hepatitis viruses but has proven rather ineffective against HIV in comparison to triple therapy with the antiretroviral (ARV) drugs. Although ARVs suppress the replication of HIV, they fail to completely clear infections. Since IFNaalpha; acts by different mechanism than ARVs and been show to reduce HIV proviral loads, clinical trials are currently underway to test whether IFNaalpha;2 combined with ARVs might eradicate HIV-1 infections. IFNaalpha; is actually a family of 12 distinct proteins and each IFNaalpha; subtype has different efficacies toward different viruses. Here we use mice that contain a human immune system such that they can be infected with HIV. With this model we demonstrate that while IFNaalpha;2 is only weakly effective against HIV, IFNaalpha;14 is extremely potent. This discovery identifies IFNaalpha;14 as a more powerful IFNaalpha; subtype for use in combination therapy trials aimed toward an HIV cure.

  • The SUMO Ligase Protein Inhibitor of Activated STAT 1 (PIAS1) is a constituent PML-NB protein that contributes to the intrinsic antiviral immune response to herpes simplex virus 1 (HSV-1). [PublishAheadOfPrint]

  • Aspects of intrinsic antiviral immunity are mediated by promyelocytic leukaemia (PML)-nuclear body (PML-NB) constituent proteins. During herpesvirus infection, these antiviral proteins are independently recruited to nuclear domains that contain infecting viral genomes to cooperatively promote viral genome silencing. Central to the execution of this particular antiviral response is the small ubiquitin-like modifier (SUMO) signalling pathway. However, the participating SUMOylation enzymes are not fully characterized. We identify the SUMO ligase Protein Inhibitor of Activated STAT1 (PIAS1) as a constituent PML-NB protein. We show that PIAS1 localizes at PML-NBs in a SUMO interaction motif (SIM)-dependent manner that requires SUMOylated or SUMOylation competent PML. Following infection with herpes simplex virus 1 (HSV-1), PIAS1 is recruited to nuclear sites associated with viral genome entry in a SIM-dependent manner, consistent with the SIM-dependent recruitment mechanisms of other well characterized PML-NB proteins. In contrast to Daxx and Sp100, however, the recruitment of PIAS1 is enhanced by PML. PIAS1 promotes the stable accumulation of SUMO1 at nuclear sites associated with HSV-1 genome entry, whereas the accumulation of other evaluated PML-NB proteins occurs independently of PIAS1. We show that PIAS1 cooperatively contributes to HSV-1 restriction through mechanisms that are additive to those of PML and cooperative with those of PIAS4. The antiviral mechanisms of PIAS1 are counteracted by ICP0, the HSV-1 SUMO-targeted ubiquitin ligase, which disrupts the recruitment of PIAS1 to nuclear domains that contain infecting HSV-1 genomes through mechanisms that do not directly result in PIAS1 degradation.

    IMPORTANCE Adaptive, innate, and intrinsic immunity cooperatively and efficiently restrict the propagation of viral pathogens. Intrinsic immunity mediated by constitutively expressed cellular proteins represents the first line of intracellular defence against infection. PML-NB constituent proteins mediate aspects of intrinsic immunity to restrict herpes simplex virus 1 (HSV-1), as well as other viruses. These proteins repress viral replication through mechanisms that rely on SUMO signalling. However, the participating SUMOylation enzymes are not known. We identify the SUMO ligase PIAS1 as a constituent PML-NB antiviral protein. This finding distinguishes a SUMO ligase that may mediate signalling events important in PML-NB-mediated intrinsic immunity. Moreover, this research complements the recent identification of PIAS4 as an intrinsic antiviral factor, supporting a role for PIAS proteins as both positive and negative regulators of host immunity to virus infection.

  • Depletion of Alveolar Macrophages Does Not Prevent Hantavirus Disease Pathogenesis in Golden Syrian Hamsters [PublishAheadOfPrint]

  • Andes virus (ANDV) is associated with a lethal vascular leak syndrome in humans termed hantavirus pulmonary syndrome (HPS). The mechanism for the massive vascular leakage associated with HPS is poorly understood, however dysregulation of components of the immune response is often suggested as a possible cause. Alveolar macrophages are found in the alveoli of the lung and represent the first line of defense to many airborne pathogens. To determine whether alveolar macrophages play a role in HPS pathogenesis, alveolar macrophages were depleted in an adult rodent model of HPS that closely resembles human HPS. Syrian hamsters were treated, intratracheally, with clodronate-encapsulated liposomes or control liposomes and were then challenged with ANDV. Treatment with clodronate-encapsulated liposomes resulted in significant reduction in alveolar macrophages but depletion did not prevent pathogenesis or prolong disease. Depletion also did not significantly reduce the amount of virus in the lung of ANDV-infected hamsters but altered neutrophil recruitment, MIP-1aalpha; and MIP-2 chemokine expression and VEGF levels in hamster BAL early after intranasal challenge. These data demonstrate that alveolar macrophages may play a limited protective role early after exposure to aerosolized ANDV, but do not directly contribute to hantavirus disease pathogenesis in the hamster model of HPS.

    IMPORTANCE Hantaviruses continue to cause disease worldwide for which there are no FDA licensed vaccines, effective post-exposure prophylactics or therapeutics. Much of this can be attributed to a poor understanding of the mechanism of hantavirus disease pathogenesis. Hantavirus disease has long been considered an immune-mediated disease; however, by directly manipulating the Syrian hamster model, we continue to eliminate individual immune cell types. As the most numerous immune cell present in the respiratory tract, alveolar macrophages are poised to defend against hantavirus infection; but, those antiviral responses may also contribute to hantavirus disease. Here, we demonstrate that, like our prior T and B cell studies, alveolar macrophages neither prevent hantavirus infection nor cause hantavirus disease. While these studies reflect pathogenesis in the hamster model, they should help us rule-out specific cell types and prompt us to consider other potential mechanisms of disease in an effort to improve the outcome of human HPS.

  • A cluster of paralytic poliomyelitis cases due to transmission of slightly diverged Sabin-2 vaccine virus [PublishAheadOfPrint]

  • Four cases of acute flaccid paralysis caused by slightly evolved (Sabin-like) vaccine polioviruses of serotype 2 were registered in July-August, 2010 in an orphanage of Biysk (Altai Region, Russia). The Biysk cluster of vaccine-associated paralytic poliomyelitis (VAPP) had several uncommon, if not unique, features (1). Until this outbreak, Sabin-like viruses (in distinction with more markedly evolved vaccine-derived polioviruses, VDPV) were reported to cause only sporadic cases of VAPP. Consequently, VAPP were not considered to require outbreak-type responses. However, the Biysk outbreak completely blurred the borderline between Sabin-like viruses and VDPV in epidemiological terms (2). The outbreak demonstrated a very high disease/infection ratio, apparently exceeding even that reported for wild polioviruses. The viral genome structures did not provide any substantial hints as to the underlying reason(s) for such pathogenicity (3). The replacement of intestinal poliovirus lineages by other Sabin-like lineages during short intervals after the disease onsets was observed in two patients. Again, the sequences of the respective genomes provided no clues to explain these events (4). The polioviruses isolated from the patients and their contacts demonstrated a striking heterogeneity as well as rapid and uneven evolution of the whole genomes and their parts, apparently due to extensive interpersonal contacts in a relatively small closed community, multiple bottlenecking and recombination. Altogether, the results demonstrate several new aspects of pathogenicity, epidemiology, and evolution of vaccine-related polioviruses and underscore several serious gaps in understanding these problems.

    IMPORTANCE. The oral poliovirus vaccine largely contributed to the nearly complete disappearance of poliovirus-caused poliomyelitis. Being generally safe, it can, in some cases, result in a paralytic disease. Two types of such outcomes are distinguished: caused by slightly diverged (Sabin-like) viruses, on the one hand, and by significantly diverged VDPVs, on the other. This classification is based on the number of mutations in the viral genome region encoding a viral structural protein. Until now, only sporadic poliomyelitis cases due to Sabin-like polioviruses were described, and, in distinction with the VDPV-triggered outbreaks, they did not require broad-scale epidemiological responses. Here, an unusual outbreak of poliomyelitis caused by a Sabin-like virus is reported, which had an exceptionally high disease/infection ratio. This outbreak blurred the borderline between Sabin-like polioviruses and VDPV both in pathogenicity and kind of responses required as well as underscores important gaps in understanding pathogenicity, epidemiology, and evolution of vaccine-derived polioviruses.

  • Characterization of epitope-specific anti-RSV antibody responses after natural infection and after vaccination with formalin-inactivated RSV. [PublishAheadOfPrint]

  • Antibodies against the fusion (F) protein of respiratory syncytial virus (RSV) play an important role in the protective immune response against this important respiratory virus. Little is known, however, about antibody levels against multiple F-specific epitopes induced by infection or after vaccination against RSV, while this is important to guide the evaluation of (novel) vaccines. In this study we analyzed antibody levels against RSV proteins and F-specific epitopes in human sera and in sera of vaccinated and experimentally-infected cotton rats; and the correlation thereof with virus neutralization. Analysis of human sera revealed substantial diversity in antibody levels against F-, G (attachment)-, and F-specific epitopes between individuals. The highest correlation with virus neutralization was observed for antibodies recognizing prefusion-specific antigenic site OOslash;. Nevertheless, our results indicate that high levels of antibodies targeting other parts of the F protein can also mediate a potent antiviral antibody response. In agreement herewith, sera of experimentally infected cotton rats contained high neutralizing activity although lacking antigenic site OOslash;-specific antibodies. Strikingly, vaccination with formalin-inactivated (FI)-RSV exclusively resulted in the induction of poorly neutralizing antibodies against postfusion-specific antigenic site I, although antigenic sites I, II and IV were efficiently displayed in FI-RSV. The apparent immunodominance of antigenic site I in FI-RSV likely explains the low levels of neutralizing antibodies upon vaccination and challenge, and may play a role in the vaccination-induced enhancement of disease observed with such preparations.

    IMPORTANCE RSV is an importance cause of hospitalization in infants. The development of a vaccine against RSV has been hampered by the disastrous results obtained with FI-RSV vaccine preparations in the 1960s that resulted in vaccination-induced enhancement of disease. To get a better understanding of the antibody repertoire induced after infection or after vaccination against RSV, we investigated antibody levels against fusion (F), attachment (G) protein and F-specific epitopes in human and animal sera. The results indicate the importance of prefusion-specific antigenic site OOslash; antibodies as well as of antibodies targeting other epitopes in virus neutralization. However, vaccination of cotton rats with FI-RSV specifically resulted in the induction of weakly-neutralizing, antigenic site I-specific antibodies, which may play a role in the enhancement of disease observed after vaccination with such preparations.

  • A single amino acid substitution in the nonstructural protein 2CATPase of poliovirus causes conditional defects in encapsidation and uncoating [PublishAheadOfPrint]

  • The specificity of encapsidation of C-cluster enteroviruses depends on an interaction between capsid proteins and nonstructural protein 2CATPase. In particular, residue N252 of poliovirus 2CATPase interacts with VP3 of coxsackie virus A20, in the context of a chimeric virus. Poliovirus 2CATPase has important roles both in RNA replication and encapsidation. In this study we searched for additional sites in 2CATPase, near N252, that are required for encapsidation. Accordingly, segments adjacent to N252 were analyzed by combining triple and single alanine mutations to identify residues required for function. Two triple alanine mutants exhibited defects in RNA replication. The remaining two mutations, located in secondary structures in a predicted three-dimensional model of 2CATPase, caused lethal growth phenotypes. Most single alanine mutants, derived from the lethal variants, were either quasi-infectious and yielded variants with wt or ts growth phenotypes or had a lethal growth phenotype due to defective RNA replication. The K259A mutation, mapping to an alpha helix in the predicted structure of 2CATPase, resulted in a cold-sensitive virus. In vivo protein synthesis and virus production were strikingly delayed at 33ddeg;C relative to the wt suggesting a defect in uncoating. Studies with a reporter virus indicated that this mutant is also defective in encapsidation at 33ddeg;C. Cell imaging confirmed a much-reduced production of K259A mature virus at 33ddeg;C relative to the wt. In conclusion, we have for the first time linked a cold sensitive encapsidation defect in 2CATPase (K259A) to a subsequent delay in uncoating of the virus particle at 33ddeg;C during the next cycle of infection.

    IMPORTANCE Enterovirus morphogenesis, which involves the encapsidation of newly made virion RNA, is a process still poorly understood. Elucidation of this process is important for future drug development for a large variety of diseases caused by these agents. We have previously shown that the specificity of encapsidation of poliovirus and of C-cluster coxsackie viruses, which are prototypes of enteroviruses, is dependent on an interaction of capsid proteins with the multifunctional nonstructural protein 2CATPase. In this study we have searched for residues in poliovirus 2CATPase, near a presumed capsid interacting site, that are important for encapsidation. An unusual cold sensitive mutant of 2CATPase possessed a defect at 37ddeg;C in encapsidation and subsequently in uncoating during the next cycle of infection at 33ddeg;C. These studies not only reveal a new site in 2CATPase that is involved in encapsidation, but also identify a link between encapsidation and uncoating.

  • Diminished antiviral innate response to Adenovirus vectors in cGAS/STING deficient mice minimally impacts adaptive immunity [PublishAheadOfPrint]

  • Infection by adenovirus, a nonenveloped DNA virus, induces an antiviral innate and adaptive immune response. Studies in transformed human and murine cell lines using shRNA knockdown strategies identified cyclic guanine adenine synthase (cGAS) as a pattern recognition receptor (PRR) that contributes to the anti-adenoviral response. Here we demonstrate how the cGAS/STING cascade influences the antiviral innate and adaptive immune response in a murine knockout model. Using knockout bone marrow derived dendritic cells (BMDC) and macrophage (BMMO), we determined cGAS and STING are essential to induction of the anti-adenoviral response in these antigen-presenting cells (APCs) in vitro. We next determined how the cGAS/STING cascade impacts the antiviral response following a systemic administration of rAd5V. Infection of cGAS-/- and STING-/- mice results in a compromised early antiviral innate response compared to WT controls: significantly lower levels of IFNbbeta; secretion, low levels of proinflammatory chemokine induction, and reduced levels of antiviral transcript induction in hepatic tissue. At 24 hours postinfection, levels of viral DNA and reporter gene expression in the liver were similar in all strains. At 28 days postinfection, clearance of infected hepatocytes in cGAS or STING knockout mice was comparable to wt C57BL/6 mice. Levels of neutralizing anti-Ad5V antibody were modestly reduced in cGAS infected mice. These data support a dominant role for the cGAS/STING cascade in the early innate antiviral inflammatory response to Adenovirus vectors. However, loss of the cGAS/STING pathway did not affect viral clearance, and cGAS deficiency had a modest influence on the magnitude of the antiviral humoral immune response to Adenovirus infections.

    Importance: The detection of a viral infection by host sentinel immune cells contributes to activation of a complex and varied antiviral innate and adaptive immune response, which limits viral replication, spread, and susceptibility to infection. In this study we have characterized how the cGAS/STING DNA sensing cascade contributes to early detection of Adenovirus infections. cGAS influences APC activation and early innate antiviral inflammatory immune responses, but adaptive immune pathways associated with viral clearance and anti-Ad antibody production was minimally influenced by loss of the cGAS PRR signaling cascade.

  • Multiple Natural Substitutions in Avian Influenza A Virus PB2 Facilitate Efficient Replication in Human Cells [PublishAheadOfPrint]

  • A strong restriction of the avian influenza A virus polymerase in mammalian cells generally limits viral host-range switching. Although substitutions like E627K in the PB2 polymerase subunit can facilitate polymerase activity to allow replication in mammals, many human H5N1 and H7N9 viruses lack this adaptive substitution. Here, several previously unknown, naturally occurring, adaptive substitutions in PB2 were identified by bioinformatics, and their enhancing activity verified using in vitro assays. Adaptive substitutions enhanced polymerase activity and virus replication in mammalian cells for avian H5N1 and H7N9 viruses, but not for a partially human-adapted H5N1 virus. Adaptive substitutions towards basic amino acids were frequent and were mostly clustered in a putative RNA exit channel in a polymerase crystal structure. Phylogenetic analysis demonstrated divergent dependency of influenza viruses on adaptive substitutions. The novel adaptive substitutions found in this study increase basic understanding of influenza virus host adaptation and will help in surveillance efforts.

    IMPORTANCE Influenza viruses from birds jump the species barrier into humans relatively frequently. Such influenza virus zoonoses may pose public health risks if the virus adapts to humans and becomes a pandemic threat. Relatively few amino acid substitutions nndash; most notably in the receptor binding site of hemagglutinin and at positions 591 and 627 in the polymerase protein PB2 nndash; have been identified in pandemic influenza strains as determinants of host adaptation, to facilitate efficient virus replication and transmission in humans. Here, we show that substantial numbers of amino acid substitutions are functionally compensating for the lack of the above-mentioned mutations in PB2, and could facilitate influenza virus emergence in humans.

  • Optimization of the Solubility of HIV-1-Neutralizing Antibody 10E8 through Somatic Variation and Structure-Based Design [PublishAheadOfPrint]

  • Extraordinary antibodies capable of near pan-neutralization of HIV-1 have been identified. One of the broadest is antibody 10E8, which recognizes the membrane-proximal external region (MPER) of HIV-1 and neutralizes ggt;95% of circulating HIV-1 strains. If delivered passively, 10E8 might serve to prevent or to treat HIV-1 infection. Antibody 10E8, however, is markedly less soluble than other antibodies. Here, we describe the use of both structural biology and somatic variation to develop optimized versions of 10E8 with increased solubility. From the structure of 10E8, we identified a prominent hydrophobic patch; reversion of four hydrophobic residues in this patch to their hydrophilic germline counterparts resulted in a ~10-fold decrease in turbidity. We also used somatic variants of 10E8, identified previously by next-generation sequencing, to optimize heavy and light chains; this process yielded several improved variants. Of these, variant 10E8v4 with 26 changes versus the parent 10E8 was the most soluble, with a paratope we showed crystallographically to be virtually identical to that of 10E8, a potency on a panel of 200-HIV-1 isolates also similar to that of 10E8, and a half-life in rhesus macaques of ~10 days. An anomaly in 10E8v4 size-exclusion chromatography that appeared to be related to conformational isomerization was resolved by engineering an inter-chain disulfide. Thus, by combining a structure-based approach with natural variation in potency and solubility from the 10E8 lineage, we successfully created variants of 10E8, which retained the potency and extraordinary neutralization breadth of the parent 10E8, but with substantially increased solubility.

    Importance Antibody 10E8 could be used to prevent HIV-1 infection, if manufactured and delivered economically. It suffers, however, from issues of solubility, which impede manufacturing. We hypothesized that the physical characteristic of 10E8 could be improved through rational design, without compromising breadth and potency. We used structural biology to identify hydrophobic patches on 10E8, which did not appear to be involved in 10E8 function. Reversion of hydrophobic residues in these patches to their hydrophilic germline counterparts increased solubility. Next, clues from somatic variants of 10E8, identified by next generation sequencing, were incorporated. A combination of structure-based design and somatic variant optimization led to 10E8v4, with substantially improved solubility and similar potency versus the parent 10E8. The co-crystal structure of antibody 10E8v4 with its HIV-1 epitope was highly similar to that with the parent 10E8, despite 26 alterations in sequence and substantially improved solubility. Antibody 10E8v4 may be suitable for manufacturing.

  • Gem: Emerging role of PML nuclear bodies in innate immune signaling [PublishAheadOfPrint]

  • Research of the last two decades has demonstrated that a specific organelle of the cell nucleus, termed PML nuclear body (PML-NBs) or nuclear domain 10 (ND10), is frequently modified during viral infection. This correlates with antagonization of a direct repressive function of individual PML-NB components like the PML, hDaxx, Sp100 or ATRX protein that are able to act as cellular restriction factors. Recent studies now reveal an emerging role of PML-NBs as co-regulatory structures of both type-I as well as type-II interferon responses. This emphasizes that targeting of PML-NBs by viral regulatory proteins has evolved as a strategy to compromise intrinsic antiviral defense and innate immune responses.

  • Intracellular distributions of capsid-associated pUL77 of HCMV and interactions with packaging proteins and pUL93 [PublishAheadOfPrint]

  • DNA packaging into procapsids is a common, multistep process during viral maturation in herpesviruses. In HCMV, proteins involved in this process are the terminase subunits pUL56 and pUL89, which are responsible for site-specific cleavage and insertion of the DNA into the procapsid via the portal protein pUL104. However, additional viral proteins are required for the DNA packaging process. We have shown previously that the capsid-associated pUL77 encodes for another potential player during capsid maturation.

    Pulse-chase experiments revealed that pUL77 is stably expressed during HCMV infection. Time course analysis demonstrated that pUL77 is expressed early-late in the infectious cycle. The sequence of pUL77 was analysed to find nuclear localisation sequences (NLS), revealing a monopartite NLSm at the N-terminus and a bipartite NLSb in the middle of pUL77. The potential NLSs were inserted into plasmid pHM829 which encodes a chimeric protein with sszlig;-galactosidase and GFP. In contrast to pUL56, neither NLSm nor NLSb was sufficient for nuclear import. Furthermore, we investigated by co-immunoprecipitation whether packaging proteins as well as pUL93, the homologue protein of HSV-1 pUL17, are interaction partners of pUL77. The interactions between pUL77 and packaging proteins as well as pUL93 could be verified.

    Importance We showed that the capsid-associated pUL77 encodes for another potential player during capsid maturation of HCMV. The protein UL77 (pUL77) is a conserved core protein of HCMV. This study demonstrates for the first time that pUL77 has an early-late expression kinetic during the infectious cycle and an intrinsic potential for nuclear translocation. According to its proposed functions in stabilization of the capsid and anchoring the encapsidated DNA during packaging interaction with further DNA packaging proteins are required. We identified physical interactions with the terminase subunits, pUL56 and pUL89, and another postulated packaging protein, pUL93, in infected as well as transfected cells.

  • HIV-1 Tat protein activates both MyD88 and TRIF pathways to induce TNF-{alpha} and IL-10 in human monocytes [PublishAheadOfPrint]

  • In this study, we show that HIV-1 Tat protein interacts, with rapid kinetics, to engage the TLR4 pathway leading to the production of pro-inflammatory and anti-inflammatory cytokines. The pre-treatment of human monocytes with Tat protein for 10 to 30 min suffices to irreversibly engage the activation of TLR4 pathway leading to the production of TNF-aalpha; and IL-10, two cytokines strongly implicated in the chronic activation and dysregulation of the immune system during HIV-1 infection.

    Therefore, this study analysed whether HIV-1 Tat protein was able to activate these two pathways separately or simultaneously. Using three complementary approaches, including mice deficient for each adaptor MyD88, TIRAP/MAL and TRIF, biochemical analysis and specific si-RNA, we demonstrated: i) that Tat was able to activate both MyD88 and TRIF pathways, ii) the capacity of Tat to induce TIRAP/MAL degradation, iii) the crucial role of MyD88 pathway in the production of Tat-induced TNF-aalpha; and IL-10, iv) a reduction, but not abrogation, of IL-10 and TNF-aalpha; by Tat-stimulated macrophages from mice deficient in TIRAP/MAL and v) the crucial role of the TRIF pathway in Tat-induced IL-10.

    Further, we showed that, downstream of the MyD88 and TRIF pathways, Tat protein activated PKC-bbeta;II isoform, P38 and ERK1/2 MAP-kinases and NF-B in a TLR4-dependent manner.

    Collectively, our data show that, by recruiting TLR4 pathway with rapid kinetics, the HIV-1 Tat protein leads to the engagement of both MyD88 and TRIF pathways and to the activation of PKC, MAP kinases and NF-B signalling, to induce the production of TNF-aalpha; and IL-10.

    Importance In this study, we demonstrated that by recruiting TLR4 pathway with rapid kinetics, the HIV-1 Tat protein leads to the engagement of both MyD88 and TRIF pathways and to the activation of PKC-bbeta;II, MAP kinases and NF-B signalling, to induce the production of TNF-aalpha; and IL-10, two cytokines strongly implicated in the chronic activation and dysregulation of the immune system during HIV-1 infection. Thus, it could be interesting to target Tat as a pathogenic factor early after HIV-1 infection. This could be achieved either by vaccination approaches including Tat as an immunogen in potential candidate vaccines or by developing molecules capable of neutralizing the effect of the Tat protein.

  • The Essential Human Cytomegalovirus Proteins pUL77 and pUL93 are Structural Components Necessary for Viral Genome Encapsidation [PublishAheadOfPrint]

  • Several essential viral proteins are proposed to participate in genome encapsidation of human cytomegalovirus (HCMV), among them pUL77 and pUL93 which remain largely uncharacterized. To gain insight into their properties, we generated an HCMV mutant expressing a pUL77-mGFP fusion protein and a pUL93-specific antibody. Immunoblotting demonstrated that both proteins are incorporated into capsids and virions. Conversely to data suggesting internal translation initiation sites within the UL93 ORF, we provide evidence that pUL93 synthesis commences at the first start codon. In infected cells pUL77-mGFP was found in nuclear replication compartments and dot-like structures, co-localizing with capsid proteins. Immunogold labelling of nuclear capsids revealed that pUL77 is present on A, B and C capsids. Pull-down of pUL77-mGFP revealed co-purification of pUL93, indicating interaction between these proteins, which still occurred when capsid formation was prevented. Correct subnuclear distribution of pUL77-mGFP required pUL93 as well as the major capsid protein (and thus probably the presence of capsids), but not the tegument protein pp150 or the encapsidation protein pUL52, demonstrating that pUL77 nuclear targeting occurs independently of the formation of DNA-filled capsids. When pUL77 or pUL93 were missing, generation of unit-length genomes was not observed and only empty B capsids were produced. Taken together, these results show that pUL77 and pUL93 are capsid constituents needed for HCMV genome encapsidation. Therefore, the task of pUL77 seems to differ from that of its aalpha;-herpesvirus orthologue pUL25, which exerts its function subsequent to genome cleavage-packaging.

    IMPORTANCE The essential HCMV proteins pUL77 and pUL93 were suggested to be involved in viral genome cleavage-packaging, but are poorly characterized both biochemically and functionally. By producing a monoclonal antibody against pUL93 and generating an HCMV mutant in which pUL77 is fused to a fluorescent protein, we show that pUL77 and pUL93 are capsid constituents, with pUL77 being similarly abundant on all capsid types. Each protein is required for genome encapsidation, as the absence of either pUL77 or pUL93 results in a genome packaging defect with the formation of empty capsids only. This distinguishes pUL77 from its aalpha;-herpesvirus orthologue pUL25, which is enriched on DNA-filled capsids and exerts its function after the viral DNA is packaged. Our data for the first time describe an HCMV mutant with a fluorescent capsid and provide insight into the roles of pUL77 and pUL93, thus contributing to a better understanding of the HCMV encapsidation network.

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