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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

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

  • Editorial Board [Masthead]

  • Assessment of the Internal Genes of Influenza A (H7N9) Virus Contributing to High Pathogenicity in Mice [Pathogenesis and Immunity]

  • The recently identified H7N9 influenza A virus has caused severe economic losses and worldwide public concern. Genetic analysis indicates that its six internal genes all originated from H9N2 viruses. However, the H7N9 virus is more highly pathogenic in humans than H9N2, which suggests that the internal genes of H7N9 have mutated. To analyze which H7N9 virus internal genes contribute to its high pathogenicity, a series of reassortants was generated by reverse genetics, with each virus containing a single internal gene of the typical A/Anhui/1/2013 (H7N9) (AH-H7N9) virus in the genetic background of the A/chicken/Shandong/lx1023/2007 (H9N2) virus. The replication ability, polymerase activity, and pathogenicity of these viruses were then evaluated in vitro and in vivo. These recombinants displayed high genetic compatibility, and the H7N9-derived PB2, M, and NP genes were identified as the virulence genes for the reassortants in mice. Further investigation confirmed that the PB2 K627 residue is critical for the high pathogenicity of the H7N9 virus and the reassortant containing the H7N9-derived PB2 segment (H9N2-AH/PB2). Notably, the H7N9-derived PB2 gene displayed greater compatibility with the H9N2 genome than that of H7N9, endowing the H9N2-AH/PB2 reassortant with greater viability and virulence than the parental H7N9 virus. In addition, the H7N9 virus, with the exception of the H9N2 reassortants, could effectively replicate in human A549 cells. Our results indicate that PB2, M, and NP are the key virulence genes, together with the surface hemagglutinin (HA) and neuraminidase (NA) proteins, contributing to the high infectivity of the H7N9 virus in humans.

    IMPORTANCE To date, the novel H7N9 influenza A virus has caused 437 human infections, with approximately 30% mortality. Previous work has primarily focused on the two viral surface proteins, HA and NA, but the contribution of the six internal genes to the high pathogenicity of H7N9 has not been systematically studied. Here, the H9N2 virus was used as a genetic backbone to evaluate the virulence genes of H7N9 virus in vitro and in vivo. Our data indicate that the PB2, M, and NP genes play important roles in viral infection in mice and, together with HA and NA, contribute to the high infectivity of the H7N9 virus in humans.

  • Contact-Induced Mitochondrial Polarization Supports HIV-1 Virological Synapse Formation [Virus-Cell Interactions]

  • Rapid HIV-1 spread between CD4 T lymphocytes occurs at retrovirus-induced immune cell contacts called virological synapses (VS). VS are associated with striking T cell polarization and localized virus budding at the site of contact that facilitates cell-cell spread. In addition to this, spatial clustering of organelles, including mitochondria, to the contact zone has been previously shown. However, whether cell-cell contact specifically induces dynamic T cell remodeling during VS formation and what regulates this process remain unclear. Here, we report that contact between an HIV-1-infected T cell and an uninfected target T cell specifically triggers polarization of mitochondria concomitant with recruitment of the major HIV-1 structural protein Gag to the site of cell-cell contact. Using fixed and live-cell imaging, we show that mitochondrial and Gag polarization in HIV-1-infected T cells occurs within minutes of contact with target T cells, requires the formation of stable cell-cell contacts, and is an active, calcium-dependent process. We also find that perturbation of mitochondrial polarization impairs cell-cell spread of HIV-1 at the VS. Taken together, these data suggest that HIV-1-infected T cells are able to sense and respond to contact with susceptible target cells and undergo dynamic cytoplasmic remodeling to create a synaptic environment that supports efficient HIV-1 VS formation between CD4 T lymphocytes.

    IMPORTANCE HIV-1 remains one of the major global health challenges of modern times. The capacity of HIV-1 to cause disease depends on the virus's ability to spread between immune cells, most notably CD4 T lymphocytes. Cell-cell transmission is the most efficient way of HIV-1 spread and occurs at the virological synapse (VS). The VS forms at the site of contact between an infected cell and an uninfected cell and is characterized by polarized assembly and budding of virions and clustering of cellular organelles, including mitochondria. Here, we show that cell-cell contact induces rapid recruitment of mitochondria to the contact site and that this supports efficient VS formation and consequently cell-cell spread. Additionally, we observed that cell-cell contact induces a mitochondrion-dependent increase in intracellular calcium, indicative of cellular signaling. Taken together, our data suggest that VS formation is a regulated process and thus a potential target to block HIV-1 cell-cell spread.

  • Tetramer Enrichment Reveals the Presence of Phenotypically Diverse Hepatitis C Virus-Specific CD8+ T Cells in Chronic Infection [Pathogenesis and Immunity]

  • Virus-specific CD8+ T cells are rarely detectable ex vivo by conventional methods during chronic hepatitis C virus (HCV) infection. In this study, however, we were able to detect and characterize HCV-specific CD8+ T cells in all chronically HCV genotype 1a-infected, HLA-A*02:01-positive patients analyzed by performing major histocompatibility complex (MHC) class I tetramer enrichment. Two-thirds of these enriched HCV-specific CD8+ T-cell populations displayed an effector memory phenotype, whereas, surprisingly, one-third displayed a naive-like phenotype despite ongoing viral replication. CD8+ T cells with an effector memory phenotype could not expand in vitro, suggesting exhaustion of these cells. Interestingly, some of the naive-like CD8+ T cells proliferated vigorously upon in vitro priming, whereas others did not. These differences were linked to the corresponding viral sequences in the respective patients. Indeed, naive-like CD8+ T cells from patients with the consensus sequence in the corresponding T-cell epitope did not expand in vitro. In contrast, in patients displaying sequence variations, we were able to induce HCV-specific CD8+ T-cell proliferation, which may indicate infection with a variant virus. Collectively, these data reveal the presence of phenotypically and functionally diverse HCV-specific CD8+ T cells at very low frequencies that are detectable in all chronically infected patients despite viral persistence.

    IMPORTANCE In this study, we analyzed CD8+ T-cell responses specific for HLA-A*02:01-restricted epitopes in chronically HCV-infected patients, using MHC class I tetramer enrichment. Importantly, we could detect HCV-specific CD8+ T-cell populations in all patients. To further characterize these HCV-specific CD8+ T-cell populations that are not detectable using conventional techniques, we performed phenotypic, functional, and viral sequence analyses. These data revealed different mechanisms for CD8+ T-cell failure in HCV infection, including T-cell exhaustion, viral escape, and functional impairment of naive-like HCV-specific CD8+ T cells.

  • Merkel Cell Polyomavirus Small T Antigen Mediates Microtubule Destabilization To Promote Cell Motility and Migration [Virus-Cell Interactions]

  • Merkel cell carcinoma (MCC) is an aggressive skin cancer of neuroendocrine origin with a high propensity for recurrence and metastasis. Merkel cell polyomavirus (MCPyV) causes the majority of MCC cases due to the expression of the MCPyV small and large tumor antigens (ST and LT, respectively). Although a number of molecular mechanisms have been attributed to MCPyV tumor antigen-mediated cellular transformation or replication, to date, no studies have investigated any potential link between MCPyV T antigen expression and the highly metastatic nature of MCC. Here we use a quantitative proteomic approach to show that MCPyV ST promotes differential expression of cellular proteins implicated in microtubule-associated cytoskeletal organization and dynamics. Intriguingly, we demonstrate that MCPyV ST expression promotes microtubule destabilization, leading to a motile and migratory phenotype. We further highlight the essential role of the microtubule-associated protein stathmin in MCPyV ST-mediated microtubule destabilization and cell motility and implicate the cellular phosphatase catalytic subunit protein phosphatase 4C (PP4C) in the regulation of this process. These findings suggest a possible molecular mechanism for the highly metastatic phenotype associated with MCC.

    IMPORTANCE Merkel cell polyomavirus (MCPyV) causes the majority of cases of Merkel cell carcinoma (MCC), an aggressive skin cancer with a high metastatic potential. However, the molecular mechanisms leading to virally induced cancer development have yet to be fully elucidated. In particular, no studies have investigated any potential link between the virus and the highly metastatic nature of MCC. We demonstrate that the MCPyV small tumor antigen (ST) promotes the destabilization of the host cell microtubule network, which leads to a more motile and migratory cell phenotype. We further show that MCPyV ST induces this process by regulating the phosphorylation status of the cellular microtubule-associated protein stathmin by its known association with the cellular phosphatase catalytic subunit PP4C. These findings highlight stathmin as a possible biomarker of MCC and as a target for novel antitumoral therapies.

  • Differentiation of Neurons Restricts Arbovirus Replication and Increases Expression of the Alpha Isoform of IRF-7 [Virus-Cell Interactions]

  • Susceptibility to alphavirus infection is age dependent, and host maturation is associated with decreased virus replication and less severe encephalitis. To identify factors associated with maturation-dependent restriction of virus replication, we studied AP-7 rat olfactory bulb neuronal cells, which can differentiate in vitro. Differentiation was associated with a 150- to 1,000-fold decrease in replication of the alphaviruses Sindbis virus and Venezuelan equine encephalitis virus, as well as La Crosse bunyavirus. Differentiation delayed synthesis of SINV RNA and protein but did not alter the susceptibility of neurons to infection or virion maturation. Additionally, differentiation slowed virus-induced translation arrest and death of infected cells. Differentiation of uninfected AP-7 neurons was associated with changes in expression of antiviral genes. Expression of key transcription factors was increased, including interferon regulatory factor 3 and 7 (IRF-3 and IRF-7) and STAT-1, suggesting that neuronal maturation may enhance the capacity for antiviral signaling upon infection. IRF-7 produced by undifferentiated AP-7 neurons was exclusively the short dominant negative -isoform, while that produced by differentiated neurons was the full-length aalpha;-isoform. A similar switch in IRF-7 isoforms also occurred in the brains of maturing C57BL/6J mice. Silencing of IRF expression did not improve virus multiplication in differentiated neurons. Therefore, neuronal differentiation is associated with upregulation of transcription factors that activate antiviral signaling, but this alone does not account for maturation-dependent restriction of virus replication.

    IMPORTANCE Viral encephalomyelitis is an important cause of age-dependent morbidity and mortality. Because mature neurons are not readily regenerated, recovery from encephalitis suggests that mature neurons utilize unique antiviral mechanisms to block infection and/or clear virus. To identify maturational changes in neurons that may improve outcome, we compared immature and mature cultured neurons for susceptibility to three encephalitic arboviruses and found that replication of Old World and New World alphaviruses and a bunyavirus was reduced in mature compared to immature neurons. Neuronal maturation was associated with increased baseline expression of interferon regulatory factor 3 and 7 mRNAs and production of distinct isoforms of interferon regulatory factor 7 protein. Overall, our studies identified maturational changes in neurons that likely contribute to assembly of immunoregulatory factors prior to infection, a more rapid antiviral response, increased resistance to virus infection, and improved survival.

  • GHSC70 Is Involved in the Cellular Entry of Nervous Necrosis Virus [Virus-Cell Interactions]

  • Nervous necrosis virus (NNV) is a devastating pathogen of cultured marine fish and has affected more than 40 fish species. NNV belongs to the betanodaviruses of Nodaviridae and is a nonenveloped icosahedral particle with 2 single-stranded positive-sense RNAs. To date, knowledge regarding NNV entry into the host cell remains limited, and no NNV-specific receptor protein has been published. Using grouper fin cell line GF-1 and purified NNV capsid protein in a virus overlay protein binding assay (VOPBA), grouper heat shock cognate protein 70 (GHSC70) and grouper voltage-dependent anion selective channel protein 2 (GVDAC2) were investigated as NNV receptor protein candidates. We cloned and sequenced the genes for GHSC70 and GVDAC2 and expressed them in Escherichia coli for antiserum preparation. Knockdown of the expression of GHSC70 and GVDAC2 genes with specific short interfering RNAs (siRNAs) significantly downregulated viral RNA expression in NNV-infected GF-1 cells. By performing an immunoprecipitation assay, we confirmed that GHSC70 interacted with NNV capsid protein, while VDAC2 did not. Immunofluorescence staining and flow cytometry analysis revealed the presence of the GHSC70 protein on the cell surface. After a blocking assay, we detected the NNV RNA2 levels after 1 h of adsorption to GF-1 cells; the level was significantly lower in the cells pretreated with the GHSC70 antiserum than in nontreated cells. Therefore, we suggest that GHSC70 participates in the NNV entry of GF-1 cells, likely functioning as an NNV receptor or coreceptor protein.

    IMPORTANCE Fish nodavirus has caused mass mortality of more than 40 fish species worldwide and resulted in huge economic losses in the past 20 years. Among the four genotypes of fish nodaviruses, the red-spotted grouper nervous necrosis virus (RGNNV) genotype exhibits the widest host range. In our previous study, we developed monoclonal antibodies with high neutralizing efficiency against grouper NNV in GF-1 cells, indicating that NNV-specific receptor(s) may exist on the GF-1 cell membrane. However, no NNV receptor protein has been published. In this study, we found GHSC70 to be an NNV receptor (or coreceptor) candidate through VOBPA and provided several lines of evidence demonstrating that GHSC70 protein has a role in the NNV entry step of GF-1 cells. To the best of our knowledge, this is the first report identifying grouper HSC70 and its role in NNV entry into GF-1 cells.

  • Venezuelan Equine Encephalitis Virus Variants Lacking Transcription Inhibitory Functions Demonstrate Highly Attenuated Phenotype [Vaccines and Antiviral Agents]

  • Alphaviruses represent a significant public health threat worldwide. They are transmitted by mosquitoes and cause a variety of human diseases ranging from severe meningoencephalitis to polyarthritis. To date, no efficient and safe vaccines have been developed against any alphavirus infection. However, in recent years, significant progress has been made in understanding the mechanism of alphavirus replication and virus-host interactions. These data have provided the possibility for the development of new rationally designed alphavirus vaccine candidates that combine efficient immunogenicity, high safety, and inability to revert to pathogenic phenotype. New attenuated variants of Venezuelan equine encephalitis virus (VEEV) designed in this study combine a variety of characteristics that independently contribute to a reduction in virulence. These constructs encode a noncytopathic VEEV capsid protein that is incapable of interfering with the innate immune response. The capsid-specific mutations strongly affect neurovirulence of the virus. In other constructs, they were combined with changes in control of capsid translation and an extensively mutated packaging signal. These modifications also affected the residual neurovirulence of the virus, but it remained immunogenic, and a single immunization protected mice against subsequent infection with epizootic VEEV. Similar approaches of attenuation can be applied to other encephalitogenic New World alphaviruses.

    IMPORTANCE Venezuelan equine encephalitis virus (VEEV) is an important human and animal pathogen, which causes periodic outbreaks of highly debilitating disease. Despite a continuous public health threat, no safe and efficient vaccine candidates have been developed to date. In this study, we applied accumulated knowledge about the mechanism of VEEV replication, RNA packaging, and interaction with the host to design new VEEV vaccine candidates that demonstrate exceptionally high levels of safety due to a combination of extensive modifications in the viral genome. The introduced mutations did not affect RNA replication or structural protein synthesis but had deleterious effects on VEEV neuroinvasion and virulence. In spite of dramatically reduced virulence, the designed mutants remained highly immunogenic and protected mice against subsequent infection with epizootic VEEV. Similar methodologies can be applied for attenuation of other encephalitogenic New World alphaviruses.

  • Topical Herpes Simplex Virus 2 (HSV-2) Vaccination with Human Papillomavirus Vectors Expressing gB/gD Ectodomains Induces Genital-Tissue-Resident Memory CD8+ T Cells and Reduces Genital Disease and Viral Shedding after HSV-2 Challenge [Vaccines and Antiviral Agents]

  • No herpes simplex virus 2 (HSV-2) vaccine has been licensed for use in humans. HSV-2 glycoproteins B (gB) and D (gD) are targets of neutralizing antibodies and T cells, but clinical trials involving intramuscular (i.m.) injection of HSV-2 gB and gD in adjuvants have not been effective. Here we evaluated intravaginal (ivag) genetic immunization of C57BL/6 mice with a replication-defective human papillomavirus pseudovirus (HPV PsV) expressing HSV-2 gB (HPV-gB) or gD (HPV-gD) constructs to target different subcellular compartments. HPV PsV expressing a secreted ectodomain of gB (gBsec) or gD (gDsec), but not PsV expressing a cytoplasmic or membrane-bound form, induced circulating and intravaginal-tissue-resident memory CD8+ T cells that were able to secrete gamma interferon (IFN-) and tumor necrosis factor alpha (TNF-aalpha;) as well as moderate levels of serum HSV neutralizing antibodies. Combined immunization with HPV-gBsec and HPV-gDsec (HPV-gBsec/gDsec) vaccines conferred longer survival after vaginal challenge with HSV-2 than immunization with HPV-gBsec or HPV-gDsec alone. HPV-gBsec/gDsec ivag vaccination was associated with a reduced severity of genital lesions and lower levels of viral shedding in the genital tract after HSV-2 challenge. In contrast, intramuscular vaccination with a soluble truncated gD protein (gD2t) in alum and monophosphoryl lipid A (MPL) elicited high neutralizing antibody titers and improved survival but did not reduce genital lesions and viral shedding. Vaccination combining ivag HPV-gBsec/gDsec and i.m. gD2t-alum-MPL improved survival and reduced genital lesions and viral shedding. Finally, high levels of circulating HSV-2-specific CD8+ T cells, but not serum antibodies, correlated with reduced viral shedding. Taken together, our data underscore the potential of HPV PsV as a platform for a topical mucosal vaccine to control local manifestations of primary HSV-2 infection.

    IMPORTANCE Genital herpes is a highly prevalent chronic disease caused by HSV infection. To date, there is no licensed vaccine against HSV infection. This study describes intravaginal vaccination with a nonreplicating HPV-based vector expressing HSV glycoprotein antigens. The data presented in this study underscore the potential of HPV-based vectors as a platform for the induction of genital-tissue-resident memory T cell responses and the control of local manifestations of primary HSV infection.

  • Slaying the Trojan Horse: Natural Killer Cells Exhibit Robust Anti-HIV-1 Antibody-Dependent Activation and Cytolysis against Allogeneic T Cells [Pathogenesis and Immunity]

  • Many attempts to design prophylactic human immunodeficiency virus type 1 (HIV-1) vaccines have focused on the induction of neutralizing antibodies (Abs) that block infection by free virions. Despite the focus on viral particles, virus-infected cells, which can be found within mucosal secretions, are more infectious than free virus both in vitro and in vivo. Furthermore, assessment of human transmission couples suggests infected seminal lymphocytes might be responsible for a proportion of HIV-1 transmissions. Although vaccines that induce neutralizing Abs are sought, only some broadly neutralizing Abs efficiently block cell-to-cell transmission of HIV-1. As HIV-1 vaccines need to elicit immune responses capable of controlling both free and cell-associated virus, we evaluated the potential of natural killer (NK) cells to respond in an Ab-dependent manner to allogeneic T cells bearing HIV-1 antigens. This study presents data measuring Ab-dependent anti-HIV-1 NK cell responses to primary and transformed allogeneic T-cell targets. We found that NK cells are robustly activated in an anti-HIV-1 Ab-dependent manner against allogeneic targets and that tested target cells are subject to Ab-dependent cytolysis. Furthermore, the educated KIR3DL1+ NK cell subset from HLA-Bw4+ individuals exhibits an activation advantage over the KIR3DL1nndash; subset that contains both NK cells educated through other receptor/ligand combinations and uneducated NK cells. These results are intriguing and important for understanding the regulation of Ab-dependent NK cell responses and are potentially valuable for designing Ab-dependent therapies and/or vaccines.

    IMPORTANCE NK cell-mediated anti-HIV-1 antibody-dependent functions have been associated with protection from infection and disease progression; however, their role in protecting from infection with allogeneic cells infected with HIV-1 is unknown. We found that HIV-1-specific ADCC antibodies bound to allogeneic cells infected with HIV-1 or coated with HIV-1 gp120 were capable of activating NK cells and/or trigging cytolysis of the allogeneic target cells. This suggests ADCC may be able to assist in preventing infection with cell-associated HIV-1. In order to fully utilize NK cell-mediated Ab-dependent effector functions, it might also be important that educated NK cells, which hold the highest activation potential, can become activated against targets bearing HIV-1 antigens and expressing the ligands for self-inhibitory receptors. Here, we show that with Ab-dependent stimulation, NK cells expressing inhibitory receptors can mediate robust activation against targets expressing the ligands for those receptors.

  • Complex T-Cell Receptor Repertoire Dynamics Underlie the CD8+ T-Cell Response to HIV-1 [Pathogenesis and Immunity]

  • Although CD8+ T cells are important for the control of HIV-1 in vivo, the precise correlates of immune efficacy remain unclear. In this study, we conducted a comprehensive analysis of viral sequence variation and T-cell receptor (TCR) repertoire composition across multiple epitope specificities in a group of antiretroviral treatment-naive individuals chronically infected with HIV-1. A negative correlation was detected between changes in antigen-specific TCR repertoire diversity and CD8+ T-cell response magnitude, reflecting clonotypic expansions and contractions related to alterations in cognate viral epitope sequences. These patterns were independent of the individual, as evidenced by discordant clonotype-specific transitions directed against different epitopes in single subjects. Moreover, long-term asymptomatic HIV-1 infection was characterized by evolution of the TCR repertoire in parallel with viral replication. Collectively, these data suggest a continuous bidirectional process of adaptation between HIV-1 and virus-specific CD8+ T-cell clonotypes orchestrated at the TCR-antigen interface.

    IMPORTANCE We describe a relation between viral epitope mutation, antigen-specific T-cell expansion, and the repertoire of responding clonotypes in chronic HIV-1 infection. This work provides insights into the process of coadaptation between the human immune system and a rapidly evolving lentivirus.

  • The Human CD8+ T Cell Responses Induced by a Live Attenuated Tetravalent Dengue Vaccine Are Directed against Highly Conserved Epitopes [Vaccines and Antiviral Agents]

  • The incidence of infection with any of the four dengue virus serotypes (DENV1 to -4) has increased dramatically in the last few decades, and the lack of a treatment or vaccine has contributed to significant morbidity and mortality worldwide. A recent comprehensive analysis of the human T cell response against wild-type DENV suggested an human lymphocyte antigen (HLA)-linked protective role for CD8+ T cells. We have collected one-unit blood donations from study participants receiving the monovalent or tetravalent live attenuated DENV vaccine (DLAV), developed by the U.S. National Institutes of Health. Peripheral blood mononuclear cells from these donors were screened in gamma interferon enzyme-linked immunosorbent spot assays with pools of predicted, HLA-matched, class I binding peptides covering the entire DENV proteome. Here, we characterize for the first time CD8+ T cell responses after live attenuated dengue vaccination and show that CD8+ T cell responses in vaccinees were readily detectable and comparable to natural dengue infection. Interestingly, whereas broad responses to structural and nonstructural (NS) proteins were observed after monovalent vaccination, T cell responses following tetravalent vaccination were, dramatically, focused toward the highly conserved NS proteins. Epitopes were highly conserved in a vast variety of field isolates and able to elicit multifunctional T cell responses. Detailed knowledge of the T cell response will contribute to the identification of robust correlates of protection in natural immunity and following vaccination against DENV.

    IMPORTANCE The development of effective vaccination strategies against dengue virus (DENV) infection and clinically significant disease is a task of high global public health value and significance, while also being a challenge of significant complexity. A recent efficacy trial of the most advanced dengue vaccine candidate, demonstrated only partial protection against all four DENV serotypes, despite three subsequent immunizations and detection of measurable neutralizing antibodies to each serotype in most subjects. These results challenge the hypothesis that seroconversion is the only reliable correlate of protection. Here, we show that CD8+ T cell responses in vaccinees were readily detectable and comparable to natural dengue virus infection. Detailed knowledge of the T cell response may further contribute to the identification of robust correlates of protection in natural immunity and vaccination against DENV.

  • Quantitative Proteomics Identifies Host Factors Modulated during Acute Hepatitis E Virus Infection in the Swine Model [Virus-Cell Interactions]

  • Hepatitis E virus (HEV) causes acute enterically transmitted hepatitis. In industrialized countries, it is a zoonotic disease, with swine being the major reservoir of human HEV contamination. The occurrence and severity of the disease are variable, with clinical symptoms ranging from asymptomatic to self-limiting acute hepatitis, chronic infection, or fulminant hepatitis. In the absence of a robust cell culture system or small-animal models, the HEV life cycle and pathological process remain unclear. To characterize HEV pathogenesis and virulence mechanisms, a quantitative proteomic analysis was carried out to identify cellular factors and pathways modulated during acute infection of swine. Three groups of pigs were inoculated with three different strains of swine HEV to evaluate the possible role of viral determinants in pathogenesis. Liver samples were analyzed by a differential proteomic approach, two-dimensional difference in gel electrophoresis, and 61 modulated proteins were identified by mass spectroscopy. The results obtained show that the three HEV strains replicate similarly in swine and that they modulate several cellular pathways, suggesting that HEV impairs several cellular processes, which can account for the various types of disease expression. Several proteins, such as heterogeneous nuclear ribonucleoprotein K, apolipoprotein E, and prohibitin, known to be involved in other viral life cycles, were upregulated in HEV-infected livers. Some differences were observed between the three strains, suggesting that HEV's genetic variability may induce variations in pathogenesis. This comparative analysis of the liver proteome modulated during infection with three different strains of HEV genotype 3 provides an important basis for further investigations on the factors involved in HEV replication and the mechanism of HEV pathogenesis.

    IMPORTANCE Hepatitis E virus (HEV) is responsible for acute hepatitis, with clinical symptoms ranging from asymptomatic to self-limiting acute hepatitis, chronic infection, or fulminant hepatitis. In industrialized countries, HEV is considered an emerging zoonotic disease, with swine being the principal reservoir for human contamination. The viral and cellular factors involved in the replication and/or pathogenesis of HEV are still not fully known. Here we report that several cellular pathways involved in cholesterol and lipid metabolism or cell survival were modulated during HEV infection in the swine model. Moreover, we observed a difference between the different swine strains, suggesting that HEV's genetic variability could play a role in pathogenesis. We also identified some proteins known to be involved in other viral cycles. Our study provides insight into the mechanisms modulated during HEV infection and constitutes a useful reference for future work on HEV pathogenesis and virulence.

  • Rhabdovirus-Based Vaccine Platforms against Henipaviruses [Vaccines and Antiviral Agents]

  • The emerging zoonotic pathogens Hendra virus (HeV) and Nipah virus (NiV) are in the genus Henipavirus in the family Paramyxoviridae. HeV and NiV infections can be highly fatal to humans and livestock. The goal of this study was to develop candidate vaccines against henipaviruses utilizing two well-established rhabdoviral vaccine vector platforms, recombinant rabies virus (RABV) and recombinant vesicular stomatitis virus (VSV), expressing either the codon-optimized or the wild-type (wt) HeV glycoprotein (G) gene. The RABV vector expressing the codon-optimized HeV G showed a 2- to 3-fold increase in incorporation compared to the RABV vector expressing wt HeV G. There was no significant difference in HeV G incorporation in the VSV vectors expressing either wt or codon-optimized HeV G. Mice inoculated intranasally with any of these live recombinant viruses showed no signs of disease, including weight loss, indicating that HeV G expression and incorporation did not increase the neurotropism of the vaccine vectors. To test the immunogenicity of the vaccine candidates, we immunized mice intramuscularly with either one dose of the live vaccines or 3 doses of 10 mmu;g chemically inactivated viral particles. Increased codon-optimized HeV G incorporation into RABV virions resulted in higher antibody titers against HeV G compared to inactivated RABV virions expressing wt HeV G. The live VSV vectors induced more HeV G-specific antibodies as well as higher levels of HeV neutralizing antibodies than the RABV vectors. In the case of killed particles, HeV neutralizing serum titers were very similar between the two platforms. These results indicated that killed RABV with codon-optimized HeV G should be the vector of choice as a dual vaccine in areas where rabies is endemic.

    IMPORTANCE Scientists have been tracking two new viruses carried by the Pteropid fruit bats: Hendra virus (HeV) and Nipah virus (NiV). Both viruses can be fatal to humans and also pose a serious risk to domestic animals. A recent escalation in the frequency of outbreaks has increased the need for a vaccine that prevents HeV and NiV infections. In this study, we performed an extensive comparison of live and killed particles of two recombinant rhabdoviral vectors, rabies virus and vesicular stomatitis virus (VSV), expressing wild-type or codon-optimized HeV glycoprotein, with the goal of developing a candidate vaccine against HeV. Based on our data from the presented mouse immunogenicity studies, we conclude that a killed RABV vaccine would be highly effective against HeV infections and would make an excellent vaccine candidate in areas where both RABV and henipaviruses pose a threat to human health.

  • An Interleukin-1 Beta-Encoding Retrovirus Exhibits Enhanced Replication In Vivo [Pathogenesis and Immunity]

  • Interleukin-1 beta (IL-1bbeta;) is an inflammatory cytokine that is secreted in response to inflammasome activation by innate microbe-sensing pathways. Although some retroviruses can trigger IL-1bbeta; secretion through the DNA-sensing molecule IFI16, the effect of IL-1bbeta; on the course of infection is unknown. To test whether IL-1bbeta; secretion affects retroviral replication in vivo, I constructed a novel murine leukemia virus strain (FMLV-IL-1bbeta;) that encodes the mature form of IL-1bbeta;. This virus replicated with kinetics similar to that of wild-type virus in tissue culture but caused a dramatically more aggressive infection of both C57BL/6 and BALB/c mice. By 7 days postinfection (PI), mice infected with FMLV-IL-1bbeta; exhibited splenomegaly and viral loads 300-fold higher than those in mice infected with wild-type FMLV. Furthermore, the enlarged spleens of FMLV-IL-1bbeta;-infected mice correlated with a large expansion of Gr-1+ CD11b+ myeloid-derived suppressor cells, as well as elevated levels of immune activation. Although FMLV-IL-1bbeta; infection was controlled by C57BL/6 mice by 14 days p.i., FMLV-IL-1bbeta; was able to establish a significant persistent infection and immune activation in BALB/c mice. These results demonstrate that IL-1bbeta; secretion is a powerful positive regulator of retroviral infection and that FMLV-IL-1bbeta; represents a new model of proinflammatory retroviral infection.

    IMPORTANCE Interleukin-1 beta (IL-1bbeta;) is an inflammatory cytokine released in response to activation of innate pathogen-sensing pathways during microbial infection. To examine the potential impact of IL-1bbeta; on retroviral replication in vivo, I constructed a novel mouse retrovirus strain (FMLV-IL-1bbeta;) that encodes IL-1bbeta; and promotes abundant IL-1bbeta; secretion from infected cells. This virus replicates with normal kinetics in cultured cells but displays a dramatically enhanced ability to replicate in mice and caused persistent infection and immune activation in the BALB/c strain of mice. These results establish IL-1bbeta; as a positive regulator of retroviral replication and suggest that targeting this pathway may have therapeutic benefits in infections with proinflammatory retroviruses. This virus can also be used to further study the impact of inflammatory pathways on retroviral infection.

  • Nonstructural Protein 5A (NS5A) and Human Replication Protein A Increase the Processivity of Hepatitis C Virus NS5B Polymerase Activity In Vitro [Genome Replication and Regulation of Viral Gene Expression]

  • The precise role(s) and topological organization of different factors in the hepatitis C virus (HCV) RNA replication complex are not well understood. In order to elucidate the role of viral and host proteins in HCV replication, we have developed a novel in vitro replication system that utilizes a rolling-circle RNA template. Under close-to-physiological salt conditions, HCV NS5B21, an RNA-dependent RNA polymerase, has poor affinity for the RNA template. Human replication protein A (RPA) and HCV NS5A recruit NS5B21 to the template. Subsequently, NS3 is recruited to the replication complex by NS5B21, resulting in RNA synthesis stimulation by helicase. Both RPA and NS5A(S25-C447), but not NS5A(S25-K215), enabled the NS5B21-NS3 helicase complex to be stably associated with the template and synthesize RNA product in a highly processive manner in vitro. This new in vitro HCV replication system is a useful tool that may facilitate the study of other replication factors and aid in the discovery of novel inhibitors of HCV replication.

    IMPORTANCE The molecular mechanism of hepatitis C virus (HCV) replication is not fully understood, but viral and host proteins collaborate in this process. Using a rolling-circle RNA template, we have reconstituted an in vitro HCV replication system that allows us to interrogate the role of viral and host proteins in HCV replication and delineate the molecular interactions. We showed that HCV NS5A(S25-C447) and cellular replication protein A (RPA) functionally cooperate as a processivity factor to stimulate HCV replication by HCV NS5B21 polymerase and NS3 helicase. This system paves the way to test other proteins and may be used as an assay for discovery of HCV inhibitors.

  • Insight into the Mechanism of Inhibition of Adeno-Associated Virus by the Mre11/Rad50/Nbs1 Complex [Genome Replication and Regulation of Viral Gene Expression]

  • Adeno-associated virus (AAV) is a dependent virus of the family Parvoviridae. The gene expression and replication of AAV and derived recombinant AAV (rAAV) vectors are severely limited (ggt;10-fold) by the cellular DNA damage-sensing complex made up of Mre11, Rad50, and Nbs1 (MRN). The AAV genome does not encode the means to circumvent this block to productive infection but relies on coinfecting helper virus to do so. Using adenovirus helper proteins E1B55k and E4orf6, which enhance the transduction of AAV via degradation of MRN, we investigated the mechanism through which this DNA damage complex inhibits gene expression from rAAV. We tested the substrate specificity of inhibition and the contribution of different functions of the MRN complex. Our results demonstrate that both single- and double-stranded rAAV vectors are inhibited by MRN, which is in contrast to the predominant model that inhibition is the result of a block to second-strand synthesis. Exploring the contribution of known functions of MRN, we found that inhibition of rAAV does not require downstream DNA damage response factors, including signaling kinases ATM and ATR. The nuclease domain of Mre11 appears to play only a minor role in inhibition, while the DNA binding domain makes a greater contribution. Additionally, mutation of the inverted terminal repeat of the rAAV genome, which has been proposed to be the signal for interaction with MRN, is tolerated by the mechanism of inhibition. These results articulate a model of inhibition of gene expression in which physical interaction is more important than enzymatic activity and several key downstream damage repair factors are dispensable.

    IMPORTANCE Many viruses modulate the host DNA damage response (DDR) in order to create a cellular environment permissive for infection. The MRN complex is a primary sensor of damage in the cell but also responds to invading viral genomes, often posing a block to infection. AAV is greatly inhibited by MRN and dependent on coinfecting helper virus, such as adenovirus, to remove this factor. Currently, the mechanism through which MRN inhibits AAV and other viruses is poorly understood. Our results reform the predominant model that inhibition of rAAV by MRN is due to limiting second-strand DNA synthesis. Instead, a novel mechanism of inhibition of gene expression independent of a block in rAAV DNA synthesis or downstream damage factors is indicated. These findings have clear implications for understanding this restriction to transduction of AAV and rAAV vectors, which have high therapeutic relevance and likely translate to other viruses that must navigate the DDR.

  • Activation of p90 Ribosomal S6 Kinases by ORF45 of Kaposi's Sarcoma-Associated Herpesvirus Is Critical for Optimal Production of Infectious Viruses [Virus-Cell Interactions]

  • We have previously shown that ORF45, an immediate-early and tegument protein of Kaposi's sarcoma-associated herpesvirus (KSHV), causes sustained activation of p90 ribosomal S6 kinases (RSKs) and extracellular regulated kinase (ERK) (E. Kuang, Q. Tang, G. G. Maul, and F. Zhu, J Virol 82:1838nndash;1850, 2008, http://dx.doi.org/10.1128/JVI.02119-07). We now have identified the critical region of ORF45 that is involved in RSK interaction and activation. Alanine scanning mutagenesis of this region revealed that a single F66A point mutation abolished binding of ORF45 to RSK or ERK and, consequently, its ability to activate the kinases. We introduced the F66A mutation into BAC16 (a bacterial artificial chromosome clone containing the entire infectious KSHV genome), producing BAC16-45F66A. In parallel, we also repaired the mutation and obtained a revertant, BAC16-45A66F. The reconstitution of these mutants in iSLK cells demonstrated that the ORF45-F66A mutant failed to cause sustained ERK and RSK activation during lytic reactivation, resulting in dramatic differences in the phosphoproteomic profile between the wild-type virus-infected cells and the mutant virus-infected cells. ORF45 mutation or deletion also was accompanied by a noticeable decreased in viral gene expression during lytic reactivation. Consequently, the ORF45-F66A mutant produced significantly fewer infectious progeny virions than the wild type or the revertant. These results suggest a critical role for ORF45-mediated RSK activation in KSHV lytic replication.

    IMPORTANCE KSHV is the causative agent of three human malignancies. KSHV pathogenesis is intimately linked to its ability to modulate the host cell microenvironment and to facilitate efficient production of progeny viral particles. We previously described the mechanism by which the KSHV lytic protein ORF45 activates the cellular kinases ERK and RSK. We now have mapped the critical region of ORF45 responsible for binding and activation of ERK/RSK to a single residue, F66. We mutated this amino acid of ORF45 (F66A) and introduced the mutation into a newly developed bacterial artificial chromosome containing the KSHV genome (BAC16). This system has provided us with a useful tool to characterize the functions of ORF45-activated RSK upon KSHV lytic reactivation. We show that viral gene expression and virion production are significantly reduced by F66A mutation, indicating a critical role for ORF45-activated RSK during KSHV lytic replication.

  • Compensatory Substitutions in the HIV-1 Capsid Reduce the Fitness Cost Associated with Resistance to a Capsid-Targeting Small-Molecule Inhibitor [Vaccines and Antiviral Agents]

  • The HIV-1 capsid plays multiple roles in infection and is an emerging therapeutic target. The small-molecule HIV-1 inhibitor PF-3450074 (PF74) blocks HIV-1 at an early postentry stage by binding the viral capsid and interfering with its function. Selection for resistance resulted in accumulation of five amino acid changes in the viral CA protein, which collectively reduced binding of the compound to HIV-1 particles. In the present study, we dissected the individual and combinatorial contributions of each of the five substitutions Q67H, K70R, H87P, T107N, and L111I to PF74 resistance, PF74 binding, and HIV-1 infectivity. Q67H, K70R, and T107N each conferred low-level resistance to PF74 and collectively conferred strong resistance. The substitutions K70R and L111I impaired HIV-1 infectivity, which was partially restored by the other substitutions at positions 67 and 107. PF74 binding to HIV-1 particles was reduced by the Q67H, K70R, and T107N substitutions, consistent with the location of these positions in the inhibitor-binding pocket. Replication of the 5Mut virus was markedly impaired in cultured macrophages, reminiscent of the previously reported N74D CA mutant. 5Mut substitutions also reduced the binding of the host protein CPSF6 to assembled CA complexes in vitro and permitted infection of cells expressing the inhibitory protein CPSF6-358. Our results demonstrate that strong resistance to PF74 requires accumulation of multiple substitutions in CA to inhibit PF74 binding and compensate for fitness impairments associated with some of the sequence changes.

    IMPORTANCE The HIV-1 capsid is an emerging drug target, and several small-molecule compounds have been reported to inhibit HIV-1 infection by targeting the capsid. Here we show that resistance to the capsid-targeting inhibitor PF74 requires multiple amino acid substitutions in the binding pocket of the CA protein. Three changes in CA were necessary to inhibit binding of PF74 while maintaining viral infectivity. Replication of the PF74-resistant HIV-1 mutant was impaired in macrophages, likely owing to altered interactions with host cell factors. Our results suggest that HIV-1 resistance to capsid-targeting inhibitors will be limited by functional constraints on the viral capsid protein. Therefore, this work enhances the attractiveness of the HIV-1 capsid as a therapeutic target.

  • Identification of TRIM27 as a Novel Degradation Target of Herpes Simplex Virus 1 ICP0 [Virus-Cell Interactions]

  • The herpes simplex virus 1 (HSV-1) immediate early protein ICP0 performs many functions during infection, including transactivation of viral gene expression, suppression of innate immune responses, and modification and eviction of histones from viral chromatin. Although these functions of ICP0 have been characterized, the detailed mechanisms underlying ICP0's complex role during infection warrant further investigation. We thus undertook an unbiased proteomic approach to identifying viral and cellular proteins that interact with ICP0 in the infected cell. Cellular candidates resulting from our analysis included the ubiquitin-specific protease USP7, the transcriptional repressor TRIM27, DNA repair proteins NBN and MRE11A, regulators of apoptosis, including BIRC6, and the proteasome. We also identified two HSV-1 early proteins involved in nucleotide metabolism, UL39 and UL50, as novel candidate interactors of ICP0. Because TRIM27 was the most statistically significant cellular candidate, we investigated the relationship between TRIM27 and ICP0. We observed rapid, ICP0-dependent loss of TRIM27 during HSV-1 infection. TRIM27 protein levels were restored by disrupting the RING domain of ICP0 or by inhibiting the proteasome, arguing that TRIM27 is a novel degradation target of ICP0. A mutant ICP0 lacking E3 ligase activity interacted with endogenous TRIM27 during infection as demonstrated by reciprocal coimmunoprecipitation and supported by immunofluorescence data. Surprisingly, ICP0-null mutant virus yields decreased upon TRIM27 depletion, arguing that TRIM27 has a positive effect on infection despite being targeted for degradation. These results illustrate a complex interaction between TRIM27 and viral infection with potential positive or negative effects of TRIM27 on HSV under different infection conditions.

    IMPORTANCE During productive infection, a virus must simultaneously redirect multiple cellular pathways to replicate itself while evading detection by the host's defenses. To orchestrate such complex regulation, viruses, including herpes simplex virus 1 (HSV-1), rely on multifunctional proteins such as the E3 ubiquitin ligase ICP0. This protein regulates various cellular pathways concurrently by targeting a diverse set of cellular factors for degradation. While some of these targets have been previously identified and characterized, we undertook a proteomic screen to identify additional targets of this activity to further characterize ICP0's role during infection. We describe a set of candidate interacting proteins of ICP0 identified through this approach and our characterization of the most statistically significant result, the cellular transcriptional repressor TRIM27. We present TRIM27 as a novel degradation target of ICP0 and describe the relationship of these two proteins during infection.

  • G2/M Cell Cycle Arrest Correlates with Primate Lentiviral Vpr Interaction with the SLX4 Complex [Virus-Cell Interactions]

  • The accessory gene vpr, common to all primate lentiviruses, induces potent G2/M arrest in cycling cells. A recent study showed that human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) mediates this through activation of the SLX4/MUS81/EME1 exonuclease complex that forms part of the Fanconi anemia DNA repair pathway. To confirm these observations, we have examined the G2/M arrest phenotypes of a panel of simian immunodeficiency virus (SIV) Vpr proteins. We show that SIV Vpr proteins differ in their ability to promote cell cycle arrest in human cells. While this is dependent on the DCAF1/DDB1/CUL4 ubiquitin ligase complex, interaction with human DCAF1 does not predict G2/M arrest activity of SIV Vpr in human cells. In all cases, SIV Vpr-mediated cell cycle arrest in human cells correlated with interaction with human SLX4 (huSLX4) and could be abolished by small interfering RNA (siRNA) depletion of any member of the SLX4 complex. In contrast, all but one of the HIV/SIV Vpr proteins tested, including those that lacked activity in human cells, were competent for G2/M arrest in grivet cells. Correspondingly, here cell cycle arrest correlated with interaction with the grivet orthologues of the SLX4 complex, suggesting a level of host adaptation in these interactions. Phylogenetic analyses strongly suggest that G2/M arrest/SLX4 interactions are ancestral activities of primate lentiviral Vpr proteins and that the ability to dysregulate the Fanconi anemia DNA repair pathway is an essential function of Vpr in vivo.

    IMPORTANCE The Vpr protein of HIV-1 and related viruses is essential for the virus in vivo. The ability of Vpr to block the cell cycle at mitotic entry is well known, but the importance of this function for viral replication is unclear. Recent data have shown that HIV-1 Vpr targets the Fanconi anemia DNA repair pathway by interacting with and activating an endonuclease complex, SLX4/MUS81/EME1, that processes interstrand DNA cross-links. Here we show that the ability of a panel of SIV Vpr proteins to mediate cell cycle arrest correlates with species-specific interactions with the SLX4 complex in human and primate cells. The results of these studies suggest that the SLX4 complex is a conserved target of primate lentiviral Vpr proteins and that the ability to dysregulate members of the Fanconi anemia DNA repair pathway is essential for HIV/SIV replication in vivo.

  • Phosphorylation of Herpes Simplex Virus 1 dUTPase Regulates Viral Virulence and Genome Integrity by Compensating for Low Cellular dUTPase Activity in the Central Nervous System [Pathogenesis and Immunity]

  • A mutation in herpes simplex virus 1 dUTPase (vdUTPase), which precluded its phosphorylation at Ser-187, decreased viral neurovirulence and increased mutation frequency in progeny virus genomes in the brains of mice where endogenous cellular dUTPase activity was relatively low, and overexpression of cellular dUTPase restored viral neurovirulence and mutation frequency altered by the mutation. Thus, phosphorylation of vdUTPase appeared to regulate viral virulence and genome integrity by compensating for low cellular dUTPase activity in vivo.

    IMPORTANCE Many DNA viruses encode a homolog of host cell dUTPases, which are known to function in accurate replication of cellular DNA genomes. The viral dUTPase activity has long been assumed to play a role in viral replication by preventing mutations in progeny virus genomes if cellular dUTPase activity was not sufficient. Here, we showed that a mutation in herpes simplex virus 1 dUTPase, which precluded its phosphorylation at Ser-187 and reduced its activity, decreased viral neurovirulence and increased mutation frequency in progeny virus genomes in the brains of mice where endogenous cellular dUTPase activity was relatively low. In contrast, overexpression of cellular dUTPase restored viral neurovirulence and mutation frequency altered by the mutation in the brains of mice. This is the first report, to our knowledge, directly showing that viral dUTPase activity regulates viral genome integrity and pathogenicity by compensating for insufficient cellular dUTPase activity in vivo.

  • Perturbation in the Conserved Methyltransferase-Polymerase Interface of Flavivirus NS5 Differentially Affects Polymerase Initiation and Elongation [Genome Replication and Regulation of Viral Gene Expression]

  • The flavivirus NS5 is a natural fusion of a methyltransferase (MTase) and an RNA-dependent RNA polymerase (RdRP). Analogous to DNA-dependent RNA polymerases, the NS5 polymerase initiates RNA synthesis through a de novo mechanism and then makes a transition to a processive elongation phase. However, whether and how the MTase affects polymerase activities through intramolecular interactions remain elusive. By solving the crystal structure of the Japanese encephalitis virus (JEV) NS5, we recently identified an MTase-RdRP interface containing a set of six hydrophobic residues highly conserved among flaviviruses. To dissect the functional relevance of this interface, we made a series of JEV NS5 constructs with mutations of these hydrophobic residues and/or with the N-terminal first 261 residues and other residues up to the first 303 residues deleted. Compared to the wild-type (WT) NS5, full-length NS5 variants exhibited consistent up- or downregulation of the initiation activities in two types of polymerase assays. Five representative full-length NS5 constructs were then tested in an elongation assay, from which the apparent single-nucleotide incorporation rate constant was estimated. Interestingly, two constructs exhibited different elongation kinetics from the WT NS5, with an effect rather opposite to what was observed at initiation. Moreover, constructs with MTase and/or the linker region (residues 266 to 275) removed still retained polymerase activities, albeit at overall lower levels. However, further removal of the N-terminal extension (residues 276 to 303) abolished regular template-directed synthesis. Together, our data showed that the MTase-RdRP interface is relevant in both polymerase initiation and elongation, likely with different regulation mechanisms in these two major phases of RNA synthesis.

    IMPORTANCE The flavivirus NS5 is very unique in having a methyltransferase (MTase) placed on the immediate N terminus of its RNA-dependent RNA polymerase (RdRP). We recently solved the crystal structure of the full-length NS5, which revealed a conserved interface between MTase and RdRP. Building on this discovery, here we carried out in vitro polymerase assays to address the functional relevance of the interface interactions. By explicitly probing polymerase initiation and elongation activities, we found that perturbation in the MTase-RdRP interface had different impacts on different phases of synthesis, suggesting that the roles and contribution of the interface interactions may change upon phase transitions. By comparing the N-terminal-truncated enzymes with the full-length NS5, we collected data to indicate the indispensability to regular polymerase activities of a region that was functionally unclarified previously. Taken together, we provide biochemical evidence and mechanistic insights for the cross talk between the two enzyme modules of flavivirus NS5.

  • Entry Mechanisms of Herpes Simplex Virus 1 into Murine Epidermis: Involvement of Nectin-1 and Herpesvirus Entry Mediator as Cellular Receptors [Virus-Cell Interactions]

  • Skin keratinocytes represent a primary entry site for herpes simplex virus 1 (HSV-1) in vivo. The cellular proteins nectin-1 and herpesvirus entry mediator (HVEM) act as efficient receptors for both serotypes of HSV and are sufficient for disease development mediated by HSV-2 in mice. How HSV-1 enters skin and whether both nectin-1 and HVEM are involved are not known. We addressed the impact of nectin-1 during entry of HSV-1 into murine epidermis and investigated the putative contribution of HVEM. Using ex vivo infection of murine epidermis, we showed that HSV-1 entered the basal keratinocytes of the epidermis very efficiently. In nectin-1-deficient epidermis, entry was strongly reduced. Almost no entry was observed, however, in nectin-1-deficient keratinocytes grown in culture. This observation correlated with the presence of HVEM on the keratinocyte surface in epidermis and with the lack of HVEM expression in nectin-1-deficient primary keratinocytes. Our results suggest that nectin-1 is the primary receptor in epidermis, while HVEM has a more limited role. For primary murine keratinocytes, on which nectin-1 acts as a single receptor, electron microscopy suggested that HSV-1 can enter both by direct fusion with the plasma membrane and via endocytic vesicles. Thus, we concluded that nectin-1 directs internalization into keratinocytes via alternative pathways. In summary, HSV-1 entry into epidermis was shown to strongly depend on the presence of nectin-1, but the restricted presence of HVEM can potentially replace nectin-1 as a receptor, illustrating the flexibility employed by HSV-1 to efficiently invade tissue in vivo.

    IMPORTANCE Herpes simplex virus (HSV) can cause a range of diseases in humans, from uncomplicated mucocutaneous lesions to life-threatening infections. The skin is one target tissue of HSV, and the question of how the virus overcomes the protective skin barrier and penetrates into the tissue to reach its receptors is still open. Previous studies analyzing entry into cells grown in vitro revealed nectin-1 and HVEM as HSV receptors. To explore the contributions of nectin-1 and HVEM to entry into a natural target tissue, we established an ex vivo infection model. Using nectin-1- or HVEM-deficient mice, we demonstrated the distinct involvement of nectin-1 and HVEM for HSV-1 entry into epidermis and characterized the internalization pathways. Such advances in understanding the involvement of receptors in tissue are essential preconditions for unraveling HSV invasion of skin, which in turn will allow the development of antiviral reagents.

  • Structure-Function Relationships Underlying the Replication Fidelity of Viral RNA-Dependent RNA Polymerases [Genome Replication and Regulation of Viral Gene Expression]

  • Viral RNA-dependent RNA polymerases are considered to be low-fidelity enzymes, providing high mutation rates that allow for the rapid adaptation of RNA viruses to different host cell environments. Fidelity is tuned to provide the proper balance of virus replication rates, pathogenesis, and tissue tropism needed for virus growth. Using our structures of picornaviral polymerase-RNA elongation complexes, we have previously engineered more than a dozen coxsackievirus B3 polymerase mutations that significantly altered virus replication rates and in vivo fidelity and also provided a set of secondary adaptation mutations after tissue culture passage. Here we report a biochemical analysis of these mutations based on rapid stopped-flow kinetics to determine elongation rates and nucleotide discrimination factors. The data show a spatial separation of fidelity and replication rate effects within the polymerase structure. Mutations in the palm domain have the greatest effects on in vitro nucleotide discrimination, and these effects are strongly correlated with elongation rates and in vivo mutation frequencies, with faster polymerases having lower fidelity. Mutations located at the top of the finger domain, on the other hand, primarily affect elongation rates and have relatively minor effects on fidelity. Similar modulation effects are seen in poliovirus polymerase, an inherently lower-fidelity enzyme where analogous mutations increase nucleotide discrimination. These findings further our understanding of viral RNA-dependent RNA polymerase structure-function relationships and suggest that positive-strand RNA viruses retain a unique palm domain-based active-site closure mechanism to fine-tune replication fidelity.

    IMPORTANCE Positive-strand RNA viruses represent a major class of human and animal pathogens with significant health and economic impacts. These viruses replicate by using a virally encoded RNA-dependent RNA polymerase enzyme that has low fidelity, generating many mutations that allow the rapid adaptation of these viruses to different tissue types and host cells. In this work, we use a structure-based approach to engineer mutations in viral polymerases and study their effects on in vitro nucleotide discrimination as well as virus growth and genome replication fidelity. These results show that mutation rates can be drastically increased or decreased as a result of single mutations at several key residues in the polymerase palm domain, and this can significantly attenuate virus growth in vivo. These findings provide a pathway for developing live attenuated virus vaccines based on engineering the polymerase to reduce virus fitness.

  • Profiling and Characterization of Influenza Virus N1 Strains Potentially Resistant to Multiple Neuraminidase Inhibitors [Vaccines and Antiviral Agents]

  • Neuraminidase inhibitors (NAIs) have been widely used to control influenza virus infection, but their increased use could promote the global emergence of resistant variants. Although various mutations associated with NAI resistance have been identified, the amino acid substitutions that confer multidrug resistance with undiminished viral fitness remain poorly understood. We therefore screened a known mutation(s) that could confer multidrug resistance to the currently approved NAIs oseltamivir, zanamivir, and peramivir by assessing recombinant viruses with mutant NA-encoding genes (catalytic residues R152K and R292K, framework residues E119A/D/G, D198N, H274Y, and N294S) in the backbones of the 2009 pandemic H1N1 (pH1N1) and highly pathogenic avian influenza (HPAI) H5N1 viruses. Of the 14 single and double mutant viruses recovered in the backbone of pH1N1, four variants (E119D, E119A/D/G-H274Y) exhibited reduced inhibition by all of the NAIs and two variants (E119D and E119D-H274Y) retained the overall properties of gene stability, replicative efficiency, pathogenicity, and transmissibility in vitro and in vivo. Of the nine recombinant H5N1 viruses, four variants (E119D, E119A/D/G-H274Y) also showed reduced inhibition by all of the NAIs, though their overall viral fitness was impaired in vitro and/or in vivo. Thus, single mutations or certain combination of the established mutations could confer potential multidrug resistance on pH1N1 or HPAI H5N1 viruses. Our findings emphasize the urgency of developing alternative drugs against influenza virus infection.

    IMPORTANCE There has been a widespread emergence of influenza virus strains with reduced susceptibility to neuraminidase inhibitors (NAIs). We screened multidrug-resistant viruses by studying the viral fitness of neuraminidase mutants in vitro and in vivo. We found that recombinant E119D and E119A/D/G/-H274Y mutant viruses demonstrated reduced inhibition by all of the NAIs tested in both the backbone of the 2009 H1N1 pandemic (pH1N1) and highly pathogenic avian influenza H5N1 viruses. Furthermore, E119D and E119D-H274Y mutants in the pH1N1 background maintained overall fitness properties in vitro and in vivo. Our study highlights the importance of vigilance and continued surveillance of potential NAI multidrug-resistant influenza virus variants, as well as the development of alternative therapeutics.

  • Interaction of NS2 with AIMP2 Facilitates the Switch from Ubiquitination to SUMOylation of M1 in Influenza A Virus-Infected Cells [Virus-Cell Interactions]

  • Influenza A viruses (IAVs) rely on host factors to support their life cycle, as viral proteins hijack or interact with cellular proteins to execute their functions. Identification and understanding of these factors would increase our knowledge of the molecular mechanisms manipulated by the viruses. In this study, we searched for novel binding partners of the influenza A virus NS2 protein, the nuclear export protein responsible for overcoming host range restriction, by a yeast two-hybrid screening assay and glutathione S-transferase-pulldown and coimmunoprecipitation assays and identified AIMP2, a potent tumor suppressor that usually functions to regulate protein stability, as one of the major NS2-binding candidates. We found that the presence of NS2 protected AIMP2 from ubiquitin-mediated degradation in NS2-transfected cells and AIMP2 functioned as a positive regulator of IAV replication. Interestingly, AIMP2 had no significant effect on NS2 but enhanced the stability of the matrix protein M1. Further, we provide evidence that AIMP2 recruitment switches the modification of M1 from ubiquitination to SUMOylation, which occurs on the same attachment site (K242) on M1 and thereby promotes M1-mediated viral ribonucleoprotein complex nuclear export to increase viral replication. Collectively, our results reveal a new mechanism of AIMP2 mediation of influenza virus replication.

    IMPORTANCE Although the ubiquitination of M1 during IAV infection has been observed, the precise modification site and the molecular consequences of this modification remain obscure. Here, we demonstrate for the first time that ubiquitin and SUMO compete for the same lysine (K242) on M1 and the interaction of NS2 with AIMP2 facilitates the switch of the M1 modification from ubiquitination to SUMOylation, thus increasing viral replication.

  • Expression of Interferon Gamma by a Recombinant Rabies Virus Strongly Attenuates the Pathogenicity of the Virus via Induction of Type I Interferon [Pathogenesis and Immunity]

  • Previous animal model experiments have shown a correlation between interferon gamma (IFN-) expression and both survival from infection with attenuated rabies virus (RABV) and reduction of neurological sequelae. Therefore, we hypothesized that rapid production of murine IFN- by the rabies virus itself would induce a more robust antiviral response than would occur naturally in mice. To test this hypothesis, we used reverse engineering to clone the mouse IFN- gene into a pathogenic rabies virus backbone, SPBN, to produce the recombinant rabies virus designated SPBN. Morbidity and mortality were monitored in mice infected intranasally with SPBN or SPBN(nndash;) control virus to determine the degree of attenuation caused by the expression of IFN-. Incorporation of IFN- into the rabies virus genome highly attenuated the virus. SPBN has a 50% lethal dose (LD50) more than 100-fold greater than SPBN(nndash;). In vitro and in vivo mouse experiments show that SPBN infection enhances the production of type I interferons. Furthermore, knockout mice lacking the ability to signal through the type I interferon receptor (IFNARnndash;/nndash;) cannot control the SPBN infection and rapidly die. These data suggest that IFN- production has antiviral effects in rabies, largely due to the induction of type I interferons.

    IMPORTANCE Survival from rabies is dependent upon the early control of virus replication and spread. Once the virus reaches the central nervous system (CNS), this becomes highly problematic. Studies of CNS immunity to RABV have shown that control of replication begins at the onset of T cell entry and IFN- production in the CNS prior to the appearance of virus-neutralizing antibodies. Moreover, antibody-deficient mice are able to control but not clear attenuated RABV from the CNS. We find here that IFN- triggers the early production of type I interferons with the expected antiviral effects. We also show that engineering a lethal rabies virus to express IFN- directly in the infected tissue reduces rabies virus replication and spread, limiting its pathogenicity in normal and immunocompromised mice. Therefore, vector delivery of IFN- to the brain may have the potential to treat individuals who would otherwise succumb to infection with rabies virus.

  • Selective Acquisition of Host-Derived ICAM-1 by HIV-1 Is a Matrix-Dependent Process [Virus-Cell Interactions]

  • HIV-1 acquires an impressive number of foreign components during its formation. Despite all previous efforts spent studying the nature and functionality of virus-anchored host molecules, the exact mechanism(s) through which such constituents are acquired by HIV-1 is still unknown. However, in the case of ICAM-1, one of the most extensively studied transmembrane proteins found associated with mature virions, the Pr55Gag precursor polyprotein appears to be a potential interaction partner. We investigated and characterized at the molecular level the process of ICAM-1 incorporation using initially a Pr55Gag-based virus-like particle (VLP) model. Substitution of various domains of Pr55Gag, such as the nucleocapsid, SP2, or p6, had no effect on the acquisition of ICAM-1. We found that the structural matrix protein (MA) is mandatory for ICAM-1 incorporation within VLPs, and we confirmed this novel observation with the replication-competent HIV-1 molecular clone NL4.3. Additional studies suggest that the C-terminal two-thirds of MA, and especially 13 amino acids positioned inside the fifth aalpha;-helix, are important. Moreover, based on three-dimensional (3D) modeling of protein-protein interactions (i.e., protein-protein docking) and further validation by a virus capture assay, we found that a series of acidic residues in the MA domain interact with basic amino acids located in the ICAM-1 cytoplasmic tail. Our findings provide new insight into the molecular mechanism governing the acquisition of ICAM-1, a host molecule known to enhance HIV-1 infectivity in a significant manner. Altogether, these observations offer a new avenue for the development of antiviral therapeutics that are directed at a target of host origin.

    IMPORTANCE Intercellular adhesion molecule 1 (ICAM-1) is a cell surface host component known to be efficiently inserted within emerging HIV-1 particles. It has been demonstrated that host-derived ICAM-1 molecules act as a strong attachment factor and increase HIV-1 infectivity substantially. Despite previous efforts spent studying virus-associated host molecules, the precise mechanism(s) through which such constituents are inserted within emerging HIV-1 particles still remains obscure. Previous data suggest that the Pr55Gag precursor polyprotein appears as a potential interaction partner with ICAM-1. In the present study, we demonstrate that the HIV-1 matrix domain plays a key role in the ICAM-1 incorporation process. Some observations were confirmed with whole-virus preparations amplified in primary human cells, thereby providing physiological significance to our data.

  • Novel Mode of ISG15-Mediated Protection against Influenza A Virus and Sendai Virus in Mice [Pathogenesis and Immunity]

  • ISG15 is a diubiquitin-like modifier and one of the most rapidly induced genes upon type I interferon stimulation. Hundreds of host proteins and a number of viral proteins have been shown to be ISGylated, and understanding how these modifications affect the interferon response and virus replication has been of considerable interest. ISG15nndash;/nndash; mice exhibit increased susceptibility to viral infection, and in the case of influenza B virus and vaccinia virus, ISG15 conjugation has been shown to restrict virus replication in vivo. A number of studies have also found that ISG15 is capable of antagonizing replication of some viruses in tissue culture. However, recent findings have demonstrated that ISG15 can protect mice from Chikungunya virus infection without affecting the virus burden. In order to better understand the function of ISG15 in vivo, we characterized the pathogenesis of influenza A virus and Sendai virus in ISG15nndash;/nndash; mice. We found that ISG15 protects mice from virus induced lethality by a conjugation-dependent mechanism in both of these models. However, surprisingly, we found that ISG15 had minimal effect on virus replication and did not have an obvious role in the modulation of the acute immune response to infection. Instead, we observed an increase in the number of diseased small airways in mice lacking ISG15. This ability of ISG15 to protect mice in a conjugation-dependent, but nonantiviral, manner from respiratory virus infection represents a previously undescribed role for ISG15 and demonstrates the importance of further characterization of ISG15 in vivo.

    IMPORTANCE It has previously been demonstrated that ISG15nndash;/nndash; mice are more susceptible to a number of viral infections. Since ISG15 is one of the most strongly induced genes after type I interferon stimulation, analysis of ISG15 function has largely focused on its role as an antiviral molecule during acute infection. Although a number of studies have shown that ISG15 does have a small effect on virus replication in tissue culture, few studies have confirmed this mechanism of protection in vivo. In these studies we have found that while ISG15nndash;/nndash; mice are more susceptible to influenza A virus and Sendai virus infections, ISGylation does not appear to mediate this protection through the direct inhibition of virus replication or the modulation of the acute immune response. Thus, in addition to showing a novel mode of ISG15 mediated protection from virus infection, this study demonstrates the importance of studying the role of ISG15 in vivo.

  • Variations in pH Sensitivity, Acid Stability, and Fusogenicity of Three Influenza Virus H3 Subtypes [Virus-Cell Interactions]

  • Influenza A virus strains adapt to achieve successful entry into host species. Entry is mediated by the viral membrane protein hemagglutinin (HA), which triggers membrane fusion and genome release under acidic conditions in the endosome. In addition to changes in the receptor binding domain, the acid stability of HA has been linked to the successful transmission of virus between avian and human hosts. However, to fully understand the connection between changes in HA and host tropism, additional factors relevant to HA structure-function and membrane fusion are also likely to be important. Using single-particle-tracking (SPT) techniques, individual membrane fusion events can be observed under specific conditions, which provide detailed information regarding HA pH sensitivity and acid stability and the rate and extent of membrane fusion. This provides a comparative way to characterize and distinguish influenza virus fusion properties among virus strains. We used SPT to quantify the fusion properties of three H3 influenza strains: A/Aichi/68/H3N2 (X:31), A/Udorn/72/H3N2 (Udorn), and A/Brisbane/07/H3N2 (Brisbane). The rate of fusion for the most clinically relevant strain, Brisbane, is generally insensitive to decreasing pH, while the fusion of the egg-adapted strains Udorn and X:31 is strongly dependent on pH (and is faster) as the pH decreases. All strains exhibit similar acid stability (the length of time that they remain fusogenic in an acidic environment) at higher pHs, but the egg-adapted strains become less acid stable at lower pHs. Thus, it appears that the laboratory-adapted H3 strains tested may have evolved to compensate for the faster HA deactivation at low pH, with a commensurate increase in the rate of fusion and number of proteins facilitating fusion, relative to the Brisbane strain.

    IMPORTANCE The ability of influenza virus to release its genome under different acidic conditions has recently been linked to the transmission of influenza virus between different species. However, it is yet to be determined how acid-induced membrane fusion varies with virus strain and influences tropism. The results presented here are the results of an intra-H3-subtype study of acid stability and fusion kinetics. Using a single-particle-tracking (SPT) technique, we show here that the highest pH that initiates fusion is not necessarily the pH at which the kinetics of fusion is fastest and most abundant for a given strain. Strains exhibit different fusion behaviors, as evidenced by their unique kinetic trends; pH sensitivities, as evidenced by the differences when the first fusion events commence; and HA stabilities, as evidenced by the length of time that virions can persist in an acidic environment and still be fusion competent.

  • Identification of a Neutralizing Epitope within Antigenic Domain 5 of Glycoprotein B of Human Cytomegalovirus [Pathogenesis and Immunity]

  • Human cytomegalovirus (HCMV) is an important, ubiquitous pathogen that causes severe clinical disease in immunocompromised individuals, such as organ transplant recipients and infants infected in utero. The envelope glycoprotein B (gB) of HCMV is a major antigen for the induction of virus-neutralizing antibodies. We have begun to define target structures within gB that are recognized by virus-neutralizing antibodies. Antigenic domain 5 (AD-5) of gB has been identified as an important target for neutralizing antibodies in studies using human monoclonal antibodies (MAbs). Anti-AD-5 MAbs share a target site on gB, despite originating from different, healthy, HCMV-infected donors. Mutational analysis of AD-5 identified tyrosine 280 in combination with other surface-exposed residues (the YNND epitope) as critical for antibody binding. The YNND epitope is strictly conserved among different HCMV strains. Recombinant viruses carrying YNND mutations in AD-5 were resistant to virus-neutralizing MAbs. Competition enzyme-linked immunosorbent assays (ELISAs) with human HCMV-convalescent-phase sera from unselected donors confirmed the conserved antibody response for the YNND epitope in HCMV-infected individuals and, because a significant fraction of the gB AD-5 response was directed against the YNND epitope, further argued that this epitope is a major target of anti-AD-5 antibody responses. In addition, affinity-purified polyclonal anti-AD-5 antibodies prepared from individual sera showed reactivity to AD-5 and neutralization activity toward gB mutant viruses that were similar to those of AD-5-specific MAbs. Taken together, our data indicate that the YNND epitope represents an important target for anti-gB antibody responses as well as for anti-AD-5 virus-neutralizing antibodies.

    IMPORTANCE HCMV is a major global health concern, and a vaccine to prevent HCMV disease is a widely recognized medical need. Glycoprotein B of HCMV is an important target for neutralizing antibodies and hence an interesting molecule for intervention strategies, e.g., vaccination. Mapping the target structures of neutralizing antibodies induced by naturally occurring HCMV infection can aid in defining the properties required for a protective capacity of vaccine antigens. The data presented here extend our knowledge of neutralizing epitopes within gB to include AD-5. Collectively, our data will contribute to optimal vaccine design and development of antibody-based therapies.

  • Autographa californica Multiple Nucleopolyhedrovirus ORF11 Is Essential for Budded-Virus Production and Occlusion-Derived-Virus Envelopment [Genome Replication and Regulation of Viral Gene Expression]

  • ORF11 (ac11) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved gene with unknown function. To determine the role of ac11 in the baculovirus life cycle, an ac11 knockout mutant of AcMNPV, Ac11KO, was constructed. Northern blot and 5' rapid amplification of cDNA ends (RACE) analyses revealed that ac11 is an early gene in the life cycle. Microscopy, titration assays, and Western blot analysis revealed that budded viruses (BVs) were not produced in Ac11KO-transfected Sf9 cells. However, quantitative PCR (qPCR) analysis demonstrated that the deletion of ac11 did not affect viral DNA replication. Furthermore, electron microscopy revealed that there was no nucleocapsid in the cytoplasm or plasma membrane of Ac11KO-transfected cells, which demonstrates that the defect in BV production in Ac11KO-transfected cells is due to the inefficient egress of nucleocapsids from the nucleus to the cytoplasm. In addition, electron microscopy observations showed that the nucleocapsids in the nucleus were not enveloped to form occlusion-derived viruses (ODVs) and that their subsequent embedding into occlusion bodies (OBs) was also blocked in Ac11KO-transfected cells, demonstrating that ac11 is required for ODV envelopment. These results therefore demonstrate that ac11 is an early gene that is essential for BV production and ODV envelopment.

    IMPORTANCE Baculoviruses have been extensively used not only as specific, environmentally benign insecticides but also as helper-independent protein expression vectors. Although the function of baculovirus genes in viral replication has been studied by using gene knockout technology, the functions of more than one-third of viral genes, which include some highly conserved genes, are still unknown. In this study, ac11 was proven to play a crucial role in BV production and ODV envelopment. These results will lead to a better understanding of baculovirus infection cycles.

  • Superinfection with Woodchuck Hepatitis Virus Strain WHVNY of Livers Chronically Infected with Strain WHV7 [Genome Replication and Regulation of Viral Gene Expression]

  • The determinants of the maintenance of chronic hepadnaviral infection are yet to be fully understood. A long-standing unresolved argument in the hepatitis B virus (HBV) research field suggests that during chronic hepadnaviral infection, cell-to-cell spread of hepadnavirus is at least very inefficient (if it occurs at all), virus superinfection is an unlikely event, and chronic hepadnavirus infection can be maintained exclusively via division of infected hepatocytes in the absence of virus spread. Superinfection exclusion was previously shown for duck HBV, but it was not demonstrated for HBV or HBV-related woodchuck hepatitis virus (WHV). Three woodchucks, which were chronically infected with the strain WHV7 and already developed WHV-induced hepatocellular carcinomas (HCCs), were superinfected with another WHV strain, WHVNY. Six weeks after the superinfection, the woodchucks were sacrificed and tissues of the livers and HCCs were examined. The WHVNY superinfection was demonstrated by using WHV strain-specific PCR assays and (i) finding WHVNY relaxed circular DNA in the serum samples collected from all superinfected animals during weeks one through six after the superinfection, (ii) detecting replication-derived WHVNY RNA in the tissue samples of the livers and HCCs collected from three superinfected woodchucks, and (iii) finding WHVNY DNA replication intermediates in tissues harvested after the superinfection. The results are consistent with the occurrence of continuous but inefficient hepadnavirus cell-to-cell spread and superinfection during chronic infection and suggest that the replication space occupied by the superinfecting hepadnavirus in chronically infected livers is limited. The findings are discussed in the context of the mechanism of chronic hepadnavirus infection.

    IMPORTANCE This study aimed to better understand the determinants of the maintenance of chronic hepadnavirus infection. The generated data suggest that in the livers chronically infected with woodchuck hepatitis virus, (i) hepadnavirus superinfection and cell-to-cell spread likely continue to occur and (ii) the virus spread is apparently inefficient, which is consistent with the interpretation that a limited number of cells in the livers facilitates the spread of hepadnavirus. The limitations of the cell-to-cell virus spread most likely are mediated at the level of the cells and do not reflect the properties of the virus. Our results further advance the understanding of the mechanism of chronic hepadnavirus infection. The significance of the continuous but limited hepadnavirus spread and superinfection for the maintenance of the chronic state of infection should be further evaluated in follow-up studies in order to determine whether blocking the virus spread would facilitate the suppression of chronic hepadnavirus infection.

  • Murine Cytomegalovirus Is Not Restricted to the House Mouse Mus musculus domesticus: Prevalence and Genetic Diversity in the European House Mouse Hybrid Zone [Genetic Diversity and Evolution]

  • Murine cytomegalovirus (MCMV) is a betaherpesvirus of the house mouse, Mus musculus domesticus. It is a common infectious agent of wild mice and a highly studied pathogen of the laboratory mouse. Betaherpesviruses are specific to their hosts, and it is not known if other Mus taxa carry MCMV or if it is restricted to M. m. domesticus. We sampled mice over a 145-km transect of Bavaria-Bohemia crossing a hybrid zone between M. m. domesticus and Mus musculus musculus in order to investigate the occurrence of MCMV in two Mus subspecies and to test the limits of the specificity of the virus for its host. We hypothesized that if the two subspecies carry MCMV and if the virus is highly specific to its host, divergent MCMV lineages would have codiverged with their hosts and would have a geographical distribution constrained by the host genetic background. A total of 520 mice were tested by enzyme-linked immunosorbent assay (ELISA) and/or nested PCR targeting the M94 gene. Seropositive and PCR-positive individuals were found in both Mus subspecies. Seroprevalence was high, at 79.4%, but viral DNA was detected in only 41.7% of mice. Sequencing revealed 20 haplotypes clustering in 3 clades that match the host genetic structure in the hybrid zone, showing 1 and 2 MCMV lineages in M. m. domesticus and M. m. musculus, respectively. The estimated time to the most recent common ancestor (1.1 million years ago [Mya]) of the MCMVs matches that of their hosts. In conclusion, MCMV has coevolved with these hosts, suggesting that its diversity in nature may be underappreciated, since other members of the subgenus Mus likely carry different MCMVs.

    IMPORTANCE Murine cytomegalovirus (MCMV) is a betaherpesvirus of the house mouse, Mus musculus domesticus, an important lab model for human cytomegalovirus (HCMV) infection. The majority of lab studies are based on only two strains of MCMVs isolated from M. m. domesticus, Smith and K181, the latter derived from repeated passage of Smith in mouse submaxillary glands. The presence of MCMV in other members of the Mus subgenus had not even been investigated. By screening mouse samples collected in the European house mouse hybrid zone between M. m. domesticus and M. m. musculus, we show that MCMV is not restricted to the M. m. domesticus subspecies and that MCMVs likely codiverged with their Mus hosts. Thus, the diversity of MCMV in nature may be seriously underappreciated, since other members of the subgenus Mus likely carry their own MCMV lineages.

  • Kaposi's Sarcoma-Associated Herpesvirus-Encoded Replication and Transcription Activator Impairs Innate Immunity via Ubiquitin-Mediated Degradation of Myeloid Differentiation Factor 88 [Virus-Cell Interactions]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) is a human gammaherpesvirus with latent and lytic reactivation cycles. The mechanism by which KSHV evades the innate immune system to establish latency has not yet been precisely elucidated. Toll-like receptors (TLRs) are the first line of defense against viral infections. Myeloid differentiation factor 88 (MyD88) is a key adaptor that interacts with all TLRs except TLR3 to produce inflammatory factors and type I interferons (IFNs), which are central components of innate immunity against microbial infection. Here, we found that KSHV replication and transcription activator (RTA), which is an immediate-early master switch protein of viral cycles, downregulates MyD88 expression at the protein level by degrading MyD88 through the ubiquitin (Ub)-proteasome pathway. We identified the interaction between RTA and MyD88 in vitro and in vivo and demonstrated that RTA functions as an E3 ligase to ubiquitinate MyD88. MyD88 also was repressed at the early stage of de novo infection as well as in lytic reactivation. We also found that RTA inhibited lipopolysaccharide (LPS)-triggered activation of the TLR4 pathway by reducing IFN production and NF-B activity. Finally, we showed that MyD88 promoted the production of IFNs and inhibited KSHV LANA-1 gene transcription. Taken together, our results suggest that KSHV RTA facilitates the virus to evade innate immunity through the degradation of MyD88, which might be critical for viral latency control.

    IMPORTANCE MyD88 is an adaptor for all TLRs other than TLR3, and it mediates inflammatory factors and IFN production. Our study demonstrated that the KSHV RTA protein functions as an E3 ligase to degrade MyD88 through the ubiquitin-proteasome pathway and block the transmission of TLRs signals. Moreover, we found that KSHV inhibited MyD88 expression during the early stage of de novo infection as well as in lytic reactivation. These results provide a potential mechanism for the virus to evade innate immunity.

  • A 2.5-Kilobase Deletion Containing a Cluster of Nine MicroRNAs in the Latency-Associated-Transcript Locus of the Pseudorabies Virus Affects the Host Response of Porcine Trigeminal Ganglia during Established Latency [Virus-Cell Interactions]

  • The alphaherpesvirus pseudorabies virus (PrV) establishes latency primarily in neurons of trigeminal ganglia when only the transcription of the latency-associated transcript (LAT) locus is detected. Eleven microRNAs (miRNAs) cluster within the LAT, suggesting a role in establishment and/or maintenance of latency. We generated a mutant (M) PrV deleted of nine miRNA genes which displayed properties that were almost identical to those of the parental PrV wild type (WT) during propagation in vitro. Fifteen pigs were experimentally infected with either WT or M virus or were mock infected. Similar levels of virus excretion and host antibody response were observed in all infected animals. At 62 days postinfection, trigeminal ganglia were excised and profiled by deep sequencing and quantitative RT-PCR. Latency was established in all infected animals without evidence of viral reactivation, demonstrating that miRNAs are not essential for this process. Lower levels of the large latency transcript (LLT) were found in ganglia infected by M PrV than in those infected by WT PrV. All PrV miRNAs were expressed, with highest expression observed for prv-miR-LLT1, prv-miR-LLT2 (in WT ganglia), and prv-miR-LLT10 (in both WT and M ganglia). No evidence of differentially expressed porcine miRNAs was found. Fifty-four porcine genes were differentially expressed between WT, M, and control ganglia. Both viruses triggered a strong host immune response, but in M ganglia gene upregulation was prevalent. Pathway analyses indicated that several biofunctions, including those related to cell-mediated immune response and the migration of dendritic cells, were impaired in M ganglia. These findings are consistent with a function of the LAT locus in the modulation of host response for maintaining a latent state.

    IMPORTANCE This study provides a thorough reference on the establishment of latency by PrV in its natural host, the pig. Our results corroborate the evidence obtained from the study of several LAT mutants of other alphaherpesviruses encoding miRNAs from their LAT regions. Neither PrV miRNA expression nor high LLT expression levels are essential to achieve latency in trigeminal ganglia. Once latency is established by PrV, the only remarkable differences are found in the pattern of host response. This indicates that, as in herpes simplex virus, LAT functions as an immune evasion locus.

  • The Putative Herpes Simplex Virus 1 Chaperone Protein UL32 Modulates Disulfide Bond Formation during Infection [Structure and Assembly]

  • During DNA encapsidation, herpes simplex virus 1 (HSV-1) procapsids are converted to DNA-containing capsids by a process involving activation of the viral protease, expulsion of the scaffold proteins, and the uptake of viral DNA. Encapsidation requires six minor capsid proteins (UL6, UL15, UL17, UL25, UL28, and UL33) and one viral protein, UL32, not found to be associated with capsids. Although functions have been assigned to each of the minor capsid proteins, the role of UL32 in encapsidation has remained a mystery. Using an HSV-1 variant containing a functional hemagglutinin-tagged UL32, we demonstrated that UL32 was synthesized with true late kinetics and that it exhibited a previously unrecognized localization pattern. At 6 to 9 h postinfection (hpi), UL32 accumulated in viral replication compartments in the nucleus of the host cell, while at 24 hpi, it was additionally found in the cytoplasm. A newly generated UL32-null mutant was used to confirm that although B capsids containing wild-type levels of capsid proteins were synthesized, these procapsids were unable to initiate the encapsidation process. Furthermore, we showed that UL32 is redox sensitive and identified two highly conserved oxidoreductase-like C-X-X-C motifs that are essential for protein function. In addition, the disulfide bond profiles of the viral proteins UL6, UL25, and VP19C and the viral protease, VP24, were altered in the absence of UL32, suggesting that UL32 may act to modulate disulfide bond formation during procapsid assembly and maturation.

    IMPORTANCE Although functions have been assigned to six of the seven required packaging proteins of HSV, the role of UL32 in encapsidation has remained a mystery. UL32 is a cysteine-rich viral protein that contains C-X-X-C motifs reminiscent of those in proteins that participate in the regulation of disulfide bond formation. We have previously demonstrated that disulfide bonds are required for the formation and stability of the viral capsids and are also important for the formation and stability of the UL6 portal ring. In this report, we demonstrate that the disulfide bond profiles of the viral proteins UL6, UL25, and VP19C and the viral protease, VP24, are altered in cells infected with a newly isolated UL32-null mutant virus, suggesting that UL32 acts as a chaperone capable of modulating disulfide bond formation. Furthermore, these results suggest that proper regulation of disulfide bonds is essential for initiating encapsidation.

  • Basic Motifs Target PSGL-1, CD43, and CD44 to Plasma Membrane Sites Where HIV-1 Assembles [Structure and Assembly]

  • HIV-1 incorporates various host membrane proteins during particle assembly at the plasma membrane; however, the mechanisms mediating this incorporation process remain poorly understood. We previously showed that the HIV-1 structural protein Gag localizes to the uropod, a rear-end structure of polarized T cells, and that assembling Gag copatches with a subset, but not all, of the uropod-directed proteins, i.e., PSGL-1, CD43, and CD44, in nonpolarized T cells. The latter observation suggests the presence of a mechanism promoting virion incorporation of these cellular proteins. To address this possibility and identify molecular determinants, in the present study we examined coclustering between Gag and the transmembrane proteins in T and HeLa cells using quantitative two-color superresolution localization microscopy. Consistent with the findings of the T-cell copatching study, we found that basic residues within the matrix domain of Gag are required for Gagnndash;PSGL-1 coclustering. Notably, the presence of a polybasic sequence in the PSGL-1 cytoplasmic domain significantly enhanced this coclustering. We also found that polybasic motifs present in the cytoplasmic tails of CD43 and CD44 also promote their coclustering with Gag. ICAM-1 and ICAM-3, uropod-directed proteins that do not copatch with Gag in T cells, and CD46, a non-uropod-directed protein, showed no or little coclustering with Gag. However, replacing their cytoplasmic tails with the cytoplasmic tail of PSGL-1 significantly enhanced their coclustering with Gag. Altogether, these results identify a novel mechanism for host membrane protein association with assembling HIV-1 Gag in which polybasic sequences present in the cytoplasmic tails of the membrane proteins and in Gag are the major determinants.

    IMPORTANCE Nascent HIV-1 particles incorporate many host plasma membrane proteins during assembly. However, it is largely unknown what mechanisms promote the association of these proteins with virus assembly sites within the plasma membrane. Notably, our previous study showed that HIV-1 structural protein Gag colocalizes with a group of uropod-directed transmembrane proteins, PSGL-1, CD43, and CD44, at the plasma membrane of T cells. The results obtained in the current study using superresolution localization microscopy suggest the presence of a novel molecular mechanism promoting the association of PSGL-1, CD43, and CD44 with assembling HIV-1 which relies on polybasic sequences in HIV-1 Gag and in cytoplasmic domains of the transmembrane proteins. This information advances our understanding of virion incorporation of host plasma membrane proteins, some of which modulate virus spread positively or negatively, and suggests a possible new strategy to enrich HIV-1-based lentiviral vectors with a desired transmembrane protein.

  • Proinflammatory Effects of Interferon Gamma in Mouse Adenovirus 1 Myocarditis [Pathogenesis and Immunity]

  • Adenoviruses are frequent causes of pediatric myocarditis. Little is known about the pathogenesis of adenovirus myocarditis, and the species specificity of human adenoviruses has limited the development of animal models, which is a significant barrier to strategies for prevention or treatment. We have developed a mouse model of myocarditis following mouse adenovirus 1 (MAV-1) infection to study the pathogenic mechanisms of this important cause of pediatric myocarditis. Following intranasal infection of neonatal C57BL/6 mice, we detected viral replication and induction of interferon gamma (IFN-) in the hearts of infected mice. MAV-1 caused myocyte necrosis and induced substantial cellular inflammation that was composed predominantly of CD3+ T lymphocytes. Depletion of IFN- during acute infection reduced cardiac inflammation in MAV-1-infected mice without affecting viral replication. We observed decreased contractility during acute infection of neonatal mice, and persistent viral infection in the heart was associated with cardiac remodeling and hypertrophy in adulthood. IFN- is a proinflammatory mediator during adenovirus-induced myocarditis, and persistent adenovirus infection may contribute to ongoing cardiac dysfunction.

    IMPORTANCE Studying the pathogenesis of myocarditis caused by different viruses is essential in order to characterize both virus-specific and generalized factors that contribute to disease. Very little is known about the pathogenesis of adenovirus myocarditis, which is a significant impediment to the development of treatment or prevention strategies. We used MAV-1 to establish a mouse model of human adenovirus myocarditis, providing the means to study host and pathogen factors contributing to adenovirus-induced cardiac disease during acute and persistent infection. The MAV-1 model will enable fundamental studies of viral myocarditis, including IFN- modulation as a therapeutic strategy.

  • Nucleocapsid Protein from Fig Mosaic Virus Forms Cytoplasmic Agglomerates That Are Hauled by Endoplasmic Reticulum Streaming [Virus-Cell Interactions]

  • Although many studies have demonstrated intracellular movement of viral proteins or viral replication complexes, little is known about the mechanisms of their motility. In this study, we analyzed the localization and motility of the nucleocapsid protein (NP) of Fig mosaic virus (FMV), a negative-strand RNA virus belonging to the recently established genus Emaravirus. Electron microscopy of FMV-infected cells using immunogold labeling showed that NPs formed cytoplasmic agglomerates that were predominantly enveloped by the endoplasmic reticulum (ER) membrane, while nonenveloped NP agglomerates also localized along the ER. Likewise, transiently expressed NPs formed agglomerates, designated NP bodies (NBs), in close proximity to the ER, as was the case in FMV-infected cells. Subcellular fractionation and electron microscopic analyses of NP-expressing cells revealed that NBs localized in the cytoplasm. Furthermore, we found that NBs moved rapidly with the streaming of the ER in an actomyosin-dependent manner. Brefeldin A treatment at a high concentration to disturb the ER network configuration induced aberrant accumulation of NBs in the perinuclear region, indicating that the ER network configuration is related to NB localization. Dominant negative inhibition of the class XI myosins, XI-1, XI-2, and XI-K, affected both ER streaming and NB movement in a similar pattern. Taken together, these results showed that NBs localize in the cytoplasm but in close proximity to the ER membrane to form enveloped particles and that this causes passive movements of cytoplasmic NBs by ER streaming.

    IMPORTANCE Intracellular trafficking is a primary and essential step for the cell-to-cell movement of viruses. To date, many studies have demonstrated the rapid intracellular movement of viral factors but have failed to provide evidence for the mechanism or biological significance of this motility. Here, we observed that agglomerates of nucleocapsid protein (NP) moved rapidly throughout the cell, and we performed live imaging and ultrastructural analysis to identify the mechanism of motility. We provide evidence that cytoplasmic protein agglomerates were passively dragged by actomyosin-mediated streaming of the endoplasmic reticulum (ER) in plant cells. In virus-infected cells, NP agglomerates were surrounded by the ER membranes, indicating that NP agglomerates form the basis of enveloped virus particles in close proximity to the ER. Our work provides a sophisticated model of macromolecular trafficking in plant cells and improves our understanding of the formation of enveloped particles of negative-strand RNA viruses.

  • A View of the E2-CD81 Interface at the Binding Site of a Neutralizing Antibody against Hepatitis C Virus [Virus-Cell Interactions]

  • Hepatitis C virus (HCV) glycoprotein E2 is considered a major target for generating neutralizing antibodies against HCV, primarily due to its role of engaging host entry factors, such as CD81, a key cell surface protein associated with HCV entry. Based on a series of biochemical analyses in combination with molecular docking, we present a description of a potential binding interface formed between the E2 protein and CD81. The virus side of this interface includes a hydrophobic helix motif comprised of residues W437LAGLF442, which encompasses the binding site of a neutralizing monoclonal antibody, mAb41. The helical conformation of this motif provides a structural framework for the positioning of residues F442 and Y443, serving as contact points for the interaction with CD81. The cell side of this interface likewise involves a surface-exposed hydrophobic helix, namely, the D-helix of CD81, which coincides with the binding site of 1D6, a monoclonal anti-CD81 antibody known to block HCV entry. Our illustration of this virus-host interface suggests an important role played by the W437LAGLF442 helix of the E2 protein in the hydrophobic interaction with the D-helix of CD81, thereby facilitating our understanding of the mechanism for antibody-mediated neutralization of HCV.

    IMPORTANCE Characterization of the interface established between a virus and host cells can provide important information that may be used for the control of virus infections. The interface that enables hepatitis C virus (HCV) to infect human liver cells has not been well understood because of the number of cell surface proteins, factors, and conditions found to be associated with the infection process. Based on a series of biochemical analyses in combination with molecular docking, we present such an interface, consisting of two hydrophobic helical structures, from the HCV E2 surface glycoprotein and the CD81 protein, a major host cell receptor recognized by all HCV strains. Our study reveals the critical role played by hydrophobic interactions in the formation of this virus-host interface, thereby contributing to our understanding of the mechanism for antibody-mediated neutralization of HCV.

  • Separate Cellular Localizations of Human T-Lymphotropic Virus 1 (HTLV-1) Env and Glucose Transporter Type 1 (GLUT1) Are Required for HTLV-1 Env-Mediated Fusion and Infection [Virus-Cell Interactions]

  • Interaction of the envelope glycoprotein (Env) of human T-lymphotropic virus 1 (HTLV-1) with the glucose transporter type 1 (GLUT1) expressed in target cells is essential for viral entry. This study found that the expression level of GLUT1 in virus-producing 293T cells was inversely correlated with HTLV-1 Env-mediated fusion activity and infectivity. Chimeric studies between GLUT1 and GLUT3 indicated that the extracellular loop 6 (ECL6) of GLUT1 is important for the inhibition of cell-cell fusion mediated by Env. When GLUT1 was translocated into the plasma membrane from intracellular storage sites by bafilomycin A1 (BFLA1) treatment in 293T cells, HTLV-1 Env-mediated cell fusion and infection also were inhibited without the overexpression of GLUT1, indicating that the localization of GLUT1 in intracellular compartments rather than in the plasma membrane is crucial for the fusion activity of HTLV-1 Env. Immunoprecipitation and laser scanning confocal microscopic analyses indicated that under normal conditions, HTLV-1 Env and GLUT1 do not colocalize or interact. BFLA1 treatment induced this colocalization and interaction, indicating that GLUT1 normally accumulates in intracellular compartments separate from that of Env. Western blot analyses of FLAG-tagged HTLV-1 Env in virus-producing cells and the incorporation of HTLV-1 Env in virus-like particles (VLPs) indicate that the processing of Env is inhibited by either overexpression of GLUT1 or BFLA1 treatment in virus-producing 293T cells. This inhibition probably is due to the interaction of the Env with GLUT1 in intracellular compartments. Taken together, separate intracellular localizations of GLUT1 and HTLV-1 Env are required for the fusion activity and infectivity of HTLV-1 Env.

    IMPORTANCE The deltaretrovirus HTLV-1 is a causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although HTLV-1 is a complex retrovirus that has accessory genes, no HTLV-1 gene product has yet been shown to regulate its receptor GLUT1 in virus-producing cells. In this study, we found that a large amount of GLUT1 or translocation of GLUT1 to the plasma membrane from intracellular compartments in virus-producing cells enhances the colocalization and interaction of GLUT1 with HTLV-1 Env, leading to the inhibition of cell fusion activity and infectivity. The results of our study suggest that GLUT1 normally accumulates in separate intracellular compartments from Env, which is indeed required for the proper processing of Env.

  • Identification of Residues in the Human Respiratory Syncytial Virus Fusion Protein That Modulate Fusion Activity and Pathogenesis [Pathogenesis and Immunity]

  • Human respiratory syncytial virus (RSV) lower respiratory tract infection can result in inflammation and mucus plugging of airways. RSV strain A2-line19F induces relatively high viral load and mucus in mice. The line 19 fusion (F) protein harbors five unique residues compared to the non-mucus-inducing strains A2 and Long, at positions 79, 191, 357, 371, and 557. We hypothesized that differential fusion activity is a determinant of pathogenesis. In a cell-cell fusion assay, line 19 F was more fusogenic than Long F. We changed the residues unique to line 19 F to the corresponding residues in Long F and identified residues 79 and 191 together as responsible for high fusion activity. Surprisingly, mutation of residues 357 or 357 with 371 resulted in gain of fusion activity. Thus, we generated RSV F mutants with a range of defined fusion activity and engineered these into recombinant viruses. We found a clear, positive correlation between fusion activity and early viral load in mice; however, we did not detect a correlation between viral loads and levels of airway mucin expression. The F mutant with the highest fusion activity, A2-line19F-K357T/Y371N, induced high viral loads, severe lung histopathology, and weight loss but did not induce high levels of airway mucin expression. We defined residues 79/191 as critical for line 19 F fusion activity and 357/371 as playing a role in A2-line19F mucus induction. Defining the molecular basis of the role of RSV F in pathogenesis may aid vaccine and therapeutic strategies aimed at this protein.

    IMPORTANCE Human respiratory syncytial virus (RSV) is the most important lower respiratory tract pathogen of infants for which there is no vaccine. Elucidating mechanisms of RSV pathogenesis is important for rational vaccine and drug design. We defined specific amino acids in the fusion (F) protein of RSV strain line 19 critical for fusion activity and elucidated a correlation between fusion activity and viral load in mice. Further, we identified two distinct amino acids in F as contributing to the mucogenic phenotype of the A2-line19F virus. Taken together, these results illustrate a role for RSV F in virulence.

  • Human Cytomegalovirus UL97 Phosphorylates the Viral Nuclear Egress Complex [Structure and Assembly]

  • Herpesvirus nucleocapsids exit the host cell nucleus in an unusual process known as nuclear egress. The human cytomegalovirus (HCMV) UL97 protein kinase is required for efficient nuclear egress, which can be explained by its phosphorylation of the nuclear lamina component lamin A/C, which disrupts the nuclear lamina. We found that a dominant negative lamin A/C mutant complemented the replication defect of a virus lacking UL97 in dividing cells, validating this explanation. However, as complementation was incomplete, we investigated whether the HCMV nuclear egress complex (NEC) subunits UL50 and UL53, which are required for nuclear egress and recruit UL97 to the nuclear rim, are UL97 substrates. Using mass spectrometry, we detected UL97-dependent phosphorylation of UL50 residue S216 (UL50-S216) and UL53-S19 in infected cells. Moreover, UL53-S19 was specifically phosphorylated by UL97 in vitro. Notably, treatment of infected cells with the UL97 inhibitor maribavir or infection with a UL97 mutant led to a punctate rather than a continuous distribution of the NEC at the nuclear rim. Alanine substitutions in both UL50-S216 and UL53-S19 resulted in a punctate distribution of the NEC in infected cells and also decreased virus production and nuclear egress in the absence of maribavir. These results indicate that UL97 phosphorylates the NEC and suggest that this phosphorylation modulates nuclear egress. Thus, the UL97-NEC interaction appears to recruit UL97 to the nuclear rim both for disruption of the nuclear lamina and phosphorylation of the NEC.

    IMPORTANCE Human cytomegalovirus (HCMV) causes birth defects and it can cause life-threatening diseases in immunocompromised patients. HCMV assembles in the nucleus and then translocates to the cytoplasm in an unusual process termed nuclear egress, an attractive target for antiviral therapy. A viral enzyme, UL97, is important for nuclear egress. It has been proposed that this is due to its role in disruption of the nuclear lamina, which would otherwise impede nuclear egress. In validating this proposal, we showed that independent disruption of the lamina can overcome a loss of UL97, but only partly, suggesting additional roles for UL97 during nuclear egress. We then found that UL97 phosphorylates the viral nuclear egress complex (NEC), which is essential for nuclear egress, and we obtained evidence that this phosphorylation modulates this process. Our results highlight a new role for UL97, the mutual dependence of the viral NEC and UL97 during nuclear egress, and differences among herpesviruses.

  • The Sheep Tetherin Paralog oBST2B Blocks Envelope Glycoprotein Incorporation into Nascent Retroviral Virions [Virus-Cell Interactions]

  • Bone marrow stromal cell antigen 2 (BST2) is a cellular restriction factor with a broad antiviral activity. In sheep, the BST2 gene is duplicated into two paralogs termed oBST2A and oBST2B. oBST2A impedes viral exit of the Jaagsiekte sheep retroviruses (JSRV), most probably by retaining virions at the cell membrane, similar to the "tethering" mechanism exerted by human BST2. In this study, we provide evidence that unlike oBST2A, oBST2B is limited to the Golgi apparatus and disrupts JSRV envelope (Env) trafficking by sequestering it. In turn, oBST2B leads to a reduction in Env incorporation into viral particles, which ultimately results in the release of virions that are less infectious. Furthermore, the activity of oBST2B does not seem to be restricted to retroviruses, as it also acts on vesicular stomatitis virus glycoproteins. Therefore, we suggest that oBST2B exerts antiviral activity using a mechanism distinct from the classical tethering restriction observed for oBST2A.

    IMPORTANCE BST2 is a powerful cellular restriction factor against a wide range of enveloped viruses. Sheep possess two paralogs of the BST2 gene called oBST2A and oBST2B. JSRV, the causative agent of a transmissible lung cancer of sheep, is known to be restricted by oBST2A. In this study, we show that unlike oBST2A, oBST2B impairs the normal cellular trafficking of JSRV envelope glycoproteins by sequestering them within the Golgi apparatus. We also show that oBST2B decreases the incorporation of envelope glycoprotein into JSRV viral particles, which in turn reduces virion infectivity. In conclusion, oBST2B exerts a novel antiviral activity that is distinct from those of BST2 proteins of other species.

  • The HIV-1 gp120 CD4-Bound Conformation Is Preferentially Targeted by Antibody-Dependent Cellular Cytotoxicity-Mediating Antibodies in Sera from HIV-1-Infected Individuals [Pathogenesis and Immunity]

  • Recent studies have linked antibody Fc-mediated effector functions with protection or control of human immunodeficiency type 1 (HIV-1) and simian immunodeficiency (SIV) infections. Interestingly, the presence of antibodies with potent antibody-dependent cellular cytotoxicity (ADCC) activity in the Thai RV144 vaccine trial was suggested to correlate with decreased HIV-1 acquisition risk. These antibodies recently were found to recognize HIV envelope (Env) epitopes exposed upon Env-CD4 interaction. CD4 downregulation by Nef and Vpu, as well as Vpu-mediated BST-2 antagonism, were reported to modulate exposure of those CD4-induced HIV-1 Env epitopes and were proposed to play a role in reducing the susceptibility of infected cells to ADCC mediated by this class of antibodies. Here, we report the high prevalence of antibodies recognizing CD4-induced HIV-1 Env epitopes in sera from HIV-1-infected individuals, which correlated with their ability to mediate ADCC responses against HIV-1-infected cells, exposing these Env epitopes at the cell surface. Furthermore, our results indicate that Env variable regions V1, V2, V3, and V5 do not represent a major determinant for ADCC responses mediated by sera from HIV-1-infected individuals. Altogether, these findings suggest that HIV-1 tightly controls the exposure of certain Env epitopes at the surface of infected cells in order to prevent elimination by Fc-effector functions.

    IMPORTANCE Here, we identified a particular conformation of HIV-1 Env that is specifically targeted by ADCC-mediating antibodies present in sera from HIV-1-infected individuals. This observation suggests that HIV-1 developed sophisticated mechanisms to minimize the exposure of these epitopes at the surface of infected cells.

  • Uneven Genetic Robustness of HIV-1 Integrase [Genetic Diversity and Evolution]

  • Genetic robustness (tolerance of mutation) may be a naturally selected property in some viruses, because it should enhance adaptability. Robustness should be especially beneficial to viruses like HIV-1 that exhibit high mutation rates and exist in immunologically hostile environments. Surprisingly, however, the HIV-1 capsid protein (CA) exhibits extreme fragility. To determine whether fragility is a general property of HIV-1 proteins, we created a large library of random, single-amino-acid mutants in HIV-1 integrase (IN), covering ggt;40% of amino acid positions. Despite similar degrees of sequence variation in naturally occurring IN and CA sequences, we found that HIV-1 IN was significantly more robust than CA, with random nonsilent IN mutations only half as likely to cause lethal defects. Interestingly, IN and CA were similar in that a subset of mutations with high in vitro fitness were rare in natural populations. IN mutations of this type were more likely to occur in the buried interior of the modeled HIV-1 intasome, suggesting that even very subtle fitness effects suppress variation in natural HIV-1 populations. Lethal mutations, in particular those that perturbed particle production, proteolytic processing, and particle-associated IN levels, were strikingly localized at specific IN subunit interfaces. This observation strongly suggests that binding interactions between particular IN subunits regulate proteolysis during HIV-1 virion morphogenesis. Overall, use of the IN mutant library in conjunction with structural models demonstrates the overall robustness of IN and highlights particular regions of vulnerability that may be targeted in therapeutic interventions.

    IMPORTANCE The HIV-1 integrase (IN) protein is responsible for the integration of the viral genome into the host cell chromosome. To measure the capacity of IN to maintain function in the face of mutation, and to probe structure/function relationships, we created a library of random single-amino-acid IN mutations that could mimic the types of mutations that naturally occur during HIV-1 infection. Previously, we measured the robustness of HIV-1 capsid in this manner and determined that it is extremely intolerant of mutation. In contrast to CA, HIV-1 IN proved relatively robust, with far fewer mutations causing lethal defects. However, when we subsequently mapped the lethal mutations onto a model of the structure of the multisubunit IN-viral DNA complex, we found the lethal mutations that caused virus morphogenesis defects tended to be highly localized at subunit interfaces. This discovery of vulnerable regions of HIV-1 IN could inform development of novel therapeutics.

  • Clonal Evolution of CD8+ T Cell Responses against Latent Viruses: Relationship among Phenotype, Localization, and Function [Cellular Response to Infection]

  • Human cytomegalovirus (hCMV) infection is characterized by a vast expansion of resting effector-type virus-specific T cells in the circulation. In mice, interleukin-7 receptor aalpha; (IL-7Raalpha;)-expressing cells contain the precursors for long-lived antigen-experienced CD8+ T cells, but it is unclear if similar mechanisms operate to maintain these pools in humans. Here, we studied whether IL-7Raalpha;-expressing cells obtained from peripheral blood (PB) or lymph nodes (LNs) sustain the circulating effector-type hCMV-specific pool. Using flow cytometry and functional assays, we found that the IL-7Raalpha;+ hCMV-specific T cell population comprises cells that have a memory phenotype and lack effector features. We used next-generation sequencing of the T cell receptor to compare the clonal repertoires of IL-7Raalpha;+ and IL-7Raalpha;nndash; subsets. We observed limited overlap of clones between these subsets during acute infection and after 1 year. When we compared the hCMV-specific repertoire between PB and paired LNs, we found many identical clones but also clones that were exclusively found in either compartment. New clones that were found in PB during antigenic recall were only rarely identical to the unique LN clones. Thus, although PB IL-7Raalpha;-expressing and LN hCMV-specific CD8+ T cells show typical traits of memory-type cells, these populations do not seem to contain the precursors for the novel hCMV-specific CD8+ T cell pool during latency or upon antigen recall. IL-7Raalpha;+ PB and LN hCMV-specific memory cells form separate virus-specific compartments, and precursors for these novel PB hCMV-specific CD8+ effector-type T cells are possibly located in other secondary lymphoid tissues or are being recruited from the naive CD8+ T cell pool.

    IMPORTANCE Insight into the self-renewal properties of long-lived memory CD8+ T cells and their location is crucial for the development of both passive and active vaccination strategies. Human CMV infection is characterized by a vast expansion of resting effector-type cells. It is, however, not known how this population is maintained. We here investigated two possible compartments for effector-type cell precursors: circulating acute-phase IL-7Raalpha;-expressing hCMV-specific CD8+ T cells and lymph node (LN)-residing hCMV-specific (central) memory cells. We show that new clones that appear after primary hCMV infection or during hCMV reactivation seldom originate from either compartment. Thus, although identical clones may be maintained by either memory population, the precursors of the novel clones are probably located in other (secondary) lymphoid tissues or are recruited from the naive CD8+ T cell pool.

  • Bindarit, an Inhibitor of Monocyte Chemotactic Protein Synthesis, Protects against Bone Loss Induced by Chikungunya Virus Infection [Pathogenesis and Immunity]

  • The recent global resurgence of arthritogenic alphaviruses, in particular chikungunya virus (CHIKV), highlights an urgent need for the development of therapeutic intervention strategies. While there has been significant progress in defining the pathophysiology of alphaviral disease, relatively little is known about the mechanisms involved in CHIKV-induced arthritis or potential therapeutic options to treat the severe arthritic symptoms associated with infection. Here, we used microcomputed tomographic (mmu;CT) and histomorphometric analyses to provide previously undescribed evidence of reduced bone volume in the proximal tibial epiphysis of CHIKV-infected mice compared to the results for mock controls. This was associated with a significant increase in the receptor activator of nuclear factor-B ligand/osteoprotegerin (RANKL/OPG) ratio in infected murine joints and in the serum of CHIKV patients. The expression levels of the monocyte chemoattractant proteins (MCPs), including MCP-1/CCL2, MCP-2/CCL8, and MCP-3/CCL7, were also highly elevated in joints of CHIKV-infected mice, accompanied by increased cellularity within the bone marrow in tibial epiphysis and ankle joints. Both this effect and CHIKV-induced bone loss were significantly reduced by treatment with the MCP inhibitor bindarit. Collectively, these findings demonstrate a unique role for MCPs in promoting CHIKV-induced osteoclastogenesis and bone loss during disease and suggest that inhibition of MCPs with bindarit may be an effective therapy for patients affected with alphavirus-induced bone loss.

    IMPORTANCE Arthritogenic alphaviruses, including chikungunya virus (CHIKV) and Ross River virus (RRV), cause worldwide outbreaks of polyarthritis, which can persist in patients for months following infection. Previous studies have shown that host proinflammatory soluble factors are associated with CHIKV disease severity. Furthermore, it is established that chemokine (C-C motif) ligand 2 (CCL2/MCP-1) is important in cellular recruitment and inducing bone-resorbing osteoclast (OC) formation. Here, we show that CHIKV replicates in bone and triggers bone loss by increasing the RANKL/OPG ratio. CHIKV infection results in MCP-induced cellular infiltration in the inflamed joints, and bone loss can be ameliorated by treatment with an MCP-inhibiting drug, bindarit. Taken together, our data reveal a previously undescribed role for MCPs in CHIKV-induced bone loss: one of recruiting monocytes/OC precursors to joint sites and thereby favoring a pro-osteoclastic microenvironment. This suggests that bindarit may be an effective treatment for alphavirus-induced bone loss and arthritis in humans.

  • Comprehensive Molecular Detection of Tick-Borne Phleboviruses Leads to the Retrospective Identification of Taxonomically Unassigned Bunyaviruses and the Discovery of a Novel Member of the Genus Phlebovirus [Genetic Diversity and Evolution]

  • Until the recent emergence of two human-pathogenic tick-borne phleboviruses (TBPVs) (severe fever with thrombocytopenia syndrome virus [SFTSV] and Heartland virus), TBPVs have been neglected as causative agents of human disease. In particular, no studies have addressed the global distribution of TBPVs, and consequently, our understanding of the mechanism(s) underlying their evolution and emergence remains poor. In order to provide a useful tool for the ecological and epidemiological study of TBPVs, we have established a simple system that can detect all known TBPVs, based on conventional reverse transcription-PCR (RT-PCR) with degenerate primer sets targeting conserved regions of the viral L genome segment. Using this system, we have determined that several viruses that had been isolated from ticks decades ago but had not been taxonomically identified are novel TBPVs. Full-genome sequencing of these viruses revealed a novel fourth TBPV cluster distinct from the three known TBPV clusters (i.e., the SFTS, Bhanja, and Uukuniemi groups) and from the mosquito/sandfly-borne phleboviruses. Furthermore, by using tick samples collected in Zambia, we confirmed that our system had enough sensitivity to detect a new TBPV in a single tick homogenate. This virus, tentatively designated Shibuyunji virus after the region of tick collection, grouped into a novel fourth TBPV cluster. These results indicate that our system can be used as a first-line screening approach for TBPVs and that this kind of work will undoubtedly lead to the discovery of additional novel tick viruses and will expand our knowledge of the evolution and epidemiology of TBPVs.

    IMPORTANCE Tick-borne phleboviruses (TBPVs) have been largely neglected until the recent emergence of two virulent viruses, severe fever with thrombocytopenia syndrome virus and Heartland virus. Little is known about the global distribution of TBPVs or how these viruses evolved and emerged. A major hurdle to study the distribution of TBPVs is the lack of tools to detect these genetically divergent phleboviruses. In order to address this issue, we have developed a simple, rapid, and cheap RT-PCR system that can detect all known TBPVs and which led to the identification of several novel phleboviruses from previously uncharacterized tick-associated virus isolates. Our system can detect virus in a single tick sample and novel TBPVs that are genetically distinct from any of the known TBPVs. These results indicate that our system will be a useful tool for the surveillance of TBPVs and will facilitate understanding of the ecology of TBPVs.

  • Activation of ATP Citrate Lyase by mTOR Signal Induces Disturbed Lipid Metabolism in Hepatitis B Virus Pre-S2 Mutant Tumorigenesis [Transformation and Oncogenesis]

  • The development of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) has been found to be associated with disturbed lipid metabolism. To elucidate the role of lipid metabolism in HBV tumorigenesis, we investigated the dynamic pattern of lipid metabolism in HBV pre-S2 mutant-induced tumorigenesis. Lipid and gene expression profiles were analyzed in an in vitro culture system and in transgenic mouse livers harboring HBV pre-S2 mutant. The pre-S2 mutant transgenic livers showed a biphasic pattern of lipid accumulation, starting from mild fatty change in early (1 month) transgenic livers, which subsided and then, remarkably, increased in HCC tissues. This biphasic pattern was synchronized with ATP citrate lyase (ACLY) activation. Further analyses revealed that the pre-S2 mutant initiated an endoplasmic reticulum (ER) stress-dependent mammalian target of rapamycin (mTOR) signalling cascade. The pre-S2 mutant-induced mTOR signal activated the sterol regulatory element binding transcription factor 1 (SREBF1) to upregulate ACLY, which then activated the fatty acid desaturase 2 (FADS2), mediated through ACLY-dependent histone acetylation. Such an ER stress-dependent mTOR signal cascade also is important for the proliferation of hepatocytes in vitro and is further validated in HBV-related HCC tissues.

    IMPORTANCE Aberrations of lipid metabolism frequently occur in chronic HBV infection. Our results provide a potential mechanism of disturbed lipid metabolism in HBV pre-S2 mutant-induced tumorigenesis, which should be valuable for the design of HCC chemoprevention in high-risk HBV carriers.

  • Autophagy Restricts HIV-1 Infection by Selectively Degrading Tat in CD4+ T Lymphocytes [Virus-Cell Interactions]

  • Autophagy is a ubiquitous mechanism involved in the lysosomal-mediated degradation of cellular components when they are engulfed in vacuoles called autophagosomes. Autophagy is also recognized as an important regulator of the innate and adaptive immune responses against numerous pathogens, which have, therefore, developed strategies to block or use the autophagy machinery to their own benefit. Upon human immunodeficiency virus type 1 (HIV-1) infection, viral envelope (Env) glycoproteins induce autophagy-dependent apoptosis of uninfected bystander CD4+ T lymphocytes, a mechanism likely contributing to the loss of CD4+ T cells. In contrast, in productively infected CD4+ T cells, HIV-1 is able to block Env-induced autophagy in order to avoid its antiviral effect. To date, nothing is known about how autophagy restricts HIV-1 infection in CD4+ T lymphocytes. Here, we report that autophagy selectively degrades the HIV-1 transactivator Tat, a protein essential for viral transcription and virion production. We demonstrated that this selective autophagy-mediated degradation of Tat relies on its ubiquitin-independent interaction with the p62/SQSTM1 adaptor. Taken together, our results provide evidence that the anti-HIV effect of autophagy is specifically due to the degradation of the viral transactivator Tat but that this process is rapidly counteracted by the virus to favor its replication and spread.

    IMPORTANCE Autophagy is recognized as one of the most ancient and conserved mechanisms of cellular defense against invading pathogens. Cross talk between HIV-1 and autophagy has been demonstrated depending on the virally challenged cell type, and HIV-1 has evolved strategies to block this process to replicate efficiently. However, the mechanisms by which autophagy restricts HIV-1 infection remain to be elucidated. Here, we report that the HIV-1 transactivator Tat, a protein essential for viral replication, is specifically degraded by autophagy in CD4+ T lymphocytes. Both Tat present in infected cells and incoming Tat secreted from infected cells are targeted for autophagy degradation through a ubiquitin-independent interaction with the autophagy receptor p62/SQSTM1. This study is the first to demonstrate that selective autophagy can be an antiviral process by degrading a viral transactivator. In addition, the results could help in the design of new therapies against HIV-1 by specifically targeting this mechanism.

  • Persistent Hepatitis C Virus Infection Impairs Ribavirin Antiviral Activity through Clathrin-Mediated Trafficking of Equilibrative Nucleoside Transporter 1 [Vaccines and Antiviral Agents]

  • Ribavirin (RBV) continues to be an important component of interferon-free hepatitis C treatment regimens, as RBV alone does not inhibit hepatitis C virus (HCV) replication effectively; the reason for this ineffectiveness has not been established. In this study, we investigated the RBV resistance mechanism using a persistently HCV-infected cell culture system. The antiviral activity of RBV against HCV was progressively impaired in the persistently infected culture, whereas interferon lambda 1 (IFN-1), a type III IFN, showed a strong antiviral response and induced viral clearance. We found that HCV replication in persistently infected cultures induces an autophagy response that impairs RBV uptake by preventing the expression of equilibrative nucleoside transporter 1 (ENT1). The Huh-7.5 cell line treated with an autophagy inducer, Torin 1, downregulated membrane expression of ENT1 and terminated RBV uptake. In contrast, the autophagy inhibitors hydroxychloroquine (HCQ), 3-methyladenine (3-MA), and bafilomycin A1 (BafA1) prevented ENT1 degradation and enhanced RBV antiviral activity. The HCV-induced autophagy response, as well as treatment with Torin 1, degrades clathrin heavy chain expression in a hepatoma cell line. Reduced expression of the clathrin heavy chain by HCV prevents ENT1 recycling to the plasma membrane and forces ENT1 to the lysosome for degradation. This study provides a potential mechanism for the impairment of RBV antiviral activity in persistently HCV-infected cell cultures and suggests that inhibition of the HCV-induced autophagy response could be used as a strategy for improving RBV antiviral activity against HCV infection.

    IMPORTANCE The results from this work will allow a review of the competing theories of antiviral therapy development in the field of HCV virology. Ribavirin (RBV) remains an important component of interferon-free hepatitis C treatment regimens. The reason why RBV alone does not inhibit HCV replication effectively has not been established. This study provides a potential mechanism for why RBV antiviral activity is impaired in persistently HCV-infected cell cultures and suggests that inhibition of the HCV-induced autophagy response could be used as a strategy to increase RBV antiviral activity against HCV infection. Therefore, it is anticipated that this work would generate a great deal of interest, not only among virologists but also among the general public.

  • Identification of Capsid Mutations That Alter the Rate of HIV-1 Uncoating in Infected Cells [Virus-Cell Interactions]

  • After viral fusion with the cell membrane, the conical capsid of HIV-1 disassembles by a process called uncoating. We recently utilized the cyclosporine (CsA) washout assay, in which TRIM-CypA-mediated restriction of viral replication is used to detect the state of the viral capsid, to study the kinetics of uncoating in HIV-1-infected cells. Here we have extended this analysis to examine the effects of p24 capsid protein (p24CA) mutations and cellular environment on the kinetics of uncoating in infected cells. We found that p24CA mutations can significantly increase (A92E), delay (E45A and N74D), or have no effect (G94D) on the rate of uncoating and that these alterations are not due to changes in reverse transcription. Inhibition of reverse transcription delayed uncoating kinetics to an extent similar to that of the wild-type virus with all the p24CA mutant viruses tested. In addition, we observed differences in uncoating in two cell lines, which suggests that the cellular environment can differentially impact the disassembly of wild-type and mutant capsids. Collectively, these experiments suggest that viral and cellular factors are important for the process of uncoating. Finally, these data support the model whereby early steps in reverse transcription facilitate HIV-1 uncoating.

    IMPORTANCE The HIV-1 capsid is a cone-shaped structure, composed of the HIV-1-encoded protein p24CA, which contains the viral RNA and other proteins needed for infection. After the virus enters a target cell, this capsid must disassemble by a process called uncoating. Uncoating is required for HIV-1 infection to progress, but the details of how this process occurs is not known. In this study, we used an in vivo assay to examine the uncoating process in HIV-1-infected cells. We determined that p24CA mutations could increase or decrease the rate of uncoating and that this rate varied in different cell lines. We also found that reverse transcription of the viral RNA altered the process of uncoating before the p24CA mutations. Collectively, these experiments provide a better understanding of how viral and cellular factors are involved with a poorly understood step in HIV-1 infection.

  • Role of ATM in the Formation of the Replication Compartment during Lytic Replication of Epstein-Barr Virus in Nasopharyngeal Epithelial Cells [Virus-Cell Interactions]

  • Epstein-Barr virus (EBV), a type of oncogenic herpesvirus, is associated with human malignancies. Previous studies have shown that lytic reactivation of EBV in latently infected cells induces an ATM-dependent DNA damage response (DDR). The involvement of ATM activation has been implicated in inducing viral lytic gene transcription to promote lytic reactivation. Its contribution to the formation of a replication compartment during lytic reactivation of EBV remains poorly defined. In this study, the role of ATM in viral DNA replication was investigated in EBV-infected nasopharyngeal epithelial cells. We observed that induction of lytic infection of EBV triggers ATM activation and localization of DDR proteins at the viral replication compartments. Suppression of ATM activity using a small interfering RNA (siRNA) approach or a specific chemical inhibitor profoundly suppressed replication of EBV DNA and production of infectious virions in EBV-infected cells induced to undergo lytic reactivation. We further showed that phosphorylation of Sp1 at the serine-101 residue is essential in promoting the accretion of EBV replication proteins at the replication compartment, which is crucial for replication of viral DNA. Knockdown of Sp1 expression by siRNA effectively suppressed the replication of viral DNA and localization of EBV replication proteins to the replication compartments. Our study supports an important role of ATM activation in lytic reactivation of EBV in epithelial cells, and phosphorylation of Sp1 is an essential process downstream of ATM activation involved in the formation of viral replication compartments. Our study revealed an essential role of the ATM-dependent DDR pathway in lytic reactivation of EBV, suggesting a potential antiviral replication strategy using specific DDR inhibitors.

    IMPORTANCE Epstein-Barr virus (EBV) is closely associated with human malignancies, including undifferentiated nasopharyngeal carcinoma (NPC), which has a high prevalence in southern China. EBV can establish either latent or lytic infection depending on the cellular context of infected host cells. Recent studies have highlighted the importance of the DNA damage response (DDR), a surveillance mechanism that evolves to maintain genome integrity, in regulating lytic EBV replication. However, the underlying molecular events are largely undefined. ATM is consistently activated in EBV-infected epithelial cells when they are induced to undergo lytic reactivation. Suppression of ATM inhibits replication of viral DNA. Furthermore, we observed that phosphorylation of Sp1 at the serine-101 residue, a downstream event of ATM activation, plays an essential role in the formation of viral replication compartments for replication of virus DNA. Our study provides new insights into the mechanism through which EBV utilizes the host cell machinery to promote replication of viral DNA upon lytic reactivation.

  • Amplified and Persistent Immune Responses Generated by Single-Cycle Replicating Adenovirus Vaccines [Vaccines and Antiviral Agents]

  • Replication-competent adenoviral (RC-Ad) vectors generate exceptionally strong gene-based vaccine responses by amplifying the antigen transgenes they carry. While they are potent, they also risk causing adenovirus infections. More common replication-defective Ad (RD-Ad) vectors with deletions of E1 avoid this risk but do not replicate their transgene and generate markedly weaker vaccine responses. To amplify vaccine transgenes while avoiding production of infectious progeny viruses, we engineered "single-cycle" adenovirus (SC-Ad) vectors by deleting the gene for IIIa capsid cement protein of lower-seroprevalence adenovirus serotype 6. In mouse, human, hamster, and macaque cells, SC-Ad6 still replicated its genome but prevented genome packaging and virion maturation. When used for mucosal intranasal immunization of Syrian hamsters, both SC-Ad and RC-Ad expressed transgenes at levels hundreds of times higher than that of RD-Ad. Surprisingly, SC-Ad, but not RC-Ad, generated higher levels of transgene-specific antibody than RD-Ad, which notably climbed in serum and vaginal wash samples over 12 weeks after single mucosal immunization. When RD-Ad and SC-Ad were tested by single sublingual immunization in rhesus macaques, SC-Ad generated higher gamma interferon (IFN-) responses and higher transgene-specific serum antibody levels. These data suggest that SC-Ad vectors may have utility as mucosal vaccines.

    IMPORTANCE This work illustrates the utility of our recently developed single-cycle adenovirus (SC-Ad6) vector as a new vaccine platform. Replication-defective (RD-Ad6) vectors produce low levels of transgene protein, which leads to minimal antibody responses in vivo. This study shows that replicating SC-Ad6 produces higher levels of luciferase and induces higher levels of green fluorescent protein (GFP)-specific antibodies than RD in a permissive Syrian hamster model. Surprisingly, although a replication-competent (RC-Ad6) vector produces more luciferase than SC-Ad6, it does not elicit comparable levels of anti-GFP antibodies in permissive hamsters. When tested in the larger rhesus macaque model, SC-Ad6 induces higher transgene-specific antibody and T cell responses. Together, these data suggest that SC-Ad6 could be a more effective platform for developing vaccines against more relevant antigens. This could be especially beneficial for developing vaccines for pathogens for which traditional replication-defective adenovirus vectors have not been effective.

  • A Newly Isolated Reovirus Has the Simplest Genomic and Structural Organization of Any Reovirus [Genetic Diversity and Evolution]

  • A total of 2,691 mosquitoes representing 17 species was collected from eight locations in southwest Cameroon and screened for pathogenic viruses. Ten isolates of a novel reovirus (genus Dinovernavirus) were detected by culturing mosquito pools on Aedes albopictus (C6/36) cell cultures. A virus that caused overt cytopathic effects was isolated, but it did not infect vertebrate cells or produce detectable disease in infant mice after intracerebral inoculation. The virus, tentatively designated Fako virus (FAKV), represents the first 9-segment, double-stranded RNA (dsRNA) virus to be isolated in nature. FAKV appears to have a broad mosquito host range, and its detection in male specimens suggests mosquito-to-mosquito transmission in nature. The structure of the T=1 FAKV virion, determined to subnanometer resolution by cryoelectron microscopy (cryo-EM), showed only four proteins per icosahedral asymmetric unit: a dimer of the major capsid protein, one turret protein, and one clamp protein. While all other turreted reoviruses of known structures have at least two copies of the clamp protein per asymmetric unit, FAKV's clamp protein bound at only one conformer of the major capsid protein. The FAKV capsid architecture and genome organization represent the most simplified reovirus described to date, and phylogenetic analysis suggests that it arose from a more complex ancestor by serial loss-of-function events.

    IMPORTANCE We describe the detection, genetic, phenotypic, and structural characteristics of a novel Dinovernavirus species isolated from mosquitoes collected in Cameroon. The virus, tentatively designated Fako virus (FAKV), is related to both single-shelled and partially double-shelled viruses. The only other described virus in this genus was isolated from cultured mosquito cells. It was previously unclear whether the phenotypic characteristics of that virus were reflective of this genus in nature or were altered during serial passaging in the chronically infected cell line. FAKV is a naturally occurring single-shelled reovirus with a unique virion architecture that lacks several key structural elements thought to stabilize a single-shelled reovirus virion, suggesting what may be the minimal number of proteins needed to form a viable reovirus particle. FAKV evolved from more complex ancestors by losing a genome segment and several virion proteins.

  • APOBEC3A Functions as a Restriction Factor of Human Papillomavirus [Pathogenesis and Immunity]

  • Human papillomaviruses (HPVs) are small DNA viruses causally associated with benign warts and multiple cancers, including cervical and head-and-neck cancers. While the vast majority of people are exposed to HPV, most instances of infection are cleared naturally. However, the intrinsic host defense mechanisms that block the early establishment of HPV infections remain mysterious. Several antiviral cytidine deaminases of the human APOBEC3 (hA3) family have been identified as potent viral DNA mutators. While editing of HPV genomes in benign and premalignant cervical lesions has been demonstrated, it remains unclear whether hA3 proteins can directly inhibit HPV infection. Interestingly, recent studies revealed that HPV-positive cervical and head-and-neck cancers exhibited higher rates of hA3 mutation signatures than most HPV-negative cancers. Here, we report that hA3A and hA3B expression levels are highly upregulated in HPV-positive keratinocytes and cervical tissues in early stages of cancer progression, potentially through a mechanism involving the HPV E7 oncoprotein. HPV16 virions assembled in the presence of hA3A, but not in the presence of hA3B or hA3C, have significantly decreased infectivity compared to HPV virions assembled without hA3A or with a catalytically inactive mutant, hA3A/E72Q. Importantly, hA3A knockdown in human keratinocytes results in a significant increase in HPV infectivity. Collectively, our findings suggest that hA3A acts as a restriction factor against HPV infection, but the induction of this restriction mechanism by HPV may come at a cost to the host by promoting cancer mutagenesis.

    IMPORTANCE Human papillomaviruses (HPVs) are highly prevalent and potent human pathogens that cause ggt;5% of all human cancers, including cervical and head-and-neck cancers. While the majority of people become infected with HPV, only 10 to 20% of infections are established as persistent infections. This suggests the existence of intrinsic host defense mechanisms that inhibit viral persistence. Using a robust method to produce infectious HPV virions, we demonstrate that hA3A, but not hA3B or hA3C, can significantly inhibit HPV infectivity. Moreover, hA3A and hA3B were coordinately induced in HPV-positive clinical specimens during cancer progression, likely through an HPV E7 oncoprotein-dependent mechanism. Interestingly, HPV-positive cervical and head-and-neck cancer specimens were recently shown to harbor significant amounts of hA3 mutation signatures. Our findings raise the intriguing possibility that the induction of this host restriction mechanism by HPV may also trigger hA3A- and hA3B-induced cancer mutagenesis.

  • T Cell Epitope Clustering in the Highly Immunogenic BZLF1 Antigen of Epstein-Barr Virus [Cellular Response to Infection]

  • Polymorphism in the human leukocyte antigen (HLA) loci ensures that the CD8+ T cell response to viruses is directed against a diverse range of antigenic epitopes, thereby minimizing the impact of virus escape mutation across the population. The BZLF1 antigen of Epstein-Barr virus is an immunodominant target for CD8+ T cells, but the response has been characterized only in the context of a limited number of HLA molecules due to incomplete epitope mapping. We have now greatly expanded the number of defined CD8+ T cell epitopes from BZLF1, allowing the response to be evaluated in a much larger proportion of the population. Some regions of the antigen fail to be recognized by CD8+ T cells, while others include clusters of overlapping epitopes presented by different HLA molecules. These highly immunogenic regions of BZLF1 include polymorphic sequences, such that up to four overlapping epitopes are impacted by a single amino acid variation common in different regions of the world. This focusing of the immune response to limited regions of the viral protein could be due to sequence similarity to human proteins creating "immune blind spots" through self-tolerance. This study significantly enhances the understanding of the immune response to BZLF1, and the precisely mapped T cell epitopes may be directly exploited in vaccine development and adoptive immunotherapy.

    IMPORTANCE Epstein-Barr virus (EBV) is an important human pathogen, associated with several malignancies, including nasopharyngeal carcinoma and Hodgkin lymphoma. T lymphocytes are critical for virus control, and clinical trials aimed at manipulating this arm of the immune system have demonstrated efficacy in treating these EBV-associated diseases. These trials have utilized information on the precise location of viral epitopes for T cell recognition, for either measuring or enhancing responses. In this study, we have characterized the T cell response to the highly immunogenic BZLF1 antigen of EBV by greatly expanding the number of defined T cell epitopes. An unusual clustering of epitopes was identified, highlighting a small region of BZLF1 that is targeted by the immune response of a high proportion of the world's population. This focusing of the immune response could be utilized in developing vaccines/therapies with wide coverage, or it could potentially be exploited by the virus to escape the immune response.

  • High-Throughput RNA Sequencing-Based Virome Analysis of 50 Lymphoma Cell Lines from the Cancer Cell Line Encyclopedia Project [Virus-Cell Interactions]

  • Using high-throughput RNA sequencing data from 50 common lymphoma cell culture models from the Cancer Cell Line Encyclopedia project, we performed an unbiased global interrogation for the presence of a panel of 740 viruses and strains known to infect human and other mammalian cells. This led to the findings of previously identified infections by Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV), and human T-lymphotropic virus type 1 (HTLV-1). In addition, we also found a previously unreported infection of one cell line (DEL) with a murine leukemia virus. High expression of murine leukemia virus (MuLV) transcripts was observed in DEL cells, and we identified four transcriptionally active integration sites, one being in the TNFRSF6B gene. We also found low levels of MuLV reads in a number of other cell lines and provided evidence suggesting cross-contamination during sequencing. Analysis of HTLV-1 integrations in two cell lines, HuT 102 and MJ, identified 14 and 66 transcriptionally active integration sites with potentially activating integrations in immune regulatory genes, including interleukin-15 (IL-15), IL-6ST, STAT5B, HIVEP1, and IL-9R. Although KSHV and EBV do not typically integrate into the genome, we investigated a previously identified integration of EBV into the BACH2 locus in Raji cells. This analysis identified a BACH2 disruption mechanism involving splice donor sequestration. Through viral gene expression analysis, we detected expression of stable intronic RNAs from the EBV BamHI W repeats that may be part of long transcripts spanning the repeat region. We also observed transcripts at the EBV vIL-10 locus exclusively in the Hodgkin's lymphoma cell line, Hs 611.T, the expression of which were uncoupled from other lytic genes. Assessment of the KSHV viral transcriptome in BCP-1 cells showed expression of the viral immune regulators, K2/vIL-6, K4/vIL-8-like vCCL1, and K5/E2-ubiquitin ligase 1 that was significantly higher than expression of the latency-associated nuclear antigen. Together, this investigation sheds light into the virus composition across these lymphoma model systems and provides insights into common viral mechanistic principles.

    IMPORTANCE Viruses cause cancer in humans. In lymphomas the Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV) and human T-lymphotropic virus type 1 are major contributors to oncogenesis. We assessed virus-host interactions using a high throughput sequencing method that facilitates the discovery of new virus-host associations and the investigation into how the viruses alter their host environment. We found a previously unknown murine leukemia virus infection in one cell line. We identified cellular genes, including cytokine regulators, that are disrupted by virus integration, and we determined mechanisms through which virus integration causes deregulation of cellular gene expression. Investigation into the KSHV transcriptome in the BCP-1 cell line revealed high-level expression of immune signaling genes. EBV transcriptome analysis showed expression of vIL-10 transcripts in a Hodgkin's lymphoma that was uncoupled from lytic genes. These findings illustrate unique mechanisms of viral gene regulation and to the importance of virus-mediated host immune signaling in lymphomas.

  • Impact and Regulation of Lambda Interferon Response in Human Metapneumovirus Infection [Pathogenesis and Immunity]

  • Human metapneumovirus (hMPV) is a respiratory paramyxovirus that is distributed worldwide and induces significant airway morbidity. Despite the relevance of hMPV as a pathogen, many aspects of the immune response to this virus are still largely unknown. In this report, we focus on the antiviral immune response, which is critical for viral clearance and disease resolution. Using in vitro and in vivo systems, we show that hMPV is able to induce expression of lambda interferon 1 (IFN-1), IFN-2, IFN-3, and IFN-4. The induction of IFN- expression by hMPV was dependent on interferon regulatory factor 7 (IRF-7) expression but not on IRF-3 expression. Treatment of hMPV-infected mice with IFN- reduced the disease severity, lung viral titer, and inflammatory response in the lung. Moreover, the IFN- response induced by the virus was regulated by the expression of the hMPV G protein. These results show that type III interferons (IFN-s) play a critical protective role in hMPV infection.

    IMPORTANCE Human metapneumovirus (hMPV) is a pathogen of worldwide importance. Despite the relevance of hMPV as a pathogen, critical aspects of the immune response induced by this virus remain unidentified. Interferons (IFNs), including IFN-, the newest addition to the interferon family, constitute an indispensable part of the innate immune response. Here, we demonstrated that IFN- exhibited a protective role in hMPV infection in vitro and in an experimental mouse model of infection.

  • Structure of Acidic pH Dengue Virus Showing the Fusogenic Glycoprotein Trimers [Structure and Assembly]

  • Flaviviruses undergo large conformational changes during their life cycle. Under acidic pH conditions, the mature virus forms transient fusogenic trimers of E glycoproteins that engage the lipid membrane in host cells to initiate viral fusion and nucleocapsid penetration into the cytoplasm. However, the dynamic nature of the fusogenic trimer has made the determination of its structure a challenge. Here we have used Fab fragments of the neutralizing antibody DV2-E104 to stop the conformational change of dengue virus at an intermediate stage of the fusion process. Using cryo-electron microscopy, we show that in this intermediate stage, the E glycoproteins form 60 trimers that are similar to the predicted "open" fusogenic trimer.

    IMPORTANCE The structure of a dengue virus has been captured during the formation of fusogenic trimers. This was accomplished by binding Fab fragments of the neutralizing antibody DV2-E104 to the virus at neutral pH and then decreasing the pH to 5.5. These trimers had an "open" conformation, which is distinct from the "closed" conformation of postfusion trimers. Only two of the three E proteins within each spike are bound by a Fab molecule at domain III. Steric hindrance around the icosahedral 3-fold axes prevents binding of a Fab to the third domain III of each E protein spike. Binding of the DV2-E104 Fab fragments prevents domain III from rotating by about 130ddeg; to the postfusion orientation and thus precludes the stem region from "zipping" together the three E proteins along the domain II boundaries into the "closed" postfusion conformation, thus inhibiting fusion.

  • Modulation of Type I Interferon-Associated Viral Sensing during Acute Simian Immunodeficiency Virus Infection in African Green Monkeys [Pathogenesis and Immunity]

  • Natural hosts of simian immunodeficiency virus (SIV), such as African green monkeys (AGMs), do not progress to AIDS when infected with SIV. This is associated with an absence of a chronic type I interferon (IFN-I) signature. It is unclear how the IFN-I response is downmodulated in AGMs. We longitudinally assessed the capacity of AGM blood cells to produce IFN-I in response to SIV and herpes simplex virus (HSV) infection. Phenotypes and functions of plasmacytoid dendritic cells (pDCs) and other mononuclear blood cells were assessed by flow cytometry, and expression of viral sensors was measured by reverse transcription-PCR. pDCs displayed low BDCA-2, CD40, and HLA-DR expression levels during AGM acute SIV (SIVagm) infection. BDCA-2 was required for sensing of SIV, but not of HSV, by pDCs. In acute infection, AGM peripheral blood mononuclear cells (PBMCs) produced less IFN-I upon SIV stimulation. In the chronic phase, the production was normal, confirming that the lack of chronic inflammation is not due to a sensing defect of pDCs. In contrast to stimulation by SIV, more IFN-I was produced upon HSV stimulation of PBMCs isolated during acute infection, while the frequency of AGM pDCs producing IFN-I upon in vitro stimulation with HSV was diminished. Indeed, other cells started producing IFN-I. This increased viral sensing by non-pDCs was associated with an upregulation of Toll-like receptor 3 and IFN--inducible protein 16 caused by IFN-I in acute SIVagm infection. Our results suggest that, as in pathogenic SIVmac infection, SIVagm infection mobilizes bone marrow precursor pDCs. Moreover, we show that SIV infection modifies the capacity of viral sensing in cells other than pDCs, which could drive IFN-I production in specific settings.

    IMPORTANCE The effects of HIV/SIV infections on the capacity of plasmacytoid dendritic cells (pDCs) to produce IFN-I in vivo are still incompletely defined. As IFN-I can restrict viral replication, contribute to inflammation, and influence immune responses, alteration of this capacity could impact the viral reservoir size. We observed that even in nonpathogenic SIV infection, the frequency of pDCs capable of efficiently sensing SIV and producing IFN-I was reduced during acute infection. We discovered that, concomitantly, cells other than pDCs had increased abilities for viral sensing. Our results suggest that pDC-produced IFN-I upregulates viral sensors in bystander cells, the latter gaining the capacity to produce IFN-I. These results indicate that in certain settings, cells other than pDCs can drive IFN-I-associated inflammation in SIV infection. This has important implications for the understanding of persistent inflammation and the establishment of viral reservoirs.

  • Interaction of Infectious Spleen and Kidney Necrosis Virus ORF119L with PINCH Leads to Dominant-Negative Inhibition of Integrin-Linked Kinase and Cardiovascular Defects in Zebrafish [Pathogenesis and Immunity]

  • Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the Megalocytivirus genus, Iridoviridae family, causing a severe systemic disease with high mortality in mandarin fish (Siniperca chuatsi) in China and Southeast Asia. At present, the pathogenesis of ISKNV infection is still not fully understood. Based on a genome-wide bioinformatics analysis of ISKNV-encoded proteins, we found that ISKNV open reading frame 119L (ORF119L) is predicted to encode a three-ankyrin-repeat (3ANK)-domain-containing protein, which shows high similarity to the dominant negative form of integrin-linked kinase (ILK); i.e., viral ORF119L lacks the ILK kinase domain. Thus, we speculated that viral ORF119L might affect the host ILK complex. Here, we demonstrated that viral ORF119L directly interacts with particularly interesting Cys-His-rich protein (PINCH) and affects the host ILK-PINCH interaction in vitro in fathead minnow (FHM) cells. In vivo ORF119L overexpression in zebrafish (Danio rerio) embryos resulted in myocardial dysfunctions with disintegration of the sarcomeric Z disk. Importantly, ORF119L overexpression in zebrafish highly resembles the phenotype of endogenous ILK inhibition, either by overexpressing a dominant negative form of ILK or by injecting an ILK antisense morpholino oligonucleotide. Intriguingly, ISKNV-infected mandarin fish develop disorganized sarcomeric Z disks in cardiomyocytes. Furthermore, phosphorylation of AKT, a downstream effector of ILK, was remarkably decreased in ORF119L-overexpressing zebrafish embryos. With these results, we show that ISKNV ORF119L acts as a domain-negative inhibitor of the host ILK, providing a novel mechanism for the megalocytivirus pathogenesis.

    IMPORTANCE Our work is the first to show the role of a dominant negative inhibitor of the host ILK from ISKNV (an iridovirus). Mechanistically, the viral ORF119L directly binds to the host PINCH, attenuates the host PINCH-ILK interaction, and thus impairs ILK signaling. Intriguingly, ORF119L-overexpressing zebrafish embryos and ISKNV-infected mandarin fish develop similar disordered sarcomeric Z disks in cardiomyocytes. These findings provide a novel mechanism for megalocytivirus pathogenesis.

  • CD4+ T Cell Help Is Dispensable for Protective CD8+ T Cell Memory against Mousepox Virus following Vaccinia Virus Immunization [Pathogenesis and Immunity]

  • It has been shown in various infection models that CD4+ T cell help (TH) is necessary for the conditioning, maintenance, and/or recall responses of memory CD8+ T cells (CD8M). Yet, in the case of vaccinia virus (VACV), which constitutes the vaccine used to eradicate smallpox and is a candidate vector for other infectious diseases, the issue is controversial because different groups have shown either TH dependence or independence of CD8M conditioning, maintenance, and/or recall response. In agreement with some of these groups, we show that TH plays a role in, but is not essential for, the maintenance, proliferation, and effector differentiation of polyclonal memory CD8+ T cells after infection with wild-type VACV strain Western Reserve. More important, we show that unhelped and helped anti-VACV memory CD8+ T cells are similarly efficient at protecting susceptible mice from lethal mousepox, the mouse equivalent of human smallpox. Thus, TH is not essential for the conditioning and maintenance of memory CD8+ T cells capable of mounting a recall response strong enough to protect from a lethal natural pathogen. Our results may partly explain why the VACV vaccine is so effective.

    IMPORTANCE We used vaccinia virus (VACV)mmdash;a gold standard vaccinemmdash;as the immunogen and ectromelia virus (ECTV) as the pathogen to demonstrate that the conditioning and maintenance of anti-VACV memory CD8+ T cells and their ability to protect against an orthopoxvirus (OPV) infection in its natural host can develop in the absence of CD4+ T cell help. Our results provide important insight to our basic knowledge of the immune system. Further, because VACV is used as a vaccine in humans, our results may help us understand how this vaccine induces protective immunity in this species. In addition, this work may partly explain why VACV is so effective as a vaccine.

  • Polyreactivity and Autoreactivity among HIV-1 Antibodies [Pathogenesis and Immunity]

  • It is generally acknowledged that human broadly neutralizing antibodies (bNAbs) capable of neutralizing multiple HIV-1 clades are often polyreactive or autoreactive. Whereas polyreactivity or autoreactivity has been proposed to be crucial for neutralization breadth, no systematic, quantitative study of self-reactivity among nonneutralizing HIV-1 Abs (nNAbs) has been performed to determine whether poly- or autoreactivity in bNAbs is a consequence of chronic antigen (Ag) exposure and/or inflammation or a fundamental property of neutralization. Here, we use protein microarrays to assess binding to ggt;9,400 human proteins and find that as a class, bNAbs are significantly more poly- and autoreactive than nNAbs. The poly- and autoreactive property is therefore not due to the infection milieu but rather is associated with neutralization. Our observations are consistent with a role of heteroligation for HIV-1 neutralization and/or structural mimicry of host Ags by conserved HIV-1 neutralization sites. Although bNAbs are more mutated than nNAbs as a group, V(D)J mutation per se does not correlate with poly- and autoreactivity. Infrequent poly- or autoreactivity among nNAbs implies that their dominance in humoral responses is due to the absence of negative control by immune regulation. Interestingly, four of nine bNAbs specific for the HIV-1 CD4 binding site (CD4bs) (VRC01, VRC02, CH106, and CH103) bind human ubiquitin ligase E3A (UBE3A), and UBE3A protein competitively inhibits gp120 binding to the VRC01 bNAb. Among these four bNAbs, avidity for UBE3A was correlated with neutralization breadth. Identification of UBE3A as a self-antigen recognized by CD4bs bNAbs offers a mechanism for the rarity of this bNAb class.

    IMPORTANCE Eliciting bNAbs is key for HIV-1 vaccines; most Abs elicited by HIV-1 infection or immunization, however, are strain specific or nonneutralizing, and unsuited for protection. Here, we compare the specificities of bNAbs and nNAbs to demonstrate that bNAbs are significantly more poly- and autoreactive than nNAbs. The strong association of poly- and autoreactivity with bNAbs, but not nNAbs from infected patients, indicates that the infection milieu, chronic inflammation and Ag exposure, CD4 T-cell depletion, etc., alone does not cause poly- and autoreactivity. Instead, these properties are fundamentally linked to neutralization breadth, either by the requirement for heteroligation or the consequence of host mimicry by HIV-1. Indeed, we show that human UBE3A shares an epitope(s) with HIV-1 envelope recognized by four CD4bs bNAbs. The poly- and autoreactivity of bNAbs surely contribute to the rarity of membrane-proximal external region (MPER) and CD4bs bNAbs and identify a roadblock that must be overcome to induce protective vaccines.

  • Shift in Monocyte Apoptosis with Increasing Viral Load and Change in Apoptosis-Related ISG/Bcl2 Family Gene Expression in Chronically HIV-1-Infected Subjects [Pathogenesis and Immunity]

  • Although monocytes and macrophages are targets of HIV-1-mediated immunopathology, the impact of high viremia on activation-induced monocyte apoptosis relative to monocyte and macrophage activation changes remains undetermined. In this study, we determined constitutive and oxidative stress-induced monocyte apoptosis in uninfected and HIV+ individuals across a spectrum of viral loads (n = 35; range, 2,243 to 1,355,998 HIV-1 RNA copies/ml) and CD4 counts (range, 26 to 801 cells/mm3). Both constitutive apoptosis and oxidative stress-induced apoptosis were positively associated with viral load and negatively associated with CD4, with an elevation in apoptosis occurring in patients with more than 40,000 (4.6 log) copies/ml. As expected, expression of Rb1 and interferon-stimulated genes (ISGs), plasma soluble CD163 (sCD163) concentration, and the proportion of CD14++ CD16+ intermediate monocytes were elevated in viremic patients compared to those in uninfected controls. Although CD14++ CD16+ frequencies, sCD14, sCD163, and most ISG expression were not directly associated with a change in apoptosis, sCD14 and ISG expression showed an association with increasing viral load. Multivariable analysis of clinical values and monocyte gene expression identified changes in IFI27, IFITM2, Rb1, and Bcl2 expression as determinants of constitutive apoptosis (P = 3.77 x 10nndash;5; adjusted R2 = 0.5983), while changes in viral load, IFITM2, Rb1, and Bax expression were determinants of oxidative stress-induced apoptosis (P = 5.59 x 10nndash;5; adjusted R2 = 0.5996). Our data demonstrate differential activation states in monocytes between levels of viremia in association with differences in apoptosis that may contribute to greater monocyte turnover with high viremia.

    IMPORTANCE This study characterized differential monocyte activation, apoptosis, and apoptosis-related gene expression in low- versus high-level viremic HIV-1 patients, suggesting a shift in apoptosis regulation that may be associated with disease state. Using single and multivariable analysis of monocyte activation parameters and gene expression, we supported the hypothesis that monocyte apoptosis in HIV disease is a reflection of viremia and activation state with contributions from gene expression changes within the ISG and Bcl2 gene families. Understanding monocyte apoptosis response may inform HIV immunopathogenesis, retention of infected macrophages, and monocyte turnover in low- or high-viral-load states.

  • Efficient Infectious Cell Culture Systems of the Hepatitis C Virus (HCV) Prototype Strains HCV-1 and H77 [Virus-Cell Interactions]

  • The first discovered and sequenced hepatitis C virus (HCV) genome and the first in vivo infectious HCV clones originated from the HCV prototype strains HCV-1 and H77, respectively, both widely used in research of this important human pathogen. In the present study, we developed efficient infectious cell culture systems for these genotype 1a strains by using the HCV-1/SF9_A and H77C in vivo infectious clones. We initially adapted a genome with the HCV-1 5'UTR-NS5A (where UTR stands for untranslated region) and the JFH1 NS5B-3'UTR (5-5A recombinant), including the genotype 2a-derived mutations F1464L/A1672S/D2979G (LSG), to grow efficiently in Huh7.5 cells, thus identifying the E2 mutation S399F. The combination of LSG/S399F and reported TNcc(1a)-adaptive mutations A1226G/Q1773H/N1927T/Y2981F/F2994S promoted adaptation of the full-length HCV-1 clone. An HCV-1 recombinant with 17 mutations (HCV1cc) replicated efficiently in Huh7.5 cells and produced supernatant infectivity titers of 104.0 focus-forming units (FFU)/ml. Eight of these mutations were identified from passaged HCV-1 viruses, and the A970T/I1312V/C2419R/A2919T mutations were essential for infectious particle production. Using CD81-deficient Huh7 cells, we further demonstrated the importance of A970T/I1312V/A2919T or A970T/C2419R/A2919T for virus assembly and that the I1312V/C2419R combination played a major role in virus release. Using a similar approach, we found that NS5B mutation F2994R, identified here from culture-adapted full-length TN viruses and a common NS3 helicase mutation (S1368P) derived from viable H77C and HCV-1 5-5A recombinants, initiated replication and culture adaptation of H77C containing LSG and TNcc(1a)-adaptive mutations. An H77C recombinant harboring 19 mutations (H77Ccc) replicated and spread efficiently after transfection and subsequent infection of naive Huh7.5 cells, reaching titers of 103.5 and 104.4 FFU/ml, respectively.

    IMPORTANCE Hepatitis C virus (HCV) was discovered in 1989 with the cloning of the prototype strain HCV-1 genome. In 1997, two molecular clones of H77, the other HCV prototype strain, were shown to be infectious in chimpanzees, but not in vitro. HCV research was hampered by a lack of infectious cell culture systems, which became available only in 2005 with the discovery of JFH1 (genotype 2a), a genome that could establish infection in Huh7.5 cells. Recently, we developed in vitro infectious clones for genotype 1a (TN), 2a (J6), and 2b (J8, DH8, and DH10) strains by identifying key adaptive mutations. Globally, genotype 1 is the most prevalent. Studies using HCV-1 and H77 prototype sequences have generated important knowledge on HCV. Thus, the in vitro infectious clones developed here for these 1a strains will be of particular value in advancing HCV research. Moreover, our findings open new avenues for the culture adaptation of HCV isolates of different genotypes.

  • The Megavirus Chilensis Cu,Zn-Superoxide Dismutase: the First Viral Structure of a Typical Cellular Copper Chaperone-Independent Hyperstable Dimeric Enzyme [Virus-Cell Interactions]

  • Giant viruses able to replicate in Acanthamoeba castellanii penetrate their host through phagocytosis. After capsid opening, a fusion between the internal membranes of the virion and the phagocytic vacuole triggers the transfer in the cytoplasm of the viral DNA together with the DNA repair enzymes and the transcription machinery present in the particles. In addition, the proteome analysis of purified mimivirus virions revealed the presence of many enzymes meant to resist oxidative stress and conserved in the Mimiviridae. Megavirus chilensis encodes a predicted copper, zinc superoxide dismutase (Cu,Zn-SOD), an enzyme known to detoxify reactive oxygen species released in the course of host defense reactions. While it was thought that the metal ions are required for the formation of the active-site lid and dimer stabilization, megavirus chilensis SOD forms a very stable metal-free dimer. We used electron paramagnetic resonance (EPR) analysis and activity measurements to show that the supplementation of the bacterial culture with copper and zinc during the recombinant expression of Mg277 is sufficient to restore a fully active holoenzyme. These results demonstrate that the viral enzyme's activation is independent of a chaperone both for disulfide bridge formation and for copper incorporation and suggest that its assembly may not be as regulated as that of its cellular counterparts. A SOD protein is encoded by a variety of DNA viruses but is absent from mimivirus. As in poxviruses, the enzyme might be dispensable when the virus infects Acanthamoeba cells but may allow megavirus chilensis to infect a broad range of eukaryotic hosts.

    IMPORTANCE Mimiviridae are giant viruses encoding more than 1,000 proteins. The virion particles are loaded with proteins used by the virus to resist the vacuole's oxidative stress. The megavirus chilensis virion contains a predicted copper, zinc superoxide dismutase (Cu,Zn-SOD). The corresponding gene is present in some megavirus chilensis relatives but is absent from mimivirus. This first crystallographic structure of a viral Cu,Zn-SOD highlights the features that it has in common with and its differences from cellular SODs. It corresponds to a very stable dimer of the apo form of the enzyme. We demonstrate that upon supplementation of the growth medium with Cu and Zn, the recombinant protein is fully active, suggesting that the virus's SOD activation is independent of a copper chaperone for SOD generally used by eukaryotic SODs.

  • Mutation V111I in HIV-2 Reverse Transcriptase Increases the Fitness of the Nucleoside Analogue-Resistant K65R and Q151M Viruses [Vaccines and Antiviral Agents]

  • Infection with HIV-2 can ultimately lead to AIDS, although disease progression is much slower than with HIV-1. HIV-2 patients are mostly treated with a combination of nucleoside reverse transcriptase (RT) inhibitors (NRTIs) and protease inhibitors designed for HIV-1. Many studies have described the development of HIV-1 resistance to NRTIs and identified mutations in the polymerase domain of RT. Recent studies have shown that mutations in the connection and RNase H domains of HIV-1 RT may also contribute to resistance. However, only limited information exists regarding the resistance of HIV-2 to NRTIs. In this study, therefore, we analyzed the polymerase, connection, and RNase H domains of RT in HIV-2 patients failing NRTI-containing therapies. Besides the key resistance mutations K65R, Q151M, and M184V, we identified a novel mutation, V111I, in the polymerase domain. This mutation was significantly associated with mutations K65R and Q151M. Sequencing of the connection and RNase H domains of the HIV-2 patients did not reveal any of the mutations that were reported to contribute to NRTI resistance in HIV-1. We show that V111I does not strongly affect drug susceptibility but increases the replication capacity of the K65R and Q151M viruses. Biochemical assays demonstrate that V111I restores the polymerization defects of the K65R and Q151M viruses but negatively affects the fidelity of the HIV-2 RT enzyme. Molecular dynamics simulations were performed to analyze the structural changes mediated by V111I. This showed that V111I changed the flexibility of the 110-to-115 loop region, which may affect deoxynucleoside triphosphate (dNTP) binding and polymerase activity.

    IMPORTANCE Mutation V111I in the HIV-2 reverse transcriptase enzyme was identified in patients failing therapies containing nucleoside analogues. We show that the V111I change does not strongly affect the sensitivity of HIV-2 to nucleoside analogues but increases the fitness of viruses with drug resistance mutations K65R and Q151M.

  • Simian Hemorrhagic Fever Virus Cell Entry Is Dependent on CD163 and Uses a Clathrin-Mediated Endocytosis-Like Pathway [Virus-Cell Interactions]

  • Simian hemorrhagic fever virus (SHFV) causes a severe and almost uniformly fatal viral hemorrhagic fever in Asian macaques but is thought to be nonpathogenic for humans. To date, the SHFV life cycle is almost completely uncharacterized on the molecular level. Here, we describe the first steps of the SHFV life cycle. Our experiments indicate that SHFV enters target cells by low-pH-dependent endocytosis. Dynamin inhibitors, chlorpromazine, methyl-bbeta;-cyclodextrin, chloroquine, and concanamycin A dramatically reduced SHFV entry efficiency, whereas the macropinocytosis inhibitors EIPA, blebbistatin, and wortmannin and the caveolin-mediated endocytosis inhibitors nystatin and filipin III had no effect. Furthermore, overexpression and knockout study and electron microscopy results indicate that SHFV entry occurs by a dynamin-dependent clathrin-mediated endocytosis-like pathway. Experiments utilizing latrunculin B, cytochalasin B, and cytochalasin D indicate that SHFV does not hijack the actin polymerization pathway. Treatment of target cells with proteases (proteinase K, papain, aalpha;-chymotrypsin, and trypsin) abrogated entry, indicating that the SHFV cell surface receptor is a protein. Phospholipases A2 and D had no effect on SHFV entry. Finally, treatment of cells with antibodies targeting CD163, a cell surface molecule identified as an entry factor for the SHFV-related porcine reproductive and respiratory syndrome virus, diminished SHFV replication, identifying CD163 as an important SHFV entry component.

    IMPORTANCE Simian hemorrhagic fever virus (SHFV) causes highly lethal disease in Asian macaques resembling human illness caused by Ebola or Lassa virus. However, little is known about SHFV's ecology and molecular biology and the mechanism by which it causes disease. The results of this study shed light on how SHFV enters its target cells. Using electron microscopy and inhibitors for various cellular pathways, we demonstrate that SHFV invades cells by low-pH-dependent, actin-independent endocytosis, likely with the help of a cellular surface protein.

  • Malawi Polyomavirus Is a Prevalent Human Virus That Interacts with Known Tumor Suppressors [Virus-Cell Interactions]

  • Malawi polyomavirus (MWPyV) is a recently identified human polyomavirus. Serology for MWPyV VP1 indicates that infection frequently occurs in childhood and reaches a prevalence of 75% in adults. The MWPyV small T antigen (ST) binds protein phosphatase 2A (PP2A), and the large T antigen (LT) binds pRb, p107, p130, and p53. However, the MWPyV LT was less stable than the simian virus 40 (SV40) LT and was unable to promote the growth of normal cells. This report confirms that MWPyV is a widespread human virus expressing T antigens with low transforming potential.

  • Swine Interferon-Inducible Transmembrane Proteins Potently Inhibit Influenza A Virus Replication [Cellular Response to Infection]

  • Human interferon-inducible transmembrane proteins (IFITMs) were identified as restriction factors of influenza A virus (IAV). Given the important role of pigs in the zoonotic cycle of IAV, we cloned swine IFITMs (swIFITMs) and found two IFITM1-like proteins, one homologue of IFITM2, and a homologue of IFITM3. We show that swIFITM2 and swIFITM3 localize to endosomes and display potent antiviral activities. Knockdown of swIFITMs strongly reduced virus inhibition by interferon, establishing the swIFITMs as potent restriction factors in porcine cells.

  • Expression of Neuronal CXCL10 Induced by Rabies Virus Infection Initiates Infiltration of Inflammatory Cells, Production of Chemokines and Cytokines, and Enhancement of Blood-Brain Barrier Permeability [Pathogenesis and Immunity]

  • It has been shown that enhancement of blood-brain barrier (BBB) permeability is modulated by the expression of chemokines/cytokines and reduction of tight junction (TJ) proteins in the brains of mice infected with rabies virus (RABV). Since CXCL10 was found to be the most highly expressed chemokine, its temporal and spatial expression were determined in the present study. The expression of the chemokine CXCL10 was initially detected in neurons as early as 3 days postinfection (p.i.) in the brains of RABV-infected mice, after which it was detected in microglia (6 days p.i.) and astrocytes (9 days p.i.). Neutralization of CXCL10 by treatment with anti-CXCL10 antibodies reduced gamma interferon (IFN-) production and Th17 cell infiltration, as well as restoring TJ protein expression and BBB integrity. Together, these data suggest that it is the neuronal CXCL10 that initiates the cascade that leads to the activation of microglia/astrocytes, infiltration of inflammatory cells, expression of chemokines/cytokines, reduction of TJ protein expression, and enhancement of the BBB permeability.

  • Virulence Determinants in the PB2 Gene of a Mouse-Adapted H9N2 Virus [Pathogenesis and Immunity]

  • The molecular bases of adaptation and pathogenicity of H9N2 influenza virus in mammals are largely unknown. Here, we show that a mouse-adapted PB2 gene with a phenylalanine-to-leucine mutation (F404L) mainly contributes to enhanced polymerase activity, replication, and pathogenicity of H9N2 in mice and also increases the virulence of the H5N1 and 2009 pandemic H1N1 influenza viruses. Therefore, we defined a novel pathogenic determinant, providing further insights into the pathogenesis of influenza viruses in mammals.

  • Correction for Acharya et al., Heavy Chain-Only IgG2b Llama Antibody Effects Near-Pan HIV-1 Neutralization by Recognizing a CD4-Induced Epitope That Includes Elements of Coreceptor- and CD4-Binding Sites [Author Corrections]

  • Correction for Kwok et al., Genomic Diversity of Epstein-Barr Virus Genomes Isolated from Primary Nasopharyngeal Carcinoma Biopsy Samples [Author Corrections]

  • Correction for Chakrabarti et al., Robust Neutralizing Antibodies Elicited by HIV-1 JRFL Envelope Glycoprotein Trimers in Nonhuman Primates [Author Corrections]

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    JVI Accepts: Articles Published Ahead of Print

  • Inhibition of reactive oxygen species production ameliorates inflammation induced by influenza A viruses via up-regulation of SOCS1 and SOCS3 [PublishAheadOfPrint]

  • Highly pathogenic avian influenza infection is associated with severe mortality in both humans and poultry. The mechanisms of disease pathogenesis and immunity are poorly understood although recent evidence suggests that cytokine/chemokine dysregulation contributes to disease severity following H5N1 infection. Influenza A virus infection causes a rapid influx of inflammatory cells, resulting in increased reactive oxygen species production, cytokine expression and acute lung injury. Pro-inflammatory stimuli are known to induce intracellular reactive oxygen species by activating NADPH oxidase activity. We therefore hypothesised that inhibition of this activity would restore host cytokine homeostasis following avian influenza virus infection. A panel of airway epithelial and immune cells from mammalian and avian species were infected with A/Puerto Rico/8/1934 H1N1 virus, low pathogenic avian influenza H5N3 virus (A/duck/Victoria/0305-2/2012), highly pathogenic avian influenza H5N1 virus (A/chicken/Vietnam/0008/2004), or low pathogenic avian influenza H7N9 virus (A/Anhui/1/2013). Quantitative real-time reverse transcriptase PCR showed that H5N1 and H7N9 viruses significantly stimulated cytokine (IL-6, IFN-bbeta;, CXCL10 and CCL5) production. Among the influenza-induced cytokines, CCL5 was identified as a potential marker for overactive immunity. Apocynin, a Nox2 inhibitor, inhibited influenza-induced cytokines and reactive oxygen species production, although viral replication was not significantly altered in vitro. Interestingly, apocynin treatment significantly increased influenza virus-induced mRNA and protein expression of SOCS1 and SOCS3, enhancing negative regulation of cytokine signaling. These findings suggest that apocynin or its derivatives (targeting host responses) could be used in combination with anti-viral strategies (targeting viruses), as therapeutic agents to ameliorate disease severity in susceptible species.

    IMPORTANCE Highly pathogenic avian influenza virus infection causes severe morbidity and mortality in both humans and poultry. Wide-spread antiviral resistance necessitates the need for the development of additional novel therapeutic measures to modulate overactive host immune responses following infection. Disease severity following avian influenza virus infection can be attributed in part to hyper-induction of inflammatory mediators such as cytokines, chemokines and reactive oxygen species. Our study shows that highly pathogenic avian influenza H5N1 virus and low pathogenic avian influenza H7N9 virus (both associated with human fatalities) promote inactivation of FoxO3 and down-regulation of the TAM receptor tyrosine kinase, Tyro3, leading to augmentation of the inflammatory cytokine response. Inhibition of influenza-induced reactive oxygen species with apocynin activated FoxO3 and stimulated SOCS1 and SOCS3 proteins, restoring cytokine homeostasis. We conclude that modulation of host immune responses with antioxidant and/or anti-inflammatory agents in combination with antiviral therapy may have important therapeutic benefits.

  • Measles virus mutants possessing the fusion protein with enhanced fusion activity spread effectively in neuronal cells without causing strong cytopathology, but not in other cells [PublishAheadOfPrint]

  • Subacute sclerosing panencephalitis (SSPE) is caused by persistent measles virus (MV) infection in the central nervous system (CNS). Since human neurons, its main target cells, do not express known MV receptors (signaling lymphocyte activation molecule (SLAM) and nectin 4), it remains to be understood how MV infects and spreads in them. We have recently reported that fusion-enhancing substitutions in the extracellular domain of the MV fusion (F) protein (T461I and S103I/N462S/N465S), which are found in multiple SSPE virus isolates, promote MV spread in human neuroblastoma cell lines and brains of suckling hamsters. In this study, we show that hyperfusogenic viruses with these substitutions also spread efficiently in human primary neuron cultures without inducing syncytia. These substitutions were found to destabilize the pre-fusion conformation of the F protein trimer, thereby enhancing fusion activity. However, these hyperfusogenic viruses exhibited stronger cytopathology and produced lower titers at later time points in SLAM- or nectin 4-expressing cells, compared with the wild-type MV. Although these viruses spread efficiently in the brains of SLAM knock-in mice, they did not in the spleens. Taken together, the results suggest that enhanced fusion activity is beneficial for MV to spread in neuronal cells where no cytopathology occurs, but detrimental to other types of cells due to strong cytopathology. Acquisition of enhanced fusion activity through substitutions in the extracellular domain of the F protein may be crucial for MV's extensive spread in the CNS and development of SSPE.

    IMPORTANCE Subacute sclerosing panencephalitis (SSPE) is a fatal disease caused by persistent measles virus (MV) infection in the central nervous system (CNS). Its cause is not well understood, and no effective therapy is currently available. Recently, we have reported that enhanced fusion activity of MV through the mutations in its fusion protein is a major determinant of efficient virus spread in human neuronal cells and brains of suckling hamsters. In this study, we show that those mutations render the conformation of the fusion protein less stable, thereby making it hyperfusogenic. Our results also show that enhanced fusion activity is beneficial for MV to spread in the CNS, but rather detrimental to other types of cells in peripheral tissues, which are strongly damaged by the virus. Our findings provide important insight into the mechanism for the development of SSPE after MV infection.

  • MVA-infected Dendritic Cells Present CD4+ T-Cell Epitopes by Endogenous MHC Class II Presentation Pathways [PublishAheadOfPrint]

  • CD4+ T-lymphocytes play a central role in the immune system and mediate their function after recognition of their respective antigens presented on MHCII molecules on antigen presenting cells (APCs). Conventionally, phagocytosed antigens are loaded on MHCII for stimulation of CD4+ T-cells. Certain epitopes, however, can be directly processed from intracellular antigens and are presented on MHCII (endogenous MHCII presentation). Here we characterized the MHCII antigen presentation pathways being possibly involved in the immune response upon vaccination with MVA (modified vaccinia virus Ankara), a promising live viral vaccine vector. We established CD4+ T-cell lines specific for MVA-derived epitopes as tools for in vitro analysis of MHCII antigen processing and presentation in MVA-infected APCs. We provide evidence that infected APCs are able to directly transfer endogenous viral proteins into the MHCII pathway to efficiently activate CD4+ T-cells. By using knockout mice and chemical inhibitory compounds we further elucidate the molecular basis showing that among the various subcellular pathways investigated proteasomes and autophagy are key players in the endogenous MHCII presentation during MVA infection. Interestingly, although proteasomal processing plays an important role neither TAP nor LAMP-2 were found to be involved in the peptide transport.

    Defining the molecular mechanism of MHCII presentation during MVA infection provides a basis to improve MVA-based vaccination strategies aiming for enhanced CD4+ T-cell activation by targeting antigens into the responsible pathways.

    Importance Statement This work contributes significantly to our understanding of the immunogenic properties of pathogens by deciphering antigen processing pathways contributing to efficient activation of antigen-specific CD4+ T-cells. We identified autophagosome formation, proteasomal activity and lysosomal integrity to be crucial for endogenous CD4+ T-cell activation. Since poxvirus vectors such as MVA are already used in clinical trials as recombinant vaccines, the data provide important information for the future design of optimized poxviral vaccines for the study of advanced immunotherapy options.

  • Clustered microRNAs of the Epstein-Barr Virus Cooperatively Downregulate an Epithelial Cell-Specific Metastasis Suppressor [PublishAheadOfPrint]

  • The Epstein-Barr virus (EBV) encodes its own microRNAs (miRNAs); however, their biological roles remain elusive. The commonly used EBV B95-8 strain lacks a 12 kb genomic region, known as BamHI A Rightward Transcripts (BART) locus, where a number of BART miRNAs are encoded. Here, bacterial artificial chromosome (BAC) technology was used to generate an EBV B95-8 strain in which the 12 kb region was fully restored at its native locus [BART(+) virus)]. Epithelial cells were stably infected with either the parental B95-8 virus or the BART(+) virus, and BART miRNA expression was successfully reconstituted in the BART(+) virus-infected cells. Microarray analyses of cellular gene expression identified N-myc downstream regulated gene 1 (NDRG1) as a putative target of BART miRNAs. The NDRG1 protein was barely expressed in B-cells, highly expressed in epithelial cells, including primary epithelial cells, and strongly donwregulated in the BART(+) virus-infected epithelial cells of various origins. Although in vitro reporter assays identified BART22 as being responsible for the NDRG1 downregulation, EBV genetic analyses revealed that BART22 was not solely responsible; rather, the entire BART miRNA cluster 2 was responsible for the downregulation. Immunohistochemical analyses revealed that the expression level of the NDRG1 protein was downregulated significantly in EBV-positive nasopharyngeal carcinoma specimens. Considering that NDRG1 encodes an epithelial differentiation marker and a suppressor of metastasis, these data implicate a causative relationship between BART miRNA expression and epithelial carcinogenesis in vivo.

    Importance EBV-related epithelial cancers, such as nasopharyngeal carcinomas and EBV-positive gastric cancers, encompass more than 80% of EBV-related malignancies. Although it is known that they express high levels of virally-encoded BART miRNAs, how these miRNAs contribute to EBV-mediated epithelial carcinogenesis remains unknown. Although a number of screenings have been performed to identify targets of viral miRNAs, many targets likely have not been identified, especially in case of epithelial cell infection. This is the first study to use EBV genetics to perform unbiased screens of cellular genes that are differentially expressed in viral miRNA-positive and -negative epithelial cells. The result indicates that multiple EBV-encoded miRNAs cooperatively downregulate NDRG1, an epithelial differentiation marker and suppressor of metastasis. The experimental system described in this study should be useful for further clarifying the mechanism of EBV-mediated epithelial carcinogenesis.

  • Acanthamoeba polyphaga mimivirus prevents amoebal encystment-mediating serine proteinase expression and circumvents cell encystment [PublishAheadOfPrint]

  • Acanthamoeba is a genus of free-living amoebas distributed worldwide. Few studies have explored the interactions between these protozoa and their infecting giant virus, Acanthamoeba polyphaga mimivirus (APMV). Here we show that once the amoebal encystment is triggered, trophozoites become significantly resistant to APMV. Otherwise, upon infection, APMV is able to interfere with the expression of a serine proteinase related to amoebal encystment and the encystment can no longer be triggered.

  • Nanobody binding to a conserved epitope promoted human norovirus particle disassembly [PublishAheadOfPrint]

  • Human noroviruses are icosahedral single-stranded RNA viruses. The capsid protein is divided into shell (S) and protruding (P) domains, which are connected by a flexible hinge region. There are numerous genetically and antigenically distinct noroviruses and the dominant strains evolve every other year. Vaccine and antiviral development is hampered by the difficulties in growing human norovirus in cell culture and the continually evolving strains. Here, we show the X-ray crystal structures of human norovirus P domains in complex with two different Nanobodies. One Nanobody, Nano-85, was broadly reactive, while the other, Nano-25, was strain specific. We showed that both Nanobodies bound to the lower region on the P domain and had nanomolar affinities. The Nano-85 binding site mainly compromised of highly conserved amino acids among the genetically distinct genogroup II noroviruses. Several of the conserved residues were also recognized by a broadly reactive monoclonal antibody, which suggested this region contained a dominant epitope. Superposition of the P domain Nanobody complex structures into a cryo-EM particle structure revealed that both Nanobodies bound at occluded sites on the particles. The flexible hinge region, which contained ~10-12 amino acids, likely permitted a certain degree of P domain movement on the particles in order to accommodate the Nanobodies. Interestingly, the Nano-85 binding interaction with intact particles caused the particles to disassemble in vitro. Altogether, these results suggested that the highly conserved Nano-85 binding epitope contained a trigger mechanism for particle disassembly. Principally, this epitope represents a potential site of norovirus vulnerability.

    IMPORTANCE We characterized two different Nanobodies (Nano-85 and Nano-25) that bind to human noroviruses. Both Nanobodies bound with high affinities to the lower region of the P domain, which was occluded on intact particles. Nano-25 was specific for GII.10, whereas Nano-85 bound several different GII genotypes, including GII.4, GII.10, and GII.12. We showed that Nano-85 was able to detect norovirus virions in clinical stool specimens using a sandwich ELISA. Importantly, we found that Nano-85 binding to intact particles caused the particles to disassemble. We believe that with further testing, Nano-85 will not only work as a diagnostic reagent in norovirus detection systems, but could also function as a broadly reactive GII norovirus antiviral.

  • Structure of an Enteric Pathogen Bovine Parvovirus [PublishAheadOfPrint]

  • Bovine Parvovirus (BPV), the causative agent of respiratory and gastrointestinal disease in cows, is the type member of the Bocaparvovirus genus of the Parvoviridae. Towards efforts to obtain a template for the development of vaccines and small molecule inhibitors for this pathogen, the structure of the BPV capsid, assembled from the major capsid viral protein 2 (VP2), was determined using X-ray crystallography and cryo-electron microscopy and three-dimensional image reconstruction to 3.2 and 8.8 AAring; resolution, respectively. The VP2 region ordered in the crystal structure, residues 39-536, conserves the parvoviral eight-stranded jellyroll motif and an aalpha;A helix. The BPV capsid displays common parvovirus features: a channel at and depressions surrounding the 5-fold axes, and protrusions surrounding the 3-fold axes. However, rather than a depression centered at the 2-fold axes, a raised surface loop divides this feature in BPV. Additional observed density in the capsid interior in the cryo-reconstructed map, compared to the crystal structure, is interpreted as ten additional N-terminal residues, 29-38 that radially extends the channel under the 5-fold axis, as observed for Human Bocavirus 1. Surface loops of varying lengths and conformations extend from the core jellyroll motif of VP2. These confer the unique surface topology of the BPV capsid, making it strikingly different from HBoV1 as well as the type members of other Parvovirinae genera for which structures have been determined. For the type members, structurally analogous regions to those decorating the BPV capsid surface serve as determinants of receptor recognition, tissue and host tropism, pathogenicity, and antigenicity.

    Importance Bovine parvovirus (BPV), identified in the 1960s in diarrheic calves, is a type member of the Bocaparvovirus genus of the non-enveloped, ssDNA Parvoviridae family. Recent isolation of human bocaparvoviruses from children with severe respiratory and gastrointestinal infections has generated interest in understanding the lifecycle and pathogenesis of these emerging viruses. We have determined high-resolution structure of the BPV capsid assembled from its predominant capsid protein VP2, known to be involved in a myriad of functions during host cell entry, pathogenesis, and antigenicity for other Parvovirinae members. Our results indicate the conservation of the core secondary structural elements and the location of the N-terminal residues for the known bocaparvovirus capsid structures. However, surface loops with high variability in sequence and conformation give BPV a unique capsid surface topology. Similar analogous regions in other Parvovirinae type members are important as determinants of receptor recognition, tissue and host tropism, pathogenicity, and antigenicity.

  • Improving Neutralization Potency and Breadth by Combining Broadly Reactive HIV-1 Antibodies Targeting Major Neutralization Epitopes [PublishAheadOfPrint]

  • The isolation of broadly neutralizing HIV-1 monoclonal antibodies (mAbs) to distinct epitopes on the viral envelope glycoprotein (Env) provides the potential to use combinations of mAbs for prevention and treatment of HIV-1 infection. Since many of these mAbs have been isolated in the last few years, the potency and breadth of mAb combinations has not been well characterized. In two parallel experiments, we examined the in vitro neutralizing activities of double, triple and quadruple mAb combinations targeting four distinct epitopes, including the CD4-binding site, the V1V2-glycan region, the V3-glycan supersite and the gp41 membrane-proximal external region (MPER), using a panel of 125 Env-pseudotyped viruses. All mAb combinations showed substantially improved neutralization breadth compared to the corresponding single mAbs, while the neutralization potency of individual mAbs was maintained. At an IC50 cutoff of 1 mmu;g/ml per antibody, double mAb combinations neutralized 89-98% of viruses, and triple combinations neutralized 98-100%. Overall the improvement of neutralization breadth was closely predicted by an additive effect model and explained by complementary neutralization profiles of antibodies recognizing distinct epitopes. Subtle but consistent favorable interactions were observed in some mAb combinations whereas less favorable interactions were observed on a small subset of viruses that are highly sensitive to V3-glycan mAbs. These data demonstrate favorable in vitro combinations of broadly neutralizing HIV-1 mAbs and suggest that such combinations could have utility for HIV-1 prevention and treatment.

    Importance Over the last five years, numerous broadly reactive HIV-1 neutralizing mAbs have been isolated from B-cells of HIV-1 infected donors. Each of these mAbs binds to one of the major vulnerable sites (epitopes) on the surface of the viral envelope glycoprotein. Since antibodies to distinct viral epitopes could theoretically act together to provide greater potency and breadth of virus neutralization, we tested physical mixtures of double, triple and quadruple combinations of neutralizing mAbs targeting four major epitopes on the HIV-1 Env. When tested together, antibody combinations showed substantially improved neutralization breadth compared to single mAbs. This improvement could be explained by the complementary neutralization profiles of individual mAbs. We further demonstrated that each antibody maintained its full neutralization potency when used in combination with other mAbs. These data provided a rationale for clinical use of antibody based combinations for HIV-1 prevention and therapy.

  • COMMD1/Murr1 reinforces HIV-1 latent infection through I{kappa}B-{alpha} stabilization. [PublishAheadOfPrint]

  • The transcription factor NF-B is important for HIV-1 transcription initiation in HIV-1 primary infection and reactivation in HIV-1 latent infected cells. However, comparative analysis of the regulation and function of NF-B in HIV-1 latent infected cells has not been explored. Here we show that expression of IB-aalpha;, an endogenous inhibitor of NF-B, is enhanced by HIV-1 latent infection via induction of host-derived factor COMMD1/Murr1 in myeloid cells but not in lymphoid cells using 4 sets of HIV-1 latent infected cells and their respective parental cells. IB-aalpha; protein was stabilized by COMMD1, which attenuated NF-B signaling during Toll-like receptor ligands and TNFaalpha; treatment, and enhanced HIV-1 latency in HIV-1 latent infected cells. Activation of PI3K and JAK pathway is involved in COMMD1 induction in HIV-1 latent infected cells. Our findings indicate that COMMD1 induction is the inhibition mechanism of NF-B in HIV-1 latent infected cells, which contributes to innate immune deficiency and reinforcing the HIV-1 latency. Thus, COMMD1 might be a double-edged sword, beneficial in primary infection but not beneficial in latent infection when considering HIV-1 eradication.

    IMPORTANCE HIV-1 latency poses a major barrier to viral eradication at the era of combination of anti-retroviral therapy (cART). In this study, we found that COMMD1/Murr1, previously identified as an HIV-1 restriction factor, inhibits the proteasomal degradation of IB-aalpha; via increasing the interaction with IB-aalpha; in myeloid HIV-1 latent infected cells. IB-aalpha; protein was stabilized by COMMD1, which attenuated NF-B signaling during innate immune response and enhanced HIV-1 latency in HIV-1 latent infected cells. Activation of PI3K and JAK pathway is involved in COMMD1 induction in HIV-1 latent infected cells. Thus, host-derived factor COMMD1 is beneficial in suppressing primary infection, but it enhances latent infection, indicating a double-edged sword for HIV-1 eradication.

  • Structural basis for development of avian virus capsids to display influenza virus proteins for induction of protective immunity [PublishAheadOfPrint]

  • Bioengineering of viruses and virus-like particles (VLP) is a well-established approach in the development of new and improved vaccines to viral and bacterial pathogens. We report here that the capsid of a major avian pathogen, infectious bursal disease virus (IBDV), can accommodate heterologous proteins to induce protective immunity. The structural units of the ~70-nm-diameter T=13 IBDV capsid are trimers of VP2, which is made as a precursor (pVP2). The pVP2 C-terminal domain has an amphipathic aalpha;-helix that controls VP2 polymorphism. In the absence of VP3 scaffolding protein, pVP2 intermediates bearing this aalpha;-helix assemble into genuine VLP only when expressed with an N-terminal His tag (protein HT-VP2-466). HT-VP2-466 capsids are optimal for protein insertion, as they are large enough (~78,000 nm3 cargo space) and are assembled from a single protein. We explored HT-VP2-466-based chimeric capsids, initially using enhanced green fluorescent protein (EGFP). VLP assembly yield was efficient when we coexpressed EGFP-HT-VP2-466 and HT-VP2-466 from two recombinant baculoviruses. The native EGFP structure (~240 copies/virion) was successfully inserted in a functional form, as VLP were fluorescent, and three-dimensional cryo-electron microscopy showed EGFP molecules incorporated at the inner capsid surface. Immunization of mice with purified EGFP-VLP particles elicited anti-EGFP antibodies. We also inserted hemagglutinin (HA) and matrix (M2) protein epitopes derived from the mouse-adapted A/PR/8/34 influenza virus and engineered several HA- and M2-derived chimeric capsids. Mice immunized with VLP containing the HA stalk, an M2 fragment, or both antigens developed full protection against viral challenge.

    Importance Virus-like particles (VLP) are multimeric protein cages that mimic the infectious virus capsid and are potential candidates for non-living vaccines that induce long-lasting protection. Chimeric VLP can display or include foreign antigens, which could be a conserved epitope to elicit broadly neutralizing antibodies, or several variable epitopes effective against a large number of viral strains. We report the biochemical, structural and immunological characterization of chimeric VLP derived from infectious bursal disease virus (IBDV), an important poultry pathogen. To test the potential of IBDV VLP as a vaccine vehicle, we used the enhanced green fluorescent protein and two fragments derived from hemagglutinin and the M2 matrix protein of the human murine-adapted influenza virus. The IBDV capsid protein fused to influenza virus peptides formed assemblies able to protect mice against viral challenge. Our studies establish the basis for a new generation of multivalent IBDV-based vaccines.

  • SARS-CoV vaccines formulated with delta inulin adjuvants provide enhanced virus protection while ameliorating lung eosinophilic immunopathology [PublishAheadOfPrint]

  • Although the SARS coronavirus (SARS-CoV) epidemic was controlled by non-vaccine measures, coronaviruses remain a major threat to human health. Design of optimal coronavirus vaccines therefore remains a priority. Such vaccines present major challenges; coronavirus immunity often wanes rapidly, individuals needing to be protected include the elderly, and vaccines may exacerbate rather than prevent coronavirus lung immunopathology. To address these issues, we compared in a murine model a range of recombinant spike protein or inactivated whole virus vaccine candidates alone or adjuvanted with either alum, CpG or Advaxttrade;, a new delta inulin-based polysaccharide adjuvant. While all vaccines protected against lethal infection, addition of adjuvant significantly increased serum neutralizing antibody titers and reduced lung virus titers on day 3 post-challenge. Whereas unadjuvanted or alum-formulated vaccines were associated with significantly increased lung eosinophilic immunopathology on day 6 post-challenge, this was not seen in mice immunized with vaccines formulated with delta inulin adjuvant. Protection against eosinophilic immunopathology by vaccines containing delta inulin adjuvants correlated better with enhanced T-cell IFN- recall responses rather than reduced IL4 responses, suggesting immunopathology predominantly reflects an inadequate vaccine-induced Th1 response. This study highlights the critical importance for development of effective and safe coronavirus vaccines of selection of adjuvants based on ability to induce durable IFN- responses.

    Importance Coronaviruses such as SARS-CoV and MERS-CoV cause high case fatality rates and remain major human public health threats, creating a need for effective vaccines. Whilst coronavirus antigens that induce protective neutralizing antibodies have been identified, coronavirus vaccines present a unique problem in that immunized individuals when infected by virus can develop lung eosinophilic pathology, a problem that is further exacerbated by formulation of SARS-CoV vaccines with alum adjuvants. This study shows that formulation of SARS-CoV spike protein or inactivated whole virus vaccines with novel delta inulin-based polysaccharide adjuvants enhances neutralizing antibody titers and protection against clinical disease but at the same time also protects against development of lung eosinophilic immunopathology. It also shows that immunity achieved with delta inulin adjuvants is long-lived thereby overcoming the natural tendency for rapidly waning coronavirus immunity. Thus delta inulin adjuvants may offer a unique ability to develop safer and more effective coronavirus vaccines.

  • LMP1 promotes expression of IGF1 to selectively activate IGF1R and drive cell proliferation [PublishAheadOfPrint]

  • Epstein-Barr Virus (EBV) is a gammaherpes virus that infects the majority of the human population and it is linked to the development of multiple cancers including nasopharyngeal carcinoma. Latent membrane protein 1 (LMP1) is considered the primary oncoprotein of EBV and in epithelial cells it induces the expression and activation, or phosphorylation, of the epidermal growth factor receptor kinase. To identify effects on additional kinases, an unbiased screen of receptor tyrosine kinases potentially activated by LMP1 was performed. Using a protein array, it was determined that LMP1 selectively activates Insulin-like Growth Factor 1 Receptor (IGF1R). This activation takes place in fibroblast, epithelial, and nasopharyngeal cell lines that expressed LMP1 stably and transiently. Of note, LMP1 altered the phosphorylation, but not the expression, of IGF1R. LMP1 mutants with defective signaling domains revealed that the C-Terminal Activating Region 2 domain of LMP1 increased the mRNA expression and the secretion of the ligand IGF1, which promoted phosphorylation of IGF1R. IGF1R phosphorylation was dependent upon activation of canonical NF-B signaling and was suppressed by IBaalpha; and a dominant negative form of TRAF6. Inhibition of IGF1R activation with two small molecule inhibitors, AG1024 and PPP, or with shRNA directed against IGF1R selectively reduced proliferation, foci formation, and Akt activation in LMP1-positive cells but did not impair LMP1-induced cell migration. Expression of constitutively active Akt rescued cell proliferation in the presence of IGF1R inhibitors. These findings suggest that LMP1-mediated activation of IGF1R contributes to the ability of LMP1 to transform epithelial cells.

    Importance: EBV is linked to the development of multiple cancers in both lymphoid and epithelial cells, including nasopharyngeal carcinoma. Nasopharyngeal carcinoma is a major cancer that develops in specific populations with nearly 80,000 new cases reported annually. LMP1 is consistently expressed in early lesions and continues to be detected within 50-80% of these cancers at later stages. It is therefore of paramount importance to understand the mechanisms through which LMP1 alters cell growth and contributes to tumorigenesis. This study is the first to determine that LMP1 activates the IGF1R tyrosine kinase by regulating expression of the ligand IGF1. Additionally, the data in this paper reveal that specific targeting of IGF1R selectively impacts LMP1-positive cells. These findings suggest that therapies directed against IGF1R may specifically impair the growth of EBV-infected cells.

  • The stress granule protein G3BP1 recruits PKR to promote multiple innate immune antiviral responses [PublishAheadOfPrint]

  • Stress granules (SGs) are cytoplasmic storage sites containing translationally silenced mRNPs that can be released to resume translation after stress subsides. We previously showed that poliovirus 3C proteinase cleaves the SG-nucleating protein G3BP1, blocking the ability of cells to form SGs late in infection. Many other viruses also target G3BP1 and inhibit SG formation but reasons why these functions evolved are unclear. Previously, we also showed a link between G3BP1-induced SGs and PKR-mediated translational control, but the mechanism of PKR interplay with SG and antiviral consequences are unknown. Here we show that G3BP1 exhibits antiviral activity against several enteroviruses whereas truncated G3BP1 that cannot form SGs does not. G3BP1-induced SGs are linked to activation of innate immune transcriptional responses through NFB and JNK. The G3BP1-induced SGs also recruit PKR and other antiviral proteins. We show that the PxxP domain within G3BP1 is essential for the recruitment of PKR to SGs, for eIF2aalpha; phosphorylation driven by PKR, and for nucleating SGs of normal composition. We also show that deletion of the PxxP domain in G3BP1 compromises its antiviral activity. These findings tie PKR activation to its recruitment to SGs by G3BP1 and indicate G3BP1 promotes innate immune responses at both transcriptional and translational levels and integrates cellular stress responses and innate immunity.

    IMPORTANCE Stress granules appear during virus infection and their importance is not well understood. Previously it was assumed that they were nonfunctional artifacts associated with cellular stress. PKR is a well-known antiviral protein, however its regulation in cells is not well understood. Our work links cellular stress granules with activation of PKR and other innate immune pathways through the activity of G3BP1, a critical stress granule component. The ability of stress granules and G3BP1 to activate PKR and other innate immune transcriptional responses indicates that G3BP1 is an antiviral protein. This work helps to refine a longstanding paradigm indicating stress granules are inert structures, and explains why G3BP1 is subverted by many viruses to promote a productive infection.

  • Ataxia telangiectasia mutated (ATM) kinase mediates NF{kappa}B Ser 276 phosphorylation and IFN expression via the IRF7-RIG-I amplification loop in paramyxovirus virus infection [PublishAheadOfPrint]

  • RSV is a primary etiological agent of childhood lower respiratory tract disease. Molecular patterns induced by active infection trigger a coordinated retinoic acid-inducible gene (RIG)-I-toll like receptor (TLR) signaling response to induce inflammatory cytokines and anti-viral mucosal interferons. Recently we discovered a nuclear oxidative stress-sensitive pathway mediated by the DNA-damage response protein, ATM, in cytokine-induced NFB/RelA Ser 276 phosphorylation. Here we, observe that ATM silencing results in enhanced ssRNA replication of RSV and Sendai Viruses, due to decreased expression and secretion of type nndash;I and nndash;III interferons (IFNs), despite maintenance of IRF3-dependent IFN-stimulated genes (ISGs). In addition to enhanced oxidative stress, RSV replication enhances foci of phosphorylated histone 2AX variant (H2AX), Ser 1981 phosphorylation of ATM, and IKK/NEMO-dependent ATM nuclear export indicating activation of the DNA-damage response. ATM deficient cells show defective RSV-induced mitogen and stress-activated kinase (MSK)-1 Ser 376 phosphorylation and reduced RelA Ser 276 phosphorylation, whose formation is required for IRF7 expression. We observe that RelA binds inducibly binds the native IFN regulatory factor (IRF)-7 promoter in an ATM-dependent manner, and IRF7 inducibly binds to the endogenous retinoic acid inducible gene (RIG)-I promoter. Ectopic IRF7 expression restores RIG-I expression and type I/III IFN expression in ATM-silenced cells. We conclude that paramyxoviruses trigger the DNA-damage response, a pathway required for MSK1 activation of phospho Ser 276 RelA formation to trigger the IRF7-RIG-I amplification loop necessary for mucosal IFN production. These data provide molecular pathogenesis for defects in cellular innate immunity patients with homozygous ATM mutations.

    IMPORTANCE RNA virus infections trigger cellular response pathways to limit spread to adjacent tissues. This "innate immune response" is mediated by germline-encoded pattern recognition receptors that trigger activation of two, largely independent, intracellular NF-B and IRF3 transcription factors. Downstream, expression of protective anti-viral interferons is amplified by positive feedback loops mediated by inducible interferon response factors (IRFs) and retinoic acid inducible gene (RIG-I). Our results indicate that a nuclear oxidative stress and DNA-damage sensing factor, ATM, is required to mediate a cross-talk pathway between NF-B and IRF7 through mediating phosphorylation of NF-B. Our studies provide further information about the defects in cellular- and innate immunity in patients with inherited ATM mutations.

  • Role of Cdk1 in the p53-independent abrogation of the postmitotic checkpoint by human papillomavirus E6 [PublishAheadOfPrint]

  • Specific types of human papillomavirus (HPV) are strongly associated with the development of cervical carcinoma. The HPV E6 oncoprotein from HPV degrades p53 and abrogates cell cycle checkpoints. Nonetheless, functional p53 has been observed in cervical cancer. We have previously identified a p53-independent function of E6 in attenuating the postmitotic G1-like checkpoint that can lead to polyploidy, an early event during cervical carcinogenesis that predisposes cells to aneuploidy. How E6 promotes cell cycle progression in the presence of p53 and its target, p21, remains a mystery. In this study, we examined the expression of cell cycle-related genes in cells expressing wild-type and the mutant E6 that is defective in p53 degradation but competent in abrogating the postmitotic checkpoint. Our results demonstrated an increase in the steady-state levels of G1- and G2-related cyclins/Cdks in E6-expressing keratinocytes. Interestingly, only Cdk1 remained active in E6 mutant-expressing cells while bypassing the postmitotic checkpoint. Furthermore, the down-regulation of Cdk1 impaired the ability of both wild-type and mutant E6 to induce polyploidy. Our study thus demonstrated an important role for Cdk1, which binds p21 with lower affinity than Cdk2, in abrogating the postmitotic checkpoint in E6-expressing cells. We further show that E2F1 is important for E6 to up-regulate Cdk1. Moreover, reduced nuclear p21 localization in the E6 mutant-expressing cells was observed. These findings shed light on the mechanisms by which HPV induces genomic instability and hold promise for the identification of drug targets.

    Importance HPV infection is strongly associated with the development of cervical carcinoma. HPV encodes an E6 oncoprotein that degrades the tumor suppressor p53 and abrogates cell cycle checkpoints. Nonetheless, functional p53 has been observed in cervical cancer. We have recently demonstrated a p53-independent abrogation of the postmitotic checkpoint by HPV E6 that induces polyploidy. However, the mechanism is not known. In this study, we provide evidence that Cdk1 plays an important role in this process. Previously, Cdk2 was thought to be essential for the G1/S transition, while Cdk1 only compensated its function in the absence of Cdk2. Our studies have demonstrated a novel role of Cdk1 at the postmitotic G1-like checkpoint in the presence of Cdk2. These findings shed light on the mechanisms by which HPV induces genomic instability and hold promise for the identification of drug targets.

  • Delayed inflammatory and cell death responses are associated with reduced pathogenicity in Lujo virus-infected cynomolgus macaques [PublishAheadOfPrint]

  • To identify host factors associated with arenavirus virulence, we used a cynomolgus macaque model to evaluate the pathogenesis of Lujo virus (LUJV), a recently emerged arenavirus that caused an outbreak of severe viral hemorrhagic fever in southern Africa. In contrast to human cases, LUJV caused mild, non-lethal illness in macaques. We then compared this to opposed clinical outcomes during arenavirus infection, specifically to samples obtained from macaques infected with three highly pathogenic lines of Lassa virus (LASV), the causative agent of Lassa fever (LF). We assessed gene expression in peripheral blood mononuclear cells (PBMC), and determined genes that significantly changed expression relative to uninfected animals over the course of infection. We detected a 72-hour delay in induction of host responses to infection during LUJV infection compared to the animals infected with LASV. This included genes associated with inflammatory and antiviral responses, and was particularly apparent among groups of genes promoting cell death. We also observed early differential expression of a subset of genes specific to LUJV infection that accounts for the delayed inflammatory response. Cell-type enrichment analysis suggested that host response induction delay and LUJV-specific profile may be due to a different proportion of natural killer cells responding in LUJV infection compared to the LASV-infected animals. Together, these data indicate that delayed pro-inflammatory and pro-apoptotic host responses to arenavirus infection could ameliorate disease severity. This conclusion provides insight into the cellular and molecular mechanisms of arenaviral hemorrhagic fever, as well as suggests potential strategies for therapeutic development.

    Importance Old World arenaviruses are significant human pathogens that are often associated with high mortality. However, mechanisms underlying disease severity and virulence in arenavirus hemorrhagic fever are largely unknown, particularly regarding host responses that contribute to pathogenicity. This study describes a comparison between Lujo and Lassa virus infection in cynomolgus macaques. Lujo-infected macaques developed only mild illness, while Lassa-infected macaques developed severe illness consistent with Lassa fever. We determined that mild disease is associated with a delay in host expression of genes linked to virulence, such as those causing inflammation and cell death, and with distinct cell types that may mediate this delay. This is the first study to associate the timing and directionality of gene expression with arenaviral pathogenicity and disease outcome, and evokes new potential approaches for developing effective therapeutics for treating these deadly emerging pathogens.

  • A Human herpesvirus 6A encoded miRNA: A role in viral lytic replication [PublishAheadOfPrint]

  • Human herpesvirus 6A (HHV-6A), a member of the betaherpesvirus family, is associated with several human diseases. Like all herpesviruses, HHV-6A establishes a life-long, latent infection in its host. Reactivation of HHV-6A is frequent within the immuno-suppressed and immuno-compromised populations and results in lytic viral replication within multiple organs, often leading to severe disease. MicroRNAs (miRNAs) are key regulators of multiple cellular processes that regulate the translation of specific transcripts. miRNAs encoded by herpesviruses play important roles in modulating the host cell, thereby facilitating a suitable environment for productive viral infection and/or latency. Currently there are approximately 150 known human herpesvirus-encoded miRNAs, although miRNA(s) encoded by HHV-6A have yet to be reported. We hypothesized that HHV-6A, like other members of the human herpesvirus family, encodes miRNAs, which function to promote viral infection. We utilized deep sequencing of small RNA species isolated from cells harboring HHV-6A to identify five novel small non-coding RNA species that originate from the viral genome, one of which has the characteristics of a viral miRNA. These RNAs are expressed during productive infection of either BAC derived virus in Jjhan cells or wild type HHV-6A U1102 strain infections of HSB2 cells and are associated with the RISC machinery. Growth analyses of mutant viruses that lack each individual miRNA revealed that a viral miRNA candidate (miR-U86) targets the HHV-6A IE gene U86, thereby regulating lytic replication. The identification and biological characterization of this HHV-6A specific miRNA is the first step to defining how the virus regulates its life cycle.

    IMPORTANCE A majority of the human population is infected with human herpesvirus 6A (HHV-6A), a betaherpesvirus family member. Infections usually occur in young children and upon resolution, the virus remains in a latent state within the host. Importantly, during times of weakened immune responses, the virus can reactivate and is correlated with significant disease states. Viruses encode many different types of factors that both undermine the host antiviral response as well as regulate viral replication including small RNA species called microRNAs (miRNAs). Here we report that HHV-6A encodes at least one miRNA, which we named miR-U86. We have characterized the requirement of this viral miRNA and its impact on the viral lifecycle and found that it functions to regulate a viral protein important for efficient viral replication. Our data suggest that viral miRNAs are important for HHV-6A and that they may serve as an important therapeutic target to inhibit the virus.

  • Mutations within the pathogenic region of HSV-1 gK signal sequences alter cell surface expression and neurovirulence [PublishAheadOfPrint]

  • To investigate the role of the signal sequences of HSV-1 gK on virus replication and viral pathogenesis, we constructed recombinant viruses with and without mutations within the signal sequences of gK. These recombinant viruses expressed two additional copies of the mutated (MgK) or native form (NgK) of gK gene in place of the latency associated transcript (LAT) with a myc epitope tag, to facilitate detection, at their 3rrsquo; end. The replication of MgK virus was similar to that of NgK both in vitro and in vivo as well as in the trigeminal ganglia (TG) of latently-infected mice. The levels of gB and gK transcripts in the corneas, TG, and brains of infected mice on days 3 and 5 post infection were markedly virus- and time-dependent as well as tissue-specific. Mutation in the signal sequence of gK in MgK virus blocked cell surface expression of gK-myc in rabbit skin (RS) cells, increased LD50 and decreased corneal scarring in ocularly infected mice as compared with the NgK or revertant (RgK) virus. MgK and NgK viruses and not the RgK virus had reduced extent of explant reactivation at the lower dose of ocular infection but not at the higher dose. However, time of reactivation was not affected by overexpression of the different forms of gK. Taken together, these results strongly suggest that the 8mer peptide (ITAYGLVL) within the signal sequence of gK promotes cell surface expression of gK in infected cells and ocular pathogenesis in infected mice.

    Significance In this study we have shown for the first time that mutations within the signal sequence of gK blocked cell surface expression of inserted recombinant gK in vitro. Furthermore, this blockage in cell surface expression was correlated with higher LD50 and lower CS in vivo. Thus, these studies point to a key role for the 8mer within the signal sequence of gK in HSV-1-induced pathogenicity.

  • Characterization of novel transcripts of human papillomavirus type 16 using CAGE technology [PublishAheadOfPrint]

  • We have performed cap-analysis gene expression (CAGE) sequencing to identify the regulatory networks that orchestrate genome-wide transcription in different grade HPV16-positive cervical cell lines: W12E, SiHa and CaSki. Additionally, a CIN1 lesion was assessed for identifying the transcriptome expression profile. Here we have precisely identified a novel antisense non-coding viral transcript in HPV16. Concluding, CAGE sequencing should pave the way for understanding a diversity of viral transcript expression.

  • N-Linked Glycosylation Protects Gammaretroviruses Against Deamination by APOBEC3 Proteins [PublishAheadOfPrint]

  • Retroviruses are pathogens with rapid infection cycles that can be a source of disease, genome instability and tumor development in their hosts. Host intrinsic restriction factors, such as APOBEC3 (A3) proteins, are constitutively expressed and dedicated to interfering with the replication cycle of retroviruses. To survive, propagate and persist, retroviruses must counteract these restriction factors, often by way of virus-encoded accessory proteins. Glyco-Gag, also called gPr80, is a gammaretrovirus-encoded protein that inhibits the antiretroviral activity of mouse A3 (mA3). Here we show that gPr80 exerts two distinct inhibitory effects on mA3: one that antagonizes deamination-independent restriction while the other inhibits its deaminase activity. More specifically, we find that the number of N-glycosylated residues in gPr80 inversely correlates with the sensitivity of a gammaretrovirus to deamination by mouse A3 and also, surprisingly, by human A3G. Finally, our work highlights that retroviruses which have successfully integrated into the mouse germline generally express a gPr80 with fewer glycosylation sites in contrast to exogenous retroviruses. This observation supports that modulation of A3 deamination intensity could be a desirable attribute for retroviruses to increase genetic diversification and avoid immune detection. Overall, we present here the first description of how gammaretroviruses employ post-translational modification to antagonize and modulate the activity of a host-encoded retroviral restriction factor.

    Importance APOBEC3 proteins are host factors that have a major role in protecting humans and other mammals against retroviruses. These enzymes hinder their replication and intensely mutate their DNA, thereby inactivating viral progeny and the spread of infection. Here we describe a new way that some retroviruses use to protect themselves against the mutator activity of APOBEC3 proteins. We show that gammaretroviruses expressing an accessory protein called glyco-Gag, or gPr80, use the host's posttranslational machinery, and more specifically N-linked glycosylation, as a way to modulate their sensitivity to mutations by APOBEC3 proteins. By carefully controlling the amount of mutations caused by APOBEC3 proteins, gammaretroviruses can find a balance that helps them evolve and persist.

  • microRNA-mediated transformation by the Kaposi's Sarcoma-associated herpesvirus Kaposin locus [PublishAheadOfPrint]

  • The human oncogenic Kaposi's Sarcoma-associated herpesvirus (KSHV) expresses a set of ~20 viral miRNAs. miR-K10a stands out among these miRNAs because its entire stem-loop precursor overlaps the coding sequence for the Kaposin (Kap) A/C proteins. The ectopic expression of KapA has been reported to lead to transformation of rodent fibroblasts. However, these experiments inadvertently also introduced miR-K10a, which raises the question whether the transforming activity of the locus could in fact be due to miR-K10a expression. To answer this question, we have uncoupled miR-K10a and KapA expression. Our experiments revealed that miR-K10a alone transformed cells with similar efficiency as its co-expression with KapA. Maintenance of the transformed phenotype was conditional upon continued miR-K10a but not KapA protein expression, consistent with its dependence on miRNA-mediated changes in gene expression. Importantly, miR-K10a taps into an evolutionary conserved network of miR-142-3p targets, several of which are expressed in 3T3 cells and are also known inhibitors of cellular transformation. In summary, our studies of miR-K10a serve as an example of an unsuspected function of a miRNA whose precursor is embedded within a coding transcript. In addition, our identification of conserved miR-K10a targets that limit transformation will point the way to a better understanding of the role of this miRNA in KSHV-associated tumors.

    Importance Kaposi's Sarcoma-associated herpesvirus (KSHV) is a human tumor virus. The viral Kaposin locus has known oncogenic potential, which has previously been ascribed to the encoded KapA protein. Here we show that the virally encoded miR-K10a miRNA, whose precursor overlaps the KapA coding region, may account for the oncogenic properties of this locus. Our data suggest that miR-K10a mimics the cellular miRNA miR-142-3p and thereby represses several known inhibitors of oncogenic transformation. Our work demonstrates that functional properties ascribed to a coding region may in fact be carried out by an embedded non-coding element and sheds light on the functions of viral miR-K10a.

  • The progressive adaptation of a Georgian isolate of African swine fever virus to Vero cells leads to a gradual attenuation of virulence in swine corresponding with major modifications of the viral genome. [PublishAheadOfPrint]

  • African swine fever virus (ASFV) causes a contagious and often lethal disease of feral and domestic swine. Experimental vaccines derived from naturally occurring, genetically modified or cell culture-adapted ASFV have been evaluated but no commercial vaccine is available to control African Swine Fever (ASF). We report here the genotypic and phenotypic analysis of viruses obtained at different passages during the process of adaptation of a virulent ASFV field isolate from the Republic of Georgia (ASFV-G) to grow in cultured cell lines. ASFV-G virus was successively passaged 110 times in Vero cells. Viruses obtained at passages 30, 60, 80 and 110 were evaluated in vitro for the ability to replicate in Vero cells and primary swine macrophages cultures and in vivo for assessing virulence in swine. Replication of ASFV-G in Vero cells increased with successive passages, corresponding with a decreased replication in primary swine macrophages cultures. In vivo, progressive loss of virus virulence was observed with increased passages in Vero cells and complete attenuation of ASFV-G was observed at passage 110. Infection of swine with the fully attenuated virus did not confer protection against challenge with virulent parental ASFV-G. Full-length sequence analysis of each of these viruses revealed significant deletions that gradually accumulate into specific areas at the right and left variable ends of the genome. Mutations that result in amino acid substitutions and frame-shift mutations were also observed though in a rather limited number of genes. The potential importance of these genetic changes in virus adaptation/attenuation is discussed.

    Importance The main problem for controlling ASF is the lack of vaccines. Attempts to produce vaccines by adaptation of ASFV to cultured cell lines have been made. These attempts led to the production of attenuated viruses that conferred only homologous protection. Specifics regarding adaptation of these isolates to cell cultures have been insufficiently described. Details like passage numbers required to obtain attenuated viruses, genetic modifications introduced into the virus genomes along passages, and the extent of attenuation and induced protective efficacy are not readily available. In this report we have assessed those changes that lead to decreased growth in swine macrophages and to attenuation in swine. Loss of virulence, probably associated with limited replication in vivo may lead to the lack of protective immunity in swine observed after challenge. This report provides valuable information that can be used towards the understanding of ASFV gene function, virus attenuation, and protection against infection.

  • Functional characterization of nuclear trafficking signals in Pseudorabies Virus pUL31 [PublishAheadOfPrint]

  • The herpesviral nuclear egress complex (NEC), consisting of pUL31 and pUL34 homologs, mediates efficient translocation of newly synthesized capsids from the nucleus to the cytosol. The tail-anchored membrane protein pUL34 is autonomously targeted to the nuclear envelope, while pUL31 is recruited to the inner nuclear membrane (INM) by interaction with pUL34. A nuclear localization signal (NLS) in several pUL31 homologs suggests importin-mediated translocation of the protein. Here we demonstrate that deletion or mutation of the NLS in pseudorabies virus (PrV) pUL31 resulted in an exclusive cytosolic localization indicating active nuclear export. Deletion or mutation of a predicted nuclear export signal (NES) in mutants lacking a functional NLS resulted in diffuse nuclear and cytosolic localization indicating that both signals are functional. pUL31 molecules lacking the complete NLS or NES were not recruited to the INM by pUL34, while the site-specifically mutated proteins formed the NEC and partially complemented the defect of the UL31-deletion mutant. Our data demonstrate that the N-terminus of pUL31 encompassing the NLS is required for efficient nuclear targeting but not for pUL34 interaction, while the C-terminus containing the NES, but not necessarily the NES itself, is required for complex formation and efficient budding of viral capsids at the INM. Moreover, pUL31-NLS displayed a dominant negative effect on PrV-wild type replication probably by diverting pUL34 to cytoplasmic membranes.

    Importance: The molecular details of nuclear egress of herpesvirus capsids are still enigmatic. Although the key players, homologs of herpes simplex virus pUL34 and pUL31, which interact and form the heterodimeric nuclear egress complex, are well known, the molecular details of this interaction and the successive budding, vesicle formation and scission from the inner nuclear membrane as well as capsid release into the cytoplasm remain largely obscure. Here we show that classical cellular targeting signals for nuclear import and export are important for proper localization and function of the NEC, thus regulating herpesvirus nuclear egress.

  • Lentiviral Nef proteins manipulate T cells in a subset-specific manner [PublishAheadOfPrint]

  • The role of the accessory viral Nef protein as a multi-functional manipulator of the host cell that is required for effective replication of HIV and SIV in vivo is well established. It is unknown, however, whether Nef manipulates all or just specific subsets of CD4+ T cells that are the main targets of virus infection and differ substantially in their state of activation and importance for a functional immune system. Here, we analyzed the effect of Nef proteins differing in their TCR-CD3 downmodulation function in HIV infected human lymphoid aggregate cultures and peripheral blood mononuclear cells. We found that Nef efficiently downmodulates TCR-CD3 in naiiuml;ve and memory CD4+ T cells and protects the latter against apoptosis. In contrast, highly proliferative CD45RA+CD45RO+CD4+ T cells were main producers of infectious virus but largely refractory to TCR-CD3 downmodulation. Such T cell subset-specific differences were also observed for Nef-mediated modulation of CD4 but not for enhancement of virion infectivity. Our results indicate that Nef predominantly modulates surface receptors on CD4+ T cell subsets that are not already fully permissive for viral replication. As a consequence, Nef-mediated downmodulation of TCR-CD3 that distinguishes most primate lentiviruses from HIV-1 and its vpu containing simian precursors may promote a selective preservation of central memory CD4+ T cells that are critical for the maintenance of a functional immune system.

    IMPORTANCE The Nef proteins of human and simian immunodeficiency viruses manipulate infected CD4+ T cells in multiple ways to promote viral replication and immune evasion in vivo. Here, we show that some effects of Nef are subset-specific. Downmodulation of CD4 and TCR-CD3 is highly effective in central memory CD4+ T cells and the latter Nef function protects this T cell subset against apoptosis. In contrast, highly activated/proliferating CD4+ T cells are largely refractory to receptor downmodulation but main producers of infectious HIV-1. Nef-mediated enhancement of virion infectivity, however, was observed in all T cell subsets examined. Our results provide new insights into how primate lentiviruses manipulate their target cells and suggest that the TCR-CD3 downmodulation function of Nef may promote a selective preservation of memory CD4+ T cells that are critical for immune function but has little effect on activated/proliferating CD4+T cells that are main targets for viral replication.

  • HSV-1 gN partners with gM to modulate the viral fusion machinery [PublishAheadOfPrint]

  • Herpes simplex virus type 1 (HSV-1) capsids are assembled in the nucleus, where they incorporate the viral genome. They then transit through the two nuclear membranes and are wrapped by a host-derived envelope. In the process, several HSV-1 proteins are targeted to the nuclear membranes but their roles in viral nuclear egress are unclear. Among them, glycoprotein M (gM), a known modulator of virus-induced membrane fusion, is distributed on both the inner and outer nuclear membranes at the early stages of the infection, when no other viral glycoproteins are yet present there. Later on, it is found on perinuclear virions and ultimately redirected to the TGN, where it cycles with the cell surface. In contrast, transfected gM is only found at the TGN and cell surface, hinting at an interaction with other viral proteins. Interestingly, many herpesvirus gM analogs interact with their gN counterparts, which typically alters their intracellular localization. To better understand how HSV-1 gM localization is regulated, we evaluated its ability to bind gN and discovered it does so in both transfected and infected cells, an interaction strongly weakened by the deletion of the gM amino terminus. Functionally, while gN had no impact on gM localization, gM redirected gN from the ER to the TGN. Most excitingly, gN overexpression stimulated the formation of syncytia in the context of an infection by a non-syncytial strain, hinting that gM and gN not only physically but also functionally interact and that gN modulates gM's activity on membrane fusion.

    Importance HSV-1 gM is an important modulator of virally-induced cell-cell fusion and viral entry, a process that is likely finely modulated in time and space. Up to now, little was known of the proteins that regulate gM's activity. In parallel, gM is found in various intracellular locations at different moments ranging from nuclear membranes, perinuclear virions, the TGN, cell surface and on mature extracellular virions. In transfected cells, it is however only found on the TGN and cell surface, hinting that its localization is modulated by other viral proteins. The present study identifies HSV-1 gN as a binding partner for gM, in agreement with their analogs in other herpesviruses, but most excitingly shows that gN modulates gM's impact on HSV-1 induced membrane fusion. These findings open up new research avenues on the viral fusion machinery.

  • Epstein-Barr virus Type 2 latently infects T-cells inducing an atypical activation characterized by expression of lymphotactic cytokines [PublishAheadOfPrint]

  • Epstein-Barr virus (EBV) is a well-established B-cell tropic virus associated with various lymphoproliferative diseases of both B-cell and non-B-cell origin. Indeed, EBV is associated with a number of T-cell lymphomas; however, in vitro studies utilizing prototypical EBV type 1 (EBV-1) laboratory strains have generally failed to readily infect mature T-cells in culture. The difficulties performing in vitro T-cell experiments have left questions regarding the role of EBV in the pathogenesis of EBVnndash;positive T-cell lymphoproliferative diseases largely unresolved. We report here that the EBV type 2 (EBV-2) strain displays a unique cell tropism for T-cells. In remarkable contrast to EBV-1, EBV-2 readily infects primary T-cells in vitro, demonstrating a propensity for CD8+ T-cells. EBV-2 infection of purified T-cells results in expression of latency genes and ultimately leads to T-cell activation, substantial proliferation, and profound alteration of cytokine expression. The pattern of cytokine production is strikingly skewed towards chemokines with roles in lymphocyte migration, demonstrating that EBV-2 has the ability to modulate normal T-cell process. Collectively, these novel findings identify a previously unknown cell population potentially utilized by EBV-2 to establish latency and lay the foundation for further studies to elucidate the role of EBV in the pathogenesis of T-cell lymphoproliferative diseases.

    Importance: The ability of EBV to infect T-cells is made apparent by its association with a variety of T-cell lymphoproliferative disorders. However studies to elucidate the pathogenic role of EBV in these diseases have been limited by the inability to conduct in vitro T-cell infection experiments. Here we report that EBV-2 isolates, compromised in the capacity to immortalize B-cells, infect CD3+ T-cells ex vivo and propose a working model of EBV-2 persistence where alteration of T-cell functions resulting from EBV-2 infection enhances the establishment of latency in B-cells. If indeed EBV-2 utilizes T-cells to establish a persistent infection this could explain one mechanism for the association of EBV with T-cell lymphomas. The novel finding that EBV-2 infects T-cells in culture will provide a model to understand the role EBV plays in the development of T-cell lymphomas.

  • Phosphatidylserine-Specific Phospholipase A1 is involved in Hepatitis C Virus Assembly through NS2 Complex Formation [PublishAheadOfPrint]

  • Several members of the phospholipase family have been reported to be involved in hepatitis C virus (HCV) replication. Here, we identified another phospholipase, phosphatidylserine-specific phospholipase A1 (PLA1A), as a host factor involved in HCV assembly. PLA1A was upregulated by HCV infection; and PLA1A knockdown significantly reduced J399EM (genotype 2a) HCV propagation at the assembly but not the entry, RNA replication, and protein translation steps of the life cycle. Protein localization and interaction analysis further reveal a role of PLA1A in the interaction of NS2-E2 and NS2-NS5A, as the formation of the NS2-E2 and NS2-NS5A complexes was weakened in the absence of PLA1A. In addition, PLA1A stabilized the NS2/NS5A-dotted structure during infection. These data suggest that PLA1A plays an important role of bridging the membrane-associated NS2-E2 complex and the NS5A-associated replication complex via its interaction with E2, NS2 and NS5A, which leads to a coordinating interaction between the structural and non-structural proteins and facilitates viral assembly.

    Importance Hepatitis C virus (HCV) genomic replication is driven by the replication complex and occurs at the membranous web, while the lipid droplet is the organelle in which virion assembly is initiated. In this study, we identified phosphatidylserine-specific phospholipase A1 (PLA1A), a member of phospholipase A 1 family, as a novel host factor involved in the assembly process of HCV. PLA1A which is induced by HCV infection at late infection stage interacts with HCV E2, NS2 and NS5A proteins and enhance and stabilize the NS2-E2 and NS2-NS5A complex formation, which is essential for viral assembly. Thus, PLA1A is an important host factor which is involved in the initiation of the viral assembly in close proximity to Core-decorated lipid droplet through bringing together the HCV replication complex and envelope complex.

  • Expanding the Role of the Virome: Commensalism in the Gut [PublishAheadOfPrint]

  • Viruses affect host physiology beyond causing acute disease, thereby giving rise to the concept that the virome is a component of the microbiome. However, the role of the enteric virome is understudied relative to the fast pace research examining commensal bacteria in the intestine. In this Gem article, I will discuss our recent work on murine norovirus indicating that an animal virus in the intestine can provide many of the same signals to the host that have been attributed to commensal bacteria. Our findings suggest that the surge in microbiome research should incorporate examination of the enteric virome.

  • Prion Infection of Mouse Brain Reveals Multiple New Upregulated Genes Involved in Neuroinflammation or Signal Transduction [PublishAheadOfPrint]

  • Gliosis is often a pre-clinical pathological finding in neurodegenerative diseases including prion diseases, but the mechanisms facilitating gliosis and neuronal damage in these diseases are not understood. To expand our knowledge of the neuroinflammatory response in prion diseases, we assessed the expression of key genes and proteins involved in the inflammatory response and signal transduction in mouse brain at various times after scrapie infection. In brains of scrapie-infected mice at pre- and post-clinical stages, we identified 15 previously unreported differentially expressed genes related to inflammation or activation of the STAT signal transduction pathway. The majority of differentially expressed genes increased with time post-infection. In quantitative immunoblotting experiments of STAT proteins, STAT1aalpha;, phosphorylated-STAT1aalpha; (pSTAT1aalpha;), and pSTAT3 were increased between 94 and 131 dpi in brain of mice infected with strain 22L. Furthermore, a select group of STAT-associated genes was increased pre-clinically during scrapie infection, suggesting early activation of the STAT signal transduction pathway. Comparison of inflammatory markers between mice infected with scrapie strains 22L and RML indicated that the inflammatory responses and gene expression profiles in the brain were strikingly similar, even though these scrapie strains infect different brain regions. The endogenous IL-1 receptor antagonist IL-1Ra, an inflammatory marker, was newly identified as increased pre-clinically in our model, and therefore might influence scrapie pathogenesis in vivo. However, in IL-1Ra-deficient or overexpressor transgenic mice inoculated with scrapie, neither loss nor overexpression of IL-1Ra demonstrated any observable effect on gliosis, PrPres formation, disease tempo, pathology, or expression of the inflammatory genes analyzed.

    Importance: Prion infection leads to PrPres deposition, gliosis, and neuroinflammation in the CNS before signs of clinical illness. Using a scrapie mouse model of prion disease to assess various time points post-inoculation, we identified 15 unreported genes increased in the brain of scrapie-infected mice and associated with inflammation and/or JAK-STAT activation. Comparison of mice infected with two scrapie strains (22L and RML), which have dissimilar neuropathologies, indicated that the inflammatory responses and gene expression profiles in the brain were similar. Genes increased prior to clinical signs might be involved in controlling scrapie infection or in facilitating damage to host tissues. We tested the possible role of the endogenous IL-1 receptor antagonist IL-1Ra, which was increased at 70 dpi. In scrapie-infected mice deficient in or overexpressing IL-1Ra, there was no observable effect on gliosis, PrPres formation, disease tempo, pathology, or expression of inflammatory genes analyzed.

  • Structural Analysis of a Rabbit Hemorrhagic Disease Virus Binding to Histo-Blood Group Antigens [PublishAheadOfPrint]

  • Rabbit hemorrhagic disease virus (RHDV) is a member of the Caliciviridae family (Lagovirus genus). RHDV is highly contagious and attaches to epithelial cells in the digestive or respiratory tract, leading to massive lesions with high mortality rates. A new variant of RHDV (termed RHDVb) has recently emerged and previously vaccinated rabbits appear to have little protection against this new strain. Similar to human norovirus (Caliciviridae, Norovirus genus), RHDV binds histo-blood group antigens (HBGAs) and this is thought to be important for infection. Here, we report the HBGA binding site on the RHDVb capsid-protruding domain (P domain) using X-ray crystallography. The HBGA binding pocket was located in a negatively charged patch on the side of the P domain and at a dimeric interface. Residues from both monomers contributed to the HBGA binding and involved a network of direct hydrogen bonds and water-mediated interactions. An amino acid sequence alignment of different RHDV strains indicated that the residues directly interacting with the ABH-fucose of the HBGAs (Asp472, Asn474, and Ser479) were highly conserved. This result suggested that different RHDV strains could also bind HBGAs at the equivalent pocket. Moreover, several HBGA binding characteristics between RHDVb and human genogroup II norovirus were similar, which indicated a possible convergent evolution of HBGA binding interactions. Further structural studies with other RHDV strains are needed in order to better understand the HBGA binding mechanisms among the diverse RHDV strains.

    IMPORTANCE We identified, for the first time, the HBGA binding site on an RHDVb P domain using X-ray crystallography. Our results showed that RHDVb and human genogroup II noroviruses had similar HBGA binding interactions. Recently, it was discovered that synthetic HBGAs or HBGA-expressing enteric bacteria could enhance human genogroup II norovirus infection in B cells. Considering that RHDVb and genogroup II norovirus similarly interacted with HBGAs, it may be possible that a comparable cell culture system could also work with RHDVb. Taken together, these new findings will extend our understanding of calicivirus HBGA interactions and may help to elucidate the specific roles of HBGAs in calicivirus infections.

  • Analysis of the early immune response to infection by Infectious Bursal Disease Virus (IBDV) in chickens differing in their resistance to the disease [PublishAheadOfPrint]

  • Chicken whole genome gene expression arrays were used to analyse the host response to infection by Infectious Bursal Disease Virus (IBDV). Spleen and bursal tissue were examined from control and infected birds at 2, 3 and 4 days post-infection from two lines that differ in their resistance to IBDV infection. The host response was evaluated over this period and differences between susceptible and resistant chicken lines were examined. Anti-viral genes, including IFNA, IFNG, MX1, IFITM1, IFITM3 and IFITM5 were up-regulated in response to infection. Evaluation of this gene expression data has allowed us to predicted several genes as candidates for involvement in resistance to IBDV.

    IMPORTANCE Infectious bursal disease (IBD) is of economic importance to the poultry industry and thus is also important for food security. Vaccines are available but field strains of the virus are of increasing virulence. There is thus an urgent need to explore new control solutions, one of which would be to breed birds with greater resistance to IBD. A goal which is perhaps uniquely achievable with poultry, of all farm animal species, as the genetics of 85% of the 60 billion chickens produced worldwide each year is under the control of essentially two breeding companies. This is the most comprehensive study to try to identify global transcriptomic differences in the target organ of the virus between chicken lines that differ in resistance, and to predict candidate resistance genes.

  • Hepatitis B virus polymerase disrupts K63-linked ubiquitination of STING to block innate cytosolic DNA-sensing pathways [PublishAheadOfPrint]

  • The cellular innate immune system recognizing pathogen infection is essential for host defense against viruses. In parallel, viruses have developed a variety of strategies to evade the innate immunity. The hepatitis B virus (HBV), a DNA virus that causes chronic hepatitis, has been shown to inhibit the RNA helicase RIG-I-mediated interferon (IFN) induction. However, it is still unknown whether HBV could affect the host DNA-sensing pathways. Here we report that in transient HBV-transfected Huh7 cells, HBV stably producing cell line HepAD38, and HBV-infected HepaRG cells and primary human hepatocytes, HBV markedly interfered with IFN-bbeta; induction and antiviral immunity mediated by STING, which has been identified as a central factor in foreign DNA recognition and antiviral innate immunity. Screening analysis demonstrated that the viral polymerase (Pol), but not other HBV-encoded proteins, was able to inhibit STING-stimulated interferon regulatory factor 3 (IRF3) activation and IFN-bbeta; induction. Moreover, the reverse transcriptase (RT) and the ribonuclease H (RH) domains of Pol were identified to be responsible for the inhibitory effects. Furthermore, Pol was shown to physically associate with STING and dramatically decrease the K63-linked polyubiquitination of STING via its RT domain without altering the expression level of STING. Taken together, these observations suggest that besides its inherent catalytic function, Pol has a role in suppression of IFN-bbeta; production by direct interaction with STING and subsequent disruption of its K63-linked ubiquitination, providing a new mechanism for HBV to counteract the innate DNA-sensing pathways.

    Importance Although whether and how HBV infection induces the innate immune responses is still controversial, it becomes increasingly clear that HBV has developed strategies to counteract the pattern recognition receptors-mediated signaling pathways. Previous studies have shown that type I IFN induction activated by the host RNA sensors could be inhibited by HBV. However, it remains unknown whether HBV as a DNA virus utilizes evasion mechanisms against the foreign DNA-elicited antiviral signalings. In recent years, the cytosolic DNA sensor and key adaptor STING has been demonstrated to be essential in multiple foreign DNA-elicited innate immune signalings. Here, for the first time, we reported STING as a new target of HBV to antagonize the IFN induction and identified the viral polymerase responsible for the inhibitory effect, thus providing an additional molecular mechanism by which HBV evades the innate immunity and implying HBV polymerase is a multifunctional immunomodulatory protein besides its inherent catalytic function.

  • Kaposi's Sarcoma-Associated Herpesvirus Induces Nrf2 Activation in Latently-Infected Endothelial Cells Through SQSTM1 Phosphorylation and Interaction with Polyubiquitinated Keap1 [PublishAheadOfPrint]

  • Nuclear factor erythroid 2-related factor 2 (Nrf2), the cellular master regulator of the anti-oxidant response, dissociates from its inhibitor Keap1 when activated by stress signals and participates in the pathogenesis of viral infections and tumorigenesis. Early during de novo infection of endothelial cells, KSHV induces Nrf2 through an intricate mechanism involving reactive oxygen species and prostaglandin E2 (PGE2). When we investigated the Nrf2 activity during latent KSHV infection, we observed increased nuclear serine-40-phosphorylated Nrf2 in human KS lesions compared to healthy tissues. Using KSHV long-term-infected endothelial (LTC) cells as a cellular model for KS, we demonstrate that KSHV infection induces Nrf2 constitutively by extending its half-life, increasing its phosphorylation by protein kinase C (PKC) via the infection-induced COX-2/PGE2 axis, and inducing its nuclear localization. Nrf2 knockdown in LTC cells decreased expression of anti-oxidant genes and genes involved in KS pathogenesis such as NQO1, GCSH, xCT, IL6 and VEGF-A. Nrf2 activation was independent of oxidative stress but dependent on the autophagic protein sequestosome-1 (SQSTM1; p62). SQSTM1 levels were elevated in LTC cells, a consequence of protein accumulation due to decreased autophagy and Nrf2-mediated transcriptional activation. SQSTM1 was phosphorylated on serine-351 and -403, while Keap1 was polyubiquitinated with lysine-63-ubiquitin chains, modifications known to increase their mutual affinity and interaction, leading to Keap1 degradation and Nrf2 activation. The latent KSHV protein vFLIP increased SQSTM1 expression and activated Nrf2. Collectively, these results demonstrate that KSHV induces SQSTM1 to constitutively activate Nrf2 involved in the regulation of genes participating in KSHV oncogenesis.

    IMPORTANCE The transcription factor Nrf2 is activated by stress signals, including viral infection, and responds by activating the transcription of cytoprotective genes. Recently, Nrf2 has been implicated in oncogenesis and was shown to be activated during de novo KSHV infection of endothelial cells through ROS-dependent pathways. The present study was undertaken to determine the mechanism of Nrf2 activation during prolonged latent infection of endothelial cells, using an endothelial cell-line latently-infected with KSHV. We show that Nrf2 activation was elevated in KSHV latently-infected endothelial cells independently of oxidative stress, but dependent on the autophagic protein sequestosome-1 (SQSTM1), which was involved in the degradation of the Nrf2 inhibitor Keap1. Furthermore, our results indicated that the KSHV latent protein vFLIP participates in Nrf2 activation. This study suggests that KSHV hijacks the host's autophagic protein SQSTM1 to induce Nrf2 activation, thereby manipulating the infected host gene regulation to promote KS pathogenesis.

  • Virion-independent transfer of replication competent HCV RNA between permissive cells [PublishAheadOfPrint]

  • In this study we show that replication-competent subgenomic HCV RNA can be transferred to permissive Huh7 cells leading to the establishment of viral RNA replication. Further, we show that these events are mediated by exosomes rather than infectious virus particles. If similar events occur in vivo, this could represent a novel, albeit inefficient, mechanism of viral spread and immune escape.

  • Identification and functional comparison of 7TM G protein-coupled BILF1 receptors in recently discovered non-human primate lymphocryptoviruses [PublishAheadOfPrint]

  • Co-evolution of herpesviruses with their respective host has resulted in a delicate balance between virus-encoded immune evasion mechanisms and host anti-viral immunity. BILF1 encoded by the human Epstein-Barr virus (EBV) is a 7 transmembrane (7TM) viral GPCR -like protein with multiple immunomodulatory functions, including: attenuation of PKR phosphorylation, activation of G protein signaling and down-regulation of MHC class I surface expression. Herein, we explore the evolutionary and functional relationships between BILF1 receptor family members from EBV and 12 previously uncharacterized non-human primate (NHP) LCVs. Phylogenetic analysis defined 3 BILF1 clades, corresponding to LCVs of New World monkeys (Clade A), or Old World monkeys and great apes (Clade B and C). Common functional properties were suggested by a high degree of sequence conservation in functionally important regions of the BILF1 molecules. A subset of BILF1 receptors from EBV and LCVs from NHPs (chimpanzee, orang-utan, marmoset, and siamang) were selected for multi-functional analysis. All receptors exhibited constitutive signaling activity via G protein Gaalpha;i and induced activation of the NF-kB transcription factor. In contrast, only 3 of 5 were able to activate NFAT; BILF1 from chimpanzee and orang-utan were unable to activate NFAT. Similarly, although all receptors were internalized, BILF1 from chimpanzee and orang-utan displayed an altered cellular localization pattern with predominant cell surface expression. This study shows how biochemical characterization of functionally important orthologous viral proteins can be used to complement phylogenetic analysis to provide further insight into diverse microbial evolutionary relationships and immune evasion function.

    Importance Epstein-Barr virus (EBV) known as an oncovirus, is the only human herpesvirus in the genus Lymphocryptovirus (LCV). EBV uses multiple strategies to hijack infected host cells, establish persistent infection in B-cells and evade antiviral immune responses. As part of EBV's immune evasion strategy, the virus encodes a multifunctional 7 transmembrane (7TM) G protein-coupled receptor (GPCR), EBV-BILF1. In addition to multiple immune evasion-associated functions, EBV-BILF1 has transforming properties, which are linked to its high constitutive activity. We identified BILF1 receptor orthologues in 12 previously uncharacterized LCVs from non-human primates (NHP) of Old and New World origin. As 7TM receptors are excellent drug targets, our unique insight into the molecular mechanism of action of the BILF1 family and into the evolution of primate LCVs may enable validation of EBV-BILF1 as a drug target for EBV-mediated diseases, as well as facilitating the design of drugs targeting EBV-BILF1.

  • Adaptation of Pandemic H2N2 Influenza A Viruses in Humans [PublishAheadOfPrint]

  • The 1957 A/H2N2 influenza virus caused an estimated 2 million fatalities during the pandemic. Since viruses of the H2 subtype continue to infect avian species and pigs, the threat of reintroduction into humans remains. To determine factors involved in the zoonotic origin of the 1957 pandemic we performed analyses on genetic sequences of 175 newly sequenced human and avian H2N2 virus isolates and all publicly available influenza virus genomes.

  • MCL1 Enhances the Survival of CD8+ Memory T Cells after Viral Infection [PublishAheadOfPrint]

  • Viral infection results in the generation of massive numbers of activated effector CD8+ T cells that recognize viral components. Most of these are short-lived effector T cells (SLECs) that die after clearance of the virus. However, a small proportion of this population survives and forms antigen-specific memory precursor effector cells (MPECs), which ultimately develop into memory cells. These can participate in a recall response upon re-exposure to antigen even at protracted times post-infection. Here, anti-apoptotic myeloid cell leukemia 1 (MCL1) was found to prolong survival upon T cell stimulation, and mice expressing human MCL1 as a transgene exhibited a skewing in the proportion of CD8+ T cells, away from SLECs toward MPECs, during acute vaccinia virus infection. A higher frequency and total number of antigen-specific CD8+ T cells were observed in MCL1 transgenic mice. These findings show that MCL1 can shape the makeup of the CD8+ T cell response, promoting the formation of long-term memory.

    IMPORTANCE During an immune response to a virus, CD8+ T cells kill cells infected by the virus, and most die when the infection resolves. However a small proportion of cells survives and differentiates into long-lived memory cells that confer protection from re-infection by the same virus. This report shows that transgenic expression of an MCL1 protein enhances survival of memory CD8+ T cells following infection with vaccinia virus. This is important because it shows that MCL1 expression may be an important determinant for the formation of long-term CD8+ T cell memory.

  • Timing of galectin-1 exposure differentially modulates Nipah virus entry and syncytia formation in endothelial cells [PublishAheadOfPrint]

  • Nipah virus (NiV) is a deadly emerging enveloped paramyxovirus that primarily targets human endothelial cells. Endothelial cells express the innate immune effector galectin-1 that we have previously shown can bind to specific N-glycans on the NiV envelope fusion glycoprotein (F). NiV-F mediates fusion of infected endothelial cells into syncytia, resulting in endothelial disruption and hemorrhage. Galectin-1 is an endogenous carbohydrate binding protein that binds to specific glycans on NiV-F to reduce endothelial cell fusion, an effect that may reduce pathophysiologic sequelae of NiV infection. However, galectins play multiple roles in regulating host-pathogen interactions; for example, galectins can promote attachment of HIV to T cells and macrophages and attachment of HSV-1 to keratinocytes, but can also inhibit influenza entry into airway epithelial cells. Using live Nipah virus, in the present study, we demonstrate that galectin-1 can enhance NiV attachment to and infection of primary human endothelial cells by bridging glycans on the viral envelope to host cell glycoproteins. In order to exhibit an enhancing effect, galectin-1 must be present during the initial phase of virus attachment; in contrast, addition of galectin-1 post-infection results in reduced production of progeny virus and syncytia formation. Thus, galectin-1 can have dual and opposing effects on NiV infection of human endothelial cells. While various roles for galectin family members in microbial-host interactions have been described, we report opposing effects of the same galectin family member on a specific virus, with the timing of exposure during the viral life cycle determining the outcome.

    Importance Nipah virus is an emerging pathogen that targets endothelial cells lining blood vessels; the high mortality rate (up to 70%) in Nipah virus infections results from destruction of these cells and resulting catastrophic hemorrhage. Host factors that promote or prevent Nipah virus infection are not well understood. Endogenous human lectins, such as galectin-1, can function as pattern recognition receptors to reduce infection and initiate immune responses; however, lectins can also be exploited by microbes to enhance infection of host cells. We found that galectin-1, which is made by inflamed endothelial cells, can both promote Nipah virus infection of endothelial cells by "bridging" the virus to the cell, as well as reduce production of progeny virus and reduce endothelial cell fusion and damage, depending on timing of galectin-1 exposure. This is the first report of spatiotemporal opposing effects of a host lectin for a virus in one type of host cell.

  • The nucleoprotein of newly emerged H7N9 influenza A virus harbors a unique motif conferring resistance to antiviral human MxA [PublishAheadOfPrint]

  • Interferon-induced Mx proteins show strong antiviral activity against influenza A viruses. We recently demonstrated that the viral nucleoprotein (NP) determines resistance of seasonal and pandemic human influenza viruses to Mx, while avian isolates retain Mx sensitivity. We identified a surface exposed cluster of amino acids in NP of pandemic A/BM/1/1918 (H1N1) comprising isoleucine-100, proline-283 and tyrosine-313 that is essential for reduced Mx sensitivity in cell culture and in vivo. This cluster has been maintained in all descendant seasonal strains, including A/PR/8/34 (PR/8). Accordingly, two substitutions in NP of PR/8(mut) to the Mx-sensitive amino acids (P283L and Y313F) led to attenuation in Mx1-positive mice. Serial lung passages of PR/8(mut) in Mx1 mice resulted in a single exchange of tyrosine to asparagine at position 52 in NP (in close proximity to the 100/283/313-cluster), which partially compensates loss of Mx resistance in PR/8(mut). Intriguingly, NP of the newly emerged avian-origin H7N9 virus also contains an asparagine at position 52 and shows reduced Mx sensitivity. N52Y substitution in NP results in increased sensitivity of the H7N9 virus to human Mx, indicating that this residue is a determinant of Mx resistance in mammals. Our data strengthen the hypothesis that the human Mx protein represents a potent barrier against zoonotic transmission of avian influenza viruses. However, the H7N9 viruses overcome this restriction by harboring a NP that is less sensitive to Mx-mediated host defence. This might contribute to zoonotic transmission of H7N9 and to the severe to fatal outcome of H7N9 infections in humans.

    Importance: The natural host of influenza A viruses (IAV) are aquatic birds. Ocassionally, these viruses cross the species barrier, as in early 2013, when an avian H7N9 virus infected humans in China. Since then multiple transmissions of H7N9 viruses to humans occurred, leaving experts puzzled about molecular causes for such efficient crossing of the species barrier as compared to other avian influenza viruses. Mx proteins are known restriction factors preventing influenza virus replication. Unfortunately some viruses (e.g. human IAV) have developed some resistance, which is associated with specific amino acids in their nucleoprotein, the target of Mx function. Here we demonstrate that the novel H7N9 bird IAV already carries a nucleoprotein that overcomes the inhibition of viral replication by human MxA. This is the first example of an avian IAV that is naturally less sensitive to Mxnndash;mediated inhibition and might explain why H7N9 viruses transmitted efficiently to humans.

  • Dengue Virus Induces and Requires Glycolysis for Optimal Replication [PublishAheadOfPrint]

  • Viruses rely on host cellular metabolism to provide the energy and biosynthetic building blocks required for their replication. Dengue virus (DENV), a member of the Flaviviridae family, is one of the most important arthropod-borne human pathogens worldwide. We analyzed global intracellular metabolic changes associated with DENV infection of primary human cells. Our metabolic profiling data suggested that central carbon metabolism, particularly glycolysis, is strikingly altered during a time course of DENV infection. Glucose consumption is increased during DENV infection and depriving DENV-infected cells of exogenous glucose had a pronounced impact on viral replication. Furthermore, expression of both glucose transporter 1 (GLUT1) and hexokinase 2 (HK2), the first enzyme of glycolysis, are upregulated in DENV-infected cells. Pharmacologically inhibiting the glycolytic pathway dramatically reduced DENV RNA synthesis and infectious virion production, revealing a requirement for glycolysis during DENV infection. Thus, these experiments suggest that DENV induces the glycolytic pathway to support efficient viral replication. This study raises the possibility that metabolic inhibitors, such as those that target glycolysis, could be used to treat DENV infection in the future.

    Importance Approximately 400 million people are infected with dengue virus (DENV) annually and more than one-third of the global population is at risk of infection. As there are currently no effective vaccines or specific antiviral therapies for DENV, we investigated the impact DENV has on the host cellular metabolome to identify metabolic pathways that are critical for the virus life cycle. We report an essential role for glycolysis during DENV infection. DENV activates the glycolytic pathway and inhibition of glycolysis significantly blocks infectious DENV production. This study provides further evidence that viral metabolomic analyses can lead to the discovery of novel therapeutic targets to block replication of medically important human pathogens.

  • Genetic Analysis of the Localization of APOBEC3F to Human Immunodeficiency Virus Type 1 (HIV-1) Virion Cores [PublishAheadOfPrint]

  • Members of the APOBEC3 family of cytidine deaminases vary in proportion of virion-incorporated enzyme that is localized to mature retrovirus cores. We reported previously that APOBEC3F (A3F) was highly localized into mature human immunodeficiency virus type 1 (HIV-1) cores and identified that L306 in the C-terminal cytidine deaminase (CD) domain contributed to its core localization. We have now determined additional genetic determinant(s) for A3F localization to HIV-1 cores. We found that one pair of leucines in each of A3F's C-terminal and N-terminal CD domains jointly determined the degree of localization of A3F into HIV-1 virion cores. These are A3F L306/L368 (C-terminal domain) and A3F L122/L184 (N-terminal domain). Substitutions in one of these specific leucine residues in either of the two A3F CD domains (A3F L368A; L122A; L184A) decreased core localization and diminished HIV restriction, without changing virion packaging. Furthermore, double mutants in these leucine residues in each of A3F's two CD domains (A3F L368A plus L184A, or A3F L368A plus L122A) were still packaged into virions, but completely lost core localization and anti-HIV activity. HIV virion core localization of A3F is genetically separable from its virion packaging, and anti-HIV activity requires some core localization.

    Importance: Specific leucine-leucine interactions are identified as necessary for A3F's core localization and anti-HIV activity, but not for its packaging into virions. Understanding these signals may lead to novel strategies to enhance core localization that may augment effects of A3F against HIV, and perhaps of other A3s against retroviruses, parvoviruses, and hepatitis B virus.

  • Targeting cellular squalene synthase, an enzyme essential for cholesterol biosynthesis, is a potential antiviral strategy against hepatitis C virus. [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) exploits host membrane cholesterol and its metabolism for progeny virus production. Here, we examined the impact of targeting cellular squalene synthase (SQS), the first committed enzyme for cholesterol biosynthesis, on HCV production. By using the HCV JFH-1 strain and human hepatoma Huh-7.5.1-derived cells, we found that SQS inhibitors, YM-53601 and zaragozic acid A, decreased viral RNA, protein, and progeny production in HCV-infected cells without affecting cell viability. Similarly, siRNA-mediated knockdown of SQS led to significantly reduced HCV production, confirming the enzyme as an antiviral target. A metabolic labeling study demonstrated that YM-53601 suppressed the biosynthesis of cholesterol and cholesteryl esters at antiviral concentrations. Unlike YM-53601, the cholesterol esterification inhibitor Sandoz 58-035 did not exhibit an antiviral effect, suggesting that biosynthesis of cholesterol is more important for HCV production than that of cholesteryl esters. YM-53601 inhibited transient replication of a JFH-1 subgenomic replicon and entry of JFH-1 pseudoparticles, suggesting that at least suppression of viral RNA replication and entry contributes to the antiviral effect of the drug. Collectively, our findings highlight the importance of the cholesterol biosynthetic pathway in HCV production and implicate SQS as a potential target for antiviral strategies against HCV.

    IMPORTANCE Hepatitis C virus (HCV) is known to be closely associated with host cholesterol and its metabolism throughout the viral life cycle. However, impact of targeting cholesterol biosynthetic enzymes on HCV production is not fully understood. We found that squalene synthase, the first committed enzyme for cholesterol biosynthesis, is important for HCV production and propose this enzyme as a potential anti-HCV target. We provide evidence that synthesis of free cholesterol is more important for HCV production than that of esterified cholesterol, highlighting marked free cholesterol dependency of HCV production. Our findings also offer a new insight into a role of intracellular cholesterol pool that is coupled to its biosynthesis in HCV life cycle.

  • Generation and Evaluation of Clade C Simian-Human Immunodeficiency Virus Challenge Stocks [PublishAheadOfPrint]

  • The development of a panel of mucosally transmissible simian-human immunodeficiency virus (SHIV) challenge stocks from multiple virus clades would facilitate preclinical evaluation of candidate HIV-1 vaccines and therapeutics. The majority of SHIV stocks that have been generated to date have been derived from clade B HIV-1 env sequences from viruses isolated during chronic infection and typically required serial animal-to-animal adaption for establishing mucosal transmissibility and pathogenicity. To capture essential features of mucosal transmission of clade C viruses, we produced a series of SHIVs with early clade C HIV-1 env sequences from acutely HIV-1-infected individuals from South Africa. SHIV-327c and SHIV-327cRM expressed env sequences that were 99.7-100% identical to the original HIV-1 isolate and did not require in vivo passaging for mucosal infectivity. These challenge stocks infected rhesus monkeys efficiently by both intrarectal and intravaginal routes, replicated to high levels during acute infection, and established chronic setpoint viremia in 13 of 17 (76%) infected animals. The SHIV-327cRM challenge stock was also titrated for both single, high-dose intrarectal challenges and repetitive, low-dose intrarectal challenges in rhesus monkeys. These SHIV challenge stocks should facilitate the preclinical evaluation of vaccines and other interventions aimed at preventing clade C HIV-1 infection.

    Importance We describe the development of two related clade C SHIV challenge stocks. These challenge stocks should prove useful for preclinical testing of vaccines and other interventions aimed at preventing clade C HIV-1 infection.

  • Influenza M2 protein ion channel activity helps maintain the pandemic 2009 H1N1 hemagglutinin fusion competence during transport to the cell surface [PublishAheadOfPrint]

  • Influenza virus hemagglutinin (HA) envelope protein mediates virus entry by first binding to cell surface receptors, then fusing viral and endosomal membranes during endocytosis. Cleavage of the HA precursor (HA0) into a surface receptor-binding subunit (HA1) and a fusion-inducing transmembrane subunit (HA2) by host cell enzymes primes HA for fusion competence by repositioning the fusion peptide to the newly created the N-terminus of HA2. We previously reported that influenza M2 protein enhances pandemic 2009 influenza A (H1N1) (pdm09) HA-pseudovirus infectivity, but the mechanism was unclear. Here, using cell-cell fusion and HA-pseudovirus infectivity assays, we found that the ion channel function of M2 was required for enhancing HA fusion and HA-pseudovirus infectivity. The M2 activity was only needed during HA biosynthesis, and proteolysis experiments indicate that M2 proton channel activity helps to protect (H1N1)pdm09 HA from premature conformational changes as it traversed low pH compartments during transport to the cell surface. While M2 has previously been shown to protect avian influenza from H5 and H7 subtypes that have HA with polybasic cleavage motifs, this study demonstrates that M2 can protect HA from human H1N1 strains that lack a polybasic cleavage motif. This finding suggests that M2 proton channel activity may play a wider role in preserving HA fusion competence among a variety of HA subtypes, including HA from emerging strains that may have reduced HA stability.

    IMPORTANCE Influenza infects cells when the hemagglutinin surface protein (HA) undergoes irreversible pH-induced conformational changes after the virus is taken into the cell by endocytosis. HA fusion competence is primed when host cells enzymes cleave the HA precursor. The proton channel function of influenza M2 protein has previously been shown to protect avian influenza HA that contain a polybasic cleavage site from pH-induced conformation changes during biosynthesis, but this effect is less well understood for human influenza HA that lack polybasic cleavage sites. Using assays that focus on HA entry and fusion, we found that the M2 protein also protects the (H1N1)pdm09 influenza A virus HA from premature conformational changes as it transits low-pH compartments during biosynthesis. This work suggests that M2 may play a wider role in preserving HA function in a variety of influenza subtypes that infect humans and may be especially important for HA that are less stable.

  • Deletion of the ORF9p acidic cluster impairs the nuclear egress of Varicella-zoster virus capsids [PublishAheadOfPrint]

  • The protein encoded by the ORF9 is essential for Varicella-zoster virus (VZV) replication. Previous studies documented its presence in the trans-Golgi network and its involvement in secondary envelopment. In this work, we deleted the ORF9p acidic cluster, destroying its interaction with ORF47p and resulting in a nuclear accumulation of both proteins. This phenotype results to an accumulation of primary enveloped capsids in the perinuclear space, reflecting a capsid de-envelopment defect.

  • Structure-function Analysis of Vaccinia Virus H7 protein Reveals a Novel Phosphoinositide Binding Fold Essential for Poxvirus Replication [PublishAheadOfPrint]

  • Phosphoinositides and phosphoinositide-binding proteins play a critical role in membrane and protein trafficking in eukaryotes. Their critical role in replication of cytoplasmic viruses has just begun to be understood. Poxviruses, a family of large cytoplasmic DNA viruses, rely on the intracellular membranes for developing their envelope, and poxvirus morphogenesis requires enzymes from the cellular phosphoinositide metabolic pathway. However, the role of phosphoinositides in poxvirus replication remains unclear, and no poxvirus proteins show any homology to eukaryotic phosphoinositide-binding domains. Recently, a group of poxvirus proteins, termed viral-membrane assembly proteins (VMAPs), were identified as essential for poxvirus membrane biogenesis. A key component of VMAPs is the H7 protein. Here, we report the crystal structure of the H7 protein from vaccinia virus. The H7 structure displays a novel fold comprised of seven aalpha;-helices and a highly curved three-stranded antiparallel bbeta;-sheet. We identified a phosphoinositide-binding site in H7, comprised of basic residues on a surface patch and the flexible C-terminal tail. These residues were found to be essential for the viral replication and for the binding of H7 to phosphatidylinositol-3-phosphate (PI3P) and phosphatidylinositol-4-phosphate (PI4P). Our studies suggested that phosphoinositide binding by H7 plays an essential role in poxvirus membrane biogenesis.

    Importance Poxvirus viral membrane assembly proteins (VMAPs) were recently shown to be essential for poxvirus membrane biogenesis. One of the key components of VMAPs is the H7 protein. However, no known structural motifs could be identified from its sequence, and there are no homologs of H7 outside of the poxvirus family to suggest a biochemical function. We have determined the crystal structure of vaccinia virus H7 protein. The structure displays a novel fold with a distinct and positively charged surface. Our data demonstrate that H7 binds phosphatidylinositol-3-phosphate and phosphatidylinositol-4-phosphate, and the basic surface patch was indeed required for phosphoinositide binding. In addition, mutation of positively charged residues required for lipid binding disrupted VACV replication. Phosphoinositides and phosphoinositide-binding proteins play critical roles in membrane and protein trafficking in eukaryotes. Our study demonstrates that VACV H7 displays a novel fold for phosphoinositide binding, which is essential for poxvirus replication.

  • Thymic HIV-2 infection uncovers post-transcriptional control of viral replication in human thymocytes [PublishAheadOfPrint]

  • A unique HIV-host equilibrium exists in untreated HIV-2-infected individuals, characterized by low-to-undetectable viremia throughout the disease course despite the establishment of disseminated HIV-2 reservoirs at levels comparable to those observed in untreated HIV-1 infection. Although the clinical spectrum is similar in the two infections, HIV-2 is associated with a much slower rate of CD4 T-cell decline and limited impact on the mortality of infected adults. Here we investigated HIV-2 infection of the human thymus, the primary organ for T-cell production. Human thymic tissue and suspensions of total or purified CD4 single-positive thymocytes were infected with HIV-2 or HIV-1 primary isolates using either CCR5 or CXCR4 co-receptors. We found that HIV-2 infected both thymic organ cultures and thymocyte suspensions, as attested by total HIV DNA and cell-associated viral mRNA levels. Nevertheless, thymocytes featured reduced levels of intracellular Gag viral protein, irrespective of HIV-2 co-receptor tropism and cell differentiation stage, in agreement with the low viral load in culture supernatants. Our data show that HIV-2 is able to infect the human thymus but leads to reduced viral production by thymocytes, providing a new model to identify therapeutic targets for viral replication control.

    IMPORTANCE HIV-1 infects the thymus, leading to a decrease in CD4 T-cell production that contributes to the characteristic CD4 T-cell loss. HIV-2 is associated with a very slow rate of progression to AIDS, and is therefore considered a unique naturally-occurring model of attenuated HIV disease. HIV-2-infected individuals feature low-to-undetectable plasma viral load, in spite of the numbers of circulating infected T-cells being similar to those found in patients infected with HIV-1. We assessed, for the first time, the direct impact of HIV-2 infection on the human thymus. We showed that HIV-2 is able to infect the thymus, but that the HIV-2 replication cycle in thymocytes is impaired. We propose that this system will be important to devise immunotherapies that target viral production, aiding the design of future therapeutic strategies for HIV control.

  • TRIM5{alpha} restriction affects clinical outcome and disease progression in SIV infected rhesus macaques [PublishAheadOfPrint]

  • Tripartite motif-containing protein 5 alpha (TRIM5aalpha;) is considered to be a potential target for cell-based gene modification therapy against HIV-1 infection. In the present study, we used a relevant SIVsm-infected rhesus macaque model to evaluate the effect of TRIM5aalpha; restriction on clinical outcome. Disease outcome of macaques expressing a restrictive TRIM5 genotype was compared following infection with the wild type TRIM-sensitive SIVsm strain versus a virus with escape mutations in capsid. We found that TRIM5aalpha; restriction significantly delayed disease progression and improved survival rate of SIV-infected macaques, which supports the feasibility of exploiting TRIM5aalpha; as a target for gene therapy against HIV-1. Furthermore, we also found preservation of memory CD4 T cells was associated with the protection by TRIM5aalpha; restriction, suggesting memory CD4 T cells or its progenitor cells as an ideal target for gene modification. Despite a significant effect on survival, SIV escape from TRIM5aalpha; restriction was also observed, therefore, this may not be an effective stand-alone strategy and may require combination with other targets.

    Importance Recent studies suggest that it may be feasible to not only suppress viral replication with antiviral drugs but to potentially eliminate or "cure" human immunodeficiency virus (HIV) infection. One approach being explored is the use of gene therapy to introduce genes that can restrict HIV replication, including a restrictive version of the host factor TRIM5aalpha;. TRIM5 was identified as a factor that restricts HIV replication in macaque cells. The rhesus gene is polymorphic and some alleles are restrictive for primary SIVsm isolates although escape mutations arise late in infection. Introduction of these escape mutations into the parental virus conferred resistance to TRIM5 in macaques. The present study evaluated these animals for longterm outcome and found that TRIM5aalpha; restriction significantly delayed disease progression and improved survival rate of SIV-infected macaques suggesting that this could be a valid gene therapy approach that could be adapted for HIV.

  • Live-attenuated measles virus vaccine targets dendritic cells and macrophages in the muscle of non-human primates [PublishAheadOfPrint]

  • Although live-attenuated measles virus (MV) vaccines have been used successfully for over fifty years, the target cells that sustain virus replication in vivo are still unknown. We generated a reverse genetics system for the live-attenuated MV vaccine strain Edmonston-Zagreb (EZ), allowing recovery of recombinant (r)MVEZ. Three recombinant viruses were generated that contained the open reading frame encoding enhanced green fluorescent protein (EGFP) within an additional transcriptional unit (ATU) at various positions within the genome. rMVEZEGFP (1), rMVEZEGFP (3) and rMVEZEGFP (6) contained the ATU upstream of the N gene, following the P gene, and following the H gene, respectively. The viruses were compared in vitro by growth curves, which indicated that rMVEZEGFP (1) was over-attenuated. Intra-tracheal infection of cynomolgus macaques with these recombinant viruses revealed differences in immunogenicity. rMVEZEGFP (1) and rMVEZEGFP (6) did not induce satisfactory serum antibody responses, whereas both in vitro and in vivo rMVEZEGFP (3) was functionally equivalent to the commercial MVEZ-containing vaccine. Intramuscular vaccination of macaques with rMVEZEGFP (3) resulted in the identification of EGFP+ cells in the muscle at 3, 5 and 7 days post vaccination. Phenotypic characterization of these cells demonstrated that muscle cells were not infected and that dendritic cells and macrophages were the predominant target cells of live-attenuated MV.

    Importance Even though MV strain Edmonston-Zagreb has long been used as a live-attenuated vaccine (LAV) to protect against measles, nothing is known about the cells in which the virus replicates in vivo. This is vital information given the push to move towards needle-free routes of vaccination, since vaccine virus replication is essential for vaccination efficacy. We have generated a number of recombinant MV strains expressing enhanced green fluorescent protein. The virus that best mimicked the non-recombinant vaccine virus was formulated according to protocols for production of commercial vaccine virus batches, and was subsequently used to assess viral tropism in non-human primates. The virus primarily replicated in professional antigen presenting cells, which may explain why this LAV is so immunogenic and efficacious.

  • Induction of an embryonic mouse innate immune response following inoculation in utero with Minute Virus of Mice [PublishAheadOfPrint]

  • We have used an embryonic infection model system to show that MVMp the prototypic Minute Virus of Mice serotype and member of the Protoparvovirus genus, triggers a comprehensive innate immune response in the developing mouse embryo. Direct inoculation of the mid-trimester embryo in utero with MVMp results in a widespread, productive infection. During a 96 hour infection course, embryonic IFNbeta and IFNgamma transcription were induced 90- and 60-fold respectively. IFNbeta levels correlated with embryo viral burden while IFNgamma levels first increased and then decreased. Production of pro-inflammatory cytokines, IL1beta and TNFalpha also increased, but by smaller amounts, approximately 7-fold each. We observed increased levels of downstream antiviral effector molecules, PKR and phosphorylated STAT2. Finally, we showed that there is an immune cell response to the virus infection. Infected tissues in the embryo exhibited an increased density of mature leukocytes compared to the same tissues in uninfected embryos.

    The responses we observed were almost completely restricted to the infected embryos. Uninfected litter mates routinely exhibited small increases in innate immune components that rarely reached statistical significance compared to negative controls. Similarly, the placentae of infected embryos did not show any significant increase in transcription of innate immune cytokines. Since placenta has both embryonic and maternal components, we suggest minimal involvement of the dam in the response to infection.

    Importance Interaction between the small single-stranded vertebrate DNA viruses, the protoparvoviruses, and the host innate immune system has been unclear. The issue is practically important given the potential use of these viruses as oncotherapeutic agents. The data reported here stand in contrast to studies of innate immune response during protoparvovirus infection of adult hosts, which invariably reported no or minimal and sporadic induction of an interferon response during infection. We conclude that under conditions of robust and productive MVM infection, a normal murine host is able to mount a significant and broad innate immune response.

  • The P25 protein of Potato virus X is the main pathogenicity determinant responsible for systemic necrosis in PVX-associated synergisms [PublishAheadOfPrint]

  • Most plant viruses counter the RNA silencing-based antiviral defense by expressing viral suppressors of RNA silencing (VSRs). In this sense, VSRs may be regarded as virulence effectors which can be recognized by the host as avirulence (avr) factors to induce R-mediated resistance. We made use of Agrobacterium-mediated transient co-expression of VSRs in combination with Potato virus X (PVX) to recapitulate at local tissues the systemic necrosis (SN) caused by PVX-potyvirus synergistic infections in Nicotiana benthamiana. The HR-like response was associated with an enhanced accumulation of PVX subgenomic RNAs. We further show that expression of P25, the VSR of PVX, in the presence of VSR from different viruses elicited an HR-like response in Nicotiana spp. Furthermore, the expression of P25 by a Plum pox virus (PPV) vector was sufficient to induce an increase of PPV pathogenicity that led to necrotic mottling. A frameshift mutation in the P25 ORF of PVX did not lead to necrosis when co-expressed with VSRs. These findings indicate that P25 is the main PVX determinant involved in eliciting a systemic HR-like response in PVX-associated synergisms. Moreover, we show that silencing of SGT1 and RAR1 attenuated cell death in both PVX-potyvirus synergistic infection and the HR-like response elicited by P25. Our study underscores that P25 variants that have impaired ability to suppress RNA silencing cannot act as elicitors when synergized by the presence of other VSRs. These findings highlight the importance of RNA silencing suppression activity in the HR-like response elicited by VSRs in certain hosts.

    Importance The work presented here describes how the activity of the PVX suppressor P25 elicits an HR-like response in Nicotiana spp when over-expressed with other VSR proteins. This finding suggests that the SN response caused by PVX-associated synergisms is a delayed immune response triggered by P25, once it reaches a threshold level by the action of other VSRs. Moreover, this work supports the contention that the silencing suppressor activity of PVX P25 protein is a prerequisite for HR elicitation. We propose that unidentified avr determinants could be involved in other cases of viral synergisms where heterologous "helper" viruses encoding strong VSRs exacerbate the accumulation of the avr-encoding virus.

  • Mutations across murine hepatitis virus nsp4 alter virus fitness and membrane modifications [PublishAheadOfPrint]

  • A common feature of infection by positive-sense RNA virus is the modification of host-cell cytoplasmic membranes that serve as sites of viral RNA synthesis. Coronaviruses induce double membrane vesicles (DMVs), but the role of DMVs in replication and virus fitness remains unclear. Coronaviruses encode 16 nonstructural proteins (nsps), three of which - nsp3, nsp4, and nsp6 - are necessary and sufficient for DMV formation. It has been shown previously that mutations in murine hepatitis virus (MHV) nsp4 loop 1 that alter nsp4 glycosylation are associated with disrupted DMV formation and result in changes in virus replication and RNA synthesis. However, it is not known whether DMV morphology or another function of nsp4 glycosylation is responsible for effects on virus replication. In this study we tested whether mutations across nsp4, both alone and in combination with mutations that abolish nsp4 glycosylation, affected DMV formation, replication, and fitness. Residues in nsp4 distinct from glycosylation sites, particularly in the ER luminal loop 1, independently disrupted both the number and morphology of DMVs, and exacerbated DMV changes associated with loss of glycosylation. Mutations that altered DMV morphology but not glycosylation did not affect virus fitness while viruses lacking nsp4 glycosylation exhibited a loss in fitness. The results support the hypothesis that DMV morphology and numbers are not key determinants of virus fitness. The results also suggest that nsp4 glycosylation serves roles in replication in addition to the organization and stability of MHV induced double membrane vesicles.

    Importance All positive-sense RNA viruses modify host cytoplasmic membranes for viral replication complex formation. Thus, defining the mechanisms of virus-induced membrane modifications is essential for both understanding virus replication and development of novel approaches to virus inhibition. Coronavirus-induced membrane changes include double membrane vesicles (DMVs) and convoluted membranes. Three viral nonstructural proteins (nsps), nsp3, nsp4, and nsp6, are known to be required for DMV formation. It is unknown how these protein induce membrane modification or which regions of the proteins are involved in DMV formation and stability. In this study we show that mutations across nsp4 delay virus replication, disrupt DMV formation, and loss of nsp4 glycosylation is associated with a substantial fitness cost. These results support a critical role for nsp4 in DMV formation and virus fitness.

  • GB virus C co-infections in West African Ebola patients [PublishAheadOfPrint]

  • In 49 patients with known Ebolavirus (EBOV) outcomes during the ongoing outbreak in Sierra Leone, 13 were co-infected with the immunomodulatory pegivirus GB virus C (GBV-C). 53% of these GBV-C+ patients survived; in contrast, only 22% of GBV-Cmmdash; patients survived. Both survival and GBV-C status were associated with age, with older patients having lower survival rates and intermediate-age patients (21-45 years) having the highest rate of GBV-C infection. Understanding the separate and combined effects of GBV-C and age on EBOV survival could lead to new treatment and prevention strategies, perhaps through age-related pathways of immune activation.

  • Novel mechanism of regulation of Tomato bushy stunt virus replication by cellular WW-domain proteins [PublishAheadOfPrint]

  • Replication of (+)RNA viruses depends on several co-opted host proteins, but is also under the control of cell-intrinsic restriction factors (CIRFs). By using tombusviruses, small model viruses of plants, we dissect the mechanism of inhibition of viral replication by cellular WW-domain containing proteins, which act as CIRFs. By using fusion proteins between the WW-domain and the p33 replication protein, we show that the WW-domain inhibits the ability of p33 to bind to the viral RNA and to other p33 and p92 replication proteins leading to inhibition of viral replication in yeast and in a cell-free extract. Over-expression of WW-domain protein in yeast also leads to reduction of several co-opted host factors in the viral replicase complex (VRC). These host proteins, such as eEF1A, Cdc34 E2 ubiquitin-conjugating enzyme and ESCRT proteins (Bro1p and Vps4p), are known to be involved in VRC assembly. Simultaneous co-expression of pro-viral cellular factors with WW-domain protein partly neutralizes the inhibitory effect of the WW-domain protein. We propose that cellular WW-domain proteins act as CIRFs and also as regulators of tombusvirus replication by inhibiting the assembly of new membrane-bound VRCs at the late stage of infection. We suggest that tombusviruses could sense the status of the infected cells via the availability of cellular susceptibility factors versus WW-domain proteins for binding to p33 replication protein that ultimately controls the formation of new VRCs. This regulatory mechanism might explain how tombusviruses could adjust the efficiency of RNA replication to the limiting resources of the host cells during infections.

    Importance: Replication of positive-stranded RNA viruses, which are major pathogens of plants, animals and humans, is inhibited by several cell-intrinsic restriction factors (CIRFs) in infected cells. In this paper, the authors define the inhibitory roles of the cellular Rsp5 ubiquitin ligase and its WW-domain in plant-infecting tombusvirus replication in yeast cells and in vitro using purified components. The WW-domain of Rsp5 binds to the viral RNA-binding sites of p33 and p92 replication proteins and blocks the ability of these viral proteins to use the viral RNA for replication. The WW-domain also interferes with the interaction (oligomerization) of p33 and p92 that is needed for the assembly of the viral replicase. Moreover, WW-domain also inhibits the subversion of several cellular proteins into the viral replicase, which otherwise play pro-viral roles in replication. Altogether, Rsp5 is a CIRF against a tombusvirus and it possibly has regulatory function during viral replication in infected cells.

  • Pseudorabies virus triggers glycoprotein gE-mediated ERK1/2 activation and ERK1/2-dependent migratory behavior in T cells [PublishAheadOfPrint]

  • The interaction between viruses and immune cells of the host may lead to modulation of intracellular signaling pathways, and subsequent changes in cellular behavior that are of benefit for either virus or host. ERK1/2 (Extracellular signal Regulated Kinase 1/2) signaling represents one of the key cellular signaling axes. Here, using wild type and gE null virus, recombinant gE and gE-transfected cells, we show that the gE glycoprotein of the porcine Varicellovirus pseudorabies virus (PRV) triggers ERK1/2 phosphorylation in Jurkat T cells and primary porcine T lymphocytes. PRV-induced ERK1/2 signaling resulted in homotypic T cell aggregation and increased motility of T lymphocytes. Our study reveals a new function of the gE glycoprotein of PRV and suggest that PRV, through activation of ERK1/2 signaling, has a substantial impact on T cell behavior.

    Importance Herpesviruses are known to be highly successful in evading the immune system of their hosts, subverting signaling pathways of the host to their own advantage. The ERK1/2 signaling pathway, being involved in many cellular processes, represents a particularly attractive target for viral manipulation. Glycoprotein E (gE) is an important virulence factor of alphaherpesviruses, involved in viral spread. In this study, we show that gE has the previously uncharacterized ability to trigger ERK1/2 phosphorylation in T lymphocytes. We also show that virus-induced ERK1/2 signaling leads to increased migratory behavior of T cells, and that migratory T cells can spread the infection to susceptible cells. In conclusion, our results point to a novel function for gE and suggest that virus-induced ERK1/2 activation may trigger PRV-carrying T lymphocytes to migrate and infect other cells susceptible to PRV replication.

  • Analysis of human T-cell leukemia virus type 1 particles using cryo-electron tomography [PublishAheadOfPrint]

  • Human T-cell leukemia virus type 1 (HTLV-1) particle structure is poorly characterized. Here, we have used cryo-electron tomography to analyze HTLV-1 particle morphology. Particles produced from MT-2 cells were polymorphic, roughly spherical, and varied in size. Capsid cores, when present, were typically poorly defined polyhedral structures with at least one curved region contacting the inner face of the envelope membrane. However, most particles observed lacked a defined capsid core, which likely impacts HTLV-1 particle infectivity.

  • A Model Of The Tva Receptor Determinants Required For Efficient Infection By The Subgroup A Avian Sarcoma And Leukosis Virus [PublishAheadOfPrint]

  • The study of the interactions of subgroup A avian sarcoma and leucosis viruses [ASLV(A)] with the Tva receptor required to infect cells offers a powerful experimental model of retroviral entry. Several regions and specific residues in the Tva receptor have been identified previously as critical determinants of binding affinity with the ASLV(A) envelope glycoproteins and to mediate efficient infection. Two homologs of the Tva receptor have been cloned: the original quail Tva receptor that has been the basis for most of the initial ASLV(A) Tva characterization; and the chicken Tva receptor, 65% identical with the quail receptor overall but identical in the region thought critical for infection. Our previous work characterized three mutant ASLV(A) viruses that could efficiently bind and infect cells using the chicken Tva receptor homolog but not using the quail Tva homolog, the infectivity of one mutant virus ggt;500-fold less infectious with the quail Tva receptor. The mutant viruses contained mutations in the hr1 region of the surface glycoprotein. Using chimeras of the quail and chicken Tva receptors, we have identified new residues of Tva critical for binding affinity and entry of ASLV(A) viruses using the mutant glycoproteins and viruses to probe function. The quail Tva receptor required changes at residues 10, 14 and 31 to the corresponding chicken Tva residues to bind wild type and mutant ASLV(A) glycoproteins with high affinity, and recover the ability to mediate efficient infection of cells. A model of the Tva determinants critical for interacting with ASLV(A) glycoproteins is proposed.

    IMPORTANCE A detailed understanding of how retroviruses enter cells, evolve to use new receptors and maintain efficient entry is crucial for identifying new targets for combating retrovirus infection and pathogenesis, as well as for developing new approaches for targeted gene delivery. Since all retroviruses share a common envelope glycoprotein organization, they likely share a common mechanism of receptor triggering to begin the entry process. Multiple, noncontiguous interaction determinants located in the receptor and SU hypervariable domains are required for binding affinity and to restrict or broaden receptor usage. In this study further mechanistic details of the entry process are being elucidated by characterizing ASLV(A) glycoprotein interactions with the Tva receptor required for entry. The ASLV(A) envelope glycoproteins are organized into functional domains that allow changes to occur in receptor choice by mutation and/or recombination while maintaining a critical level of receptor binding affinity and ability to trigger glycoprotein conformational changes.

  • THE AMPHIPATHIC HELIX OF ADENOVIRUS CAPSID PROTEIN VI CONTRIBUTES TO PENTON RELEASE AND POST ENTRY SORTING. [PublishAheadOfPrint]

  • Nuclear delivery of the adenoviral genome requires that the capsid crosses the limiting membrane of the endocytic compartment and traverses the cytosol to reach the nucleus. This endosomal escape is initiated upon internalisation and involves a highly coordinated partial disassembly process of the entering capsid to release the membrane lytic internal capsid protein VI. Using wildtype and protein VI mutated HAdV-C5, we show that capsid stability and membrane rupture are major determinants of entry related sorting of incoming adenovirus virions. Furthermore by using electron cryo-microscopy as well as penton and protein VI specific antibodies, we show that the amphipathic helix of protein VI contributes to capsid stability by preventing premature disassembly and deployment of penton and protein VI. Thus the helix has a dual function in maintaining the metastable state of the capsid by preventing premature disassembly and mediating efficient membrane lysis to evade lysosomal targeting. Based on these findings and structural data from cryo-electron microscopy, we suggest a refined disassembly mechanism upon entry.

    Importance In this study we show the intricate connection of adenovirus particle stability and the entry dependent release of the membrane lytic capsid protein VI required for endosomal escape. We show that the amphipatic helix of the adenovirus internal protein VI is required to stabilize penton in the particle, while coinciding with penton release upon entry, release of protein VI mediates membrane lysis thereby preventing lysosomal sorting. We suggest that this dual functionality of protein VI assures an optimal disassembly process by balancing the metastable state of the mature adenovirus particle.

  • Regulatory T cells resist virus infection-induced apoptosis [PublishAheadOfPrint]

  • Regulatory T (Treg) cells are important in the maintenance of self-tolerance, and the depletion of Treg cells correlates with autoimmune development. It has been shown that IFNaalpha;bbeta; responses induced early in the infection of mice can drive memory (CD44hi) CD8 and CD4 T cells into apoptosis, and we questioned here whether the apoptosis of CD44-expressing Treg cells might be involved in the infection-associated autoimmune development. Instead, we found that Treg cells were much more resistant to apoptosis than CD44hi CD8 and CD4 T cells at day 2-3 after lymphocytic choriomeningitis virus infection, when type I IFN levels are high. The infection caused a down-regulation of the IL-7 receptor, needed for survival of conventional T cells, while increasing on Treg cell the expression of high affinity IL-2 receptor, needed for STAT5-dependent survival of Treg cells. The stably maintained Treg cells early during infection may explain the relatively low incidence of autoimmune manifestations among infected patients.

    Importance Autoimmune diseases are controlled in part by regulatory T cells (Treg) and are thought to sometimes be initiated by viral infections. We tested the hypothesis that Treg may die off at early stages of infection, when virus-induced factors kill other lymphocyte types. Instead we found that Treg resisted this cell death, perhaps reducing the tendency of viral infections to cause immune dysfunction and induce autoimmunity.

  • Structural Flexibility of a Conserved Broadly Neutralizing Epitope in Hepatitis C Virus Glycoprotein E2 [PublishAheadOfPrint]

  • Neutralizing antibodies (nAbs) targeting glycoprotein E2 are important to control hepatitis C virus (HCV) infection. One conserved antigenic site (amino acids 412-423) is disordered in the reported E2 structure, but a synthetic peptide mimicking this site forms a bbeta;-hairpin in complex with three independent nAbs. Our structure of the same peptide in complex with nAb 3/11 demonstrates a strikingly different extended conformation. We also show that residues 412-423 are essential for virus entry, but not for E2 folding. Together with the neutralizing capacity of the 3/11 Fab this indicates an unexpected structural flexibility within this epitope. NAbs 3/11 and AP33 (recognizing the extended and bbeta;-hairpin conformation, respectively) display similar neutralizing activity despite converse binding kinetics. Our results suggest that HCV utilizes conformational flexibility as immune evasion strategy contributing to the limited immunogenicity of this epitope in patients, similar to the conformational flexibility described for other enveloped and non-enveloped viruses.

    Importance Approximately 180 million people worldwide are infected with Hepatitis C virus (HCV) and neutralizing antibodies play an important role in controlling replication of this major human pathogen. We show here that one of the most conserved antigenic sites within the major glycoprotein E2 (amino acids 412-423) - that is disordered in the recently reported crystal structure of an E2 core fragment - can adopt different conformations in the context of the infectious virus particle. Recombinant Fab fragments recognizing different conformations of this antigenic site have similar neutralization activity in spite of converse kinetic binding parameters. Of note, an antibody response targeting this antigenic region is less frequent than to other more immunogenic regions in E2. Our results suggest that the observed conformational flexibility in this conserved antigenic region contributes to the evasion of the humoral host immune response, facilitating chronicity and viral spread of HCV within an infected individual.

  • The inability of wild-type rabies virus to activate dendritic cells is dependent on the glycoprotein and correlates with its low level of the de novo synthesized leader RNA. [PublishAheadOfPrint]

  • Dendritic cells (DCs) are the most efficient antigen presenting cells, playing a key role in the adaptive immune responses to viral infections. Our studies demonstrate that wild-type (wt) rabies virus (RABV) does not activate DCs. Adoptive transfer of DCs primed with wt RABV did not activate DCs, stimulate virus neutralizing antibodies (VNA), or protect recipients against challenge. However, adoptive transfer of DCs primed with laboratory-attenuated RABV resulted in DC activation, production of VNA, and protection against challenge. In vitro studies with recombinant RABV (laboratory-attenuated RABV expressing the G or the P from wt RABV) demonstrate that DC activation is dependent on the glycoprotein (G) and involves IPS-1 pathway. Furthermore, binding to and entry into DCs by wt RABV is severely blocked and the copy number of de novo synthesized leader RNA was two logs lower in DCs infected with wt than in DCs treated with laboratory-attenuated RABV. However transient transfection of DCs with synthesized leader RNA from either wt or attenuated RABV is capable of activating DCs in a dose dependent manner. Thus, the inability of wt RABV to activate DCs correlates with its low level of the de novo synthesized leader RNA.

    Importance Rabies remains a public health threat with more than 55,000 fatalities each year around the world. Since dendritic cells (DCs) play a key role in the adaptive immune responses to viral infections, we investigated the ability of rabies virus (RABV) to activate DCs. It was found that adoptive transfer of DCs primed with wild-type (wt) RABV did not activate DCs, stimulate VNA, or protect mice against lethal challenge. Yet, laboratory-attenuated RABV mediates activation of DCs via IPS-1 pathway and is G-dependent. We further show that wt RABV evades DC-mediated immune activation by inefficient binding/entry into DCs as well as reduced level of de novo synthesized leader RNA. These findings may have important implications in the development of efficient rabies vaccines.

  • Host genetic and viral determinants of HIV-1 RNA set-point among HIV-1 seroconverters from sub-Saharan Africa [PublishAheadOfPrint]

  • We quantified the collective impact of source partner HIV-1 RNA levels, human leukocyte antigen (HLA) alleles, and innate responses through toll-like receptor (TLR) alleles on HIV-1 set-point. Data came from HIV-1 seroconverters in African HIV-1 serodiscordant couple cohorts. Linear regression was used to determine associations with set-point and R2 to estimate variation explained by covariates. The strongest predictors of set-point were HLA alleles (B*53:01, B*14:01, and B*27:03) and plasma HIV-1 levels of the transmitting partner, which explained 13% and 10% of variation in set-point, respectively. HLA-A concordance between partners and TLR polymorphisms (TLR2-rs3804100, TLR7-rs179012) were also associated with set-point, explaining 6% and 5% of variation, respectively. Overall, these factors and genital factors of the transmitter (i.e. male circumcision, bacterial vaginosis and use of acyclovir) explained 46% of variation in set-point. We found that both innate and adaptive immune responses, together with plasma HIV-1 levels of the transmitting partner, explain almost half of the variation in viral load set-point.

    Importance After HIV-1 infection, uncontrolled virus replication leads to a rapid increase in HIV-1 concentrations. Once host immune responses develop, however, HIV-1 levels reach a peak and subsequently decline until they reach a stable level that may persist for years. This stable HIV-1 set-point represents an equilibrium between the virus and host responses, and is predictive of later disease progression and transmission potential. Understanding how host and virus factors interact to determine HIV-1 set-point may elucidate novel mechanisms or biological pathways for treating HIV-1 infection. We identified host and virus factors that predict HIV-1 set-point in people who recently acquired HIV-1, finding that both innate and adaptive immune responses, along with factors that likely influence HIV-1 virulence and inoculum, explain ~46% of the variation in HIV-1 set-point.

  • What DNA Viral Genomic Rearrangements Tell Us About Persistence [PublishAheadOfPrint]

  • Understanding the life cycle and pathogenesis of animal viruses requires that we have systems in which the viruses can replicate and cause disease. For the latter, we rely upon animal models or information we can obtain from studying natural infections of humans and other animals. For the former, however, we are largely dependent on the availability of cell culture systems in which viruses can be propagated to investigate the molecular mechanisms of viral replication. For many years, it was assumed that replication in culture provided an accurate description of the life cycle in the organism. In this Gem, we will discuss two viruses, polyomavirus and cytomegalovirus, in which cell culture systems have accidentally shed unique potential insights on viral replication and persistence in their hosts.

  • Identification of a role for nucleolin in rabies virus infection [PublishAheadOfPrint]

  • Rabies virus replicates in the cytoplasm of host cells, but rabies virus phosphoprotein (P-protein) undergoes active nucleocytoplasmic trafficking. Here we show that the largely nuclear P-protein isoform P3 can localise to nucleoli, and forms specific interactions with nucleolin. Importantly, depletion of nucleolin expression inhibits viral protein expression and infectious virus production by infected cells. This provides the first evidence that lyssaviruses interact with nucleolin, and that nucleolin is important to lyssavirus infection.

  • Influenza A virus uses intercellular connections to spread to neighboring cells [PublishAheadOfPrint]

  • In the extracellular environment cell-free virions seek out naiiuml;ve host cells over long distances and between organisms. This is the primary mechanism of spread for most viruses. Here we provide evidence for an alternative pathway previously undescribed for Orthomyxoviruses where the spread of influenza A virus (IAV) infectious cores to neighboring cells can occur within intercellular connections. The formation of these connections requires actin dynamics and is enhanced by viral infection. Connected cells have contiguous membranes and the core infectious viral machinery (RNP and polymerase) were present inside the intercellular connections. A live cell movie of GFP-tagged NS1 of IAV shows viral protein moving from one cell to another through an intercellular connection. Movement of tagged protein was saltatory but overall traveled only in one direction. Infectious virus cores can move from one cell to another without budding and release of cell-free virions, as evidenced by finding that whereas a neuraminidase inhibitor alone did not inhibit the development of IAV microplaques, the presence of a neuraminidase inhibitor together with drugs inhibiting actin dynamics, or the microtubule stabilizer taxol precluded microplaque formation. Similar results were also observed with parainfluenza virus 5 (PIV5), a paramyxovirus, when neutralizing antibody was used to block spread by cell-free virions. Intercellular spread of infectious core particles was unaffected or enhanced in the presence of nocodazole for IAV but inhibited for PIV5. The intercellular connections have a core of filamentous actin, which hints toward transport of virus particles through the use of a myosin motor.

    IMPORTANCE Here we describe a new method by which influenza A virus (IAV) spreads from cell to cell: IAV uses tunneling nanotubes. The formation of these connections requires actin dynamics and is enhanced by viral infection and the absence of microtubules. Connected cells appeared to have contiguous membranes and the core infectious viral machinery (RNP and polymerase) were present inside the intercellular connections. Infectious virus cores can move from one cell to another without budding and release of cell-free virions. Similar results were also observed with parainfluenza virus 5 (PIV5).

  • Hepatitis B core protein sensitizes hepatocytes to tumor necrosis factor-induced apoptosis by suppression of the phosphorylation of mitogen-activated protein kinase kinase 7 [PublishAheadOfPrint]

  • Hepatitis B, which caused by Hepatitis B virus (HBV) infection, remains a major health threat worldwide. Hepatic injury and regeneration from chronic inflammation is the main driving factor of liver fibrosis and cirrhosis in chronic hepatitis B. Pro-inflammatory tumor necrosis factor (TNF)-aalpha; has been implicated as a major inducer of liver cell death during viral hepatitis. Here, we report that in hepatoma cell lines and in primary mouse and human hepatocytes, expression of hepatitis B core (HBc) protein made cells susceptible to TNF-aalpha;-induced apoptosis. We found that receptor of activated protein kinase C (RACK) 1 interacted with HBc by tandem affinity purification and mass spectrometry. RACK1 was recently reported as a scaffold protein that facilitates the phosphorylation of mitogen-activated protein kinase kinase 7 (MKK7) by its upstream activators. Our study showed that HBc abrogated the interaction between MKK7 and RACK1 by competitively binding to RACK1, thereby downregulating TNF-aalpha;-induced phosphorylation of MKK7 and the activation of c-Jun N-terminal kinase. In line with this finding, specific knockdown of MKK7 increased the sensitivity of hepatocytes to TNF-aalpha;-induced apoptosis, while overexpression of RACK1 counteracted the pro-apoptotic activity of HBc. Capsid particle formation was not obligatory for HBc pro-apoptotic activity, as analyzed using an assembly-defective HBc mutant. In conclusion, the expression of HBc sensitized hepatocytes to TNF-aalpha;-induced apoptosis by disrupting the interaction between MKK7 and RACK1. Our study is thus a first indication of the pathogenic effects of HBc in liver injury during hepatitis B.

    Importance: Our study revealed a previously unappreciated role of HBc in TNF-aalpha;-mediated apoptosis. The pro-apoptotic activity of HBc is important for understanding hepatitis B pathogenesis. In particular, severe hepatitis-associated HBV variants may upregulate apoptosis of hepatocytes through enhanced HBc expression. Our study also found that MKK7 is centrally involved in TNF-aalpha;-induced hepatocyte apoptosis and revealed a multifaceted role for JNK signaling in this process.

  • The Enterovirus 71 procapsid binds neutralizing antibodies and rescues virus infection in vitro [PublishAheadOfPrint]

  • Enterovirus 71 (EV71) is responsible for seasonal outbreaks of hand, foot, and mouth disease in the Asia-Pacific region. The virus has the capability of causing severe disease and death, especially in young children. Although several vaccines are currently in clinical trials no vaccines or therapeutics have been approved for use. Previous structural studies have revealed that two antigenically distinct capsid forms are produced in EV71 infected cells: an expanded empty capsid, sometimes called procapsid, and the infectious virus. Specifically an immunodominant epitope of EV71 that maps to the virus canyon is structurally different between the procapsid and virus. This structure function study shows that the procapsid can sequester antibodies thus enhancing EV71 infection in vitro. The results presented here suggest that due to conformational differences between the EV71 procapsid and virus, the presence of the procapsid in natural virus infections should be considered in the future design of vaccines or therapeutics.

    IMPORTANCE In a picornavirus infection both an infectious and a non-infectious empty capsid, sometimes referred to as procapsid, are produced. It was novel to discover that the procapsid form of enterovirus 71 (EV71) was expanded and antigenically distinct from the infectious virus. Previously it had been supposed that this empty capsid was an off pathway dead end, or at best served as storage of pentameric subunits, which was later shown to be unlikely. It remains unexplained why picornaviruses evolutionarily conserve the wasteful production of so much non-infectious capsid. Here we demonstrate that the EV71 procapsid has different antigenic properties than the infectious virus. Thus the procapsid has the capacity to sequester neutralizing antibody and protect the virus, promoting or restoring a successful infection in vitro. This important observation should be considered in the future design and development of vaccines and therapeutics.

  • Non-Muscle Myosin Heavy Chain IIB Mediates Herpes Simplex Virus 1 Entry [PublishAheadOfPrint]

  • Non-muscle myosin heavy chain IIA (NMHC-IIA) has been reported to function as a herpes simplex virus 1 (HSV-1) entry co-receptor by interacting with viral envelope glycoprotein B (gB). Vertebrates have three genetically distinct isoforms of the NMHC-II, designated NMHC-IIA, NMHC-IIB and NMHC-IIC. COS cells, which are readily infected by HSV-1, do not express NMHC-IIA but do express NMHC-IIB. This observation prompted us to investigate whether NMHC-IIB might associate with HSV-1 gB and be involved in an HSV-1 entry like NMHC-IIA. In these studies, we show that: (i) NMHC-IIB co-precipitated with gB in COS-1 cells upon HSV-1 entry; (ii) a specific inhibitor of myosin light chain kinase inhibited both cell-surface expression of NMHC-IIB in COS-1 cells upon HSV-1 entry as well as HSV-1 infection as reported with NMHC-IIA; (iii) overexpression of mouse NMHC-IIB in IC21 cells significantly increased their susceptibility to HSV-1 infection; (iv) knock-down of NMHC-IIB in COS-1 cells inhibited HSV-1 infection as well as cell-cell fusion mediated by HSV-1 envelope glycoproteins. These results supported the hypothesis that, like NMHC-IIA, NMHC-IIB associated with HSV-1 gB and mediated HSV-1 entry.

    Importance Herpes simplex virus 1 (HSV-1) was reported to utilize non-muscle myosin heavy chain IIA (NMHC-IIA) as an entry co-receptor associating with gB. Vertebrates have three genetically distinct isoforms of NMHC-II. In these isoforms, NMHC-IIB is of special interest since it highly expresses in neuronal tissue, one of the most important cellular targets of HSV-1 in vivo. In this study, we demonstrated that the ability to mediate HSV-1 entry appeared to be conserved in NMHC-II isoforms. These results may provide an insight into the mechanism by which HSV-1 infects a wide variety of cell types in vivo.

  • Characterization of a proteolytically stable D-peptide that suppresses Herpes simplex virus-1 infection: Implications for the development of entry-based antiviral therapy [PublishAheadOfPrint]

  • Uncontrolled herpes simplex virus type-1 (HSV-1) infection can advance to serious conditions, including corneal blindness or fatal encephalitis. Here, we describe a highly potent anti-HSV-1 peptide (DG2) that inhibits HSV-1 entry into host cells and blocks all aspects of infection. Importantly, DG2 is highly resistant to proteases and shows minimal toxicity, paving the way for prophylactic or therapeutic application of the peptide in vivo.

  • Human Noroviruses' Fondness of Histo-Blood Group Antigens [PublishAheadOfPrint]

  • Human noroviruses are the dominant cause of outbreaks of gastroenteritis around the world. Human norovirus interacts with the polymorphic human histo-blood group antigens (HBGAs) and this interaction is thought to be important for infection. Indeed, synthetic HBGAs or HBGA-expressing enteric bacteria were shown to enhance norovirus infection in B cells. A number of studies have found a possible relationship between HBGA type and norovirus susceptibility. The genogroup II genotype 4 (GII.4) noroviruses are the dominant cluster, evolve every other year, and are thought to modify their binding interactions to different HBGA types. Here, we show the high-resolution X-ray crystal structures of the capsid protruding (P) domains from epidemic GII.4 variants in 2004, 2006, and 2012, co-crystallized with a panel of HBGA types (H type 2, Lewis Y, Lewis B, Lewis A, Lewis X, A-type, and B-type). Many of the HBGA binding interactions were found to be complex, involving capsid loop movements, alternative HBGA conformations, and HBGA rotations. We showed that a loop (residues 391-395) was elegantly repositioned in order to allow for Lewis Y binding. This loop was also slightly shifted to provide direct hydrogen and water-mediated bonds with Lewis B. We considered that the flexible loop modulated Lewis HBGA binding. The GII.4 noroviruses have dominated outbreaks over the past decade, which may be explained by their exquisite HBGA binding mechanisms, their fondness for Lewis HBGAs, and their temporal amino acid modifications.

    Importance: Our data provides a comprehensive picture of GII.4 P domain and HBGA binding interactions. The exceptionally high resolutions of our X-ray crystal structures allowed us to accurately recognize novel GII.4 P domain interactions with numerous HBGA types. We showed that the GII.4 P domain HBGA interactions involved complex binding mechanisms that were not previously observed in norovirus structural studies. Many of the GII.4 P domain HBGA interactions we identified were negative in earlier ELISA-based studies. Altogether, we showed that the GII.4 norovirus P domains could accommodate numerous HBGA types.

  • Interferon-induced Ifit proteins: Their role in viral pathogenesis [PublishAheadOfPrint]

  • A major component of the protective antiviral host defense is contributed by the intracellular actions of the proteins encoded by interferon-stimulated genes (ISG); amongst these are the Interferon-induced proteins with tetratricopeptide repeats (IFIT), consisting of four members in human and three in mouse. IFIT proteins do not have any known enzyme activity; instead, they inhibit virus replication by binding and regulating the functions of cellular and viral proteins and RNAs. Although all IFITs are comprised of multiple copies of the degenerate tetratricopeptide repeats, their distinct tertiary structures enable them to bind different partners and affect host-virus interactions differently. The recent use of Ifit knock-out mouse models has revealed novel antiviral functions of these proteins and new insights into the specificities of ISG actions. This article focuses on human and murine IFIT1 and IFIT2 by reviewing their mechanisms of action, their critical roles in protecting mice from viral pathogenesis and viral strategies to evade IFIT action.

  • Receptor recognition mechanisms of coronaviruses - a decade of structural studies [PublishAheadOfPrint]

  • Receptor recognition by viruses is the first and essential step of viral infections of host cells. It is an important determinant of viral host range and cross-species infection, and a primary target for antiviral intervention. Coronaviruses recognize a variety of host receptors, infect many hosts, and are health threats to humans and animals. The receptor-binding S1 subunit of coronavirus spike proteins contains two distinctive domains, N-terminal domain (S1-NTD) and C-terminal domain (S1-CTD), both of which can function as receptor-binding domains (RBDs). S1-NTDs and S1-CTDs from three major coronavirus genera recognize at least four protein receptors and three sugar receptors, and demonstrate a complex receptor recognition pattern. For example, highly similar coronavirus S1-CTDs within the same genus can recognize different receptors, whereas very different coronavirus S1-CTDs from different genera can recognize the same receptor. Moreover, coronavirus S1-NTDs can recognize either protein or sugar receptors. Structural studies in the past decade have elucidated many of the puzzles associated with coronavirus-receptor interactions. This article reviews the latest knowledge on the receptor recognition mechanisms of coronaviruses, and discusses how coronaviruses have evolved their complex receptor recognition pattern. It also summarizes important principles that govern receptor recognition by viruses in general.

  • The NSP3 Macrodomain Promotes Virulence in Mice With Coronavirus-induced Encephalitis [PublishAheadOfPrint]

  • All coronaviruses encode for a macrodomain containing ADP-ribose-1"-phosphatase (ADRP) activity within the N-terminus of non-structural protein 3 (nsp3). Previous work showed that mouse hepatitis virus strain A59 (MHV-A59) with a mutated catalytic site (N1348A) replicated similarly to wild-type virus but was unable to cause acute hepatitis in mice. To determine whether this attenuated phenotype is applicable to multiple disease models, we mutated the catalytic residue in the JHM strain of MHV (JHMV), which causes acute and chronic encephalomyelitis, using a newly developed bacterial artificial chromosome (BAC)-based MHV reverse genetics system. Infection of mice with the macrodomain catalytic point mutant virus (N1347A) resulted in reduced lethality, weight loss, viral titers, pro-inflammatory cytokine and chemokine expression, and immune cell infiltration in the brain when compared to mice infected with wild-type virus. Specifically, macrophages were most affected, with approximately 2.5-fold fewer macrophages at day 5 post-infection in N1347A-infected brains. Tumor necrosis factor (TNF) and interferon (IFN) signaling were not required for effective host control of mutant virus, as all N1347A-infected mice survived the infection. However, the adaptive immune system was required for protection, since N1347A virus was able to cause lethal encephalitis in RAG1 (recombination activation gene1)-/- knockout mice, although disease onset was modestly delayed. Overall, these results indicate that the BAC-based MHV reverse genetics system will be useful for studies of JHMV, and expand upon previous studies, showing that the macrodomain is critical for the ability of coronaviruses to evade the immune system and promote viral pathogenesis.

    IMPORTANCE Coronaviruses are an important cause of human and veterinary diseases worldwide. Viral processes that are conserved across a family are likely to be good targets for the development of antiviral therapeutics and vaccines. The macrodomain is a ubiquitous structural domain, and is also conserved among all coronaviruses. The coronavirus macrodomain has ADP-ribose-1"-phosphatase activity; however, its function during infection remains unclear as does why coronaviruses have maintained this enzymatic activity throughout evolution. For MHV, this domain has now been shown to promote multiple types of disease, including hepatitis and encephalitis. These data indicate that this domain is vital for the virus to replicate and cause disease. Understanding the mechanism used by this enzyme to promote viral pathogenesis will open up novel avenues for therapies, and may give further insight into the role of macrodomain proteins in the host cell, since these proteins are found in all living organisms.

  • Vaccine-induced protection against orthopoxvirus infection is mediated through the combined functions of CD4 T cell-dependent antibody and CD8 T cell responses [PublishAheadOfPrint]

  • Antibody production by B cells, in the absence of CD4 T cell help, has been shown to be necessary and sufficient for protection against secondary orthopoxvirus infections. This conclusion is based on short-term depletion of leukocyte subsets in vaccinated animals in addition to passive transfer of immune serum to naiiuml;ve hosts that are subsequently protected from lethal orthopoxvirus infection. Here we show that CD4 T cell help is necessary for neutralizing antibody production and virus control during a secondary ectromelia virus (ECTV) infection. A crucial role for CD4 T cells was revealed when depletion of this subset was extended beyond the acute phase of infection. Sustained depletion of CD4 T cells over several weeks in vaccinated animals during a secondary infection resulted in gradual diminution of B cell responses including neutralizing antibody, contemporaneous with a corresponding increase in viral load. Long-term elimination of CD8 T cells alone delayed virus clearance but prolonged depletion of both CD4 and CD8 T cells resulted in death associated with uncontrolled virus replication. In the absence of CD4 T cells, perforin- and granzyme A and B-dependent effector functions of CD8 T cells became critical. Our data therefore show that both CD4 T cell help for antibody production and CD8 T cell effector function are critical for protection against secondary OPV infection. These results are consistent with the notion that the effectiveness of the smallpox vaccine is related to its capacity to induce both B and T cell memory.

    IMPORTANCE Smallpox eradication through vaccination is one of the most successful public health endeavors of modern medicine. The use of various orthopoxvirus (OPV) models to elucidate correlates of vaccine-induced protective immunity show that antibody is critical for protection against secondary infection whereas the role of T cells is unclear. Short-term leukocyte subset depletion in vaccinated animals or transfer of immune serum to naiiuml;ve, immunocompetent hosts indicates that antibody alone is necessary and sufficient for protection. We show here that long-term depletion of CD4 T cells over several weeks in vaccinated animals during secondary OPV challenge reveals an important role for CD4 T cell-dependent antibody responses in effective virus control. Prolonged elimination of CD8 T cells alone delayed virus clearance but depletion of both T cell subsets resulted in death associated with uncontrolled virus replication. Thus, vaccinated individuals who subsequently acquire T cell deficiencies may not be protected against secondary OPV infection.

  • The level of viral infection of antigen-presenting cells is critical for the development of TMEV-induced demyelinating disease [PublishAheadOfPrint]

  • Intracerebral infection with Theiler's murine encephalomyelitis virus (TMEV) induces immune-mediated demyelinating disease in susceptible SJL/J mice but not in resistant C57BL/6 mice. Previous studies have indicated that the major histocompatibility complex (MHC) genes play the most prominent role in the development of TMEV-induced demyelinating disease. In this study, we used C57BL/6.S (B6.S) congenic mice, which carry H-2s MHC genes instead of H-2b MHC genes in conjunction with the C57BL/6 (B6) background genes. Our data show that virus-infected B6.S mice are free from disease and have significantly lower viral loads than susceptible SJL mice, particularly in the spinal cord. A strong protective Th1-type T helper response with virtually no pathogenic Th17 response was detected in B6.S mice, in contrast to the reduced Th1 and robust Th17 type responses in SJL mice. Notably, lower levels of viral infectivity in B6.S antigen-presenting cells (APCs) correlated with the disease resistance and T-cell-type response. In vitro studies using APCs from B6.S and SJL mice show that TLR2, 3, 4, and 7, but not TLR9, signaling can replace viral infection and augment the effect of viral infection in the differentiation of the pathogenic Th17 cell type. Taken together, these results strongly suggest that the viral replication levels in APCs critically affect the induction of protective vs. pathogenic Th cell types via the signaling of pattern recognition receptors for innate immune responses. Our current findings further imply that the levels of viral infectivity/replication and TLR-mediated signaling play critical roles in the pathogenesis of chronic viral diseases.

    Importance: This study indicates that innate immune cytokines produced in antigen-presenting cells stimulating the T cell immune responses during early viral infection play a critical role in determining the susceptibility of mice to the development of demyelinating disease. The level of innate immune cytokines reflects the level of initial viral infection in the antigen-presenting cells and the level determines the development of T cell types, which are either protective or pathogenic. The level of initial viral infection to the cells is controlled by a gene or genes, which are not associated with the major histocompatibility antigen complex genes. This finding has an important implication in controlling not only chronic viral infections but also infection-induced autoimmune-like diseases, which are closely associated with the pathogenic type of T cell responses.

  • Lethal co-infection of influenza virus and Streptococcus pneumoniae lowers antibody response to influenza virus in lung, and reduces germinal center B cells, T follicular helper cells and plasma cells in mediastinal lymph node [PublishAheadOfPrint]

  • Secondary Streptococcus pneumoniae infection after influenza is a significant clinical complication resulting in morbidity and sometimes mortality. Prior influenza virus infection has been demonstrated to impair the macrophage and neutrophil response to the subsequent pneumococcal infection. In contrast, how the secondary pneumococcal infection after influenza can affect the adaptive immune response to the initial influenza virus infection is less well understood. Therefore, this study focuses on how secondary pneumococcal infection after influenza may impact on the humoral immune response to the initial influenza virus infection in a lethal co-infection mouse model. When compared to mice infected with influenza virus alone, mice co-infected with influenza virus followed by pneumococcus had significant body weight loss and 100% mortality. In the lung, lethal co-infection significantly increased virus titer, bacterial cell counts and decreased the level of virus specific IgG, IgM and IgA, as well as the number of B cells, CD4 T cells and plasma cells. In spleen, lethal co-infection significantly reduced the size and weight of spleen, as well as the B cells along the follicular developmental lineage. In mediastinal lymph nodes, lethal co-infection significantly decreased germinal center B cells, T follicular helper cells and plasma cells. Adoptive transfer of influenza virus-specific immune serum to co-infected mice improved survival, suggesting the protective functions of anti-influenza virus antibodies. In conclusion, co-infection reduced the B cell response to influenza virus. This study helps us to understand the modulation of B cell response to influenza virus during the lethal co-infection.

    Importance: Secondary pneumococcal infection after influenza is an important clinical issue that often results in excess mortality. Since antibodies are key mediator of protection, this study aims to examine the antibody response to influenza virus, and demonstrated that lethal co-infection reduced the B cell response to influenza virus. This study helps to highlight the complexity of the modulation of B cell response in the context of co-infection.

  • Macropinocytosis is the Entry Mechanism of Amphotropic Murine Leukemia Virus (A-MLV) [PublishAheadOfPrint]

  • The entry mechanism of murine amphotropic retrovirus (A-MLV) has not been unambiguously determined. We show here that A-MLV does not internalize by caveolae or other pinocytic mechanism, but by macropinocytosis. Thus A-MLV infection of mouse embryonic fibroblasts deficient for caveolin or dynamin, or NIH-3T3 cells knocked-down for caveolin expression, was unaffected. Conversely, A-MLV infection of NIH-3T3 and HeLa cells was sensitive to amiloride analogues and actin-depolymerizing drugs that interfere with macropinocytosis. Further manipulation of the actin cytoskeleton through conditional expression of dominant positive or negative mutants of Rac1, PAK1, and RhoG, to increase or decrease macropinocytosis, similarly correlated with an augmented or inhibited infection with A-MLV, respectively. The same experimental perturbations only mildly or not at all affected the infection of viruses that use clathrin coated-pit endocytosis or other pathways for entry. These data agree with immunofluorescence studies and cryo-immunogold labeling for electron microscopy, which demonstrate the presence of AMLV in protrusion-rich areas of the cell surface and in cortical fluid phase (dextran)-filled macropinosomes, which also account for up to a half of the cellular uptake of the cell surface-binding lectin concanavalin A. We conclude that AMLV use macropinocytosis as the predominant entry portal into cells.

    IMPORTANCE Binding and entry of virus particles into mammalian cells are the first steps of infection. Understanding how pathogens and toxins exploit or divert endocytosis pathways has advanced our understanding of membrane trafficking pathways, which benefits development of new therapeutical schemes and methods of drug delivery. We show here that Murine Leukemia Virus (A-MLV) pseudotyped with the amphotropic (expands the host range to many mammalian cells) envelope protein gains entry into host cells by macropinocytosis. Macropinosomes form as large, fluid-filled vacuoles (up to 10 mmu;m) following collapse of cell surface protrusions and membrane scission. We use drugs or introduction of mutant proteins that affect the actin cytoskeleton and cell surface dynamics to show that macropinocytosis and A-MLV infection correlate, and provide both light and electron microscopical evidence to show the localization of A-MLV in macropinosomes. Finally, we specifically exclude some other potential entry portals including caveolae, previously suggested to internalize A-MLV.

  • Nipah Virus Attachment Glycoprotein Stalk C-terminal Region Links Receptor Binding to Fusion Triggering [PublishAheadOfPrint]

  • Membrane fusion is essential for paramyxovirus entry into target cells and for the cell-cell fusion (syncytia) that results from many paramyxoviral infections. The concerted efforts of two membrane integral viral proteins, the attachment (HN or H or G) and fusion (F) glycoproteins, mediate membrane fusion. The emergent Nipah virus (NiV) is a highly pathogenic and deadly zoonotic paramyxovirus. We recently reported that upon cell receptor ephrinB2 or ephrinB3 binding, at least two conformational changes occur in the NiV-G head, followed by one in the NiV-G stalk, that subsequently result in F triggering and F execution of membrane fusion. However, the domains and residues in NiV-G that trigger F, and the specific events that link receptor binding to F triggering, are unknown. In the present study we identified a NiV-G stalk C-terminal region (aa 159-163) that is important for multiple G functions including G tetramerization, conformational integrity, G-F interactions, receptor-induced conformational changes in G, and F triggering. Based on these results, we propose that this NiV-G region serves as an important structural and functional linker between the NiV-G head and the rest of the stalk, and is critical in propagating the F-triggering signal via specific conformational changes that open a concealed F-triggering domain(s) in the G stalk. These findings broaden our understanding of the mechanism(s) of receptor-induced paramyxovirus F triggering during viral entry and cell-cell fusion.

    Importance: The emergent deadly Nipah and Hendra viruses belong to the Henipavirus genus in the Paramyxoviridae family. NiV infections target endothelial cells and neurons and, in humans, result in 40-75% mortality rates. The broad tropism of the henipaviruses and the unavailability of therapeutics threaten the health of humans and livestock. Viral entry into host cells is the first step of henipavirus infections, which ultimately cause syncytia formation. After attaching to the host cell receptor, henipaviruses enter the target cell via direct viral-cell membrane fusion mediated by two membrane glycoproteins: the attachment protein (G) and the fusion protein (F). In this study, we identified and characterized a region in the NiV-G stalk C-terminal domain that links receptor binding to fusion triggering via several important glycoprotein functions. These findings advance our understanding of the membrane fusion triggering mechanism(s) for the henipaviruses and the paramyxoviruses.

  • SAMD9 is an innate antiviral host factor with stress response properties that can be antagonized by poxviruses [PublishAheadOfPrint]

  • We show that SAMD9 is an innate host anti-viral stress response element that participates in the formation of anti-viral granules. Poxviruses, myxoma virus and vaccinia virus specifically, utilize virus-encoded host range factor(s), such as members of the C7L superfamily, to antagonize SAMD9 to prevent the granule formation in an eIF2aalpha;-independent manner. When SAMD9 is stimulated due to failure of the viral antagonism during infection, the resulting anti-viral granules exhibit properties different from canonical stress granules.

  • Unraveling the web of viroinformatics: computational tools and databases in viral research [PublishAheadOfPrint]

  • The beginning of the second century of research in the field of virology (the first virus was discovered in 1898) was marked with its amalgamation with bioinformatics resulting in the birth of a new domain - viroinformatics. The availability of more than 100 web servers and databases embracing all or specific viruses (for example dengue, influenza, hepatitis, HIV, HFV, HPV, West Nile etc.) as well as distinct applications (comparative/diversity analysis, viral recombination, siRNA/shRNA/miRNA studies, RNA folding, protein-protein interaction, structural analysis, phylotyping/genotyping) will definitely aid the development of effective drugs and vaccines. However, the information about their access and utility is not available at any single source/platform. Therefore, a compendium of various computational tools/resources dedicated specifically to virology is presented in this article.

  • HYDROPHOBIC AND CHARGED RESIDUES IN THE C-TERMINAL ARM OF HEPATITIS C VIRUS RNA-DEPENDENT RNA POLYMERASE REGULATE INITIATION AND ELONGATION. [PublishAheadOfPrint]

  • The RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV) is essential for viral genome replication. Crystal structures of the HCV RdRp reveal two C-terminal features, a bbeta;-loop and a C-terminal arm, suitably located for involvement in positioning components of the initiation complex. Here we show that these two elements intimately regulate template and nucleotide binding, initiation and elongation. We constructed a series of bbeta;-loop and C-terminal arm mutants, which were used for in vitro analysis of RdRp de novo initiation and primer extension activities. All mutants showed a substantial decrease in initiation assays but a marked increase in primer extension activities indicating an ability to form more stable elongation complexes with long primer:template RNAs. Structural studies of the mutants indicated that these enzyme properties might be attributed to an increased flexibility in the C-terminal features resulting in a more open polymerase cleft, which likely favours the elongation process but hampers the initiation steps. A UTP co-crystal structure of one mutant shows, in contrast to wild type protein, several alternate conformations of the substrate confirming that even subtle changes in the C-terminal arm result in a more loosely organized active site and flexible binding modes of the nucleotide. We used a sub-genomic replicon system to assess the effects of the same mutations on viral replication in cells. Even the subtlest mutations either severely impaired or completely abolished the ability of the replicon to replicate, further supporting the concept that correct positioning of both the bbeta;-loop and C-terminal arm play an essential role during initiation and in HCV replication in general.

    IMPORTANCE HCV RNA polymerase is a key target for the development of directly acting agents to cure HCV infections and necessitates a thorough understanding of the functional roles of the various structural features on the RdRp. Here we show that even highly conservative changes, e.g. Tyr -ggt; Phe or Asp -ggt; Glu, in these seemingly peripheral structural features have profound effects on the initiation and elongation properties of the HCV polymerase.

  • Abortive intrabronchial infection of rhesus macaques with varicella zoster virus provides partial protection against simian varicella virus challenge. [PublishAheadOfPrint]

  • Varicella zoster virus (VZV) is a human neurotropic alphaherpesvirus and the etiological agent of varicella (chickenpox) and herpes zoster (HZ, shingles). Previously, inoculation of monkeys via the subcutaneous, intratracheal, intravenous or oral-nasal-conjunctival routes did not recapitulate all the hallmarks of VZV infection including varicella, immunity, latency and reactivation. Intrabronchial inoculation of rhesus macaques (RMs) with simian varicella virus (SVV), a homolog of VZV, recapitulates virologic and immunologic hallmarks of VZV infection in humans. Given that VZV is acquired primarily via the respiratory route, we investigated whether intrabronchial inoculation of RMs with VZV would result in a robust model. Despite the lack of varicella and viral replication in either the lungs or whole blood, all four RMs generated an immune response characterized by generation of VZV-specific antibodies and T cells. Two of 4 VZV inoculated RMs were challenged with SVV to determine cross-protection. VZV-immune RMs displayed no varicella rash, decreased SVV viral loads as well as earlier and stronger humoral and cellular immune responses compared to controls. In contrast to SVV, no VZV DNA was detected in sensory ganglia at necropsy. In summary, following an abortive VZV infection, RMs developed an adaptive immune response that confers partial protection against SVV challenge. These data suggest that a replication incompetent VZV vaccine that does not establish latency may provide sufficient protection against VZV disease and that VZV vaccination of RMs followed by SVV challenge provides a model to evaluate new vaccines and therapeutics against VZV.

    Importance Although VZV vaccine strain Oka is attenuated, it can cause mild varicella, establish latency, and in rare cases reactivate to cause herpes zoster (HZ). Moreover, studies suggest that the HZ vaccine (Zostavax) only confers short-lived immunity. Development of more efficacious vaccines would be facilitated by a robust animal model of VZV infection. Data presented in this manuscript show that intrabronchial inoculation of rhesus macaques (RMs) with VZV resulted in an abortive VZV infection. Nevertheless, all animals generated a humoral and cellular immune response that conferred partial cross-protection against simian varicella virus (SVV) challenge. Additionally, VZV DNA was not detected in the sensory ganglia suggesting that viremia might be required for the establishment of latency. Therefore, VZV vaccination of RMs followed by SVV challenge is a model that will support the development of vaccines that boost protective T cell responses against VZV.

  • Early activation of teleost B cells in response to rhabdovirus infection [PublishAheadOfPrint]

  • To date, the response of B cells to specific pathogens has been only scarcely addressed in teleost. In the current work, we have demonstrated that viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus, has the capacity to infect rainbow trout spleen IgM+ cells although the infection is not productive. Consequently, we have studied the effects of VHSV on IgM+ cell functionality comparing these effects to those elicited by a TLR3 ligand, Poly I:C. We found that Poly I:C and VHSV significantly up-regulated TLR3 and type I interferon (IFN) transcription in spleen and blood IgM+ cells. Further effects included the up-regulated transcription of the CK5B chemokine. Significant inhibition of some of these effects in presence of bafilomicyn A1 (BAF), an inhibitor of endosomal acidification, suggests the involvement of an intracellular TLR in these responses. In the case of VHSV, these transcriptional effects were dependent on viral entry into B cells and initiation of viral transcription. VHSV also provoked the activation of NF-B and the up-regulation of MHC-II cell surface expression on IgM+ cells, that along with an increased transcription of the co-stimulatory molecules CD80/86 and CD83 pointed to a VHSV-induced IgM+ cell activation towards an antigen-presenting profile. Finally, despite the moderate effects of VHSV on IgM+ cell proliferation, a consistent effect on IgM+ cell survival was detected.

    IMPORTANCE Innate immune responses to pathogens established through their recognition by PRRs have been traditionally ascribed to innate cells. However, recent evidence in mammals has revealed that innate pathogen recognition by B lymphocytes is a crucial factor in shaping the type of immune response that is mounted. In teleost, these immediate effects of viral encounter on B lymphocytes have not been addressed to date. In our study, we have demonstrated that VHSV infection provoked immediate transcriptional effects in B cells, at least partially mediated by intracellular PRR signaling. VHSV also activated NF-B and increased IgM+ cell survival. Interestingly, VHSV activated B lymphocytes towards an antigen-presenting profile, suggesting an important role of IgM+ cells in VHSV presentation. Our results provide a first description of the effects provoked by fish rhabdoviruses through their early interaction with teleost B cells.

  • Construction and Evaluation of Novel Rhesus Monkey Adenovirus Vaccine Vectors [PublishAheadOfPrint]

  • Adenovirus vectors are widely used as vaccine candidates for a variety of pathogens, including HIV-1. To date, human and chimpanzee adenoviruses have been explored in detail as vaccine vectors. The phylogeny of human and chimpanzee adenoviruses are overlapping, and both exhibit pre-existing humoral and cellular immunity in human populations worldwide. More distantly related adenoviruses may therefore offer advantages as vaccine vectors. Here we describe the primary isolation and vectorization of three novel adenoviruses from rhesus monkeys. These novel rhesus adenovirus vectors exhibited extremely low seroprevalence in sub-Saharan human populations and proved comparably immunogenic to human and chimpanzee adenovirus vaccine vectors in mice. These rhesus monkey adenoviruses phylogenetically clustered with the poorly described adenovirus species G and robustly stimulated innate immune responses. These novel adenoviruses represent a new class of candidate vaccine vectors.

    Importance Although there have been substantial efforts in the development of vaccine vectors from human and chimpanzee adenoviruses, far less is known about rhesus monkey adenoviruses. In this manuscript, we describe the isolation and vectorization of three novel rhesus monkey adenoviruses. These vectors exhibit virologic and immunologic characteristics that make them attractive as potential candidate vaccine vectors for both HIV-1 and other pathogens.

  • The HIV-1 nucleocapsid protein recruits negatively charged lipids on binding to lipid membranes [PublishAheadOfPrint]

  • The HIV-1 Gag polyprotein precursor composed of the matrix (MA), capsid (CA), nucleocapsid (NC) and p6 domains orchestrates virus assembly via interactions between MA and the cell plasma membrane (PM) on one hand, and NC and the genomic RNA on the other hand. As the Gag precursor can adopt a bent conformation, a potential interaction of the NC domain with the PM cannot be excluded during Gag assembly at the PM. To investigate the possible interaction of NC with lipid membranes in the absence of any interference from the other domains of Gag, we quantitatively characterized by fluorescence spectroscopy the binding of the mature NC protein to large unilamellar vesicles (LUVs) used as membrane models. We found that NC, either in its free form or bound to an oligonucleotide, was binding with high affinity (~ 107 M-1) to negatively charged LUVs. The number of NC binding sites, but not the binding constant, was observed to decrease with the percentage of negatively charged lipids in the LUV composition, suggesting that NC and NC/oligonucleotide complexes were able to recruit negatively charged lipids to ensure optimal binding. However, in contrast to MA, NC did not exhibit a preference for phosphatidylinositol- (4, 5)-bisphosphate. These results lead us to propose a modified Gag assembly model where the NC domain contributes to the initial binding of the bent form of Gag to the PM.

    IMPORTANCE The NC protein is a highly conserved nucleic acid binding protein that plays numerous key roles in HIV-1 replication. While accumulating evidence shows that NC either as a mature protein or a domain of the Gag precursor also interacts with host proteins, only few data are available on the possible interaction of NC with lipid membranes. Interestingly, during HIV-1 assembly, the Gag precursor is thought to adopt a bent conformation where the NC domain may interact with the plasma membrane. In this context, we quantitatively characterized the binding of NC, as a free protein or as a complex with nucleic acids, to lipid membranes and evidenced that the latter constitute a binding platform for NC. Taken together, our data suggest that the NC domain may play a role in the initial binding events of Gag to the plasma membrane during HIV-1 assembly.

  • Proteomic analysis of ubiquitin-like post-translational modifications induced by the adenovirus E4-ORF3 protein [PublishAheadOfPrint]

  • Viruses interact with and regulate many host metabolic pathways in order to advance the viral life cycle and counteract intrinsic and extrinsic antiviral responses. The human Adenovirus (Ad) early protein, E4-ORF3, forms a unique scaffold throughout the nuclei of infected cells and inhibits multiple antiviral defenses including a DNA damage response (DDR) and an interferon response. We previously reported that the Ad5 E4-ORF3 protein induces sumoylation of Mre11 and Nbs1, which are essential for the DDR, and their relocalization into E4-ORF3-induced nuclear inclusions is required for this modification to occur. In this study, we sought to analyze a global change in ubiquitin-like (Ubl) modifications, with particular focus on SUMO3, by the Ad5 E4-ORF3 protein and identify new substrates with these modifications. By a comparative proteome-wide approach utilizing immunoprecipitation/mass spectrometry, we found that Ubl modifications of 166 statistically significant lysine sites in 51 proteins are affected by E4-ORF3 and the proteome of modifications spans a diverse range of cellular functions. Ubl modifications of 92% of these identified sites were increased by E4-ORF3. We further analyzed SUMO3 conjugation of several identified proteins. Our findings demonstrate a role for the Ad5 E4-ORF3 protein as a regulator of Ubl modifications and reveal new SUMO3 substrates induced by E4-ORF3.

    IMPORTANCE The Adenovirus E4-ORF3 protein induces dynamic structural changes in the nuclei of infected cells and counteracts host antiviral responses. One of the mechanisms that accounts for this process is the relocalization and sequestration of cellular proteins into an E4-ORF3 nuclear scaffold, but little is known how this small viral protein affects diverse cellular responses. In this study, we analyzed for the first time the global pattern of Ubiquitin-like (Ubl) modifications, with particular focus on SUMO3, altered by E4-ORF3 expression. The results suggest a role for the Ad5 E4-ORF3 protein as a regulator of Ubl modifications and reveal new SUMO3 substrates targeted by E4-ORF3. Our findings propose Ubl modifications as a new mechanism by which E4-ORF3 may modulate cellular protein functions in addition to subnuclear relocalization.

  • The Large Tegument Protein pUL36 is Essential for Formation of the Capsid Vertex Specific Component at the Capsid-Tegument Interface of HSV-1. [PublishAheadOfPrint]

  • Herpesviruses have a characteristic particle structure comprising an icosahedral capsid, which contains the DNA genome and is in turn surrounded by a proteinaceous tegument layer and a lipid envelope. In herpes simplex virus the interaction between the capsid and tegument is limited to the capsid vertices and involves two minor capsid proteins, pUL17 and pUL25, and the large, inner tegument protein, pUL36. pUL17 and pUL25 form a heterodimeric structure, the capsid vertex specific component (CVSC), that lies on top of the peripentonal triplexes, while pUL36 has been reported to connect the CVSC to the penton. In this study we use virus mutants with deletions in the genes for pUL36 and another inner tegument protein, pUL37, to analyse the contributions of these proteins to CVSC structure. Using electron cryo-microscopy and icosahedral reconstruction of mutants that express pUL17 and pUL25, but not pUL36, we show that, in contrast to accepted models, the CVSC is not formed from pUL17 and pUL25 on their own but requires a contribution from pUL36. In addition, the presence of full-length pUL36 results in weak density that extends the CVSC towards the penton, suggesting, either that this extra density is formed directly by pUL36, or that pUL36 is stabilising other components of the vertex-tegument interface.

    IMPORTANCE Herpesviruses have complex particles that are formed as a result of a carefully controlled sequence of assembly steps. The nature of the interaction between two of the major particle compartments, the icosahedral capsid and the amorphous tegument, has been extensively studied, but the identity of the interacting proteins and their roles in forming the connections are still unclear. In this study we use electron microscopy and 3D reconstruction to analyse virus particles formed by mutants that do not express particular interacting proteins. We show that the largest viral protein, pUL36, which occupies the layer of tegument closest to the capsid, is essential for formation of structurally normal connections to the capsid. This demonstrates the importance of pUL36 in the initial stages of tegument addition and provides new insights into the process of virus particle assembly.

  • BGLF4 Kinase Modulates the Structure and Transport Preference of the Nuclear Pore Complex to Facilitate Nuclear Import of Epstein-Barr Virus Lytic Proteins [PublishAheadOfPrint]

  • BGLF4 kinase, the only Ser/Thr protein kinase encoded by EBV, phosphorylates multiple viral and cellular substrates to optimize the cellular environment for viral DNA replication and the nuclear egress of nucleocapsids. Previously, we found that nuclear targeting of BGLF4 is through direct interaction with the FG repeat-containing nucleoporins (FG-Nups) Nup62 and Nup153, independent of cytosolic transport factors. Here, we investigated the regulatory effects of BGLF4 on the structure and biological functions of the nuclear pore complex (NPC). In EBV positive NA cells, the distribution of FG-Nups was modified during EBV reactivation. In transfected cells, BGLF4 changed the staining pattern of Nup62 and Nup153 in a kinase activity-dependent manner. Detection with anti-phospho-Ser/Thr-Pro MPM-2 antibody demonstrated that BGLF4 induced the phosphorylation of Nup62 and Nup153. The nuclear targeting of importin-bbeta; was attenuated in the presence of BGLF4, leading to inhibition of canonical nuclear localization signal (NLS)-mediated nuclear import. An in vitro nuclear import assay revealed that BGLF4 induced the nuclear import of larger molecules. Notably, we found BGLF4 promoted the nuclear import of several non-NLS containing EBV proteins, including the viral DNA replicating enzymes BSLF1, BBLF2/3 and BBLF4 and the major capsid protein (VCA), in co-transfected cells. Data here suggest that BGLF4 interferes with the normal functions of Nup62 and Nup153 and preferentially helps the nuclear import of viral proteins for viral DNA replication and assembly. In addition, the nuclear import promoting activity was found in cells expressing the BGLF4 homologs of another two gamma-herpesviruses, but not those from alpha- and beta-herpesviruses.

    Importance During lytic replication, many EBV encoded proteins need to be transported into the nucleus, not only for viral DNA replication but also for the assembly of nucleocapsids. Because nuclear pore complexes are effective gateways that control nucleocytoplasmic traffic, most EBV proteins without canonical-NLS signals are retained in the cytoplasm until they form complexes with their NLS-containing partners for nuclear targeting. In this study, we found EBV BGLF4 protein kinase interacts with the Nup62 and Nup153 and induces redistribution of FG-Nups. BGLF4 modulates the function of the NPC to inhibit the nuclear import of host NLS-containing proteins. Simultaneously, the nuclear import of non-NLS-containing EBV lytic proteins was enhanced, possibly through phosphorylation of Nup62 and Nup153, nuclear pore dilation or microtubule reorganization. Overall, our data suggest that BGLF4-induced modification of nuclear pore transport may block nuclear targeting of cellular proteins and increase the import of viral proteins to promote viral lytic replication.

  • GBF1- and ACBD3-independent recruitment of PI4KIII{beta} to replication sites by rhinovirus 3A proteins [PublishAheadOfPrint]

  • PI4KIIIbbeta; recruitment to Golgi membranes relies on GBF1/Arf and ACBD3. Enteroviruses like poliovirus and coxsackievirus recruit PI4KIIIbbeta; to their replication sites via their 3A proteins. Here, we show that human rhinovirus (HRV) 3A also recruited PI4KIIIbbeta; to replication sites. Unlike other enterovirus 3A proteins, HRV 3A failed to bind GBF1. Although previously HRV 3A was shown to interact with ACBD3, our data suggest that PI4KIIIbbeta; recruitment occurred independently of both GBF1 and ACBD3.

  • The evolution and evolutionary relationships between extant vaccinia virus strains [PublishAheadOfPrint]

  • Although vaccinia virus (VACV) was once used as a vaccine to eradicate smallpox on a worldwide scale, the biological origins of VACV are uncertain, as are the historical relationships between the different strains once used as smallpox vaccines. Here, we sequenced additional VACV strains that represent either relatively pristine examples of old vaccines (e.g. Dryvax, Lister, and Tashkent) or which have been subjected to additional laboratory passage (e.g. IHD-W and WR). These genome sequences were compared with those previously reported for other VACV as well as other orthopoxviruses. These extant VACV do not always cluster in simple phylogenetic trees that are aligned with the known historical relationships between these strains. Rather, the pattern of deletions suggests that all existing strains likely come from a complex stock of viruses that has been passaged, distributed, and randomly sampled over time, thus obscuring simple historical or geographic links. We examined surviving non-clonal vaccine stocks, like Dryvax, which continue to harbor larger and now rare variants including one we have designated "clone DPP25". DPP25 encodes genes not found in most VACV strains including an ankyrin-F-box protein, a homolog of the variola virus (Bangladesh) B18R gene, which we show can be deleted without affecting virulence in mice. We propose a simple common mechanism by which recombination of a larger and hypothetical DPP25-like ancestral strain, combined with selection for retention of critically important genes near the terminal inverted repeat boundaries (vaccinia growth factor gene and an interferon aalpha;/bbeta; receptor homolog), could produce all known VACV variants.

    IMPORTANCE Smallpox was eradicated using a combination of intensive disease surveillance and vaccination using vaccinia virus (VACV). Interestingly, little is known about the historical relationships between different strains of vaccinia virus (VACV) and how these viruses may have evolved from a common ancestral strain. To understand these relationships additional strains were sequenced and compared to existing strains of VACV as well as other Orthopoxviruses using whole genome sequence alignments. Extant strains of VACV didn't always cluster in simple phylogenetic trees based on known historical relationships between these strains. Based on these findings it is possible that all existing strains of VACV could derive from a single complex stock of viruses that has been passaged, distributed, and sampled over time.

  • Cellular differentiation regulator BLIMP1 induces Epstein-Barr virus lytic reactivation in epithelial and B cells by activating transcription from both the R and Z promoters [PublishAheadOfPrint]

  • EBV maintains a life-long latent infection within a subset of its host's memory B cells, while lytic EBV replication takes place in plasma cells and differentiated epithelial cells. Therefore, cellular transcription factors such as BLIMP1 that are key mediators of differentiation likely contribute to the EBV latent-to-lytic switch. Previous reports showed that ectopic BLIMP1 expression induces reactivation in some EBV(+) B-cell lines and transcription from Zp, with all Z(+) cells in oral hairy leukoplakia being BLIMP1(+). Here, we examined BLIMP1's role in inducing EBV lytic-gene expression in numerous EBV(+) epithelial and B-cell lines and activating transcription from Rp. BLIMP1 addition was sufficient to induce reactivation in latently infected epithelial cells derived from gastric cancers, nasopharyngeal carcinomas, and normal oral keratinocytes (NOK) as well as some, but not all B-cell lines. BLIMP1 strongly induced transcription from Rp as well as Zp, with there being three or more synergistically acting BLIMP1-responsive elements (BRE) within Rp. BLIMP1's DNA-binding domain was required for reactivation, but BLIMP1 did not directly bind the nt -660 Rp BRE. siRNA knockdown of BLIMP1 inhibited TPA-induced lytic reactivation in NOK-Akata cells, cells that can be reactivated by R, but not Z. Thus, we conclude that BLIMP1 expression is both necessary and sufficient to induce EBV lytic replication in many (possibly all) EBV(+) epithelial-cell types, but in only a subset of EBV(+) B-cell types; it does so, at least in part, by strongly activating expression of both EBV immediately-early genes, BZLF1 and BRLF1.

    Importance: This study is the first one to show that the cellular transcription factor BLIMP1, a key player in both epithelial and B-cell differentiation, induces reactivation of the oncogenic herpes virus, Epstein-Barr virus (EBV), out of latency into lytic replication in a variety of cancerous epithelial cell types as well as in some, but not all B-cell types that contain this virus in a dormant state. The mechanism by which BLIMP1 does so involves strongly turning on expression of both of the immediate-early genes of the virus, probably by directly acting upon the promoters as part of protein complexes or indirectly by altering the expression or activities of some cellular transcription factors and signaling pathways. The fact that EBV(+) cancers usually contain mostly undifferentiated cells may be due in part to these cells dying from lytic EBV infection when they differentiate and express wild-type BLIMP1.

  • Deletion mutants of Schmallenberg virus are avirulent and protect from virus challenge [PublishAheadOfPrint]

  • Since its emergence, Schmallenberg virus (SBV), a novel insect-transmitted orthobunyavirus which predominantly infects ruminants, caused a large epidemic in European livestock. Newly developed inactivated vaccines are available, but highly efficacious and safe live vaccines are still missing. Here, the properties of novel recombinant SBV-mutants lacking the non-structural proteins NSs (rSBVNSs), NSm (rSBVNSm) or both of them (rSBVNSs/NSm) were tested in vitro and in vivo in type I interferon receptor knock-out mice (IFNAR-/-) and in a vaccination/challenge trial in cattle. As for other bunyaviruses, both non-structural proteins of SBV are not essential for viral growth in vitro. In interferon-defective BHK-21 cells rSBVNSs and rSBVNSm replicated to comparable level as the parental rSBV, the double mutant virus, however, showed a mild growth defect resulting in lower final virus titers. Additionally, both mutants with a NSs-deletion induced high levels of interferon and showed a marked growth defect in interferon-competent sheep SFT-R cells. Nevertheless, in IFNAR-/- mice, all mutants were virulent, with the highest mortality rate for rSBVNSs and a reduced virulence for the NSm-deleted virus. In cattle, SBV lacking NSm caused viraemia and seroconversion comparable to wild-type virus, while the NSs and the combined NSs/NSm deletion mutant induced no detectable virus replication or clinical disease after immunization. Furthermore, three out of four cattle immunized once with the NSs deletion mutant and all animals vaccinated with the virus lacking both non-structural proteins were fully protected against a challenge infection. Therefore, the double deletion mutant will provide the basis for further developments of safe and efficacious modified live SBV-vaccines which could be also a model for other viruses of the Simbu-serogroup and related orthobunyaviruses.

    Importance SBV induces only mild clinical signs in adult ruminants, but causes severe fetal malformation and, thereby, can have an important impact on animal welfare and production. As SBV is an insect-transmitted pathogen, vaccination will be one of the most important aspects of disease control. Here, mutant viruses lacking one or two proteins that essentially contribute to viral pathogenicity were tested as modified live vaccines in cattle. It could be demonstrated, that a novel recombinant double deletion mutant is a safe and efficacious vaccine candidate. This is the first description of a putative modified live vaccine for the complete genus Orthobunyavirus and, in addition, such a vaccine type has never been tested in cattle for any virus of the entire family Bunyaviridae. Therefore, the described vaccine represents also a first model for a broad range of related viruses, and is of high importance to the field.

  • p38MAPK and MK2 pathways are important for the differentiation-dependent HPV life cycle [PublishAheadOfPrint]

  • Amplification of human papillomaviruses (HPV) is dependent on the ATM DNA damage pathway. In cells with impaired p53 activity, DNA damage repair requires the activation of p38MAPK along with MAPKAP kinase 2 (MK2). In HPV positive cells, phosphorylation of p38 and MK2 proteins was induced along with relocalization to the cytoplasm. Treatment with MK2 or p38 inhibitors blocked HPV genome amplification identifying the p38/MK2 pathway as a key regulator of the HPV life cycle.

  • Adeno-associated virus serotypes 1 and 5 - antibody complex structures reveal evolutionary commonalities in parvovirus antigenic reactivity [PublishAheadOfPrint]

  • The clinical utility of the Adeno-associated virus (AAV) gene delivery system has been validated by the regulatory approval of an AAV serotype 1 (AAV1) vector for the treatment of lipoprotein lipase deficiency. However, neutralization from pre-existing antibodies is detrimental to AAV transduction efficiency. Hence, mapping AAV antigenic sites and engineering neutralization-escaping vectors is important for improving clinical efficacy. We report the structures for four AAV:monoclonal antibody fragment complexes: AAV1:ADK1a, AAV1:ADK1b, AAV5:ADK5a, and AAV5:ADK5b, determined by cryo-electron microscopy and image reconstruction to ~11-12 AAring; resolution. Pseudo-atomic modeling mapped the ADK1a epitope to the protrusions surrounding the icosahedral 3-fold axis, the ADK1b and ADK5a epitopes, which overlap, to the wall between depressions at the 2- and 5-fold axes (2/5-fold wall), and the ADK5b epitope spans both the 5-fold facing wall of the 3-fold protrusion and portions of the 2/5-fold wall of the capsid. Combined with the six antigenic sites previously elucidated for different AAV serotypes through structural approaches, including AAV1 and AAV5, this study identified two common AAV epitopes: one on the 3-fold protrusions and one on the 2/5-fold wall. These epitopes coincide with regions with the highest sequence and structure diversity between AAV serotypes and correspond to regions determining receptor recognition and transduction phenotypes. Significantly, these locations overlap with the two dominant epitopes reported for autonomous parvoviruses. Thus rather than amino acid sequence alone, the antigenic sites of parvoviruses appear to be dictated by structural features evolved to enable specific infectious functions.

    Importance The Adeno-associated viruses (AAVs) are promising vectors for in vivo therapeutic gene delivery with more than 20 years of intense research now realized in a number of successful human clinical trials that report therapeutic efficacy. However, a large percentage of the population has pre-existing AAV capsid antibodies and therefore must be excluded from clinical trials or re-administration of vector. This manuscript represents our continuing efforts to understand the antigenic structure of the AAVs, specifically to obtain a picture of the "polyclonal" reactivity as is the situation in humans. It describes the structures of four AAV-antibody complexes determined by cryo-electron microscopy and image reconstruction, increasing the number of mapped epitopes to four and three, respectively, for AAV1 and AAV5, two vectors currently in clinical trials. The results presented provide essential information for generating antigenic escape vectors towards overcoming a critical challenge remaining in optimization of this highly promising vector delivery system.

  • Identification of the physiological gene targets of the essential lytic replicative KSHV ORF57 protein. [PublishAheadOfPrint]

  • The KSHV ORF57 gene product is essential for lytic KSHV replication and virion production. Recombinant ORF57 null mutants fail to accumulate several lytic cycle mRNAs at wild-type levels, leading to decreased production of lytic proteins necessary for efficient replication. Several mechanisms by which ORF57 may enhance expression of lytic KSHV mRNAs have been proposed, including mRNA stabilization, mRNA nuclear export, increased polyadenylation, and transcriptional activation. ORF57 activity is also gene specific, with some genes being highly dependent on ORF57, whereas others are relatively independent. Most experiments have utilized transfection models for ORF57 and have not systematically examined the gene specificity and potential mechanisms of action of ORF57 in the context of KSHV infected cells. In this study, the KSHV genes that are most highly upregulated by ORF57 during KSHV lytic replication were identified by a combination of high-throughput deep RNA sequencing, qPCR, Northern blotting and RACE methods. Comparison of gene expression from a ORF57 KSHV recombinant, rescued ORF57 KSHV recombinant and wild-type KSHV revealed that two clusters of lytic genes are most highly dependent on ORF57 for efficient expression. Despite contiguous location in the genome and shared polyadenylation of several of the ORF57 dependent genes, ORF57 regulation was promoter and polyadenylation signal-independent, suggesting that the mRNAs are stabilized by ORF57. The eight genes identified as critically requiring ORF57 belong to both early and late lytic temporal classes, and seven are involved in DNA replication, virion assembly or viral infectivity, explaining the essential role of ORF57 in infectious KSHV production.

    IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is a human herpesvirus involved in causation of several human cancers. The KSHV ORF57 protein is required for KSHV to replicate and produce infectious virus. We have identified several KSHV genes whose expression is highly dependent on ORF57 and shown that ORF57 increases expression of these genes specifically. These genes code for proteins that are required for the virus to replicate its DNA and to infect other cells. Identifying the targets and mechanism of action of ORF57 provides further approaches to discover antiviral therapy.

  • Nuclear import of adenovirus DNA involves direct interaction of hexon with an N-terminal domain of the nucleoporin Nup214 [PublishAheadOfPrint]

  • In this study, we characterized the molecular basis for binding of adenovirus (AdV) to the cytoplasmic face of the nuclear pore complex (NPC), a key step during delivery of the viral genome into the nucleus. We used RNAi to deplete cells of either Nup214 or Nup358, the two major FG repeat nucleoporins localized on the cytoplasmic side of the NPC, and evaluated the impact on hexon binding and AdV infection. The accumulation of purified hexon trimers or partially disassembled AdV at the nuclear envelope (NE) was observed in digitonin permeabilized cells in the absence of cytosolic factors. Both in vitro hexon binding and in vivo nuclear import of the AdV genome were strongly reduced in Nup214-depleted cells but still occurred in Nup358-depleted cells, suggesting that Nup214 is a major binding site of AdV during infection. The expression of an NPC-targeted N-terminal domain of Nup214 in Nup214-depleted cells restored the binding of hexon at the NE and the nuclear import of pVII, indicating that this region is sufficient to allow AdV binding. We further narrowed the binding site to a 137 amino acid segment in N-terminal domain of Nup214. Together, our results have identified a specific region within the N-terminus of Nup214 that acts as a direct NPC binding site for AdV.

    Importance AdV, which have the largest genome of non-enveloped DNA viruses, are being extensively explored for use in gene therapy, especially in alternative treatments for cancers that are refractory to traditional therapies. In this study, we characterized the molecular basis for binding of AdV to the cytoplasmic face of the NPC, a key step for delivery of the viral genome into the nucleus. Our data indicate that a 137 amino acid region of the nucleoporin Nup214 is a binding site for the major AdV capsid protein hexon, and that this interaction is required for viral DNA import. These findings provide additional insight on how AdV exploits the nuclear transport machinery for infection. The results could promote the development of new strategies for gene transfer, and enhance understanding of the nuclear import of other viral DNA genomes, such as those of papillomavirus or hepatitis B virus that induce specific cancers.

  • Global displacement of canine parvovirus by a host-adapted variant: A structural comparison between pandemic viruses with distinct host ranges [PublishAheadOfPrint]

  • Canine parvovirus (CPV-2) emerged in 1978 and spread worldwide within two years. Subsequently, CPV-2 was completely replaced by the variant CPV-2a, which is characterized by four specific capsid (VP2) mutations. The X-ray crystal structure of the CPV-2a capsid shows that each mutation confers small local changes. The loss of a hydrogen bond and introduction of a glycine residue likely introduce flexibility to sites that control interactions with the host receptor, antibodies, and sialic acids.

  • The Focal Adhesion kinase is involved in rabies virus infection through its interaction with the viral phosphoprotein P [PublishAheadOfPrint]

  • The rabies virus (RABV) phosphoprotein P is a multifunctional protein: it plays an essential role in viral transcription and replication, and in addition RABV P has been identified as an interferon antagonist. Here, a yeast two hybrid screen revealed that RABV P interacts with the focal adhesion kinase (FAK). The binding involved the domain 106-131 corresponding to the dimerization domain of P and the C-ter domain of FAK containing the proline rich domains PRR2 and PRR3. The P-FAK interaction was confirmed in infected cells by co-immunoprecipitation and co-localization of FAK with P in Negri bodies. By alanine scanning, we identified a single mutation in the P protein that abolishes this interaction. The mutant virus containing a substitution of Arg to Ala in position 109 (PR109A) in P, which did not interact with FAK, is affected at a post-transcriptional step involving protein synthesis and viral RNA replication. Furthermore, FAK depletion inhibited viral protein expression in infected cells. This provides the first evidence of an interaction of RAV with FAK which positively regulates infection.

    Importance Rabies virus exhibits small-sized genome that encodes a limited number of viral proteins. To maintain efficient virus replication, some of them are multifunctional such as the phosphoprotein P. Indeed, we and others have shown that P establishes complex networks of interactions with host cell components. These interactions have revealed much about the role of P and about host-pathogen interactions in infected cells. Here, we identified another cellular partner of P, the Focal Adhesion Kinase (FAK). Our data shed light on the implication of FAK in RAV infection and provide evidence that P-FAK interaction has a proviral function.

  • Syntaxin 5-Dependent Retrograde Transport to the Golgi is Required for AAV Transduction. [PublishAheadOfPrint]

  • Intracellular transport of recombinant Adeno-associated virus (AAV) is still incompletely understood. In particular, the trafficking steps preceding the release of incoming AAV particles from the endosomal system into the cytoplasmmmdash;allowing subsequent nuclear import and the initiation of gene expressionmmdash;remain to be elucidated fully. Others and we previously showed that a significant proportion of viral particles are transported to the Golgi apparatus, and that Golgi disruption caused by the drug brefeldin A efficiently blocks AAV2 transduction. However, because brefeldin A is known to exert pleiotropic effects on the entire endosomal system, the functional relevance of Golgi transport for AAV transduction remains to be established definitively. Here, we show that AAV2 trafficking toward the trans-Golgi network (TGN) and the Golgi apparatus correlates with transduction efficiency and relies on a non-classical retrograde transport pathway that is independent of retromer, late endosomes and recycling endosomes. AAV2 transduction is unaffected by knock-down of syntaxins 6 and 16, which are two major effectors in the retrograde transport of both exogenous and endogenous cargo. On the other hand, inhibition of syntaxin 5 function by siRNA silencing or treatment with Retro-2cycl strongly decrease AAV2 transduction and Golgi transport. This inhibition of transduction is observed with several AAV serotypes and a number of primary and immortalized cells. Together, our data strongly suggest that syntaxin 5-mediated retrograde transport to the Golgi is a broadly conserved feature of AAV trafficking that appears to be independent of the identity of the receptors used for viral attachment.

    IMPORTANCE Gene therapy constitutes a promising approach for treatment of life-threatening conditions refractory to any other form of remedy. Adeno-associated virus (AAV) vectors are currently being evaluated for the treatment of diseases such as Duchenne Muscular Dystrophy, hemophilia, heart failure, Parkinson disease and others. Despite their promise as gene delivery vehicles, a better understanding of the biology of AAV-based vectors is necessary to improve further their efficacy. AAV vectors must reach the nucleus in order to deliver their genome, and their intracellular transport is not fully understood. Here, we dissect an important step of the intracellular journey of AAV by showing that retrograde transport of capsids to the trans-Golgi network is necessary for gene delivery. We show that the AAV trafficking route differs from known Golgi-targeted cargos, and we raise the possibility that this non-classical pathway is shared by most AAV variants, regardless of their attachment receptors.

  • Identification and mutagenesis of the AAV5 sialic acid binding region [PublishAheadOfPrint]

  • As a genus, the dependoviruses use a diverse group of cell surface carbohydrates for attachment and entry. Despite the fact that a majority of AAVs utilize sialic acid (SIA) for binding and transduction, this virus:carbohydrate interaction is poorly understood. Utilizing X-ray crystallography, two SIA binding regions were mapped for AAV5. The first site mapped to the depression in the center of the threefold axis of symmetry, while the second site was located under the HI loop close to the fivefold axis. Mutagenesis of amino acids 569 and 585 or 587 within the threefold depression resulted in elimination or alteration in SIA dependent transduction, respectively. This change in SIA binding was confirmed using glycan microarrays. Mutagenesis of the second site identified a role in transduction that was SIA independent. Further studies of the mutants at the threefold site demonstrated a change in transduction activity and cell tropism in vivo as well as resistance to neutralization by a polycloncal antibody raised against the wild type virus.

    Importance Despite that a majority of AAVs utilize sialic acid for binding and transduction, this virus: carbohydrate interaction is poorly understood. Utilizing X-ray crystallography, the sialic acid binding regions of AAV5 were identified and study using a variety of approaches. Mutagenesis of this region resulted in elimination or alteration in sialic acid dependent transduction in cell lines. This change in sialic acid glycan binding was confirmed using glycan arrays. Further study also demonstrated a change in transduction and activity and cell tropism in vivo as well as resistance to neutralization by antibodies raised against the wild type virus.

  • A live attenuated equine H3N8 influenza vaccine is highly immunogenic and efficacious in mice and ferrets [PublishAheadOfPrint]

  • Equine influenza viruses (EIV) are responsible for rapidly spreading outbreaks of respiratory disease in horses. Although natural infection of humans with EIV have not been reported, experimental inoculation of humans with these viruses can lead to a productive infection and elicit a neutralizing antibody response. Moreover, EIV have crossed the species barrier to infect dogs, pigs and camels and therefore may also pose a threat to humans. Based on serologic cross-reactivity of H3N8 EIV from different lineages and sublineages, A/equine/Georgia/1/1981 (eq/GA/81) was selected to produce a live attenuated candidate vaccine by reverse genetics with the hemagglutinin and neuraminidase genes of the eq/GA/81 wild-type (wt) virus and the six internal protein genes of the cold-adapted (ca) A/Ann Arbor/6/60 ca (H2N2) vaccine donor virus, which is the backbone of the licensed seasonal live attenuated influenza vaccine. In both mice and ferrets, intranasal administration of a single dose of the eq/GA/81 ca vaccine virus induced neutralizing antibodies and conferred complete protection from homologous wt virus challenge in the upper respiratory tract. One dose of the eq/GA/81 ca vaccine also induced neutralizing antibodies and conferred complete protection in mice and nearly complete protection in ferrets upon heterologous challenge with the H3N8 (eq/Newmarket/03) wt virus. These data support further evaluation of the eq/GA/81 ca vaccine in humans for use in the event of transmission of an equine H3N8 influenza virus to humans.

    IMPORTANCE Equine influenza viruses have crossed the species barrier to infect other mammals such as dogs, pigs and camels and therefore may also pose a threat to humans. We believe that it is important to develop vaccines against equine influenza viruses in the event that an EIV evolves, adapts and spreads in humans causing disease. We generated a live attenuated H3N8 vaccine candidate and demonstrated that the vaccine was immunogenic and protected mice and ferrets against homologous and heterologous EIV.

  • Andes Virus Nucleocapsid Protein Interrupts PKR Dimerization to Counteract The Host Interference in Viral Protein Synthesis. [PublishAheadOfPrint]

  • Pathogenic hantaviruses delay type I interferon response during early stages of viral infection. However, the robust interferon response and induction of interferon stimulated genes, observed during later stages of hantavirus infection fails to combat the virus replication in infected cells. Protein kinase R, a classical interferon stimulated gene product phosphorylates the eukaryotic translation initiation factor eIF2aalpha; and causes translation shutdown to create roadblocks for the synthesis of viral proteins. The PKR induced translation shutdown helps host cells to establish an antiviral state to interrupt virus replication. However, hantavirus infected cells do not undergo translation shutdown and fail to establish an antiviral state during the course of viral infection. Here we show for the first time that Andes virus infection induced PKR over-expression. However, the over-expressed PKR was not active due to significant inhibition of autophosphorylation. Further studies revealed that Andes virus nucleocapsid protein inhibited PKR dimerization, a critical step required for PKR autophosphorylation to attain activity. The studies reported here have established hantavirus nucleocapsid protein as a new PKR inhibitor. These studies have provided mechanistic insights for hantavirus resistance to host interferon response and have solved the puzzle for the lack of translation shutdown observed in hantavirus infected cells. The sensitivity of hantavirus replication to PKR has likely imposed a selective evolutionary pressure on hantaviruses to evade PKR antiviral response for survival. We envision that evasion of PKR antiviral response by NP has likely helped hantaviruses to exist during evolution and survive in infected hosts having multifaceted antiviral defense.

    Importance Protein kinase R, a versatile antiviral host factor shuts down the translation machinery upon activation in virus-infected cells to create hurdles for the manufacture of viral proteins. The studies reported in this manuscript reveal that hantavirus nucleocapsid protein counteracts PKR antiviral response by inhibiting PKR dimerization, required for its activation. We report the discovery of a new PKR inhibitor whose expression in hantavirus infected cells prevents the PKR induced host translation shutdown to ensure the continuous synthesis of viral proteins, required for efficient virus replication.

  • Identification of RISC-associated adenoviral miRNAs, a subset of their direct targets, and global changes in the targetome upon lytic Ad5 infection [PublishAheadOfPrint]

  • Adenoviruses encode a set of highly abundant miRNAs (mivaRNAs), which are generated by Dicer-mediated cleavage of the larger non-coding virus-associated RNAs (VA RNAs) I and II. We performed deep RNA sequencing to thoroughly investigate the relative abundance of individual single strands of mivaRNA isoforms in human A549 cells lytically infected with human adenovirus 5 (Ad5) at physiologically relevant MOIs. In addition, we investigated their relative abundance in the endogenous RNA-induced silencing complexes (RISCs). The occupation of endogenous RISCs by mivaRNAs turned out to be pronounced but not as dominant as previously inferred from experiments with AGO2-overexpressing cells infected at high MOIs. In parallel, levels of RISC-incorporated mRNAs were investigated as well. Analysis of mRNAs enriched in RISCs in Ad5-infected cells revealed that only mRNAs with complementarity to the seed sequences of mivaRNAs derived from VA RNAI but not VA RNAII were overrepresented among them, indicating that only mivaRNAs derived from VA RNAI are likely to contribute substantially to the post-transcriptional downregulation of host gene expression. Furthermore, to generate a comprehensive picture of the entire transcriptome/targetome in lytically infected cells we determined changes in cellular miRNA levels in both total RNA/RISC RNA as well, and bioinformatical analysis of mRNAs of total RNA/RISC fractions revealed a general, genome-wide, trend toward detargeting of cellular mRNAs upon infection. Lastly, we identified direct targets of both single strands of a VA RNAI-derived mivaRNA that constituted one of the 2 most abundant isoforms in RISCs of lytically infected A549 cells.

    Importance Viral and cellular miRNAs have been recognized as important players in virus-host interactions. This work provides the currently most comprehensive picture of the entire mRNA/miRNA transcriptome and of the complete RISC-targetome during lytic adenovirus infection, and thus represents the basis for a deeper understanding of the interplay between the virus and the cellular RNA interference machinery. Our data suggest that, at least in the model system that was employed, lytic infection by Ad5 is accompanied by a measurable global net-detargeting effect on cellular mRNAs, and analysis of RISC-associated viral small RNAs revealed that the VA RNAs are the only source of virus-encoded miRNAs. Moreover this work allows to assess the power of individual viral miRNAs to regulate cellular gene expression and provides a list of proven and putative direct targets of these miRNAs, which is of importance, given the fact that information about validated targets of adenovirus-encoded miRNAs is scarce.

  • Microparticles in Dengue Infection Provide a Novel Biomarker to Predict Severe Clinical Outcomes [PublishAheadOfPrint]

  • Shedding of microparticles (MPs) is a consequence of apoptotic cell death and cellular activation. Low levels of circulating MPs in blood help maintain homeostasis whereas increased MP generation is linked to many pathologic conditions. Herein, we investigated the role of MPs in dengue virus (DENV) infection. Infection of various susceptible cells by DENV led to apoptotic death and MP release. These MPs harbored a viral envelope protein and a nonstructural protein 1 (NS1) on their surfaces. Ex vivo analysis of clinical specimens of patients with different degrees of severity at multiple time points revealed that MPs generated from erythrocytes and platelets are two major MP populations in the circulation of DENV-infected patients. Elevated levels of red blood cell-derived MPs (RMPs) directly correlated with DENV disease severity whereas a significant decrease in platelet-derived MPs was associated with a bleeding tendency. Removal by mononuclear cells of complement opsonized NS1-anti NS1 immune complexes bound to erythrocytes via complement receptor 1 triggered MP shedding in vitro nndash; a process that could explain increased RMPs in severe dengue. These findings point to the multiple roles of MPs in dengue pathogenesis. They offer a potential novel biomarker candidate capable of differentiating dengue fever from the more serious dengue hemorrhagic fever.

    Importance Dengue is the most important mosquito-transmitted viral disease in the world. No vaccines or specific treatments are available. Rapid diagnosis and immediate treatment are the keys to achieve a positive outcome. Dengue virus (DENV) infection, like some other medical conditions, changes the level and composition of microparticles (MPs) - tiny bag-like structures which are normally present at low levels in the blood of healthy individuals. This study investigated how MPs in culture and patient blood are changed in response to DENV infection. Infection of cells led to programed cell death and MP release. In patients' blood, the majority of MPs originated from red blood cells and platelets. Decreased platelet-derived MPs were associated with bleeding tendency while increased red blood cell-derived MPs (RMPs) correlated with disease severity. Importantly, the level of RMPs at the early acute period could serve as a biomarker to identify potentially severe patients who require immediate care.

  • Cancer-Causing Human Papillomavirus E6 Proteins Display Major Differences in the Phospho-Regulation of their PDZ Interactions. [PublishAheadOfPrint]

  • Previous studies have shown that that the cancer-causing high risk HPV E6 oncoproteins have PDZ binding potential, an activity which is important for their ability to support the viral life cycle and to cooperate in the induction of malignancy. However, PDZ interactions are not constitutive, and can be negatively regulated by phosphorylation within the E6 PDZ binding motif (PBM). In this study we have investigated the differential regulation of the HPV E6 PBM from diverse high-risk HPV types. We show that, depending on the HPV type, PDZ binding activity can be regulated by phosphorylation with PKA or AKT, which in turn inhibits PDZ recognition. Such regulation is highly conserved between E6 proteins derived from HPV-16, HPV-18 and HPV-58, whilst being somewhat weaker or absent from other types such as HPV-31, 33 and 51. In the case of HPV31, PKA phosphorylation occurs within the core of the E6 protein and has no effect on PDZ interactions, and this demonstrates a surprising degree of heterogeneity amongst the different high risk HPV E6 oncoproteins in how they are regulated by different cellular signaling pathways.

    Importance This study demonstrates that the cancer-causing HPV E6 oncoproteins are all subject to post translational modification of their extreme C terminal PDZ binding motif through phosphorylation. However the identity of the kinase is not the same for all HPV types. This demonstrates a very important divergence between these HPVs, and it suggests that changes in cell signalling pathways will have different consequences for different high risk virus infections and their associated malignancies.

  • Dual Pro-inflammatory and Antiviral Properties of Pulmonary Eosinophils in Respiratory Syncytial Virus (RSV) Vaccine-Enhanced Disease [PublishAheadOfPrint]

  • Human respiratory syncytial virus (RSV) is a major cause of morbidity and severe lower respiratory tract disease in the elderly and very young, with some infants developing bronchiolitis, recurrent wheezing and asthma following infection. Previous studies in humans and animal models have shown that vaccination with formalin-inactivated RSV (FI-RSV) leads to prominent airway eosinophilic inflammation following RSV challenge; however, the role of pulmonary eosinophilia in the antiviral response and in disease pathogenesis is inadequately understood. In vivo studies in mice having eotaxin and/or interleukin (IL)-5 deficiency showed that FI-RSV vaccination did not lead to enhanced pulmonary disease where following challenge there was reduced pulmonary eosinophilia, inflammation, Th2-type cytokine responses and altered chemokines (TARC, CCL17) responses. In contrast to wild-type mice, RSV was recovered at high titers from the lungs of eotaxin and/or IL-5 deficient mice. Adoptive transfer of eosinophils to FI-RSV immunized eotaxin and IL-5 deficient (double deficient) mice challenged with RSV was associated with potent viral clearance that was mediated at least partly through nitric oxide. These studies show that pulmonary eosinophilia has dual outcomes: one linked to RSV-induced airway inflammation and pulmonary pathology and one with innate features that contribute to a reduction in viral load.

    Importance This study is critical to understanding the mechanisms attributable to RSV vaccine enhanced disease. This study addresses the hypothesis that IL-5 and eotaxin are critical in pulmonary eosinophil response related to FI-RSV vaccine enhanced disease. The findings suggest that, while eosinophils within a Th2-polarized inflammatory response may have pathologic features, they are also effective mediators of antiviral defence.

  • Fruits of Virus Discovery: New Pathogens and New Experimental Models [PublishAheadOfPrint]

  • The application of high-throughput sequencing has lead to a tremendous increase in the rate of discovery of viral sequences. In some instances, novel pathogens have been identified. What has been less well appreciated is that novel virus discoveries in distinct hosts have led to the establishment of unique experimental systems to define host-virus interactions. These new systems have opened new frontiers in the study of fundamental virology and infectious disease.

  • Three novel virophage genomes discovered from Yellowstone Lake metagenomes [PublishAheadOfPrint]

  • Virophages are a unique group of circular double-stranded DNA viruses that are considered parasites of giant DNA viruses, which in turn are known to infect eukaryotic hosts. In this study the genomes of three novel virophages YSLV5, YSLV6 and YSLV7 were identified from Yellowstone Lake through metagenomic analyses. The relative abundance of these three novel virophages and previously identified Yellowstone Lake virophages YSLVs 1-4 were determined in different locations of the lake, revealing that most of the sampled locations in the lake, including both mesophilic and thermophilic habitats, had multiple virophage genotypes. This likely reflects the diverse habitats or diversity of the eukaryotic hosts and their associated giant viruses that serve as putative hosts for these virophages. YSLV5 has a 29,767 bp genome with 32 predicted ORFs, YSLV6 has a 24,837 bp genome with 29 predicted ORFs, and YSLV7 has a 23,193 bp genome with 26 predicted ORFs. Based on multilocus phylogenetic analysis, YSLV6 shows a close evolutionary relationship with YSLVs 1-4, whereas YSLV5 and YSLV7 are distantly related to the others, and YSLV7 represents the fourth novel virophage lineage. In addition, the genome of YSLV5 has a G+C content of 51.1% that is much higher than all other known virophages, indicating a unique host range for YSLV5. These results suggest that virophages are abundant and have diverse genotypes that likely mirror diverse giant viral and eukaryotic hosts within the Yellowstone Lake ecosystem.

    Importance This study discovered novel virophages present within the Yellowstone Lake ecosystem using a conserved major capsid protein as a phylogenetic anchor for assembly of sequence reads from Yellowstone Lake metagenomic samples. The three novel virophage genomes (YSLV5-7) were completed by identifying specific environmental samples containing these respective virophages, and closing gaps by targeted PCR and sequencing. Most of the YSLV genotypes were associated primarily with photic zone and non-hydrothermal samples; however, YSLV5 had a unique distribution with an occurrence in vent samples similar to that in photic zone samples, and with a higher GC content that suggests a distinct host and habitat compared to other YSLVs. In addition, genome content and phylogenetic analyses indicate that YSLV5 and YSLV7 are distinct from known virophages, and that additional as-yet-uncharacterized virophages are likely present within the Yellowstone Lake ecosystem.

  • Electron cryo-tomography studies of maturing HIV-1 particles reveal the assembly pathway of the viral core [PublishAheadOfPrint]

  • To better characterize the assembly of the HIV-1 core, we have used electron cryo-tomography (ECT) to image infected cells and the viral particles cryo-preserved next to them. We observed progressive stages of virus assembly and egress including flower-like flat gag lattice assemblies, hemispherical budding profiles and virus buds linked to the plasma membrane via a thin membrane neck. The population of budded viral particles contains immature, maturation intermediate and mature core morphologies. Structural characteristics of the maturation intermediates suggest that the core assembly pathway involves the formation of a CA sheet that associates with the condensed ribonucleoprotein (RNP) complex. Our analysis also reveals a correlation between RNP localization within the viral particle and the formation of conical cores, suggesting that the RNP helps drive conical core assembly. Our findings support an assembly pathway for the HIV-1 core that begins with a small CA sheet that associates with the RNP to form the core base, followed by polymerization of the CA sheet along one side of the conical core towards the tip and then closure around the body of the cone.

    IMPORTANCE During HIV-1 assembly and release the Gag polyprotein is organized into a signature hexagonal lattice, termed the immature lattice. To become infectious, the newly budded virus must disassemble the immature lattice by proteolyzing Gag, and then reassemble the key proteolytic product, the structural protein p24 (CA), into a distinct, mature hexagonal lattice during a process termed maturation. The mature HIV-1 virus contains a conical capsid that encloses the condensed viral genome at its wide base. Mutations or small molecules that interfere with viral maturation also disrupt viral infectivity. Little is known about the assembly pathway that results in the conical core and genome encapsidation. Here we have used electron cryo-tomography to structurally characterize HIV-1 particles that are actively maturing. Based on the morphologies of core assembly intermediates, we propose that CA forms a sheet-like structure that associates with the condensed viral genome to produce the mature infectious conical core.

  • Abrogation of the interferon response promotes more efficient human cytomegalovirus replication [PublishAheadOfPrint]

  • The effect of abrogating the interferon (IFN) response on human cytomegalovirus (HCMV) replication was investigated using primary human cells engineered to either block the production of or the response to type I IFNs. In IFN-deficient cells HCMV produced larger plaques, spread and replicated more rapidly than in parental cells. These cells demonstrate the vital role of IFNs in controlling HCMV replication and provide useful tools to investigate the IFN response to HCMV.

  • Biological and protective properties of immune sera directed to influenza virus neuraminidase [PublishAheadOfPrint]

  • The envelope of influenza A viruses contains two large antigens, hemagglutinin (HA) and neuraminidase (NA). Conventional influenza virus vaccines induce neutralizing antibodies that are predominantly directed to the HA globular head, a domain which is subject to extensive antigenic drift. Antibodies directed to NA are induced at much lower levels, probably a consequence of the immunodominance of the HA antigen. Although antibodies to NA may affect virus release by inhibiting the sialidase function of the glycoprotein, this antigen has been largely neglected in past vaccine design. In this study, we characterized the protective properties of monospecific immune sera that were generated by vaccination with recombinant RNA replicon particles encoding NA. These immune sera inhibited hemagglutination in a NA subtype-specific and HA subtype-independent manner and interfered with infection of MDCK cells. In addition, they inhibited the sialidase activity of various influenza viruses of the same and even different NA subtypes. With this, the anti-NA immune sera inhibited the spreading of H5N1 highly pathogenic avian influenza virus and HA/NA-pseudotyped viruses in MDCK cells in a concentration-dependent manner. When chickens were immunized with NA-recombinant replicon particles and subsequently infected with low-pathogenic avian influenza virus, inflammatory serum markers were significantly reduced and virus shedding was limited or eliminated. These findings suggest that NA antibodies can inhibit virus dissemination by interfering with both virus attachment and egress. Our results underline the potential of high quality NA antibodies for controlling influenza virus replication and place emphasis on NA as vaccine antigen.

    Importance The neuraminidase of influenza A viruses is a sialidase which acts as a receptor-destroying enzyme facilitating the release of progeny virus from infected cells. Here, we demonstrate that monospecific anti-NA immune sera not only inhibited sialidase activity but also influenza virus hemagglutination and infection of MDCK cells, suggesting that NA antibodies can interfere with virus attachment. Inhibition of both processes, virus release and virus binding, may explain why NA antibodies efficiently blocked virus dissemination in vitro and in vivo. Anti-NA immune sera showed a broader reactivity than anti-HA sera in hemagglutination inhibition tests and demonstrated cross-subtypic activity in sialidase inhibition tests. These remarkable features of NA antibodies highlight the importance of the NA antigen for the development of next generation influenza virus vaccines.

  • A CryoEM study identifies the complete H16.V5 epitope and reveals global conformational changes initiated by binding of the neutralizing antibody fragment [PublishAheadOfPrint]

  • Human papillomavirus 16 (HPV16) is a worldwide health threat and an etiologic agent of cervical cancer. To understand the antigenic properties of HPV16 we pursued a structural study to elucidate HPV capsids and antibody interactions. The cryo-EM structures of HPV16 mature and an altered capsid particles were solved individually and as complexes with fragment of antibody (Fab) from the neutralizing antibody H16.V5. Fitted crystal structures provided a pseudo-atomic model of the virus-Fab complex, which identified a precise footprint of H16.V5, including previously unrecognized residues. The altered capsid-Fab complex map showed that binding of the Fab induced significant conformational changes than were seen in the altered capsid structure alone. These changes included more ordered surface loops, consolidated invading arm structures, and tighter inter-capsomeric connections at the capsid floor. The H16.V5 Fab preferentially bound hexavalent capsomers likely with a stabilizing effect that directly correlated with the number of bound Fabs. Additional cryoEM reconstructions of the virus-Fab complex for different incubation times and structural analysis provide a model for a hyper-stabilization of the capsomer by H16.V5 Fab and showed that the Fab distinguishes subtle differences between antigenic sites.

    IMPORTANCE Our analysis of the cryoEM reconstructions of the HPV16 capsids and virus-Fab complexes has identified the entire HPV.V5 conformational epitope and demonstrated a detailed neutralization mechanism of this clinically important monoclonal antibody against HPV16. The Fab bound and ordered the apical loops of HPV16. This conformational change was transmitted to the lower region of the capsomer resulting in enhanced inter-capsomeric interactions evidenced by the more ordered capsid floor and llsquo;invading armrrsquo; structures. This study advances the understanding of the neutralization mechanism used by H16.V5.

  • Two distinct sets of NS2A molecules are responsible for dengue virus RNA synthesis and virion assembly [PublishAheadOfPrint]

  • Flavivirus nonstructural protein 2A (NS2A) plays important roles in both viral RNA synthesis and virion assembly. The molecular details of how NS2A protein modulates the two distinct events have not been defined. To address this question, we have performed a systematic mutagenesis of NS2A using dengue virus serotype 2 (DENV-2) as a model. We identified two sets of NS2A mutations with distinct defects during a viral infection cycle. One set of NS2A mutations (D125A and G200A) selectively abolished viral RNA synthesis. Mechanistically, mutation D125A abolished viral RNA synthesis through blocking the N-terminal cleavage of NS2A protein, leading to an unprocessed NS1-NS2A protein; this result suggests that amino acid D125 (far downstream of the N-terminus of NS2A) may contribute to the recognition of host protease at the NS1-NS2A junction. The other set of NS2A mutations (G11A, E20A, E100A, Q187A, and K188A) specifically impaired virion assembly without significantly affecting viral RNA synthesis. Remarkably, mutants defective in virion assembly could be rescued by trans supplying wild-type NS2A molecules expressed from a replicative replicon, by wild-type NS2A protein expressed alone, by a mutant NS2A (G200A) that is lethal for viral RNA synthesis, or by a different mutant NS2A that is defective in virion assembly. In contrast, none of the mutants defective in viral RNA synthesis could be rescued by trans complementation. Collectively, the results indicate that two distinct sets of NS2A molecules are responsible for DENV RNA synthesis and virion assembly.

    SIGNIFICANCE Dengue virus (DENV) represents the most prevalent mosquito-borne human pathogen. Understanding the replication of DENV is essential for development of vaccine and therapeutics. Here we characterized the function of DENV-2 NS2A using a systematic mutagenesis approach. The mutagenesis results revealed two distinct sets of NS2A mutations: One set of mutations that are defective in viral RNA synthesis; another set of mutations that are defective in virion assembly. Trans complementation analysis showed that mutants defective in viral RNA synthesis could not be rescued by wild-type NS2A; in contrast, mutants defective in virion assembly could be successfully rescued by wild-type NS2A or even by a mutant NS2A that is incompetent to support viral RNA synthesis. These results support a model that two distinct sets of NS2A molecules are responsible for DENV RNA synthesis (located in the viral replication complex) and virion assembly (located in the virion assembly/budding site). The study has confirmed and extended our understanding of the two critical roles of flavivirus NS2A in viral RNA synthesis and virion assembly.

  • Ebola virus transmission in guinea pigs [PublishAheadOfPrint]

  • Ebola virus (EBOV) transmission is currently poorly characterized and thought to occur primarily by direct contact with infectious material; however transmission from swine to nonhuman primates via the respiratory tract has been documented. To establish an EBOV transmission model for performing studies with statistical significance, groups of six guinea pigs (gps) were challenged intranasally (IN) or intraperitoneally (IP) with 10,000 x LD50 of gp-adapted EBOV, and naiiuml;ve gps were then introduced as cage-mates for contact exposure at 1 day post-infection (dpi). Animals were monitored for survival and clinical signs of disease, and quantitated for virus shedding post-exposure. Changes in contact duration of naiiuml;ve gps with infected animals were evaluated for impact on transmission efficiency. Transmission was more efficient from IN compared to IP-challenged gps, with 17% versus 83% of naiiuml;ve gps surviving exposure, respectively. Virus shedding was detected beginning at 3 dpi from both IN- and IP-challenged animals. Contact duration positively correlated with transmission efficiency, and the abrogation of direct contact between infected and naiiuml;ve animals through the erection of a steel mesh is effective at stopping virus spread, provided that infectious animal bedding was absent in the cages. Histopathological and immunohistochemical findings show that IN-infected gps display enhanced lung pathology and EBOV antigen in the trachea, which support increased virus transmission from these animals. The results suggest that IN-challenged gps are more infectious to naiiuml;ve animals than their systemically-infected counterparts, and that transmission occurs through direct contact with infectious materials, including those transported through air movement over short distances.

    Importance Ebola is generally thought to be spread between humans though infectious bodily fluids. However, a study has shown that Ebola can be spread from pigs to monkeys without direct contact. Further studies have been hampered, because an economical animal model for Ebola transmission is not available. To address this, we established a transmission model in guinea pigs, and determined the mechanisms behind virus spread. The survival data, in addition to microscopic examination of lung and trachea sections, show that mucosal infection of guinea pigs is an efficient model for Ebola transmission. Virus spread is increased with longer contact times to an infected animal and is possible without direct contact between an infected and naiiuml;ve host, but can be stopped if infectious materials were absent. These results warrant consideration for the development of future strategies against Ebola transmission, and a better understanding of the parameters involved with virus spread.

  • Specificity of coxsackievirus B3 interaction with human, but not murine, decay accelerating factor (DAF): replacement of a single residue within short consensus repeat 2 prevents virus attachment [PublishAheadOfPrint]

  • Many coxsackievirus B (CVB) isolates bind to human decay accelerating factor (DAF) as well as to the coxsackievirus and adenovirus receptor (CAR). However, virus does not interact with murine DAF. To understand why CVB3 binds specifically to human DAF we constructed a series of chimeric molecules in which specific regions of the human DAF molecule were replaced by the corresponding murine sequences. We found that replacement of human with murine SCR2 ablated virus binding to human DAF, as did deletion of human SCR2. Although replacement of human SCR4 had a partial inhibitory effect, deletion of SCR4 had no effect. Within human SCR2, replacement of serine 104 (S104) with the proline residue found in murine DAF eliminated virus binding. Based on the structure of the CVB3-DAF complex determined by cryo-electron microscopy, DAF S104 is in close contact with a viral capsid residue, a threonine at VP1 position 271. Replacement of this capsid residue with larger amino acids specifically eliminated virus attachment to human DAF, but had no effect on attachment to CAR or replication in HeLa cells. Taken together, these results support the current model of virus-DAF interaction, and point to a specific role for VP1-T271 and DAF S104 at the virus-DAF interface.

    IMPORTANCE These results point to a specific role for VP1-T271 and DAF S104 at the interface between CVB3 and DAF-DAF, and demonstrate how subtle structural changes can dramatically influence virus-receptor interactions. In addition, the results support a recent pseudoatomic model of CVB3-DAF interaction obtained by cryo-electron microscopy.

  • Structure-based functional analyses of domains II and III of pseudorabies virus glycoprotein H [PublishAheadOfPrint]

  • Enveloped viruses utilize membrane fusion for entry into, and release from host cells. For entry, members of the Herpesviridae require at least three envelope glycoproteins: the homotrimeric gB, and a heterodimer of gH and gL. The crystal structures of three gH homologues including pseudorabies virus (PrV) gH revealed four conserved domains. Domain II contains a planar bbeta;-sheet (fence), and a syntaxin-like bundle of three aalpha;-helices (SLB), similar to those found in eukaryotic fusion proteins, potentially executing an important role in gH function. To test this hypothesis, we introduced targeted mutations into the PrV gH gene, which either disrupt the helices of the SLB by introduction of proline residues, or covalently join them by artificial intramolecular disulfide bonds between themselves, to the adjacent "fence" region or to domain III. Disruption of either of the three aalpha;-helices of the SLB (V250P, V275P, A298P) severely affected gH function in in vitro fusion assays and replication of corresponding PrV mutants. Considerable defects in fusion activity of gH, as well as in penetration kinetics and cell-to-cell spread of PrV mutants were also observed after disulfide-linkage of two aalpha;-helices within the SLB (A284C-S291C), or between SLB and domain III (H251C-L432C), as well as by insertions of additional cysteine pairs linking fence, SLB and domain III. In vitro fusion activity of mutated gH could be partly restored by reduction of the artificial disulfide bonds. Our results indicate that structure and flexibility of the SLB are relevant for the function of PrV gH in membrane fusion.

    Importance: Mutational analysis based on crystal structures of proteins is a powerful tool to understand protein function. Here, we continued our study of pseudorabies virus gH, a part of the core fusion machinery of herpesviruses. We previously showed that the "flap" region in domain IV of PrV gH is important for its function. We now demonstrate that mutations within domain II which interfere with integrity or flexibility of a syntaxin-like three-helix bundle also significantly impair gH function during fusion. These studies provide important insights into the structural requirements of gH for function in fusion.

  • Measles Virus Glycoprotein Complexes Preassemble Intracellularly and Relax During Transport to the Cell Surface in Preparation for Fusion [PublishAheadOfPrint]

  • Measles virus (MeV), a morbillivirus within the paramyxovirus family, expresses two envelope glycoproteins. The attachment (H) protein mediates receptor binding, followed by triggering of the fusion (F) protein, which leads to merger of the viral envelope with target cell membranes. Receptor binding by members of related paramyxovirus genera rearranges the head domains of the attachment proteins, liberating an F-contact domain within the attachment protein helical stalk. However, morbillivirus glycoproteins first assemble intracellularly prior to receptor binding, raising the question of whether alternative protein-protein interfaces are involved or an entirely distinct triggering principle is employed. To test these possibilities, we generated headless H-stem mutants of progressively shorter length. Conformationally restricted H-stems remained capable of intracellular assembly with standard F and a soluble MeV F mutant. Proteolytic maturation of F, but not the altered biochemical conditions at the cell surface, reduces the strength of glycoprotein interaction, readying the complexes for triggering. F mutants stabilized in the prefusion conformation interact with H intracellularly and at the cell surface, while destabilized F mutants interact only intracellularly, prior to F maturation. These results showcase a MeV entry machinery that functionally varies conserved motifs of the proposed paramyxovirus infection pathway. Intracellular and plasma membrane-resident MeV glycoprotein complexes employ the same protein-protein interface. F maturation prepares for complex separation after triggering, and the H head domains in pre-receptor bound conformation prevent premature stalk rearrangements and F activation. Intracellular preassembly affects MeV fusion profiles and may contribute to the high cell-to-cell fusion activity characteristic for the morbillivirus genus.

  • The ORF012 gene of Marek's disease virus (MDV) produces a spliced transcript and encodes a novel nuclear phosphoprotein essential for virus growth [PublishAheadOfPrint]

  • Marek's disease virus (MDV), an alphaherpesvirus, is the causative agent of a lethal disease in chickens characterized by generalized nerve inflammation and rapid lymphoma development. The extensive colinearity of the MDV genome with those of related herpesviruses has eased functional characterization of many MDV genes. However, MDV encodes a number of unique open reading frames (ORFs) that have not yet been investigated regarding their coding potential and the functions of their products. Among these unique ORFs are two putative ORFs, ORF011 and ORF012, which are found at the extreme left end of the MDV unique-long region. Using reverse transcription PCR we showed that ORF011 and 012 are not individual genes, but encode a single gene through mRNA splicing of a small intron resulting in the novel ORF012. We generated an ORF012-null virus using an infectious clone of MDV strain RB-1B. The deletion virus had a marked growth defect in vitro and could not be passaged in cultured cells suggesting an essential role for the ORF012 product for virus replication. Further studies revealed that p012 localized to the nucleus in transfected and infected cells and we identified by site-directed mutagenesis and GFP reporter fusion assays a nuclear localization signal (NLS) that was mapped to a 23 amino acid sequence at the protein's C-terminus. Nuclear export was blocked using leptomycin B suggesting a potential role for p012 as a nuclear/cytoplasmic shuttling protein. Finally, p012 is phosphorylated at multiple residues, a modification that could possibly regulate its subcellular distribution.

    Importance Marek's disease virus (MDV) causes a devastating oncogenic disease in chickens with high morbidity and mortality. The costs for disease prevention reach several billion US dollars annually. The functional investigation of MDV genes is necessary to understand its complex replication cycle that eventually could help to interfere with MDV and herpesviral pathogenesis. We have identified a previously unidentified phosphoprotein encoded by MDV ORF012. We were able to show experimentally that predicted splicing of the gene based on bioinformatics data does indeed occur during replication. The newly identified p012 is essential for MDV replication and localizes to the nucleus due to the presence of a transferable nuclear localization signal at its C-terminus. Our results also imply that p012 could constitute a nucleocytoplasmic shuttle protein, a feature that could prove interesting and important.

  • Tulane virus recognizes the A-type 3 and B histo-blood group antigens [PublishAheadOfPrint]

  • Tulane virus (TV), the prototype of the Recovirus genus in the calicivirus family, was isolated in the stools of rhesus monkeys and can be cultivated in vitro in monkey kidney cells. TV is genetically closely related to the Norovirus genus and recognizes the histo-blood group antigens (HBGAs) like human noroviruses (NoVs), making it a valuable surrogate for human NoVs. However, the precise structures of HBGAs recognized by TV remain elusive. In this study, we performed binding and blocking experiments on TV with extended HBGA types and showed that, while TV binds all four types (types 1 to 4) of the B antigens, it recognizes only the A-type 3 antigen among four types of A antigens tested. The requirements of HBGAs in TV replication were demonstrated by blocking of TV replication in cell culture using the A-type 3/4 and B saliva samples. Similar results were also observed in oligosaccharide-based blocking assays. Importantly, the previously reported, unexplained increase of TV replication by oligosaccharide in cell-based blocking assays has been clarified, which will facilitate the application of TV as a surrogate for human NoVs.

    Importance. Our understanding on the role of HBGAs in NoV infection has been significantly advanced in the past decade but direct evidence on HBGAs as a receptor for NoVs remains lacking due to a lack of a NoV cell culture. TV recognizes HBGAs and can replicate in vitro, providing a valuable surrogate for human NoVs. However, TV binds to some but not all saliva samples from the A positive individuals and an unexplained result on synthetic oligosaccharides in blocking TV binding have been reported. These issues now have been resolved in this study.

  • Efficient reverse genetics reveals genetic determinants of budding and fusogenic differences between Nipah and Hendra virus and enables real-time monitoring of viral spread in small animal models of henipavirus infection. [PublishAheadOfPrint]

  • Nipah (NiV) and Hendra (HeV) viruses are closely related henipaviruses of the Paramyxovirinae. Spillover from their fruit bat reservoirs can cause severe disease in humans and livestock. Despite their high sequence similarity, NiV and HeV exhibit apparent differences in receptor and tissue tropism, envelope-mediated fusogenicity, replicative fitness, and other pathophysiologic manifestations. To investigate the molecular basis for these differences, we first established a highly efficient reverse genetics system that increased rescue titers by gge;3 logs, which offset the difficulty of generating multiple recombinants under constraining BSL-4 conditions. We then substituted singly and in combination, the matrix (M), fusion (F), and attachment glycoprotein (G) genes in mCherry-expressing recombinant NiV (rNiV) with their HeV counterparts. These chimeric but isogenic rNiVs replicated well in primary human endothelial and neuronal cells, indicating efficient heterotypic complementation. The determinants of budding efficiency, fusogenicity, and replicative fitness were dissociable: HeV-M budded more efficiently than NiV-M, accounting for the early higher replicative titers of HeV-M-bearing chimeras, while the enhanced fusogenicity of NiV-G-bearing chimeras did not correlate with increased replicative fitness. Furthermore, to facilitate spatiotemporal studies on henipavirus pathogenesis, we generated a Firefly luciferase-expressing NiV and monitored virus replication and spread in infected interferon-aalpha;/bbeta; receptor knockout mice via bioluminescence imaging. While intraperitoneal inoculation resulted in neuroinvasion following systemic spread and respiratory tract replication, intranasal inoculation resulted in confined spread to regions corresponding to olfactory bulbs and salivary glands before subsequent neuroinvasion. This optimized henipavirus reverse genetics system will facilitate future investigations into the growing numbers of novel henipa-like viruses.

    Importance Nipah (NiV) and Hendra (HeV) viruses are recently emergent, zoonotic, and highly lethal pathogens with pandemic potential. Although differences have been observed between NiV and HeV replication and pathogenesis, the molecular basis for these differences has not been examined. In this study, we established a highly efficient system to reverse engineer changes into replication-competent NiV and HeV, which facilitated generation of reporter-expressing viruses and recombinant NiV-HeV chimeras substituted in the genes responsible for viral exit (M, critical for assembly and budding) and viral entry (attachment (G) and fusion (F)). These chimeras revealed differences in the budding and fusogenic properties of the M and G proteins, respectively, which help explain previously observed differences between NiV and HeV. Finally, to facilitate future in vivo studies, we monitored replication and spread of a bioluminescent reporter-expressing NiV in susceptible mice, the first time such in vivo imaging has been performed under BSL-4 conditions.

  • JC virus quasispecies analysis reveals complex viral population underlying PML and supports viral dissemination via the hematogenous route [PublishAheadOfPrint]

  • Opportunistic infection of oligodendrocytes by human JC polyomavirus may result into development of progressive multifocal encephalopathy in immune compromised individuals. Neurotropic JC virus generally harbors reorganized non-coding control region (NCCR) DNA interspersed on the viral genome between early and late coding genes. By applying 454 sequencing on NCCR DNA amplified from body fluid samples (urine, plasma and CSF) from nineteen PML patients, we attempted to reveal the composition of the JC polyomavirus population (the quasispecies i.e. the whole of the consensus and minor viral variants) contained in different body compartments and to better understand intra-patient viral dissemination. Our data demonstrate that in CSF of PML patients the JC viral population often is a complex mixture composed of multiple viral variants that contribute to the quasispecies. In contrast, urinary JC virus highly resembled archetype virus and most often did not contain minor viral variants. It also appeared that archetype JC virus sporadically can be identified in PML brain although selection of rearranged JC virus DNA is favored. Comparison of the quasispecies between different body compartments within a given patient suggested a strong correlation between the viral population in plasma and CSF whereas the viral population shed in urine appeared as unrelated. In conclusion, it is shown that the representation of viral DNA in the CSF following high level DNA replication in the brain underlying PML has hitherto been much underestimated. Our data also underscore that the hematogenous route might play a pivotal role in viral dissemination from or towards the brain.

    IMPORTANCE For the first time the JC polyomavirus population contained in different body compartments of patients diagnosed with progressive multifocal encephalopathy has been studied by deep sequencing. Two main findings came out of this work. First, it became apparent that the complexity of the viral population associated with PML has been highly underestimated so far, suggestive for a highly dynamic process of reorganization of the non-coding control region of JC polyomavirus in vivo, mainly in CSF and blood borne virus. Secondly, evidence was provided showing viral dissemination from and/or towards the brain via the hematogenous route, confirming a hypothesis that recently had been put forward in the field.

  • The murine norovirus (MNV) core subgenomic RNA promoter consists of a stable stem-loop that can direct accurate initiation of RNA synthesis [PublishAheadOfPrint]

  • All members of the Caliciviridae family of viruses produce a subgenomic RNA during infection. The subgenomic RNA typically encodes only the major and minor capsid proteins, but in murine norovirus (MNV), the subgenomic RNA also encodes the VF1 protein that functions to suppress host innate immune responses. To date, the mechanism of norovirus subgenomic RNA synthesis has not been characterized. We have previously described the presence of an evolutionarily conserved RNA stem-loop structure on the negative-sense RNA, the complementary sequence of which codes for the viral RNA-dependent RNA polymerase (NS7). The conserved stem-loop is positioned 6 nucleotides 3rrsquo; of the start site of the subgenomic RNA in all caliciviruses. We demonstrate that the conserved stem-loop is essential for MNV viability. Mutant MNV RNAs with substitutions in the stem-loop replicated poorly until they accumulated mutations that revert to restore the stem-loop sequence and/or structure. The stem-loop sequence functions in a non-coding context as it was possible to restore the replication of a MNV mutant by introducing an additional copy of the stem-loop between the NS7 and VP1 coding regions. Finally, in vitro biochemical data would suggest that stem-loop sequence is sufficient for the initiation of viral RNA synthesis by the recombinant MNV RdRp, confirming that the stem-loop forms the core of the norovirus subgenomic promoter.

    Importance Noroviruses are a significant cause of viral gastroenteritis and it is important to understand the mechanism of norovirus RNA synthesis. Herein we describe the identification of an RNA stem-loop structure that functions as the core of the norovirus subgenomic RNA promoter in cells and in vitro. This work provides new insights into the molecular mechanisms of norovirus RNA synthesis and the sequences that determine the recognition of viral RNA by the RNA dependent RNA polymerase.

  • Nature of the N-terminal Amino Acid Residue of HIV-1 RNase H is Critical for the Stability of Reverse Transcriptase in Viral Particles [PublishAheadOfPrint]

  • Reverse transcriptase (RT) of Human Immunodeficiency Virus type 1 (HIV-1) is synthesized and packaged into the virion as a part of the GagPol polyprotein. Mature RT is released by the action of viral protease. However unlike other viral proteins RT is subject to an internal cleavage event leading to the formation of two subunits in the virion nndash; a p66 subunit and a p51 subunit that lacks the RNase H domain. We have previously identified RNase H as an HIV-1 protein that has the potential to be a substrate for the N-end rule pathway that is an ubiquitin dependent proteolytic system in which the identity of the N-terminal amino acid determines the half-life of a protein. Here we examined the importance of the N-terminal amino acid residue of RNase H in the early life cycle of HIV-1. We show that changing this residue to a structurally different amino acid than the conserved residue leads to degradation of RT and in some cases integrase in the virus particle and this abolishes infectivity. Using intravirion complementation and in vitro protease cleavage assays we show that degradation of RNase H N-terminal mutant RT occurs in the absence of active viral protease in the virion. Our results also indicate the importance of the RNase H N-terminal residue in the dimerization of RT subunits.

    Importance HIV-1 proteins are initially made as part of a polyprotein that is cleaved by the viral protease into the proteins that form the virus particle. We were interested in one particular protein RNase H that is cleaved from reverse transcriptase. In particular we found that the first amino acid of RNase H never varies in over 1850 isolates of HIV-1 that we compared. When we changed the first amino acid we found that the reverse transcriptase in the virus was degraded. While other studies have implied that the viral protease can degrade mutant RT proteins we show here that this is may not be the case for our mutants. Our results suggest that presence of active viral protease is not required for the degradation of RT in RNase H N-terminal mutants suggesting a role for a cellular protease in this process.

  • Attenuated West Nile virus mutant NS1130-132QQA/175A/207A exhibits virus-induced ultrastructural changes and accumulation of protein in the Endoplasmic Reticulum [PublishAheadOfPrint]

  • We have previously shown that ablation of the three N-linked glycosylation sites in the West Nile virus NS1 protein completely attenuates mouse neuroinvasiveness (gge;1,000,000 PFU). Here we compared replication of the NS1130-132QQA/175A/207A mutant to parental NY99 strain in monkey kidney Vero cells. The results suggest that the mechanism of attenuation is due to lack of NS1 glycosylation, which blocks efficient replication, maturation and NS1 secretion from the ER, and results in changes of virus-induced ultrastructure.

  • Cyclic avian mass mortality in the northeastern United States is associated with a novel orthomyxovirus [PublishAheadOfPrint]

  • Since 1998, cyclic mortality events in common eiders (Somateria mollissima), numbering in the hundreds to thousands of dead birds, have been documented along the coast of Cape Cod, Massachusetts, USA. Although longitudinal disease investigations have uncovered potential contributing factors responsible for these outbreaks, detecting a primary etiological agent has proven enigmatic. Here we identify a novel orthomyxovirus, tentatively named Wellfleet Bay virus (WFBV), as a potential causative agent of these outbreaks. Genomic analysis of WFBV revealed that it is most closely related to members of the Quaranjavirus genus within the family Orthomyxoviridae. Similar to other members of the genus, WFBV contains an alphabaculovirus gp64-like glycoprotein, which was demonstrated to have fusion activity, and also tentatively suggests that ticks (and/or insects) may vector the virus in nature. However, in addition to the six RNA segments encoding the prototypical structural proteins identified in other quaranjaviruses, a previously unknown RNA segment (segment 7) encoding a novel protein designated as VP7 was discovered in WFBV. Although WFBV shows low to moderate levels of sequence similarity to Quaranfil virus and Johnston Atoll virus, the original members of the Quaranjavirus genus, additional antigenic and genetic analyses demonstrated that it is closely related to the recently identified Cygnet River virus (CyRV) from South Australia, suggesting that WFBV and CyRV may be geographic variants of the same virus. Although the identification of WFBV in part may resolve the enigma of these mass mortality events, the details of the ecology and epidemiology of the virus remain to be determined.

    Importance The emergence or reemergence of viral pathogens resulting in large-scale outbreaks of disease in humans and/or animals is one of the most important challenges facing biomedicine. For example, understanding how orthomyxoviruses such as novel influenza A virus reassortants and/or mutants emerge to cause epidemic or pandemic disease is at the forefront of current global health concerns. Here we describe the emergence of a novel orthomyxovirus, Wellfleet Bay virus (WFBV), which has been associated with cyclic large-scale bird die-offs in the northeastern United States. This initial characterization study provides a foundation for further research into the evolution, epidemiology, and ecology of newly emerging orthomyxoviruses, such as WFBV, and their potential impacts on animal and/or human health.

  • Wongabel rhabdovirus accessory protein U3 targets the SWI/SNF chromatin remodelling complex [PublishAheadOfPrint]

  • Wongabel virus (WONV) is an arthropod-borne rhabdovirus that infects birds. It is one of the growing array of rhabdoviruses with complex genomes that encode multiple accessory proteins of unknown function. In addition to the five canonical rhabdovirus structural protein genes (N, P, M, G and L), the 13.2 kb (-) ssRNA WONV genome contains five uncharacterised accessory genes, one overlapping the N gene (Nx or U4), three located between the P and M genes (U1 to U3) and a fifth overlapping the G gene (Gx or U5). Here we show that WONV U3 is expressed during infection in insect and mammalian cells and is required for efficient viral replication. A yeast two-hybrid screen against a mosquito cell cDNA library identified that WONV U3 interacts with the 83-aa C-terminal domain of SNF5, a component of the SWI/SNF chromatin-remodelling complex. The interaction was confirmed by affinity chromatography and nuclear co-localisation was established by confocal microscopy. Gene expression studies showed that SNF5 transcripts are up-regulated during infection in mosquito cells with WONV, as well as West Nile virus (Flaviviridae) and bovine ephemeral fever virus (Rhabdoviridae), and that SNF5 knock-down results in increased WONV replication. WONV U3 also inhibits SNF5-regulated expression of the cytokine CSF1 gene. The data suggests that WONV U3 targets the SWI/SNF complex to block the host response to infection.

    Importance The rhabdoviruses comprise a large family of RNA viruses, infecting plants, vertebrates and invertebrates. In addition to the major structural proteins (N, P, M, G and L), many rhabdoviruses encode a diverse array of accessory proteins of largely unknown function. Understanding the role of these proteins may reveal much about host-pathogen interactions in infected cells. Here we examine accessory protein U3 of Wongabel virus, an arthropod-borne rhabdovirus that infects birds. We show that U3 enters the nucleus and interacts with SNF5, a component of the chromatin remodelling complex that is upregulated in response to infection and restricts viral replication. We also show that U3 inhibits SNF5-regulated expression of the cytokine CSF-1, suggesting that it targets the chromatin remodelling complex to block the host response to infection. This study appears to provide the first evidence of a virus targeting SNF5 to inhibit host gene expression.

  • Eilat Virus Host Range Restriction is Present at Multiple Levels of Virus Life Cycle [PublishAheadOfPrint]

  • Most alphaviruses are mosquito-borne and exhibit a broad host range, infecting many different vertebrates including birds, rodents, equids, humans, and nonhuman primates. This ability of most alphaviruses to infect arthropods and vertebrates is essential for their maintenance in nature. Recently, a new alphavirus, Eilat virus (EILV), was described and in contrast to all other mosquito-borne viruses is unable to replicate in vertebrate cell lines. Investigations into the nature of its host range restriction showed the inability of genomic EILV RNA to replicate in vertebrate cells. Here, we investigated whether the EILV host range restriction is present at the entry level and further explored the viral factors responsible for the lack of genomic RNA replication. Utilizing Sindbis (SINV) and EILV chimeras, we show that the EILV vertebrate host range restriction is also manifested at the entry level. Furthermore, the EILV RNA replication restriction is independent of the 3rrsquo; untranslated genome region (UTR). Complementation experiments with SINV suggested that RNA replication is restricted by the inability of the EILV non-structural proteins to form functional replicative complexes. These data demonstrate that the EILV host range restriction is multigenic, involving at least one gene from both non-structural (nsP) and structural protein (sP) open reading frames (ORFs). As EILV groups phylogenetically within the mosquito-borne virus clade of pathogenic alphaviruses, our findings have important evolutionary implications for arboviruses.

    Importance: Our work explores the nature of host range restriction of the first "mosquito-only alphavirus", EILV. EILV is related to pathogenic mosquito-borne viruses [eastern (EEEV), western WEEV), Venezuelan equine encephalitis (VEEV) and chikungunya (CHIKV)] that cause severe disease in humans. Our data demonstrate that EILV is restricted both at entry and genomic RNA replication levels in vertebrate cells. These findings have important implications for arbovirus evolution and will help elucidate the viral factors responsible for the broad host range of pathogenic mosquito-borne alphaviruses, facilitate vaccine development, and inform potential strategies to reduce/prevent alphavirus transmission.

  • Less is More: Ebola Surface Glycoprotein Expression Levels Regulate Virus Production and Infectivity [PublishAheadOfPrint]

  • The Ebola virus surface glycoprotein (GP1,2) mediates host cell attachment and fusion, and is the primary target for host neutralizing antibodies. Expression of GP1,2 at high levels disrupts normal cell physiology, and EBOV uses an RNA editing mechanism to regulate expression of the GP gene. In this study, we demonstrate that high levels of GP1,2 expression impair production and release of EBOV VLPs, as well as infectivity of GP1,2-pseudotyped viruses. We further show that this effect is mediated through two mechanisms. First, high levels of GP1,2 expression reduce synthesis of other proteins needed for virus assembly. Second, viruses containing high levels of GP1,2 are intrinsically less infectious, possibly due to impaired receptor binding or endosomal processing. Importantly, proteolysis can rescue the infectivity of high-GP1,2 containing viruses. Taken together, our findings indicate that GP1,2 expression levels have a profound effect on factors that contribute to virus fitness, and that RNA editing may be an important mechanism employed by EBOV to regulate GP1,2 expression in order to optimize virus production and infectivity.

    IMPORTANCE The Ebola virus (EBOV), as well as other members of the Filoviridae family, causes severe hemorrhagic fever that is highly lethal with up to 90% mortality. The EBOV surface glycoprotein (GP1,2) plays important roles in virus infection and pathogenesis, and its expression is tightly regulated by an RNA editing mechanism during virus replication. Our study demonstrates that the level of GP1,2 expression profoundly affects virus particle production and release and uncovers a new mechanism by which Ebola virus infectivity is regulated by the level of GP1,2 expression. These findings extend our understanding of EBOV infection and replication in adaptation of host environments, which will aid the development of countermeasures against EBOV infection.

  • Distinct endometrial and decidual macrophage characteristics and regulation of their permissivity to HIV-1 infection by SAMHD1 [PublishAheadOfPrint]

  • In order to develop strategies to prevent HIV-1 (human immunodeficiency virus type 1) transmission, it is crucial to better characterize HIV-1 target cells in the female reproductive tract (FRT) mucosae, and to identify effective innate responses. Control of HIV-1 infection in the decidua (the uterine mucosa during pregnancy) can serve as a model to study natural mucosal protection. Macrophages (dM) are the main HIV-1 target cells in the decidua. Here we report that, in vitro, macrophages (eM) and T cells are the main HIV-1 targets in the non pregnant endometrium. As reported for dM, eM were found to have an M2-like phenotype (CD68+/CD163+/CD206+/IL-10high). However, eM and dM may belong to different subpopulations, as they differently express certain markers and secrete different amounts of pro- and anti-inflammatory cytokines. We observed strong expression of the SAMHD1 restriction factor and weak expression of its inactive form (pSAMHD1, phosphorylated at residue Thr592) in both eM and dM. Infection of macrophages from both tissues was enhanced in the presence of the viral protein Vpx, suggesting a role of SAMHD1 in the restriction of HIV-1 infection. This study and further comparisons of the decidua with non pregnant FRT mucosae should help to identify mechanisms of mucosal protection against HIV-1 infection.

    Importance The female reproductive tract mucosae are major portals of HIV-1 entry into the body. The decidua (uterine mucosa during pregnancy) can serve as a model for studying natural mucosal protection against HIV-1 transmission. A comparison of target cells and innate responses in the decidua versus the non pregnant endometrium could help to identify protective mechanisms. Here, we report for the first time that macrophages are one of the main HIV-1 target cells in the endometrium, and that infection of macrophages from both the endometrium and the decidua is restricted by SAMHD1. These findings might have implications for the development of vaccines to prevent HIV-1 mucosal transmission.

  • A lethal murine infection model for dengue virus 3 in AG129 mice deficient in Type I and II interferon receptors leads to systemic disease [PublishAheadOfPrint]

  • The mosquito-borne disease dengue (DEN) is caused by four serologically and genetically related viruses, termed DENV-1 to DENV-4. Infection with one DENV usually leads to acute illness and results in lifelong homotypic immunity, but individuals remain susceptible to infection by the other three DENVs. The lack of a small animal model that mimics systemic DEN disease without neurovirulence has been an obstacle, but DENV-2 models have been recently developed in AG129 mice (deficient in interferon aalpha;/bbeta; and receptor signaling) that resemble human disease. However, comparable DENV-1, -3 and -4 models have not been developed. We utilized a non-mouse adapted DENV-3 Thai human isolate to develop a lethal infection model in AG129 mice. Intraperitoneal inoculation of six-to-eight-week old animals with strain C0360/94 led to rapid, fatal disease. Lethal C0360-94 infection resulted in physical signs of illness, high viral loads in the spleen, liver, and large intestine, histological changes in the liver and spleen tissues, and increased serum cytokine levels. Importantly, the animals developed vascular leakage, thrombocytopenia, and leukopenia. Overall, we have developed a lethal DENV-3 murine infection model, with no evidence of neurotropic disease based on a non-mouse adapted human isolate, which can be used to investigate DEN pathogenesis and to evaluate candidate vaccines and antivirals. This suggests that murine models utilizing non-mouse adapted isolates can be obtained for all four DENVs.

    Importance Dengue (DEN) is a mosquito-borne disease caused by four DENV serotypes (DENV-1, -2 -3 and -4) that have no treatments or vaccines. Primary infection with one DENV usually leads to acute illness followed by lifelong homotypic immunity, but susceptibility to infection by the other three DENVs remains. Therefore, a vaccine needs to protect from all four DENVs simultaneously. To date a suitable animal model to mimic systemic human illness only exists for DENV-2 in immunocompromised mice using passaged viruses; however, models are still needed for the remaining serotypes. This study describes establishment of a lethal systemic DENV-3 infection model with a human isolate in immunocompromised mice and is the first report of lethal infection by a non-adapted clinical DENV isolate without evidence of neurological disease. Our DENV-3 model provides a relevant platform to test DEN vaccines and antivirals.

  • Replication of Boid Inclusion Body Disease Associated Arenaviruses is Temperature Sensitive in Both Boid and Mammalian Cells [PublishAheadOfPrint]

  • Boid inclusion body disease (BIDB) is a fatal disease of boid snakes, the etiology of which has only recently been revealed following identification of several novel arenaviruses in diseased snakes. BIBD-associated arenaviruses (BIBDAV) are genetically divergent from the "classical" Old and New World arenaviruses and also differ substantially from each other. Even though there is convincing evidence that BIBDAV are indeed the etiological agent of BIBD, the BIBDAV reservoir hosts nndash; if any exist besides boid snakes themselves nndash; are not yet known. In this report we use University of Helsinki virus (UHV) to show that BIBDAV can effectively replicate also in mammalian, including human cells provided they are cultured at 30ddeg;C. The infection induces the formation of cytoplasmic inclusion bodies (IB), comprised mainly of viral nucleoprotein (NP), similar to those observed in BIBD and in boid cell cultures. Transfer of infected cells from 30ddeg;C to 37ddeg;C ambient temperature resulted in a progressive decline in IB formation and in the amount of viral NP and RNA, suggesting that BIBDAV growth is limited at 37ddeg;C. These observations indirectly indicate that IB formation is linked to viral replication. In addition to mammalian and reptilian cells, UHV infected arthropod (tick) cells when grown at 30ddeg;C. Even though our findings suggest that BIBDAV have a high potential to cross the species barrier, their inefficient growth at mammalian body temperatures indicates that the reservoir hosts of BIBDAV are likely species with a lower body temperature such as snakes.

    IMPORTANCE The newly-discovered boid inclusion body disease-associated arenaviruses (BIBDAV) of reptiles have drastically altered the phylogeny of the family Arenavirus. Prior to their discovery, known arenaviruses were considered as mainly rodent-borne viruses, with each arenavirus species having its own reservoir host. BIBDAV have so far been demonstrated in captive boid snakes but their possible reservoir host(s) have not yet been identified. Here we show, using University of Helsinki virus as a model, that these viruses are able to infect mammalian (including human) and arthropod cells. Our results provide in vitro proof of the considerable ability of arenaviruses to cross species barrier. However, our data indicate BIBDAV growth occurs at 30ddeg;C but is inhibited at 37ddeg;C, implying that crossing of the species barrier would be hindered by the body temperature of mammalian species.

  • Expression of the epidermodysplasia verruciformis-associated genes EVER1 and EVER2 is activated by exogenous DNA and inhibited by LMP1 oncoprotein from Epstein--Barr virus [PublishAheadOfPrint]

  • EVER1 and EVER2 are mutated in epidermodysplasia verruciformis patients, who are susceptible to beta HPV infection. It is unknown whether their products control the infection of other viruses. Here, we show that the expression of both genes in B cells is activated immediately after EBV infection, whereas at later stages it is strongly repressed via activation of NF-B signaling pathway by LMP1. Ectopic expression of EVER1 impairs the ability of EBV to infect B cells.

  • Two classes of broadly neutralizing antibodies within a single lineage directed to the high-mannose patch of HIV Envelope [PublishAheadOfPrint]

  • The high-mannose patch of HIV envelope (Env) elicits broadly neutralizing antibodies (bnAbs) during natural infection relatively frequently and consequently this region has become a major target of vaccine design. However, it has also become clear that antibody recognition of the region is complex due to the quaternary nature of the bnAb epitopes and variability in neighboring loops and glycans critical to the epitopes. BnAbs against this region have some shared and some distinguishing features that are crucial to understand in order to design optimal immunogens that can induce different classes of bnAbs against this region. Here, we compare two branches of a single antibody lineage, in which all members recognize the high-mannose patch. One branch (prototype bnAb PGT128) has a 6-amino acid insertion in CDRH2 that is crucial for broad neutralization. Antibodies in this branch appear to favor a glycan site at N332 on gp120 and somatic hypermutation is required to accommodate the neighboring V1 loop glycans and glycan heterogeneity. The other branch (prototype bnAb PGT130) lacks the CDRH2 insertion. Antibodies in this branch are noticeably effective at neutralizing viruses with an alternate N334 glycan site but are less able to accommodate glycan heterogeneity. We identify a new somatic variant within this branch that is predominantly dependent on N334. The crystal structure of PGT130 offers some insight into these differences compared to PGT128. We conclude that different immunogens may be required to elicit bnAbs that have the optimal characteristics of the two branches of the lineage described.

    Importance Development of an HIV vaccine is of vital importance for control of new infections and it is thought that elicitation of HIV bnAbs will be an important component of an effective vaccine. Increasingly, bnAbs that bind to the cluster of high-mannose glycans on the HIV envelope glycoprotein, gp120, are being highlighted as important templates for vaccine design. In particular, bnAbs from donor 36 (PGTs125-131) have been shown to be extremely broad and potent. Combination of these bnAbs enhanced neutralization breadth considerably suggesting that an optimal immunogen should elicit several antibodies from this family. Here we study the evolution of this antibody family to inform immunogen design. We identify two classes of bnAb that differ in their recognition of the high-mannose patch and show that different immunogens may be required to elicit these different classes.

  • Three-dimensional structure of a protozoal dsRNA virus that infects enteric pathogen Giardia lamblia [PublishAheadOfPrint]

  • Giardia lamblia virus (GLV) is a small, nonenveloped, nonsegmented dsRNA virus infecting Giardia lamblia, the most common protozoan pathogen of the human intestine and a major agent of waterborne diarrheal disease worldwide. GLV (genus Giardiavirus) is a member of family Totiviridae, along with several other groups of protozoal or fungal viruses including Leishmania RNA viruses and Trichomonas vaginalis viruses. Interestingly, GLV is more closely related than other Totiviridae members to a group of recently discovered metazoan viruses that includes penaeid shrimp infectious myonecrosis virus (IMNV). Moreover, GLV is the only known protozoal dsRNA virus that can transmit efficiently by extracellular means, also like IMNV. In this study, we used transmission electron cryomicroscopy and icosahedral image reconstruction to examine the GLV virion at an estimated resolution of 6.0 AAring;. Its outermost diameter is 485 AAring;, making it the largest totivirus capsid analyzed to date. Structural comparisons of GLV and other totiviruses highlighted a related "T=2" capsid organization and a conserved helix-rich fold in the capsid subunits. In agreement with its unique capacity as a protozoal dsRNA virus to survive and spread through extracellular environments, GLV was found to be more thermoresistant than Trichomonas vaginalis virus 1, but no specific protein machinery to mediate cell entry, such as the fiber complexes in IMNV, could be localized. These and other structural and biochemical findings provide a basis for future work to dissect the cell entry mechanism of GLV into a "primitive" (early-branching) eukaryotic host and an important enteric pathogen of humans.

    IMPORTANCE Numerous pathogenic bacteria including Corynebacterium diphtheriae, Salmonella enterica, and Vibrio cholerae are infected with lysogenic bacteriophages that contribute significantly to bacterial virulence. In line with this phenomenon, several pathogenic protozoa including Giardia lamblia, Leishmania species, and Trichomonas vaginalis are persistently infected with dsRNA viruses, and growing evidence indicates that at least some of these protozoal viruses can likewise enhance the pathogenicity of their hosts. Understanding of these protozoal viruses, however, lags far behind that of many bacteriophages. Here we investigated the dsRNA virus that infects the widespread enteric parasite Giardia lamblia. Using electron cryomicroscopy and icosahedral image reconstruction, we determined the virion structure of Giardia lamblia virus, obtaining new information relating to its assembly, stability, functions in cell entry and transcription, and similarities and differences with other dsRNA viruses. The results of our study set the stage for further mechanistic work on the roles of these viruses in protozoal virulence.

  • Mutations in HIV-1 envelope that enhance entry with the macaque CD4 receptor alter antibody recognition by disrupting quaternary interactions within the trimer [PublishAheadOfPrint]

  • Chimeric SIV/HIV (SHIV) infection of macaques is commonly used to model HIV-1 transmission and pathogenesis in humans. Despite the fact that SHIVs encode SIV antagonists of the known macaque host restriction factors, these viruses require additional adaptation for replication in macaques to establish a persistent infection. Additional adaptation may be required in part because macaque CD4 (mCD4) is a suboptimal receptor for most HIV-1 Env variants. This requirement raises the possibility that adaptation of HIV-1 Env to the macaque host leads to selection of variants that lack important biological and antigenic properties of the viruses responsible for the HIV-1 pandemic in humans. Here, we investigated whether this adaptation process leads to changes in the antigenicity and structure of HIV-1 Env. For this purpose, we examined how two independent mutations that enhance mCD4-mediated entry, A204E and G312V, impact antibody recognition in the context of seven different parental HIV-1 Envs from diverse subtypes. We also examined HIV-1 Env variants from three SHIVs that had been adapted for increased replication in macaques. Our results indicate that these different macaque-adapted variants shared common features including resistance to antibodies directed to quaternary epitopes and sensitivity to antibodies directed to epitopes in the variable domains (V2 and V3) that are buried in the parental, un-adapted Envs. Collectively, these findings suggest that adaptation to mCD4 results in conformational changes that expose epitopes in the variable domains and disrupt quaternary epitopes in the native Env trimer.

    Importance: These findings indicate the antigenic consequences of adapting HIV-1 Env to macaque CD4. They also suggest that to best mimic HIV-1 infection in humans using the SHIV/macaque model, HIV-1 Envs should be identified that use mCD4 as a functional receptor and preserve quaternary epitopes characteristic of HIV-1 Env.

  • Activation of the chicken type I IFN response by infectious bronchitis coronavirus. [PublishAheadOfPrint]

  • Coronaviruses from both the Alpha and Betacoronavirus genera, interfere with the type I interferon (IFN) response in various ways, ensuring limited activation of the IFN response in most cell types. Of Gammacoronaviruses that mainly infect birds, little is known about activation of the host immune response. We show that the prototypical Gammacoronavirus, infectious bronchitis virus (IBV), induces a delayed activation of the IFN response in primary renal cells, tracheal epithelial cells and in a chicken cell line. Ifnbbeta; expression in fact, is delayed with respect to the peak of viral replication and accompanying accumulation of dsRNA. In addition, we demonstrate that MDA5 is the primary sensor for Gammacoronavirus infections in chicken cells. Furthermore, we provide evidence that accessory proteins 3a and 3b of IBV modulate the IFN response at the transcriptional and translational level. Finally, we show that, despite the lack of activation of the IFN response during the early phase of IBV infection, signalling of non-self dsRNA through both MDA5 and TLR3 remains intact in IBV-infected cells. Taken together, this study provides the first comprehensive analysis of host-virus interactions of a Gammacoronavirus with avian innate immune responses.

    Importance: Our results demonstrate that IBV has evolved multiple strategies to avoid activation of the type I interferon response. Taken together, the present study closes a gap in the understanding of host-IBV interaction, and paves the way for further characterization of the mechanisms underlying immune evasion strategies as well as pathogenesis of Gammacoronaviruses.

  • Complete genome characterization of recent and ancient Belgian pig group A rotaviruses and assessment of their evolutionary relationship with human rotaviruses [PublishAheadOfPrint]

  • Group A rotaviruses (RVAs) are an important cause of diarrhea in young pigs and children. An evolutionary relationship has been suggested to exist between pig and human RVAs. This hypothesis was further investigated by phylogenetic analysis of the complete genomes of six recent (G2P[27], G3P[6], G4P[7], G5P[7], G9P[13], and G9P[23]) and one historic (G1P[7]) Belgian pig RVA strains, and of all completely characterized pig RVAs around the globe. In contrast to the large diversity of genotypes found for the outer capsid proteins VP4 and VP7, a relatively conserved genotype constellation (I5-R1-C1-M1-A8-N1-T7-E1-H1) was found for the other 9 genes in most pig RVA strains. VP1, VP2, VP3, NSP2, NSP4 and NSP5 genes of porcine RVAs belonged to genotype 1, which is shared with human Wa-like RVAs. However, for most of these gene segments, pig strains clustered distantly from human Wa-like RVAs, indicating that viruses from both species have entered different evolutionary paths. However, VP1, VP2 and NSP3 genes of some archival human strains were moderately related to pig strains. Phylogenetic analysis of the VP6, NSP1 and NSP3 genes, as well as amino acid analysis of the antigenic regions of VP7 further confirm this evolutionary segregation. The present results also indicate that the species barrier is less strict for pig P[6]-strains, but that chances for successful spread of these strains in the human population are hampered by the better adaptation of pig RVAs to pig enterocytes. However, future surveillance of pig and human RVA strains is warranted.

    Importance Rotaviruses are an important cause of diarrhea in many species including pigs and humans. Our understanding of the evolutionary relationship between rotaviruses from both species is limited by the lack of genomic data of pig strains. In this study, recent and ancient Belgian pig rotavirus isolates were sequenced and their evolutionary relationship with human Wa-like strains was investigated. Our data show that Wa-like human and pig strains have entered different evolutionary paths. Our data indicate that pig P[6]-strains form the most considerable risk for interspecies transmission to humans. However, efficient spread of pig strains in the human population is most likely hampered by the adaptation of some crucial viral proteins to the cellular machinery of pig enterocytes. These data allow a better understanding of the risk for direct interspecies transmission events and the emergence of pig rotaviruses or pig-human reassortants in the human population.

  • Prevention of Measles Infection by intranasal delivery of fusion inhibitory peptides [PublishAheadOfPrint]

  • Measles virus (MV) infection is undergoing resurgence and remains one of the leading causes of death among young children worldwide despite the availability of an effective measles vaccine. MV infects its target cells by coordinated action of the MV H and the fusion (F) envelope glycoprotein; upon receptor engagement by H, the pre-fusion F undergoes a structural transition, extending and inserting into the target cell membrane and then re-folding into a post-fusion structure that fuses the viral and cell membranes. By interfering with this structural transition of F, peptides derived from the heptad-repeat (HR) regions of F can potently inhibit MV infection at the entry stage. We show here that specific features of H's interaction with its receptors modulate the susceptibility of MV F to peptide fusion inhibitors. A higher concentration of inhibitory peptides is required to inhibit F-mediated fusion when H is engaged to its nectin-4 receptor than when H is engaged to its CD150 receptor. Peptide inhibition of F may be subverted by continued engagement of receptor by H, a finding that highlights the ongoing role of H-receptor interaction after F has been activated, and helps guide the design of more potent inhibitory peptides. Intranasal administration of these peptides results in peptide accumulation in the airway epithelium with minimal systemic levels of peptide, and efficiently prevents MV infection in vivo in animal models. The results suggest an antiviral strategy for prophylaxis in vulnerable and/or immune compromised hosts.

    Importance Measles virus (MV) infection causes an acute illness that may be associated with infection of the central nervous system (CNS) and severe neurological disease. No specific treatment is available. We have shown that parenterally delivered fusion inhibitory peptides protect mice from lethal CNS MV disease. Here we show, using established small animal models of MV infection, that fusion inhibitory peptides delivered intranasally provide effective prophylaxis against MV infection. Since the fusion inhibitors are stable at room temperature, this intranasal strategy is feasible even outside of health care settings, could be used to protect individuals and communities in case of MV outbreaks, and could complement global efforts to control measles.

  • Short Conserved Sequences of HIV-1 Are Highly Immunogenic and Shift Immunodominance [PublishAheadOfPrint]

  • Cellular immunity is pivotal in HIV-1 pathogenesis, but hampered by viral sequence diversity. An approach to minimize this diversity is to focus immunity on conserved proteome sequences; therefore we selected four relatively conserved regions (Gag amino acids 148-214 and 250-335, Env 521-606, and Nef 106-148), each created in three mosaics to provide better coverage of M-group HIV-1 sequences. A conserved region vaccine (CRV) delivering genes for these four regions as equal mixtures of three mosaics (each region at a separate injection site) was compared to a whole protein vaccine (WPV) delivering equimolar amounts of genes for whole Gag, Env, and Nef as clade B consensus sequences (separate injection sites). Three rhesus macaques were vaccinated via three DNA primes and a recombinant adenovirus-5 boost (weeks 0, 4, 8, and 24 respectively). Although CRV inserts were about a fifth that of WPV, the CRV generated comparable magnitude blood CD4+ and CD8+ T lymphocyte responses against Gag, Env, and Nef. WPV responses preferentially targeted proteome areas outside the selected conserved regions in direct proportion to sequence lengths, indicating similar immunogenicities for the conserved regions versus the outside regions. The CRV yielded conserved region targeting density that was approximately five-fold that of the WPV. A similar pattern was seen in bronchoalveolar lymphocytes, but quadruple the magnitudes in blood. Overall, these findings demonstrated that the selected conserved regions are highly immunogenic, and that anatomically isolated vaccinations with these regions focuses immunodominance compared to full-length protein vaccination.

    IMPORTANCE HIV-1 sequence diversity is a major barrier limiting the capability of cellular immunity to contain infection and the ability of vaccines to match circulating viral sequences. To date, vaccines tested in humans have delivered whole proteins or genes for whole proteins, and it is unclear whether including only conserved sequences would yield sufficient cellular immunogenicity. We tested a vaccine delivering genes for four small conserved HIV-1 regions compared to a control vaccine with genes for whole Gag, Env, and Nef. Although the conserved regions ranged from 43 to 86 amino acids and comprised less than one fifth of whole Gag/Env/Nef, the vaccines elicited equivalent total magnitudes of both CD4+ and CD8+ T lymphocyte responses. These data demonstrate immunogenicity of these small conserved regions, and the potential for a vaccine to steer immunodominance towards conserved epitopes.

  • White Spot Syndrome Virus Protein Kinase 1 (PK1) Defeats the Host Cell's Iron Withholding Defense Mechanism by Interacting with Host Ferritin [PublishAheadOfPrint]

  • Iron is an essential nutrient for nearly all living organisms, including both hosts and invaders. Proteins such as ferritin regulate the iron levels in a cell, and in the event of a pathogenic invasion, the host can use an iron withholding mechanism to restrict the availability of this essential nutrient to the invading pathogens. However, pathogens use various strategies to overcome this host defense. In this study, we demonstrated that white spot syndrome virus (WSSV) protein kinase 1 (PK1) interacted with shrimp ferritin by the yeast two-hybrid system. A pull-down assay and 27-MHz quartz crystal microbalance (QCM) confirmed the interaction between PK1 and both ferritin and apoferritin. PK1 did not promote the release of iron ions from ferritin, but it prevented apoferritin from binding ferrous ions. When PK1 was overexpressed in Sf9 cells, the cellular labile iron pool (LIP) levels were elevated significantly. Immunoprecipitation and atomic absorption spectrophotometry (AAS) further showed that the number of iron ions bound by ferritin decreased significantly at 24 h post-WSSV infection. Taken together, these results suggest that PK1 prevents apoferritin from iron loading and thus stabilizes the cellular LIP levels, and that WSSV uses this novel mechanism to counteract the host cell's iron withholding defense mechanism.

    Importance Statement We show here that the white spot syndrome virus ensures the availability of iron by using a previously unreported mechanism to defeat the host cell's iron withholding defense mechanism. This defense is often implemented by ferritin, which can bind up to 4,500 iron atoms and acts to sequester free iron within the cell. WSSV's novel counter-strategy is mediated by a direct protein-protein interaction between viral protein kinase 1 (PK1) and the host ferritin. PK1 interacts with both ferritin and apoferritin, suppresses apoferritin's ability to sequester free iron ions, maintains the intracellular labile iron pool (LIP) - and thus the availability of free iron - is increased within cells.

  • Class switch recombination and somatic hypermutation of virus- neutralizing antibodies are not essential for the control of Friend retrovirus infection [PublishAheadOfPrint]

  • Toll-like receptor 7 and Myd88 are required for antiretroviral antibody and germinal center responses, but whether somatic hypermutation and class-switch recombination are required for antiretroviral immunity has not been examined. Mice deficient of activation-induced cytidine deaminase (AID) resisted Friend virus infection, produced virus-neutralizing antibodies, and controlled viremia. Passive transfer demonstrated that immune IgM from AID-deficient mice contributes to Friend virus control in the presence of virus-specific CD4+ T cells.

  • The influenza NA protein transmembrane and head domains have co-evolved [PublishAheadOfPrint]

  • Transmembrane domains (TMDs) from single-spanning membrane proteins are commonly viewed as membrane anchors for functional domains. Influenza virus neuraminidase (NA) exemplifies this concept as it retains enzymatic function upon proteolytic release from the membrane. However, the subtype 1 NA TMDs have become increasingly polar in human strains since 1918, which suggests that selection pressure exists on this domain. Here, we investigated the N1 TMD-head domain relationship by exchanging a prototypical llsquo;oldrrsquo; TMD (1933) with a llsquo;recentrrsquo; (2009) more polar TMD and an engineered hydrophobic TMD. Each exchange altered the TMD association, decreased the NA folding efficiency, and significantly reduced viral budding and replication at 37ddeg;C compared to 33ddeg;C where NA folds more efficiently. Passaging the chimera viruses at 37ddeg;C restored the NA folding efficiency, viral budding and infectivity by selecting for NA TMD mutations that correspond with their polar or hydrophobic assembly properties. These results demonstrate that single-spanning membrane protein TMDs can influence distal domain folding, as well as membrane-related processes, and suggests the NA TMD in H1N1 viruses has become more polar to maintain compatibility with the evolving enzymatic head domain.

    IMPORTANCE The neuraminidase (NA) protein from influenza A viruses (IAVs) functions to promote viral release and is one of the major surface antigens. The receptor destroying activity in NA resides in the distal head domain that is linked to the viral membrane by an N-terminal hydrophobic transmembrane domain (TMD). Over the last century, the subtype 1 NA TMDs (N1) in human H1N1 viruses have become increasingly more polar, and the head domains have changed to alter their antigenicity. Here, we provide the first evidence that an llsquo;oldrrsquo; N1 head domain from 1933 is incompatible with a llsquo;recentrrsquo; (2009) more polar N1 TMD sequence, and that during viral replication the head domain drives the selection of TMD mutations. These mutations modify the intrinsic TMD assembly to restore the head domain folding compatibility and the resultant budding deficiency. This likely explains why the N1 TMDs have become more polar and suggests the N1 TMD and head domain have co-evolved.

  • A conserved regulatory module at the C-terminus of the papillomavirus E1 helicase domain controls E1 helicase assembly [PublishAheadOfPrint]

  • Viruses frequently combine multiple activities into one polypeptide to conserve coding capacity. This strategy creates regulatory challenges to ascertain that the combined activities are compatible and do not interfere with each other. The papillomavirus E1 protein, as many other helicases, has the intrinsic ability to form hexamers and double hexamers (DH) that serve as the replicative DNA helicase. However, E1 also has the more unusual ability to generate local melting by forming a double trimer (DT) complex that can untwist the double stranded ori DNA in preparation for DH formation. Here we describe a switching mechanism that allows the papillomavirus E1 protein to form these two different kinds of oligomers and to transition between them. We show that a conserved regulatory module attached to the E1 helicase domain blocks hexamer and DH formation and promotes DT formation. In the presence of the appropriate trigger, the inhibitory effect of the regulatory module is relieved and the transition to DH formation can occur.

    Importance. This study provides a mechanistic understanding into how a multi-functional viral polypeptide can provide different, seemingly incompatible activities. A conserved regulatory sequence module attached to the AAA+ helicase domain in the papillomavirus E1 protein allows the formation of different oligomers with different biochemical activities.

  • Cognate CD4 T-cell licensing of dendritic cells heralds anti-CMV CD8 T-cell immunity after human allogeneic umbilical cord blood transplantation [PublishAheadOfPrint]

  • Reactivation of human cytomegalovirus (CMV) is hazardous to patients undergoing allogeneic cord-blood transplantation (CBT), lowering survival rates by approximately 25%. While antiviral treatment ameliorates viremia, complete viral control requires CD8+ T-cell-driven immunity. Mouse studies suggest that cognate antigen-specific CD4+ T-cell licensing of dendritic cells is required to generate effective CD8+ T-cell responses. For humans this was not fully understood. We here show that CD4+ T-cells are essential for licensing of human DCs to generate effector and memory CD8+ T-cell immunity against CMV in CBT patients. First, we show in CBT recipients that clonal expansion of CMV-pp65 specific CD4+ T-cells precedes the rise in CMV-pp65 specific CD8+ T-cells. Second, the elicitation of CMV-pp65 specific CD8+ T-cells from rare naiiuml;ve precursors in cord blood requires DC licensing by cognate CMV-pp65-specific CD4+ T-cells. Finally, also CD8+ T-cell memory responses require CD4+ T-cell-mediated licensing of DCs in our system, by secretion of IFN- by pp65-specific CD4+ T-cells. Together these data show that human DCs require the licensing by cognate antigen-specific CD4+ T-cells to elicit effective CD8+ T-cell-mediated immunity and fight off viral reactivation in CBT patients.

    Importance paragraph: Survival rates after stem cell transplantation are lowered by 25% when patients undergo reactivation of cytomegalovirus (CMV) they harbor. Immune protection against CMV is mostly executed by white blood cells called killer T-cells. We here show that for generation of optimally protective killer T-cell responses that respond to CMV, the early elicitation of help from a second branch of CMV-directed T-cells, called helper T-cells, is required.

  • Rotavirus entry, a deep journey into the cell with several exits [PublishAheadOfPrint]

  • Rotaviruses are the leading etiological agents of acute gastroenteritis in infants and young children worldwide. These nonenveloped viruses enter cells using different types of endocytosis and, depending on the virus strain, travel to different endosomal compartments before exiting to the cytosolic space. In this GEM we review the viral and cellular factors involved in the different stages of a productive virus cell entry and share with the readers the journey we have taken into the cell to learn about virus entry.

  • Chronic administration of {Delta}9-tetrahydrocannabinol induces intestinal anti-inflammatory microRNA expression during acute SIV infection of rhesus macaques [PublishAheadOfPrint]

  • Recreational and medical use of cannabis among HIV-infected individuals has increased in recent years. In SIV-infected macaques, chronic administration of 9-tetrahydrocannabinol (9-THC), inhibited viral replication, intestinal inflammation and slowed disease progression. Persistent gastrointestinal disease/inflammation has been proposed to facilitate microbial translocation, systemic immune activation and promote disease progression. Cannabinoids including 9-THC attenuated intestinal inflammation in mouse colitis models and SIV-infected rhesus macaques. To determine if the anti-inflammatory effects of 9-THC involved differential microRNA (miRNA) modulation, we profiled miRNA expression at 14, 30 and 60 days post-infection (DPI) in the intestine of uninfected macaques receiving 9-THC (n=3) and SIV-infected macaques administered either vehicle (VEH/SIV; n=4) or THC (THC/SIV; n=4). Chronic 9-THC administration to uninfected macaques significantly and positively modulated intestinal miRNA expression by increasing the total number of differentially expressed miRNAs from 14 to 60DPI. At 60DPI, ~28% of miRNAs showed decreased expression in VEH/SIV compared to none in the THC/SIV group. Furthermore, compared to the VEH/SIV group, THC selectively upregulated the expression of miR-10a, miR-24, miR-99b, miR-145, miR-149 and miR-187 previously been shown to target proinflammatory molecules. NOX4, a potent reactive oxygen species generator was confirmed as a direct miR-99b target. A significant increase in NOX4+ crypt epithelial cells was detected in VEH/SIV compared to the THC/SIV group. We speculate that miR-99b-mediated NOX4 downregulation may protect the intestinal epithelium from oxidative stress-induced damage. These results support a role for differential miRNA induction in THC-mediated suppression of intestinal inflammation. Whether similar miRNA modulation occurs in other tissues requires further investigation.

    IMPORTANCE Gastrointestinal tract (GI) disease/inflammation is a hallmark of HIV/SIV infection. Previously, we showed that chronic treatment of SIV-infected macaques with 9 tetrahydrocannabinol (9-THC) increased survival and decreased viral replication and infection induced gastrointestinal inflammation. Here, we show that chronic THC administration to SIV-infected macaques induced an anti-inflammatory microRNA expression profile in the intestine at 60DPI. These included several miRNAs bioinformatically predicted to directly target CXCL12, a chemokine known to regulate lymphocyte and macrophage trafficking into the intestine. Specifically, miR-99b was significantly upregulated in THC-treated SIV-infected macaques and confirmed to directly target NADPH oxidase 4 (NOX4), a reactive oxygen species generator known to damage intestinal epithelial cells. Elevated miR-99b expression was associated with a significantly decreased number of NOX4+ epithelial cells in the intestines of THC-treated SIV-infected macaques. Overall, our results show that selective upregulation of anti-inflammatory miRNA expression, contributes to THC-mediated suppression of gastrointestinal inflammation and maintenance of intestinal homeostasis.

  • miR-21 Attenuates Human Cytomegalovirus Replication in Neural Cells by Targeting Cdc25a [PublishAheadOfPrint]

  • Congenital human cytomegalovirus (HCMV) infection is a leading cause of birth defects, primarily manifesting as neurological disorders. HCMV infection alters expression of cellular microRNAs and induces cell-cycle arrest, which in turn modifies the cellular environment to favor virus replication. Previous observations found that HCMV infection reduces miR-21 expression in neural progenitor/stem cells (NPCs). Here, we show that infection of NPCs and U-251MG cells represses miR-21 while increasing levels of Cdc25a, a cell cycle regulator and known target of miR-21. These opposing responses to infection prompted an investigation of the relationship between miR-21, Cdc25a, and viral replication. Overexpression of miR-21 in NPCs and U-251MG cells inhibited viral gene expression, genome replication, and production of infectious progeny, while shRNA-knockdown of miR-21 in U-251MG cells increased viral gene expression. In contrast, overexpression of Cdc25a in U-251MG cells increased viral gene expression and production of infectious progeny and overcame the inhibitory effects of miR-21 overexpression. Three viral gene products, IE1, pp71, and UL26, were shown to inhibit miR-21 expression at the transcriptional level. These results suggest that Cdc25a promotes HCMV replication and elevation of Cdc25a levels post HCMV infection are due in part to HCMV-mediated repression of miR-21. Thus, miR-21 is an intrinsic antiviral factor that is modulated by HCMV infection. This suggests a role for miR-21 downregulation in the neuropathogenesis of HCMV infection of the developing CNS.

    IMPORTANCE Human cytomegalovirus (HCMV) is a ubiquitous pathogen and has very high prevalence among population, especially in China, and congenital HCMV infection is a major cause for birth defects. Elucidating virus/host interactions that govern (HCMV) replication in neuronal cells is critical to understanding the neuropathogenesis of birth defects resulting from congenital infection. In this study, we confirm that HCMV infection down regulates miR-21, but up regulates Cdc25a. Further determined the negative effects of cellular miRNA miR-21 on HCMV replication in neural progenitor/stem cells and U-251MG glioblastoma/astrocytoma cells. More importantly, our results provide the first evidence that miR-21 negatively regulates HCMV replication by targeting Cdc25a, a vital cell cycle regulator. We further found that viral gene products of IE1, pp71, and UL26 play roles in inhibiting miR-21 expression, which in turn causes increase of Cdc25a and benefits HCMV replication. Thus, miR-21 appears to be an intrinsic anti-viral factor that represents a potential target for therapeutic intervention.

  • Evidence of lymphocytic choriomeningitis virus in domestic mice in Gabon; risk of emergence of LCMV encephalitis in Central Africa [PublishAheadOfPrint]

  • Lymphocytic choriomeningitis virus (LCMV) can cause acute fatal disease in all continents but was never detected in Africa. We report the first detection of LCMV RNA in common European house mouse (Mus musculus domesticus) in Africa. Phylogenetic analyses showed close relationship with North American strains. These findings suggest appearance of LCMV acute encephalitis cases. This is a perfect example of virus dissemination by its natural host that may have dramatic public health consequences.

  • Conformational Changes in Adenovirus Hexon Subunit Responsible for Regulating Cytoplasmic Dynein Recruitment [PublishAheadOfPrint]

  • Virus capsids provide genome protection from environmental challenges, but are also poised to execute a program of compositional and conformational changes to facilitate virion entry and infection. The most abundant adenovirus 5 (AdV5) capsid protein, hexon, directly recruits the motor protein cytoplasmic dynein following virion entry. Dynein recruitment is crucial for capsid transport to the nucleus and requires transient exposure of AdV5 hexon to low pH, presumably mimicking passage through the endosomal compartment. These results suggest a pH-dependent capsid modification during early infection. The changes to the hexon structure controlling this behavior have not been explored. We report that hexon remains trimeric at low pH, but undergoes more subtle conformational changes. These are indicated by an increased sensitivity to SDS-mediated dissociation and to dispase proteolysis. Both effects are reversed at neutral pH, as is dynein binding of low pH-treated hexon. Dispase cleavage, which we find maps to a specific site within the hypervariable region 1 (HVR1) of AdV5 hexon, has no apparent effect on virion entry, but inhibits its transport to the nucleus. In addition, an AdV5 mutant containing HVR1 of AdV48 is unable to bind dynein and strongly inhibited in the post-entry transport step. These results reveal that conformational changes involving hexon HVR1 are the basis for a novel viral mechanism controlling capsid transport to the nucleus.

    IMPORTANCE The adenovirus 5 (AdV5) capsid protein hexon recruits the molecular motor protein cytoplasmic dynein in a pH-dependent manner, a function critical for efficient transport towards the nucleus and AdV5 infectivity. In this work, we describe how low pH exposure induces reversible structural changes in AdV5 hexon and how these changes affect dynein binding. In addition, we identified a pH-sensitive dispase cleavage site in hexon HVR1 which depends on the same structural changes and, furthermore, regulates dynein recruitment and capsid redistribution in infected cells. These data provide the first evidence relating the long known but subtle pH-dependent structural changes in hexon to a more recently established essential but poorly understood role in virus transport. These results have broad implications for understanding virus infectivity in general, and our ability to block the recruitment mechanism has potential therapeutic implications as well.

  • Co-circulation of two distinct genetic and antigenic lineages of proposed influenza D virus in cattle [PublishAheadOfPrint]

  • Viruses with approximately 50% homology to human influenza C virus (ICV) have recently been isolated from swine and cattle. The overall low homology to ICV, lack of antibody cross reactivity to ICV in hemagglutination inhibition (HI) and agar gel immunodiffusion assays and inability to productively reassort with ICV led to the proposal that these viruses represented a new genus of influenza, influenzavirus D (IDV). To further our understanding of the epidemiology of IDV, real time reverse transcription PCR was performed on a set of 208 samples from bovines with respiratory disease. Ten samples (4.8%) were positive and six viruses were successfully isolated in vitro. Phylogenetic analysis of full genome sequences of these six new viruses and four previously reported viruses revealed two distinct co-circulating lineages represented by D/swine/Oklahoma/1334/2011 (D/OK) and D/bovine/Oklahoma/660/2013 (D/660) which frequently reassorted with one another. Antigenic analysis using the HI assay and lineage-representative D/OK and D/660 antiserum found up to an approximate 10-fold loss in cross reactivity against heterologous clade antiserum. One isolate, D/bovine/Kansas/3-13/2011 (D/3-13), clustered with the D/660 lineage, but also had high HI titers to heterologous (D/OK) clade antiserum. Molecular modeling of the hemagglutinin esterase fusion protein of D/3-13 identified a mutation at position 212 as a possible antigenic determinant responsible for the discrepant HI results. These results suggest that IDV is common in bovines with respiratory disease and at least two genetic and antigenically distinct clades co-circulate.

    Importance: A novel bovine influenza virus was recently identified. Detailed genetic and antigenic studies led to the proposal that this virus represents a new genus of influenza, influenzavirus D (IDV). Here we show that IDV is common in clinical samples of bovine respiratory disease complex (BRDC), with prevalence similar to other established BRDC etiological agents. These results are in good agreement with near ubiquitous seroprevalence of IDV previously found. Phylogenetic analysis of complete genome sequences found evidence for two distinct co-circulating lineages of IDV which freely reassort. Significant antigenic differences were observed between the two lineages that generally agreed with the surface glycoprotein hemagglutinin esterase phylogeny. These results, in addition to the ability of IDV to infect and transmit in multiple mammalian species, warrant additional studies to determine the pathogenic potential of IDV.

  • Enhanced transgene expression from single-stranded D-sequence-substituted recombinant AAV vectors in human cell lines in vitro and in murine hepatocytes in vivo [PublishAheadOfPrint]

  • We have previously reported that the removal of a 20-nucleotide sequence, termed the D-sequence, from both ends of the inverted terminal repeats (ITRs) in the adeno-associated virus serotype 2 (AAV2) genome, significantly impairs rescue, replication, and encapsidation of the viral genomes (J. Mol. Biol., 250: 573-580, 1995; J. Virol., 70: 1668-1677, 1996). Here we describe that substitution of only one D-sequence in either ITR restores each of these functions, but DNA strands of only single-polarity are encapsidated in mature progeny virions. Since most commonly used recombinant AAV vectors contain a single-stranded (ss) DNA, which is transcriptionally-inactive, efficient transgene expression from AAV vectors is dependent upon viral second-strand DNA synthesis. We have also identified a transcription-suppressor sequence in one of the D-sequences, which shares homology with the binding site for the cellular NF-B-repressing factor (NRF). The removal of this D-sequence from, and substitution with a sequence containing putative binding sites for transcription factors in, ssAAV vectors significantly augments transgene expression both in human cell lines in vitro and in murine hepatocytes in vivo. The development of these genome-modified ssAAV vectors has implications not only in the basic biology of AAV, but also in the optimal use of these vectors in human gene therapy.

    Importance The results of the studies described here have provided not only novel insights into some of the critical steps in the life cycle of a human virus, the adeno-associated virus (AAV) that causes no known disease, but have also led to the development of novel recombinant AAV vectors which are more efficient in allowing increased level of gene expression. Thus, these studies have significant implications in the use of these novel AAV vectors in their potential use in human gene therapy.

  • Varicella-zoster virus-derived MHC class I-restricted peptide affinity is a determining factor for HLA risk profile to develop PHN [PublishAheadOfPrint]

  • Postherpetic neuralgia (PHN) is the most common complication of herpes zoster and is typified by a lingering pain that can last months or years after the characteristic herpes zoster rash disappears. It is well known that there are risk factors for the development of PHN, such as its association with certain HLA alleles. In this study, previous HLA-genotyping results were collected and subjected to a meta-analysis with increased statistical power. This work shows that the alleles HLA-A*33 and HLA-B*44 are significantly enriched in PHN patients, while HLA-A*02 and HLA-B*40 are significantly depleted. Prediction of the varicella-zoster virus (VZV) peptide affinity for these four HLA variants using two existing and one in-house-developed state-of-the-art MHC class I ligand prediction methods reveals that there is a great difference in their absolute and relative peptide binding repertoires. It was observed that HLA-A*02 displays a high affinity for approximately seven-fold higher number of VZV peptides than HLA-B*44. Further, after correction for HLA-allele specific limitations, the relative affinity for VZV peptides of HLA-A*33 and HLA-B*44 was found to be significantly less than those of HLA-A*02 and HLA-B*40. In addition, HLA peptide affinity calculations indicate strong trends for VZV to avoid high affinity peptides in some of its proteins, independent from the studied HLA allele.

    Importance The varicella-zoster virus can cause two distinct diseases: chickenpox (varicella) and shingles (herpes zoster). Varicella is a common disease in young children, while herpes zoster is more frequent in older individuals. A common complication of herpes zoster is postherpetic neuralgia, a persistent and debilitating pain that can remain months up to years after the resolution of the rash. In this study, we show that the affinity for varicella-zoster peptides of HLA variants associated with higher postherpetic neuralgia risk have a lower relative affinity for varicella-zoster peptides than variants with a lower risk. These results provide new insight into the development of postherpetic neuralgia and strongly support the hypothesis that one of its possible underlying causes is a suboptimal anti-VZV immune response due to weak HLA-binding peptide affinity.

  • Virological and immunological characterization of novel NYVAC-based HIV/AIDS vaccine candidates expressing clade C trimeric soluble gp140(ZM96) and Gag(ZM96)-Pol-Nef(CN54) as VLPs [PublishAheadOfPrint]

  • The generation of vaccines against HIV/AIDS able to induce long-lasting protective immunity remains a major goal in HIV field. The modest efficacy (31.2%) against HIV infection observed in the RV144 phase III clinical trial highlighted the need for further improvement of HIV vaccine candidates, formulation and vaccine regimen. In this study, we have generated two novel NYVAC vectors expressing HIV-1 clade C gp140(ZM96) (NYVAC-gp140) or Gag(ZM96)-Pol-Nef(CN54) (NYVAC-Gag-Pol-Nef) and defined their virological and immunological characteristics in cultured cells and in mice. Insertion of HIV genes does not affect the replication capacity of NYVAC recombinants in primary chicken embryo fibroblast cells, HIV sequences remain stable after multiple passages and HIV antigens are correctly expressed and released from cells, with Env as a trimer (NYVAC-gp140), while in NYVAC-Gag-Pol-Nef-infected cells, Gag-induced virus-like particles (VLPs) are abundant. Electron microscopy revealed that VLPs accumulated with time at the cell surface, with no interference with NYVAC morphogenesis. Both vectors trigger specific innate responses in human cells and show an attenuation profile in immunocompromised adult BALB/c and newborn CD1 mice after intracranial inoculation. Analysis of the immune responses elicited in mice after homologous NYVAC prime/NYVAC boost immunization shows that recombinant viruses induced polyfunctional Env-specific CD4 or Gag-specific CD8 T cell responses. Antibody responses against gp140 and p17/p24 were elicited. Our findings showed important insights in virus-host cell interactions of NYVAC vectors expressing HIV antigens, with activation of specific immune parameters which will help to unravel potential correlates of protection against HIV in human clinical trials with these vectors.

    Importance We have generated two novel NYVAC-based HIV vaccine candidates expressing HIV-1 clade C trimeric soluble gp140 (ZM96) or Gag(ZM96)-Pol-Nef(CN54) as VLPs. These vectors are stable, express high levels of both HIV-1 antigens, Gag-induced VLPs do not interfere with NYVAC morphogenesis, are highly attenuated in immunocompromised and newborn mice after intracranial inoculation, trigger specific innate immune responses in human cells and activate T (Env-specific CD4 and Gag-specific CD8) and B cell immune responses to the HIV antigens, leading to high antibody titers against gp140. For these reasons, these vectors can be considered as vaccine candidates against HIV/AIDS and are currently being tested in macaques and humans.

  • The Hepatitis B Virus HBx protein activates AKT to simultaneously regulate HBV replication and hepatocyte survival [PublishAheadOfPrint]

  • Chronic infection with the hepatitis B virus (HBV) is a risk factor for developing liver diseases such as hepatocellular carcinoma (HCC). HBx is a multifunctional protein encoded by the HBV genome; HBx stimulates HBV replication and is thought to play an important role in the development of HBV-associated HCC. HBx can activate the phosphatidylinositol 3 kinase (PI3K)/AKT signaling pathway in some cell lines; however, whether HBx regulates PI3K/AKT signaling in normal hepatocytes has not been evaluated. In studies described here, we assessed HBx activation of PI3K/AKT signaling in an ex vivo model of cultured primary hepatocytes and determined how this HBx activity affects HBV replication. We report that HBx activates AKT in primary hepatocytes and that activation of AKT decreases HBV replication and HBV mRNA and core protein levels. We show that the transcription factor Hepatocyte Nuclear Factor (HNF) 4aalpha; is a target of HBx-regulated AKT and link HNF4aalpha; to HBx-regulated AKT modulation of HBV transcription and replication. Although we and others have shown that HBx stimulates and is likely required for HBV replication, we now report that HBx also activates signals that can diminish the overall level of HBV replication. While this may seem counterintuitive, we show that an important effect of HBx activation of AKT is inhibition of apoptosis. Consequently, our studies suggest that HBx balances HBV replication and cell survival by stimulating signaling pathways that enhance hepatocyte survival at the expense of higher levels of HBV replication.

    Importance A chronic hepatitis B virus (HBV) infection is a common cause for the development of liver cancer. Regulation of cell-signaling pathways by the HBV HBx protein is thought to influence the development of HBV-associated liver cancer. HBx stimulates, and may be essential for HBV replication. We show that HBx activates AKT in hepatocytes to reduce HBV replication. While this seems contradictory to an essential role of HBx during HBV replication, HBx activation of AKT inhibits hepatocyte apoptosis, and this may facilitate persistent, noncytopathic HBV replication. AKT regulates HBV replication by reducing the activity of the transcription factor Hepatocyte Nuclear Factor (HNF) 4aalpha;. HBx activation of AKT may contribute to the development of liver cancer by facilitating persistent HBV replication, augmenting dedifferentiation of hepatocytes by inhibiting HNF4aalpha; functions, and activating AKT-regulated oncogenic pathways. AKT-regulated factors may provide therapeutic targets for inhibiting HBV replication and the development of HBV-associated liver cancer.

  • A Novel Factor I Activity in Nipah Virus Inhibits Human Complement Pathways Through Cleavage of C3b [PublishAheadOfPrint]

  • Complement (C') is an innate immune system that most animal viruses must face during natural infections. Given that replication and dissemination of the highly pathogenic Nipah virus (NiV) includes exposure to environments rich in C' factors, we tested the in vitro sensitivity of NiV to C'-mediated neutralization. Here we show that NiV was completely resistant to in vitro neutralization by normal human serum (NHS). Treatment of purified NiV with NHS activated C' pathways, but there was very little C3 deposition on virus particles. In in vitro reconstitution experiments, NiV particles provided time- and dose-dependent Factor I-like protease activity capable of cleaving C3b into inactive iC3b. NiV-dependent inactivation of C3b only occurred with cofactors Factor H and soluble CR1, but not with CD46. Purified NiV particles did not support C4b cleavage. Electron microscopy of purified NiV particles showed immunogold-labeling with anti-Factor I antibodies. Our results suggest a novel mechanism by which NiV evades the human C' system through a unique Factor I-like activity.

    IMPORTANCE Viruses have evolved mechanisms to limit C'-mediated neutralization, some of which involve high-jacking cellular proteins involved in control of inappropriate C' activation. Here we report a previously unknown mechanism whereby NiV provides a novel protease activity capable of in vitro cleavage and inactivation of C3b, a key component of the C' cascade. These data help to explain how an enveloped virus such as NiV can infect and disseminate through body fluids that are rich in C' activity. Disrupting the ability of NiV to recruit C' inhibitors could form the basis for the development of effective therapies and safer vaccines to combat these highly pathogenic emerging viruses.

  • Regulation of Influenza A Virus Nucleoprotein Oligomerisation by Phosphorylation [PublishAheadOfPrint]

  • In the influenza virus ribonucleoprotein complex the oligomerisation of the nucleoprotein is mediated by an interaction between the tail-loop of one molecule and the groove of the neighbouring molecule. In this study we show that phosphorylation of a serine residue (S165) within the groove of influenza A virus nucleoprotein inhibits oligomerisation and, consequently, ribonucleoprotein activity and viral growth. We propose that nucleoprotein oligomerisation in infected cells is regulated by reversible phosphorylation.

  • Discovery and characterization of auxiliary proteins encoded by Simian T-cell Lymphotropic Viruses type 3 [PublishAheadOfPrint]

  • HTLV-1 and HTLV-2 encode auxiliary proteins that play important roles in viral replication, viral latency and immune escape. The presence of auxiliary proteins-encoding ORFs in HTLV-3, the latest HTLV to be discovered is unknown. STLV-3 viruses are almost identical to HTLV-3. Given the lack of HTLV-3-infected cell lines, we took advantage of STLV-3-infected cells and of an STLV-3 molecular clone to search for the presence of auxiliary transcripts. Using RT-PCR, we first uncovered the presence of three unknown viral mRNAs encoding putative proteins of 5, 8 and 9 kDa, and confirmed the presence of the previously reported RorfII transcript. The existence of those viral mRNAs was confirmed using splice site-specific RT-PCR in ex vivo samples. We showed that p5 is distributed throughout the cell and does not colocalize with a specific organelle. p9 localization is similar to that of HTLV-1 p12 and induced a strong decrease in calreticulin signal, similar to HTLV-1 p12. Despite that p8, RorfII and Rex-3 share an N-terminal sequence that is predicted to contain a nucleolar localization signal, only p8 is found in the nucleolus. p8 location in the nucleolus is linked to a bipartite NoLS. p8, and to a lesser extent p9, repressed viral expression but did not alter Rex-3-dependent mRNA export. Using a transformation assay, we finally showed that none of the STLV-3 auxiliary proteins had the ability to induce colony formation, while both Tax3 and APH-3 promote cellular transformation. Altogether, these results complete the characterization of the newly described PTLV-3 virus.

    IMPORTANCE Together with their simian counterparts, HTLVs form the Primate T-Lymphotropic Viruses. HTLVs arose from interspecies transmission between non-human primates and humans. HTLV-1 and HTLV-2 encode auxiliary proteins that play important roles in viral replication, viral latency and immune escape. The presence of ORFs encoding auxiliary proteins in HTLV-3 or STLV-3 genomes was unknown. Using in silico analyses, ex vivo samples or in vitro experiments, we have uncovered the presence of 3 unknown viral mRNAs encoding putative proteins and confirmed the presence of a previously reported viral transcript. We characterized the intracellular localization of the four different proteins. We showed that two of them repress viral expression, but that none of them has the ability to induce colony formation. However, both Tax and the antisense encode protein APH-3 promote cell transformation. Our results allowed us to characterize for the first time 4 new retroviral proteins.

  • HIV-1 and morphine regulation of autophagy in microglia: Limited interactions in the context of HIV-1 infection and opioid abuse. [PublishAheadOfPrint]

  • Microglia are the predominant resident central nervous system (CNS) cell type productively infected by human immunodeficiency virus (HIV) type-1, and play a key role in the progression of HIV-associated dementia (HAD). Moreover, neural dysfunction and progression to HAD are accelerated in opiate drug abusers. In the present study, we examined the role of the autophagy pathway in the neuropathogenesis of HIV-1 using primary human microglial cells and determined whether opiates converge at this point. Infection of microglia with the HIV-1SF162 macrophage (M)-tropic strain resulted in increased Beclin1 expression, accompanied by an increase of LC3 protein levels and accumulation of LC3 reporter RFP+GFP+ (yellow) punctae, suggesting that HIV-1 infection triggers autophagosome formation without promoting protein degradation by the lysosome. Conversely, co-exposure with HIV-1 and morphine significantly decreased viral-induced Beclin1 expression and autophagosome formation. Exploration of the possible mechanism(s) used by morphine to disrupt the autophagic process unveiled a significant increase in intracellular pH, which coincided with a reduction in the formation of acidic vesicular organelles and in autophagolysosome formation. Small interfering (si) RNA targeting BECN1, a gene critical for autophagosome formation, significantly reduced viral replication and the viral-induced inflammatory responses. Conversely, morphine-enhanced viral replication and inflammatory responses were not affected by gene silencing with siBeclin1, suggesting that the interactive effect of morphine in HIV-1 pathogenesis is mediated through a Beclin1-independent mechanism. These novel findings may have important implications on the connections between autophagy and HIV-1 pathogenesis mediated by microglial cells in opioid-abusing individuals.

    IMPORTANCE About 50% of individuals infected with HIV-1 will develop some sort of neurocognitive impairment that cannot be prevented nor eradicated by antiretroviral therapy. The neuropathogenesis is mostly due to inflammatory responses by infected microglia, the resident immune cells of the brain. Cognitive disorders may also be associated with drugs of abuse. In fact, opioid drug users have an increased risk of developing neurocognitive disorders with increased progression to dementia. While the mechanism(s) by which opioids exacerbate the neuropathogenesis of HIV-1 are not entirely known, it is well accepted that glia are critical to opiate responses. This study gives us new insight into possible autophagic mechanism(s) in microglia that control HIV-1 replication and viral-induced inflammation in the context of opioid abuse and should greatly improve our knowledge in the pathogenesis of HIV-1 resulting from substance abuse to provide a better understanding for the design of candidate antiviral therapies targeting drug-abusing individuals.

  • Canine Distemper Virus Envelope Protein Interactions Modulated by Hydrophobic Residues in the Fusion Protein Globular Head [PublishAheadOfPrint]

  • Membrane fusion for morbillivirus cell entry relies on critical interactions between the viral fusion (F) and attachment (H) envelope glycoproteins. Through extensive mutagenesis of an F-cavity recently proposed to contribute to the interaction with the H-protein, we identified two neighboring hydrophobic residues responsible for severe F-to-H-binding and fusion-triggering deficiencies when mutated in combination. Since both residues reside on one side of the F-cavity, the data suggest that H binds the F-globular head domain sideway.

  • Interactome analysis of the human respiratory syncytial virus RNA polymerase complex identifies protein chaperones as important co-factors that promote L protein stability and RNA synthesis. [PublishAheadOfPrint]

  • The human respiratory syncytial virus (HRSV) core viral RNA polymerase comprises the large polymerase protein (L) and its co-factor the phosphoprotein (P) which associate with the viral ribonucleoprotein complex to replicate the genome and, together with the M2-1 protein, transcribe viral mRNAs. Whilst cellular proteins have long been proposed to be involved in the synthesis of HRSV RNA by associating with the polymerase complex, their characterization has been hindered by the difficulty of purifying the viral polymerase from mammalian cell culture. In this study, EGFP-tagged L and P protein expression was coupled with high affinity anti-GFP antibody-based immunoprecipitation and quantitative proteomics to identify cellular proteins that interacted with either the L or the P proteins when expressed as part of a biologically active viral RNP. Several core groups of cellular proteins were identified that interacted with each viral protein, including in both cases, protein chaperones. Ablation of chaperone activity using small molecule inhibitors confirmed previous studies, which suggested this class of proteins acted as positive viral factors. Inhibition of HSP90 chaperone function in the current study showed that HSP90 was critical for L protein function and stability, whether in the presence or absence of the P protein. Inhibition studies suggested that HSP70 also disrupted virus biology and might help the polymerase remodel the nucleocapsid to allow RNA synthesis to occur efficiently. This indicated a pro-viral role for protein chaperones in HRSV replication and demonstrates that the function of cellular proteins can be targeted as potential therapeutics to disrupt virus replication.

    Importance Human respiratory syncytial virus (HRSV) represents a major health care and economic burden, being the main cause severe respiratory infections in infants worldwide. No vaccine or effective therapy is available. This study focused on identifying those cellular proteins that potentially interact specifically with the viral proteins that are central to virus replication and transcription with a view to providing potential targets for the development of a specific, transient therapeutic, which disrupts virus biology but prevents the emergence of resistance whilst maintaining cell viability. In particular, protein chaperones (heat shock protein 70 and 90), which aid protein folding and function, were identified. The mechanism by which these chaperones contribute to virus biology was tested and this study demonstrates to the field, that cellular protein chaperones may be required for maintaining the correct folding and therefore functionality of specific proteins within the virus replication complex.

  • PDZRN3/LNX3 is a Novel Target of HPV 16 and HPV-18 E6 [PublishAheadOfPrint]

  • High-risk HPV E6 proteins have a C-terminal PDZ binding motif, through which they bind, and target for proteasome-mediated degradation, a number of PDZ-containing cellular targets. Recently, studies suggested that the RING-containing ubiquitin-protein ligase, PDZRN3, might also be an HPV E6 target. In this analysis we show that HPV-16 and HPV-18 E6 can target PDZRN3 in a PDZ and proteasome-dependent manner, and provides a connection between the HPV life-cycle and differentiation-related STAT-signalling.

  • Coxsackievirus B3 Engineered to Contain microRNA Targets for Muscle-specific microRNAs Display Attenuated Cardiotropic Virulence in Mice [PublishAheadOfPrint]

  • Coxsackievirus B3 (CVB3) is trophic for cardiac tissue and is a major causative agent for viral myocarditis, where local viral replication in the heart may lead to heart failure or even death. Recent studies show that inserting microRNA target sequences into the genomes of certain viruses can eradicate these viruses within local host tissues that specifically express the cognate microRNA. Here, we demonstrated both in vitro and in vivo that incorporating target sequences for miRNA-133 and -206 into the 5' untranslated region of the CVB3 genome ameliorated CVB3 virulence in skeletal muscle and myocardial cells that specifically expressed the cognate cellular microRNAs. Compared to wild-type CVB3, viral replication of the engineered CVB3 was attenuated in human TE671 (rhabdomyosarcoma) and L6 (skeletal muscle) cell lines in vitro that expressed high levels of miRNA-206. In the in vivo murine CVB3-infection model, viral replication of the engineered CVB3 was attenuated specifically in the heart that expressed high levels of both miRNAs, but not in certain tissues, which allowed the host to retain the ability to induce a strong and protective humoral immune response against CVB3. The results of this study suggest that a microRNA-targeting strategy to control CVB3 tissue tropism and pathogenesis may be useful for viral attenuation and vaccine development.

    Importance statement Coxsackievirus B3 (CVB3) is a major causative agent for viral myocarditis, and viral replication in the heart may lead to heart failure or even death. Limiting CVB3 replication within the heart may be a promising strategy to decrease CVB3 pathogenicity. miRNAs are ~21 nucleotidemmdash;long, tissue-specific endogenous small RNA molecules that post-transcriptionally regulate gene expression by imperfectly binding to the 3' untranslated region (UTR), 5'-UTR, or coding region within a gene. In our study, muscle specific miRNA targets (miRT) were incorporated into the CVB3 genome. Replication of the engineered viruses was restricted in the important heart tissue of infected mice, which reduced cardiac pathology and increased mouse survival. Meanwhile, replication ability was retained in other tissues, thus inducing a strong humoral immune response and providing long-term protection against CVB3 rechallenge. This study suggests that a microRNA-targeting strategy can potentially control CVB3 tissue tropism and pathogenesis, and may be useful for viral attenuation and vaccine development.

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