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

Table of Contents for this page:

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

    JVI Current Issue

  • Editorial Board [Masthead]

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

  • Hemagglutinin-Neuraminidase Balance Influences the Virulence Phenotype of a Recombinant H5N3 Influenza A Virus Possessing a Polybasic HA0 Cleavage Site [Pathogenesis and Immunity]

  • Although a polybasic HA0 cleavage site is considered the dominant virulence determinant for highly pathogenic avian influenza (HPAI) H5 and H7 viruses, naturally occurring virus isolates possessing a polybasic HA0 cleavage site have been identified that are low pathogenic in chickens. In this study, we generated a reassortant H5N3 virus that possessed the hemagglutinin (HA) gene from H5N1 HPAI A/swan/Germany/R65/2006 and the remaining gene segments from low pathogenic A/chicken/British Columbia/CN0006/2004 (H7N3). Despite possessing the HA0 cleavage site GERRRKKR/GLF, this rH5N3 virus exhibited a low pathogenic phenotype in chickens. Although rH5N3-inoculated birds replicated and shed virus and seroconverted, transmission to naive contacts did not occur. To determine whether this virus could evolve into a HPAI form, it underwent six serial passages in chickens. A progressive increase in virulence was observed with the virus from passage number six being highly transmissible. Whole-genome sequencing demonstrated the fixation of 12 nonsynonymous mutations involving all eight gene segments during passaging. One of these involved the catalytic site of the neuraminidase (NA; R293K) and is associated with decreased neuraminidase activity and resistance to oseltamivir. Although introducing the R293K mutation into the original low-pathogenicity rH5N3 increased its virulence, transmission to naive contact birds was inefficient, suggesting that one or more of the remaining changes that had accumulated in the passage number six virus also play an important role in transmissibility. Our findings show that the functional linkage and balance between HA and NA proteins contributes to expression of the HPAI phenotype.

    IMPORTANCE To date, the contribution that hemagglutinin-neuraminidase balance can have on the expression of a highly pathogenic avian influenza virus phenotype has not been thoroughly examined. Reassortment, which can result in new hemagglutinin-neuraminidase combinations, may have unpredictable effects on virulence and transmission characteristics of a virus. Our data show the importance of the neuraminidase in complementing a polybasic HA0 cleavage site. Furthermore, it demonstrates that adaptive changes selected for during the course of virus evolution can result in unexpected traits such as antiviral drug resistance.

  • The Breadth of Expandable Memory CD8+ T Cells Inversely Correlates with Residual Viral Loads in HIV Elite Controllers [Cellular Response to Infection]

  • Previous studies have shown that elite controllers with minimal effector T cell responses harbor a low-frequency, readily expandable, highly functional, and broadly directed memory population. Here, we interrogated the in vivo relevance of this cell population by investigating whether the breadth of expandable memory responses is associated with the magnitude of residual viremia in individuals achieving durable suppression of HIV infection. HIV-specific memory CD8+ T cells were expanded by using autologous epitopic and variant peptides. Viral load was measured by an ultrasensitive single-copy PCR assay. Following expansion, controllers showed a greater increase in the overall breadth of Gag responses than did untreated progressors (P = 0.01) as well as treated progressors (P = 0.0003). Nef- and Env-specific memory cells expanded poorly for all groups, and their expanded breadths were indistinguishable among groups (P = 0.9 for Nef as determined by a Kruskal-Wallis test; P = 0.6 for Env as determined by a Kruskal-Wallis test). More importantly, we show that the breadth of expandable, previously undetectable Gag-specific responses was inversely correlated with residual viral load (r = nndash;0.6; P = 0.009). Together, these data reveal a direct link between the abundance of Gag-specific expandable memory responses and prolonged maintenance of low-level viremia. Our studies highlight a CD8+ T cell feature that would be desirable in a vaccine-induced T cell response.

    IMPORTANCE Many studies have shown that the rare ability of some individuals to control HIV infection in the absence of antiretroviral therapy appears to be heavily dependent upon special HIV-specific killer T lymphocytes that are able to inhibit viral replication. The identification of key features of these immune cells has the potential to inform rational HIV vaccine design. This study shows that a special subset of killer lymphocytes, known as central memory CD8+ T lymphocytes, is at least partially involved in the durable control of HIV replication. HIV controllers maintain a large proportion of Gag-specific expandable memory CD8+ T cells involved in ongoing viral suppression. These data suggest that induction of this cell subset by future HIV vaccines may be important for narrowing possible routes of rapid escape from vaccine-induced CD8+ T cell responses.

  • Encapsidation of Host RNAs by Cucumber Necrosis Virus Coat Protein during both Agroinfiltration and Infection [Virus-Cell Interactions]

  • Next-generation sequence analysis of virus-like particles (VLPs) produced during agroinfiltration of cucumber necrosis virus (CNV) coat protein (CP) and of authentic CNV virions was conducted to assess if host RNAs can be encapsidated by CNV CP. VLPs containing host RNAs were found to be produced during agroinfiltration, accumulating to approximately 1/60 the level that CNV virions accumulated during infection. VLPs contained a variety of host RNA species, including the major rRNAs as well as cytoplasmic, chloroplast, and mitochondrial mRNAs. The most predominant host RNA species encapsidated in VLPs were chloroplast encoded, consistent with the efficient targeting of CNV CP to chloroplasts during agroinfiltration. Interestingly, droplet digital PCR analysis showed that the CNV CP mRNA expressed during agroinfiltration was the most efficiently encapsidated mRNA, suggesting that the CNV CP open reading frame may contain a high-affinity site or sites for CP binding and thus contribute to the specificity of CNV RNA encapsidation. Approximately 0.09% to 0.7% of the RNA derived from authentic CNV virions contained host RNA, with chloroplast RNA again being the most prominent species. This is consistent with our previous finding that a small proportion of CNV CP enters chloroplasts during the infection process and highlights the possibility that chloroplast targeting is a significant aspect of CNV infection. Remarkably, 6 to 8 of the top 10 most efficiently encapsidated nucleus-encoded RNAs in CNV virions correspond to retrotransposon or retrotransposon-like RNA sequences. Thus, CNV could potentially serve as a vehicle for horizontal transmission of retrotransposons to new hosts and thereby significantly influence genome evolution.

    IMPORTANCE Viruses predominantly encapsidate their own virus-related RNA species due to the possession of specific sequences and/or structures on viral RNA which serve as high-affinity binding sites for the coat protein. In this study, we show, using next-generation sequence analysis, that CNV also encapsidates host RNA species, which account for ~0.1% of the RNA packaged in CNV particles. The encapsidated host RNAs predominantly include chloroplast RNAs, reinforcing previous observations that CNV CP enters chloroplasts during infection. Remarkably, the most abundantly encapsidated cytoplasmic mRNAs consisted of retrotransposon-like RNA sequences, similar to findings recently reported for flock house virus (A. Routh, T. Domitrovic, and J. E. Johnson, Proc Natl Acad Sci U S A 109:1907nndash;1912, 2012). Encapsidation of retrotransposon sequences may contribute to their horizontal transmission should CNV virions carrying retrotransposons infect a new host. Such an event could lead to large-scale genomic changes in a naive plant host, thus facilitating host evolutionary novelty.

  • Generation of Live Attenuated Influenza Virus by Using Codon Usage Bias [Vaccines and Antiviral Agents]

  • Seasonal influenza epidemics and occasional pandemics threaten public health worldwide. New alternative strategies for generating recombinant viruses with vaccine potential are needed. Interestingly, influenza viruses circulating in different hosts have been found to have distinct codon usage patterns, which may reflect host adaptation. We therefore hypothesized that it is possible to make a human seasonal influenza virus that is specifically attenuated in human cells but not in eggs by converting its codon usage so that it is similar to that observed from avian influenza viruses. This approach might help to generate human live attenuated viruses without affecting their yield in eggs. To test this hypothesis, over 300 silent mutations were introduced into the genome of a seasonal H1N1 influenza virus. The resultant mutant was significantly attenuated in mammalian cells and mice, yet it grew well in embryonated eggs. A single dose of intranasal vaccination induced potent innate, humoral, and cellular immune responses, and the mutant could protect mice against homologous and heterologous viral challenges. The attenuated mutant could also be used as a vaccine master donor strain by introducing hemagglutinin and neuraminidase genes derived from other strains. Thus, our approach is a successful strategy to generate attenuated viruses for future application as vaccines.

    IMPORTANCE Vaccination has been one of the best protective measures in combating influenza virus infection. Current licensed influenza vaccines and their production have various limitations. Our virus attenuation strategy makes use of the codon usage biases of human and avian influenza viruses to generate a human-derived influenza virus that is attenuated in mammalian hosts. This method, however, does not affect virus replication in eggs. This makes the resultant mutants highly compatible with existing egg-based vaccine production pipelines. The viral proteins generated from the codon bias mutants are identical to the wild-type viral proteins. In addition, our massive genome-wide mutational approach further minimizes the concern over reverse mutations. The potential use of this kind of codon bias mutant as a master donor strain to generate other live attenuated viruses is also demonstrated. These findings put forward a promising live attenuated influenza vaccine generation strategy to control influenza.

  • Adenovirus E4-ORF1 Dysregulates Epidermal Growth Factor and Insulin/Insulin-Like Growth Factor Receptors To Mediate Constitutive Myc Expression [Virus-Cell Interactions]

  • The E4-ORF1 protein encoded by human adenovirus stimulates viral replication in human epithelial cells by binding and activating cellular phosphatidylinositol 3-kinase (PI3K) at the plasma membrane and cellular Myc in the nucleus. In this study, we showed that E4-ORF1 hijacks the tyrosine kinase activities of cellular epidermal growth factor receptor (EGFR) and insulin receptor (InsR)/insulin-like growth factor receptor 1 (IGF1R), as well as the lipid kinase activity of PI3K, to mediate constitutive Myc protein expression. We additionally demonstrated that EGFR contributes to constitutive Myc expression through the capacity of E4-ORF1 to induce ligand-independent EGFR activation and stimulation of the Ras/Mek/Erk pathway, the latter activity of which was conserved by human adenoviruses. Results further suggested that EGFR normally forms a complex with the cellular PDZ protein Discs Large 1 (Dlg1), a component of the Dlg1:E4-ORF1:PI3K ternary complex that mediates E4-ORF1-induced PI3K activation, and that E4-ORF1 binds the Dlg1:EGFR complex and promotes the association of EGFR with InsR and IGF1R. In addition to its role in constitutive Myc expression, InsR/IGF1R also negatively regulates EGFR autophosphorylation and EGFR-mediated Ras/Mek/Erk signaling, and data suggested that E4-ORF1 binding to Dlg1 antagonizes these activities. Collectively, our findings suggest that in human epithelial cells, E4-ORF1 targets EGFR, InsR/IGF1R, and PI3K at the plasma membrane to activate cytosolic signaling pathways that sustain Myc protein levels in the nucleus. We postulate that E4-ORF1-induced constitutive Myc expression functions to ensure the formation of nuclear E4-ORF1:Myc complexes, which have been shown to activate Myc and to enhance adenovirus replication.

    IMPORTANCE While human adenoviruses primarily produce self-limited acute infections in humans, these agents are associated with life-threatening diseases in immunocompromised patients and in otherwise healthy individuals infected with certain virulent serotypes. The adenovirus E4-ORF1 protein enhances viral replication by activating the cellular lipid kinase PI3K and the cellular transcription factor Myc. Here we report that E4-ORF1 usurps the functions of the cellular tyrosine kinase receptors EGFR and InsR/IGF1R, as well as PI3K, to sustain Myc protein expression in cells. Furthermore, sustained Myc expression depended on E4-ORF1-induced ligand-independent EGFR activation that stimulated Ras/Mek/Erk signaling, a function found to be conserved by human adenoviruses. Given the established roles of PI3K, the Ras/Mek/Erk pathway, and Myc in the adenovirus life cycle, our findings may aid in the development of safer, more effective therapeutic strategies to treat severe adenovirus infections as well as improved adenovirus vectors for use in vaccination and gene and cancer therapy.

  • Increased Immune Response Variability during Simultaneous Viral Coinfection Leads to Unpredictability in CD8 T Cell Immunity and Pathogenesis [Pathogenesis and Immunity]

  • T cell memory is usually studied in the context of infection with a single pathogen in naive mice, but how memory develops during a coinfection with two pathogens, as frequently occurs in nature or after vaccination, is far less studied. Here, we questioned how the competition between immune responses to two viruses in the same naive host would influence the development of CD8 T cell memory and subsequent disease outcome upon challenge. Using two different models of coinfection, including the well-studied lymphocytic choriomeningitis (LCMV) and Pichinde (PICV) viruses, several differences were observed within the CD8 T cell responses to either virus. Compared to single-virus infection, coinfection resulted in substantial variation among mice in the size of epitope-specific T cell responses to each virus. Some mice had an overall reduced number of virus-specific cells to either one of the viruses, and other mice developed an immunodominant response to a normally subdominant, cross-reactive epitope (nucleoprotein residues 205 to 212, or NP205). These changes led to decreased protective immunity and enhanced pathology in some mice upon challenge with either of the original coinfecting viruses. In mice with PICV-dominant responses, during a high-dose challenge with LCMV clone 13, increased immunopathology was associated with a reduced number of LCMV-specific effector memory CD8 T cells. In mice with dominant cross-reactive memory responses, during challenge with PICV increased immunopathology was directly associated with these cross-reactive NP205-specific CD8 memory cells. In conclusion, the inherent competition between two simultaneous immune responses results in significant alterations in T cell immunity and subsequent disease outcome upon reexposure.

    IMPORTANCE Combination vaccines and simultaneous administration of vaccines are necessary to accommodate required immunizations and maintain vaccination rates. Antibody responses generally correlate with protection and vaccine efficacy. However, live attenuated vaccines also induce strong CD8 T cell responses, and the impact of these cells on subsequent immunity, whether beneficial or detrimental, has seldom been studied, in part due to the lack of known T cell epitopes to vaccine viruses. We questioned if the inherent increased competition and stochasticity between two immune responses during a simultaneous coinfection would significantly alter CD8 T cell memory in a mouse model where CD8 T cell epitopes are clearly defined. We show that some of the coinfected mice have sufficiently altered memory T cell responses that they have decreased protection and enhanced immunopathology when reexposed to one of the two viruses. These data suggest that a better understanding of human T cell responses to vaccines is needed to optimize immunization strategies.

  • Vaccine-Induced Simian Immunodeficiency Virus-Specific CD8+ T-Cell Responses Focused on a Single Nef Epitope Select for Escape Variants Shortly after Infection [Vaccines and Antiviral Agents]

  • Certain major histocompatibility complex class I (MHC-I) alleles (e.g., HLA-B*27) are enriched among human immunodeficiency virus type 1 (HIV-1)-infected individuals who suppress viremia without treatment (termed "elite controllers" [ECs]). Likewise, Mamu-B*08 expression also predisposes rhesus macaques to control simian immunodeficiency virus (SIV) replication. Given the similarities between Mamu-B*08 and HLA-B*27, SIV-infected Mamu-B*08+ animals provide a model to investigate HLA-B*27-mediated elite control. We have recently shown that vaccination with three immunodominant Mamu-B*08-restricted epitopes (Vif RL8, Vif RL9, and Nef RL10) increased the incidence of elite control in Mamu-B*08+ macaques after challenge with the pathogenic SIVmac239 clone. Furthermore, a correlate analysis revealed that CD8+ T cells targeting Nef RL10 was correlated with improved outcome. Interestingly, this epitope is conserved between SIV and HIV-1 and exhibits a delayed and atypical escape pattern. These features led us to postulate that a monotypic vaccine-induced Nef RL10-specific CD8+ T-cell response would facilitate the development of elite control in Mamu-B*08+ animals following repeated intrarectal challenges with SIVmac239. To test this, we vaccinated Mamu-B*08+ animals with nef inserts in which Nef RL10 was either left intact (group 1) or disrupted by mutations (group 2). Although monkeys in both groups mounted Nef-specific cellular responses, only those in group 1 developed Nef RL10-specific CD8+ T cells. These vaccine-induced effector memory CD8+ T cells did not prevent infection. Escape variants emerged rapidly in the group 1 vaccinees, and ultimately, the numbers of ECs were similar in groups 1 and 2. High-frequency vaccine-induced CD8+ T cells focused on a single conserved epitope and therefore did not prevent infection or increase the incidence of elite control in Mamu-B*08+ macaques.

    IMPORTANCE Since elite control of chronic-phase viremia is a classic example of an effective immune response against HIV/SIV, elucidating the basis of this phenomenon may provide useful insights into how to elicit such responses by vaccination. We have previously established that vaccine-induced CD8+ T-cell responses against three immunodominant epitopes can increase the incidence of elite control in SIV-infected Mamu-B*08+ rhesus macaquesmmdash;a model of HLA-B*27-mediated elite control. Here, we investigated whether a monotypic vaccine-induced CD8+ T-cell response targeting the conserved "late-escaping" Nef RL10 epitope can increase the incidence of elite control in Mamu-B*08+ monkeys. Surprisingly, vaccine-induced Nef RL10-specific CD8+ T cells selected for variants within days after infection and, ultimately, did not facilitate the development of elite control. Elite control is, therefore, likely to involve CD8+ T-cell responses against more than one epitope. Together, these results underscore the complexity and multidimensional nature of virologic control of lentivirus infection.

  • A Conserved Gammaherpesvirus Cyclin Specifically Bypasses Host p18INK4c To Promote Reactivation from Latency [Virus-Cell Interactions]

  • Gammaherpesviruses (GHVs) carry homologs of cellular genes, including those encoding a viral cyclin that promotes reactivation from latent infection. The viral cyclin has reduced sensitivity to host cyclin-dependent kinase inhibitors in vitro; however, the in vivo significance of this is unclear. Here, we tested the genetic requirement for the viral cyclin in mice that lack the host inhibitors p27Kip1 and p18INK4c, two cyclin-dependent kinase inhibitors known to be important in regulating B cell proliferation and differentiation. While the viral cyclin was essential for reactivation in wild-type mice, strikingly, it was dispensable for reactivation in mice lacking p27Kip1 and p18INK4c. Further analysis revealed that genetic ablation of only p18INK4c alleviated the requirement for the viral cyclin for reactivation from latency. p18INK4c regulated reactivation in a dose-dependent manner so that the viral cyclin was dispensable in p18INK4c heterozygous mice. Finally, treatment of wild-type cells with the cytokine BAFF, a known attenuator of p18INK4c function in B lymphocytes, was also able to bypass the requirement for the viral cyclin in reactivation. These data show that the gammaherpesvirus viral cyclin functions specifically to bypass the cyclin-dependent kinase inhibitor p18INK4c, revealing an unanticipated specificity between a GHV cyclin and a single cyclin-dependent kinase inhibitor.

    IMPORTANCE The gammaherpesviruses (GHVs) cause lifelong infection and can cause chronic inflammatory diseases and cancer, especially in immunosuppressed individuals. Many GHVs encode a conserved viral cyclin that is required for infection and disease. While a common property of the viral cyclins is that they resist inhibition by normal cellular mechanisms, it remains unclear how important it is that the GHVs resist this inhibition. We used a mouse GHV that either contained or lacked a viral cyclin to test whether the viral cyclin lost importance when these inhibitory pathways were removed. These studies revealed that the viral cyclin was required for optimal function in normal mice but that it was no longer required following removal or reduced function of a single cellular inhibitor. These data define a very specific role for the viral cyclin in bypassing one cellular inhibitor and point to new methods to intervene with viral cyclins.

  • Dynamics of HIV-1 RNA Near the Plasma Membrane during Virus Assembly [Structure and Assembly]

  • To increase our understanding of the events that lead to HIV-1 genome packaging, we examined the dynamics of viral RNA and Gag-RNA interactions near the plasma membrane by using total internal reflection fluorescence microscopy. We labeled HIV-1 RNA with a photoconvertible Eos protein via an RNA-binding protein that recognizes stem-loop sequences engineered into the viral genome. Near-UV light exposure causes an irreversible structural change in Eos and alters its emitted fluorescence from green to red. We studied the dynamics of HIV-1 RNA by photoconverting Eos near the plasma membrane, and we monitored the population of photoconverted red-Eos-labeled RNA signals over time. We found that in the absence of Gag, most of the HIV-1 RNAs stayed near the plasma membrane transiently, for a few minutes. The presence of Gag significantly increased the time that RNAs stayed near the plasma membrane: most of the RNAs were still detected after 30 min. We then quantified the proportion of HIV-1 RNAs near the plasma membrane that were packaged into assembling viral complexes. By tagging Gag with blue fluorescent protein, we observed that only a portion, ~13 to 34%, of the HIV-1 RNAs that reached the membrane were recruited into assembling particles in an hour, and the frequency of HIV-1 RNA packaging varied with the Gag expression level. Our studies reveal the HIV-1 RNA dynamics on the plasma membrane and the efficiency of RNA recruitment and provide insights into the events leading to the generation of infectious HIV-1 virions.

    IMPORTANCE Nascent HIV-1 particles assemble on plasma membranes. During the assembly process, HIV-1 RNA genomes must be encapsidated into viral complexes to generate infectious particles. To gain insights into the RNA packaging and virus assembly mechanisms, we labeled and monitored the HIV-1 RNA signals near the plasma membrane. Our results showed that most of the HIV-1 RNAs stayed near the plasma membrane for only a few minutes in the absence of Gag, whereas most HIV-1 RNAs stayed at the plasma membrane for 15 to 60 min in the presence of Gag. Our results also demonstrated that only a small proportion of the HIV-1 RNAs, approximately 1/10 to 1/3 of the RNAs that reached the plasma membrane, was incorporated into viral protein complexes. These studies determined the dynamics of HIV-1 RNA on the plasma membrane and obtained temporal information on RNA-Gag interactions that lead to RNA encapsidation.

  • Intracellular Signaling and Desmoglein 2 Shedding Triggered by Human Adenoviruses Ad3, Ad14, and Ad14P1 [Virus-Cell Interactions]

  • We recently discovered that desmoglein 2 (DSG2) is a receptor for human adenovirus species B serotypes Ad3, Ad7, Ad11, and Ad14. Ad3 is considered to be a widely distributed human pathogen. Ad3 binding to DSG2 triggers the transient opening of epithelial junctions. Here, we further delineate the mechanism that leads to DSG2-mediated epithelial junction opening in cells exposed to Ad3 and recombinant Ad3 fiber proteins. We identified an Ad3 fiber knob-dependent pathway that involves the phosphorylation of mitogen-activated protein (MAP) kinases triggering the activation of the matrix-metalloproteinase ADAM17. ADAM17, in turn, cleaves the extracellular domain of DSG2 that links epithelial cells together. The shed DSG2 domain can be detected in cell culture supernatant and also in serum of mice with established human xenograft tumors. We then extended our studies to Ad14 and Ad14P1. Ad14 is an important research and clinical object because of the recent appearance of a new, more pathogenic strain (Ad14P1). In a human epithelial cancer xenograft model, Ad14P1 showed more efficient viral spread and oncolysis than Ad14. Here, we tested the hypothesis that a mutation in the Ad14P1 fiber knob could account for the differences between the two strains. While our X-ray crystallography studies suggested an altered three-dimensional (3D) structure of the Ad14P1 fiber knob in the F-G loop region, this did not significantly change the fiber knob affinity to DSG2 or the intracellular signaling and DSG2 shedding in epithelial cancer cells.

    IMPORTANCE A number of widely distributed adenoviruses use the epithelial junction protein DSG2 as a receptor for infection and lateral spread. Interaction with DSG2 allows the virus not only to enter cells but also to open epithelial junctions which form a physical barrier to virus spread. Our study elucidates the mechanism beyond virus-triggered junction opening with a focus on adenovirus serotype 3. Ad3 binds to DSG2 with its fiber knob domain and triggers intracellular signaling that culminates in the cleavage of the extracellular domain of DSG2, thereby disrupting DSG2 homodimers between epithelial cells. We confirmed this pathway with a second DSG2-interacting serotype, Ad14, and its recently emerged strain Ad14P1. These new insights in basic adenovirus biology can be employed to develop novel drugs to treat adenovirus infection as well as be used as tools for gene delivery into epithelial tissues or epithelial tumors.

  • Type I Interferons Triggered through the Toll-Like Receptor 3-TRIF Pathway Control Coxsackievirus A16 Infection in Young Mice [Pathogenesis and Immunity]

  • Coxsackievirus A16 (CVA16) is one of the major etiological agents of hand, foot, and mouth disease (HFMD) in children. The host defense mechanisms against CVA16 infection remain almost entirely unknown. Unlike previous observations with enterovirus 71 (EV71) infection, here we show that gamma interferon (IFN-) or invariant NK T cell deficiency does not affect disease development or the survival of CVA16-infected mice. In contrast, type I interferon receptor deficiency resulted in the development of more severe disease in mice, and the mice had a lower survival rate than wild-type mice. Similarly, a deficiency of Toll-like receptor 3 (TLR3) and TRIF, but not other pattern recognition receptors, led to the decreased survival of CVA16-infected mice. TLR3-TRIF signaling was indispensable for the induction of type I interferons during CVA16 infection in mice and protected young mice from disease caused by the infection. In particular, TRIF-mediated immunity was critical for preventing CVA16 replication in the neuronal system before disease occurred. IFN-bbeta; treatment was also found to compensate for TRIF deficiency in mice and decreased the disease severity in and mortality of CVA16-infected mice. Altogether, type I interferons induced by TLR3-TRIF signaling mediate protective immunity against CVA16 infection. These findings may shed light on therapeutic strategies to combat HFMD caused by CVA16 infection.

    IMPORTANCE Hand, foot, and mouth disease (HFMD) is a major threat to public health in the Asia-Pacific region. Both CVA16 and EV71 are major pathogens that are responsible for HFMD. The majority of research efforts have focused on the more virulent EV71, but little has been done with CVA16. Thus far, host immune responses to CVA16 infection have not yet been elucidated. The present study discovered an initial molecular mechanism underlying host protective immunity against CVA16 infection, providing the first explanation for why CVA16 and EV71 cause different clinical outcomes upon infection of humans. Therefore, different therapeutic strategies should be developed to treat HFMD cases caused by these two viruses.

  • Breast Milk of HIV-Positive Mothers Has Potent and Species-Specific In Vivo HIV-Inhibitory Activity [Pathogenesis and Immunity]

  • Despite the nutritional and health benefits of breast milk, breast milk can serve as a vector for mother-to-child HIV transmission. Most HIV-infected infants acquire HIV through breastfeeding. Paradoxically, most infants breastfed by HIV-positive women do not become infected. This is potentially attributed to anti-HIV factors in breast milk. Breast milk of HIV-negative women can inhibit HIV infection. However, the HIV-inhibitory activity of breast milk from HIV-positive mothers has not been evaluated. In addition, while significant differences in breast milk composition between transmitting and nontransmitting HIV-positive mothers have been correlated with transmission risk, the HIV-inhibitory activity of their breast milk has not been compared. This knowledge may significantly impact the design of prevention approaches in resource-limited settings that do not deny infants of HIV-positive women the health benefits of breast milk. Here, we utilized bone marrow/liver/thymus humanized mice to evaluate the in vivo HIV-inhibitory activity of breast milk obtained from HIV-positive transmitting and nontransmitting mothers. We also assessed the species specificity and biochemical characteristics of milk's in vivo HIV-inhibitory activity and its ability to inhibit other modes of HIV infection. Our results demonstrate that breast milk of HIV-positive mothers has potent HIV-inhibitory activity and indicate that breast milk can prevent multiple routes of infection. Most importantly, this activity is unique to human milk. Our results also suggest multiple factors in breast milk may contribute to its HIV-inhibitory activity. Collectively, our results support current recommendations that HIV-positive mothers in resource-limited settings exclusively breastfeed in combination with antiretroviral therapy.

    IMPORTANCE Approximately 240,000 children become infected with HIV annually, the majority via breastfeeding. Despite daily exposure to virus in breast milk, most infants breastfed by HIV-positive women do not acquire HIV. The low risk of breastfeeding-associated HIV transmission is likely due to antiviral factors in breast milk. It is well documented that breast milk of HIV-negative women can inhibit HIV infection. Here, we demonstrate, for the first time, that breast milk of HIV-positive mothers (nontransmitters and transmitters) inhibits HIV transmission. We also demonstrate that breast milk can prevent multiple routes of HIV acquisition and that this activity is unique to human milk. Collectively, our results support current guidelines which recommend that HIV-positive women in resource-limited settings exclusively breastfeed in combination with infant or maternal antiretroviral therapy.

  • Functional Comparison of Mx1 from Two Different Mouse Species Reveals the Involvement of Loop L4 in the Antiviral Activity against Influenza A Viruses [Virus-Cell Interactions]

  • The interferon-induced Mx1 gene is an important part of the mammalian defense against influenza viruses. Mus musculus Mx1 inhibits influenza A virus replication and transcription by suppressing the polymerase activity of viral ribonucleoproteins (vRNPs). Here, we compared the anti-influenza virus activity of Mx1 from Mus musculus A2G with that of its ortholog from Mus spretus. We found that the antiviral activity of M. spretus Mx1 was less potent than that of M. musculus Mx1. Comparison of the M. musculus Mx1 sequence with the M. spretus Mx1 sequence revealed 25 amino acid differences, over half of which were present in the GTPase domain and 2 of which were present in loop L4. However, the in vitro GTPase activity of Mx1 from the two mouse species was similar. Replacement of one of the residues in loop L4 in M. spretus Mx1 by the corresponding residue of A2G Mx1 increased its antiviral activity. We also show that deletion of loop L4 prevented the binding of Mx1 to influenza A virus nucleoprotein and, hence, abolished the antiviral activity of mouse Mx1. These results indicate that loop L4 of mouse Mx1 is a determinant of antiviral activity. Our findings suggest that Mx proteins from different mammals use a common mechanism to inhibit influenza A viruses.

    IMPORTANCE Mx proteins are evolutionarily conserved in vertebrates and inhibit a wide range of viruses. Still, the exact details of their antiviral mechanisms remain largely unknown. Functional comparison of the Mx genes from two species that diverged relatively recently in evolution can provide novel insights into these mechanisms. We show that both Mus musculus A2G Mx1 and Mus spretus Mx1 target the influenza virus nucleoprotein. We also found that loop L4 in mouse Mx1 is crucial for its antiviral activity, as was recently reported for primate MxA. This indicates that human and mouse Mx proteins, which have diverged by 75 million years of evolution, recognize and inhibit influenza A viruses by a common mechanism.

  • Unique Determinants of Neuraminidase Inhibitor Resistance among N3, N7, and N9 Avian Influenza Viruses [Vaccines and Antiviral Agents]

  • Human infections with avian influenza viruses are a serious public health concern. The neuraminidase (NA) inhibitors (NAIs) are the frontline anti-influenza drugs and are the major option for treatment of newly emerging influenza. Therefore, it is essential to identify the molecular markers of NAI resistance among specific NA subtypes of avian influenza viruses to help guide clinical management. NAI-resistant substitutions in NA subtypes other than N1 and N2 have been poorly studied. Here, we identified NA amino acid substitutions associated with NAI resistance among influenza viruses of N3, N7, and N9 subtypes which have been associated with zoonotic transmission. We applied random mutagenesis and generated recombinant influenza viruses carrying single or double NA substitution(s) with seven internal genes from A/Puerto Rico/8/1934 (H1N1) virus. In a fluorescence-based NA inhibition assay, we identified three categories of NA substitutions associated with reduced inhibition by NAIs (oseltamivir, zanamivir, and peramivir): (i) novel subtype-specific substitutions in or near the enzyme catalytic site (R152W, A246T, and D293N, N2 numbering), (ii) subtype-independent substitutions (E119G/V and/or D and R292K), and (iii) substitutions previously reported in other subtypes (Q136K, I222M, and E276D). Our data show that although some markers of resistance are present across NA subtypes, other subtype-specific markers can only be determined empirically.

    IMPORTANCE The number of humans infected with avian influenza viruses is increasing, raising concerns of the emergence of avian influenza viruses resistant to neuraminidase (NA) inhibitors (NAIs). Since most studies have focused on NAI-resistance in human influenza viruses, we investigated the molecular changes in NA that could confer NAI resistance in avian viruses grown in immortalized monolayer cells, especially those of the N3, N7, and N9 subtypes, which have caused human infections. We identified not only numerous NAI-resistant substitutions previously reported in other NA subtypes but also several novel changes conferring reduced susceptibility to NAIs, which are subtype specific. The findings indicate that some resistance markers are common across NA subtypes, but other markers need to be determined empirically for each subtype. The study also implies that antiviral surveillance monitoring could play a critical role in the clinical management of influenza virus infection and an essential component of pandemic preparedness.

  • Herpesvirus saimiri MicroRNAs Preferentially Target Host Cell Cycle Regulators [Transformation and Oncogenesis]

  • In latently infected marmoset T cells, Herpesvirus saimiri (HVS) expresses six microRNAs (known as miR-HSURs [H. saimiri U-rich RNAs]). The viral miR-HSURs are processed from chimeric primary transcripts, each containing a noncoding U-rich RNA (HSUR) and a pre-miRNA hairpin. To uncover the functions of miR-HSURs, we identified mRNA targets in infected cells using high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP). HITS-CLIP revealed hundreds of robust Argonaute (Ago) binding sites mediated by miR-HSURs that map to the host genome but few in the HVS genome. Gene ontology analysis showed that several pathways regulating the cell cycle are enriched among cellular targets of miR-HSURs. Interestingly, miR-HSUR4-3p represses expression of the p300 transcriptional coactivator by binding the open reading frame of its mRNA. miR-HSUR5-3p directly regulates BiP, an endoplasmic reticulum (ER)-localized chaperone facilitating maturation of major histocompatibility complex class I (MHC-I) and the antiviral response. miR-HSUR5-3p also robustly downregulates WEE1, a key negative regulator of cell cycle progression, leading to reduced phosphorylation of its substrate, cyclin-dependent kinase (Cdk1). Consistently, inhibition of miR-HSUR5-3p in HVS-infected cells decreases their proliferation. Together, our results shed light on the roles of viral miRNAs in cellular transformation and viral latency.

    IMPORTANCE Viruses express miRNAs during various stages of infection, suggesting that viral miRNAs play critical roles in the viral life cycle. Compared to protein-coding genes, the functions of viral miRNAs are not well understood. This is because it has been challenging to identify their mRNA targets. Here, we focused on the functions of the recently discovered HVS miRNAs, called miR-HSURs. HVS is an oncogenic gammaherpesvirus that causes acute T-cell lymphomas and leukemias in New World primates and transforms human T cells. A better understanding of HVS biology will help advance our knowledge of virus-induced oncogenesis. Because numerous cellular miRNAs play crucial roles in cancer, viral miRNAs from the highly oncogenic HVS might also be important for transformation. Here, we found that the miR-HSURs preferentially modulate expression of host cell cycle regulators, as well as antiviral response factors. Our work provides further insight into the functions of herpesviral miRNAs in virus-induced oncogenesis and latency.

  • Equine Herpesvirus Type 1 Enhances Viral Replication in CD172a+ Monocytic Cells upon Adhesion to Endothelial Cells [Virus-Cell Interactions]

  • Equine herpesvirus type 1 (EHV-1) is a main cause of respiratory disease, abortion, and encephalomyelopathy in horses. Monocytic cells (CD172a+) are the main carrier cells of EHV-1 during primary infection and are proposed to serve as a "Trojan horse" to facilitate the dissemination of EHV-1 to target organs. However, the mechanism by which EHV-1 is transferred from CD172a+ cells to endothelial cells (EC) remains unclear. The aim of this study was to investigate EHV-1 transmission between these two cell types. We hypothesized that EHV-1 employs specific strategies to promote the adhesion of infected CD172a+ cells to EC to facilitate EHV-1 spread. Here, we demonstrated that EHV-1 infection of CD172a+ cells resulted in a 3- to 5-fold increase in adhesion to EC. Antibody blocking experiments indicated that aalpha;4bbeta;1, aalpha;Lbbeta;2, and aalpha;Vbbeta;3 integrins mediated adhesion of infected CD172a+ cells to EC. We showed that integrin-mediated phosphatidylinositol 3-kinase (PI3K) and ERK/MAPK signaling pathways were involved in EHV-1-induced CD172a+ cell adhesion at early times of infection. EHV-1 replication was enhanced in adherent CD172a+ cells, which correlates with the production of tumor necrosis factor alpha (TNF-aalpha;). In the presence of neutralizing antibodies, approximately 20% of infected CD172a+ cells transferred cytoplasmic material to uninfected EC and 0.01% of infected CD172a+ cells transmitted infectious virus to neighboring cells. Our results demonstrated that EHV-1 infection induces adhesion of CD172a+ cells to EC, which enhances viral replication, but that transfer of viral material from CD172a+ cells to EC is a very specific and rare event. These findings give new insights into the complex pathogenesis of EHV-1.

    IMPORTANCE Equine herpesvirus type 1 (EHV-1) is a highly prevalent pathogen worldwide, causing frequent outbreaks of abortion and myeloencephalopathy, even in vaccinated horses. After primary replication in the respiratory tract, EHV-1 disseminates via cell-associated viremia in peripheral blood mononuclear cells (PBMC) and subsequently infects the endothelial cells (EC) of the pregnant uterus or central nervous system, leading in some cases to abortion and/or neurological disorders. Recently, we demonstrated that CD172a+ monocytic carrier cells serve as a "Trojan horse" to facilitate EHV-1 spread from blood to target organs. Here, we investigated the mechanism underlying the transmission of EHV-1 from CD172a+ cells to EC. We demonstrated that EHV-1 infection induces cellular changes in CD172a+ cells, promoting their adhesion to EC. We found that both cell-to-cell contacts and the secretion of soluble factors by EC activate EHV-1 replication in CD172a+ cells. This facilitates transfer of cytoplasmic viral material to EC, resulting mainly in a nonproductive infection. Our findings give new insights into how EHV-1 may spread to EC of target organs in vaccinated horses.

  • Identification and Mechanism of Action of a Novel Small-Molecule Inhibitor of Arenavirus Multiplication [Vaccines and Antiviral Agents]

  • Several arenaviruses cause hemorrhagic fever disease in humans and represent important public health problems in the regions where these viruses are endemic. In addition, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is an important neglected human pathogen. There are no licensed arenavirus vaccines and current antiarenavirus therapy is limited to the use of ribavirin that is only partially effective. Therefore, there is an unmet need for novel antiarenaviral therapeutics. Here, we report the generation of a novel recombinant LCM virus and its use to develop a cell-based high-throughput screen to rapidly identify inhibitors of LCMV multiplication. We used this novel assay to screen a library of 30,400 small molecules and identified compound F3406 (chemical name: N-[3,5-bis(fluoranyl)phenyl]-2-[5,7-bis(oxidanylidene)-6-propyl-2-pyrrolidin-1-yl-[1,3]thiazolo[4,5-d]pyrimidin-4-yl]ethanamide), which exhibited strong anti-LCMV activity in the absence of cell toxicity. Mechanism-of-action studies revealed that F3406 inhibited LCMV cell entry by specifically interfering with the pH-dependent fusion in the endosome compartment that is mediated by LCMV glycoprotein GP2 and required to release the virus ribonucleoprotein into the cell cytoplasm to initiate transcription and replication of the virus genome. We identified residue M437 within the transmembrane domain of GP2 as critical for virus susceptibility to F3406.

    IMPORTANCE Hemorrhagic fever arenaviruses (HFA) are important human pathogens that cause high morbidity and mortality in areas where these viruses are endemic. In addition, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance. Concerns posed by arenavirus infections are aggravated by the lack of U.S. Food and Drug Administration-licensed arenavirus vaccines and current antiarenaviral therapy being limited to the off-label use of ribavirin that is only partially effective. Here we describe a novel recombinant LCMV and its use to develop a cell-based assay suitable for HTS to rapidly identify inhibitors arenavirus multiplication. The concepts and experimental strategies we describe in this work provide the bases for the rapid identification and characterization of novel anti-HFA therapeutics.

  • Site-Specific Recombination by SSV2 Integrase: Substrate Requirement and Domain Functions [Virus-Cell Interactions]

  • SSV-type integrases, encoded by fuselloviruses which infect the hyperthermophilic archaea of the Sulfolobales, are archaeal members of the tyrosine recombinase family. These integrases catalyze viral integration into and excision from a specific site on the host genome. In the present study, we have established an in vitro integration/excision assay for SSV2 integrase (IntSSV2). IntSSV2 alone was able to catalyze both integration and excision reactions in vitro. A 27-bp specific DNA sequence is minimally required for the activity of the enzyme, and its flanking sequences influence the efficiency of integration by the enzyme in a sequence-nonspecific manner. The enzyme forms a tetramer through interactions in the N-terminal part (residues 1 to 80), interacts nonspecifically with DNA and performs chemical catalysis in the C-terminal part (residues 165 to 328), and appears to recognize and bind the specific site of recombination in the middle portion (residues 81 to 164). It is worth noting that an N-terminally truncated mutant of IntSSV2 (residues 81 to 328), which corresponded to the putative product of the 3'-end sequence of the IntSSV2 gene of the integrated SSV2 genome, was unable to form tetramers but possessed all the catalytic properties of full-length IntSSV2 except for the slightly reduced recombination activity. Our results suggest that, unlike integrase, SSV-type integrases alone are capable of catalyzing viral DNA recombination with the host genome in a simple and reversible fashion.

    IMPORTANCE Archaea are host to a variety of viruses. A number of archaeal viruses are able to integrate their genome into the host genome. Many known archaeal viral integrases belong to a unique type, or the SSV type, of tyrosine recombinases. SSV-type integrases catalyze viral integration into and excision from a specific site on the host genome. However, the molecular details of the recombination process have yet to be fully understood because of the lack of an established in vitro recombination assay system. Here we report an in vitro assay for integration and excision by SSV2 integrase, a member of the SSV-type integrases. We show that SSV2 integrase alone is able to catalyze both integration and excision and reveal how different parts of the target DNA and the enzyme serve their roles in these processes. Therefore, our results provide mechanistic insights into a simple recombination process catalyzed by an archaeal integrase.

  • Sinorhizobium meliloti Phage {Phi}M9 Defines a New Group of T4 Superfamily Phages with Unusual Genomic Features but a Common T=16 Capsid [Genetic Diversity and Evolution]

  • Relatively little is known about the phages that infect agriculturally important nitrogen-fixing rhizobial bacteria. Here we report the genome and cryo-electron microscopy structure of the Sinorhizobium meliloti-infecting T4 superfamily phage M9. This phage and its close relative Rhizobium phage vB_RleM_P10VF define a new group of T4 superfamily phages. These phages are distinctly different from the recently characterized cyanophage-like S. meliloti phages of the M12 group. Structurally, M9 has a T=16 capsid formed from repeating units of an extended gp23-like subunit that assemble through interactions between one subunit and the adjacent E-loop insertion domain. Though genetically very distant from the cyanophages, the M9 capsid closely resembles that of the T4 superfamily cyanophage Syn9. M9 also has the same T=16 capsid architecture as the very distant phage SPO1 and the herpesviruses. Despite their overall lack of similarity at the genomic and structural levels, M9 and S. meliloti phage M12 have a small number of open reading frames in common that appear to encode structural proteins involved in interaction with the host and which may have been acquired by horizontal transfer. These proteins are predicted to encode tail baseplate proteins, tail fibers, tail fiber assembly proteins, and glycanases that cleave host exopolysaccharide.

    IMPORTANCE Despite recent advances in the phylogenetic and structural characterization of bacteriophages, only a small number of phages of plant-symbiotic nitrogen-fixing soil bacteria have been studied at the molecular level. The effects of phage predation upon beneficial bacteria that promote plant growth remain poorly characterized. First steps in understanding these soil bacterium-phage dynamics are genetic, molecular, and structural characterizations of these groups of phages. The T4 superfamily phages are among the most complex phages; they have large genomes packaged within an icosahedral head and a long, contractile tail through which the DNA is delivered to host cells. This phylogenetic and structural study of S. meliloti-infecting T4 superfamily phage M9 provides new insight into the diversity of this family. The comparison of structure-related genes in both M9 and S. meliloti-infecting T4 superfamily phage M12, which comes from a completely different lineage of these phages, allows the identification of host infection-related factors.

  • Out of the Reservoir: Phenotypic and Genotypic Characterization of a Novel Cowpox Virus Isolated from a Common Vole [Pathogenesis and Immunity]

  • The incidence of human cowpox virus (CPXV) infections has increased significantly in recent years. Serological surveys have suggested wild rodents as the main CPXV reservoir. We characterized a CPXV isolated during a large-scale screening from a feral common vole. A comparison of the full-length DNA sequence of this CPXV strain with a highly virulent pet rat CPXV isolate showed a sequence identity of 96%, including a large additional open reading frame (ORF) of about 6,000 nucleotides which is absent in the reference CPXV strain Brighton Red. Electron microscopy analysis demonstrated that the vole isolate, in contrast to the rat strain, forms A-type inclusion (ATI) bodies with incorporated virions, consistent with the presence of complete ati and p4c genes. Experimental infections showed that the vole CPXV strain caused only mild clinical symptoms in its natural host, while all rats developed severe respiratory symptoms followed by a systemic rash. In contrast, common voles infected with a high dose of the rat CPXV showed severe signs of respiratory disease but no skin lesions, whereas infection with a low dose led to virus excretion with only mild clinical signs. We concluded that the common vole is susceptible to infection with different CPXV strains. The spectrum ranges from well-adapted viruses causing limited clinical symptoms to highly virulent strains causing severe respiratory symptoms. In addition, the low pathogenicity of the vole isolate in its eponymous host suggests a role of common voles as a major CPXV reservoir, and future research will focus on the correlation between viral genotype and phenotype/pathotype in accidental and reservoir species.

    IMPORTANCE We report on the first detection and isolation of CPXV from a putative reservoir host, which enables comparative analyses to understand the infection cycle of these zoonotic orthopox viruses and the relevant genes involved. In vitro studies, including whole-genome sequencing as well as in vivo experiments using the Wistar rat model and the vole reservoir host allowed us to establish links between genomic sequences and the in vivo properties (virulence) of the novel vole isolate in comparison to those of a recent zoonotic CPXV isolated from pet rats in 2009. Furthermore, the role of genes present only in a reservoir isolate can now be further analyzed. These studies therefore allow unique insights and conclusions about the role of the rodent reservoir in CPXV epidemiology and transmission and about the zoonotic threat that these viruses represent.

  • Middle East Respiratory Syndrome Coronavirus nsp1 Inhibits Host Gene Expression by Selectively Targeting mRNAs Transcribed in the Nucleus while Sparing mRNAs of Cytoplasmic Origin [Virus-Cell Interactions]

  • The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome CoV (SARS-CoV) represent highly pathogenic human CoVs that share a property to inhibit host gene expression at the posttranscriptional level. Similar to the nonstructural protein 1 (nsp1) of SARS-CoV that inhibits host gene expression at the translational level, we report that MERS-CoV nsp1 also exhibits a conserved function to negatively regulate host gene expression by inhibiting host mRNA translation and inducing the degradation of host mRNAs. Furthermore, like SARS-CoV nsp1, the mRNA degradation activity of MERS-CoV nsp1, most probably triggered by its ability to induce an endonucleolytic RNA cleavage, was separable from its translation inhibitory function. Despite these functional similarities, MERS-CoV nsp1 used a strikingly different strategy that selectively targeted translationally competent host mRNAs for inhibition. While SARS-CoV nsp1 is localized exclusively in the cytoplasm and binds to the 40S ribosomal subunit to gain access to translating mRNAs, MERS-CoV nsp1 was distributed in both the nucleus and the cytoplasm and did not bind stably to the 40S subunit, suggesting a distinctly different mode of targeting translating mRNAs. Interestingly, consistent with this notion, MERS-CoV nsp1 selectively targeted mRNAs, which are transcribed in the nucleus and transported to the cytoplasm, for translation inhibition and mRNA degradation but spared exogenous mRNAs introduced directly into the cytoplasm or virus-like mRNAs that originate in the cytoplasm. Collectively, these data point toward a novel viral strategy wherein the cytoplasmic origin of MERS-CoV mRNAs facilitates their escape from the inhibitory effects of MERS-CoV nsp1.

    IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic human CoV that emerged in Saudi Arabia in 2012. MERS-CoV has a zoonotic origin and poses a major threat to public health. However, little is known about the viral factors contributing to the high virulence of MERS-CoV. Many animal viruses, including CoVs, encode proteins that interfere with host gene expression, including those involved in antiviral immune responses, and these viral proteins are often major virulence factors. The nonstructural protein 1 (nsp1) of CoVs is one such protein that inhibits host gene expression and is a major virulence factor. This study presents evidence for a strategy used by MERS-CoV nsp1 to inhibit host gene expression that has not been described previously for any viral protein. The present study represents a meaningful step toward a better understanding of the factors and molecular mechanisms governing the virulence and pathogenesis of MERS-CoV.

  • Mechanism of Binding to Ebola Virus Glycoprotein by the ZMapp, ZMAb, and MB-003 Cocktail Antibodies [Vaccines and Antiviral Agents]

  • Cocktails of monoclonal antibodies (MAbs) that target the surface glycoprotein (GP) of Ebola virus (EBOV) are effective in nonhuman primate models and have been used under emergency compassionate-treatment protocols in human patients. However, the amino acids that form the detailed binding epitopes for the MAbs in the ZMapp, ZMAb, and the related MB-003 cocktails have yet to be identified. Other binding properties that define how each MAb functionally interacts with GPmmdash;such as affinity, epitope conservation, and epitope accessibilitymmdash;also remain largely unknown. To help define how each MAb interacts with GP, here we used comprehensive alanine-scanning mutagenesis (shotgun mutagenesis), neutralization escape, and whole virion binding to define each MAb's specific epitope, epitope accessibility, epitope conservation, and apparent affinity. Each of the six therapeutic MAbs binds nonidentical epitopes in the GP base, glycan cap, or mucin-like domain. Their apparent affinity, epitope complementarity, and epitope accessibility helps explain why MAbs 4G7 and 13C6 are more protective than 2G4 and 1H3. The mucin-like domain MAbs 6D8 and 13F6 bind with the strongest apparent affinity, helping to explain their effectiveness in vivo despite their inability to neutralize virus.

    IMPORTANCE Ebola virus disease (EVD) can be caused by four different filovirus family members, including Ebola virus (EBOV), which infected 10 times more people in western Africa over the last year than all previous EVD outbreaks combined, with a number of cases distributed across the globe by travelers. Cocktails of inhibitory monoclonal antibodies (MAbs), such as ZMAb, MB-003, and in particular ZMapp, have demonstrated in animal models some of the most significant therapeutic potential for treating EVD, and in 2014, 15 patients were treated with ZMapp or ZMAb under compassionate-use protocols. Here, we have defined the epitope features for the most important therapeutic MAbs against EBOV developed to date. Defining the epitopes and binding characteristics for these MAbs, as well as the commonly used reference MAb KZ52, helps explain their breadth of reactivity against different ebolavirus species, predict viral evasion against these MAbs, and design new cocktails of MAbs with improved complementarity.

  • Evolutionary Dynamics and Global Diversity of Influenza A Virus [Genetic Diversity and Evolution]

  • The increasing number of zoonotic infections caused by influenza A virus (IAV) subtypes of avian origin (e.g., H5N1 and H7N9) in recent years underscores the need to better understand the factors driving IAV evolution and diversity. To evaluate the current feasibility of global analyses to contribute to this aim, we evaluated information in the public domain to explore IAV evolutionary dynamics, including nucleotide substitution rates and selection pressures, using 14 IAV subtypes in 32 different countries over a 12-year period (2000 to 2011). Using geospatial information from 39,785 IAV strains, we examined associations between subtype diversity and socioeconomic, biodiversity, and agricultural indices. Our analyses showed that nucleotide substitution rates for 11 of the 14 evaluated subtypes tended to be higher in Asian countries, particularly in East Asia, than in Canada and the United States. Similarly, at a regional level, subtypes H5N1, H5N2, and H6N2 exhibited significantly higher substitution rates in East Asia than in North America. In contrast, the selection pressures (measured as ratios of nonsynonymous to synonymous evolutionary changes [dN/dS ratios]) acting on individual subtypes showed little geographic variation. We found that the strongest predictors for the detected subtype diversity at the country level were reporting effort (i.e., total number of strains reported) and health care spending (an indicator of economic development). Our analyses also identified major global gaps in IAV reporting (including a lack of sequences submitted from large portions of Africa and South America and a lack of geolocation information) and in broad subtype testing which, until addressed, will continue to hinder efforts to track the evolution and diversity of IAV around the world.

    IMPORTANCE In recent years, an increasing number of influenza A virus (IAV) subtypes, including H5N1, H7N9, and H10N8, have been detected in humans. High fatality rates have led to an increased urgency to better understand where and how novel pathogenic influenza virus strains emerge. Our findings showed that mutational rates of 11 commonly encountered subtypes were higher in East Asian countries than in North America, suggesting that there may be a greater risk for the emergence of novel pathogenic strains in East Asia. In assessing the potential drivers of IAV subtype diversity, our analyses confirmed that reporting effort and health care spending were the best predictors of the observed subtype diversity at the country level. These findings underscore the need to increase sampling and reporting efforts for all subtypes in many undersampled countries throughout the world.

  • Oligomerization of Mumps Virus Phosphoprotein [Genome Replication and Regulation of Viral Gene Expression]

  • The mumps virus (MuV) genome encodes a phosphoprotein (P) that is important for viral RNA synthesis. P forms the viral RNA-dependent RNA polymerase with the large protein (L). P also interacts with the viral nucleoprotein (NP) and self-associates to form a homotetramer. The P protein consists of three domains, the N-terminal domain (PN), the oligomerization domain (PO), and the C-terminal domain (PC). While PN is known to relax the NP-bound RNA genome, the roles of PO and PC are not clear. In this study, we investigated the roles of PO and PC in viral RNA synthesis using mutational analysis and a minigenome system. We found that PN and PC functions can be trans-complemented. However, this complementation requires PO, indicating that PO is essential for P function. Using this trans-complementation system, we found that P forms parallel dimers (PN to PN and PC to PC). Furthermore, we found that residues R231, K238, K253, and K260 in PO are critical for P's functions. We identified PC to be the domain that interacts with L. These results provide structure-function insights into the role of MuV P.

    IMPORTANCE MuV, a paramyxovirus, is an important human pathogen. The P protein of MuV is critical for viral RNA synthesis. In this work, we established a novel minigenome system that allows the domains of P to be complemented in trans. Using this system, we confirmed that MuV P forms parallel dimers. An understanding of viral RNA synthesis will allow the design of better vaccines and the development of antivirals.

  • Bst2/Tetherin Is Induced in Neurons by Type I Interferon and Viral Infection but Is Dispensable for Protection against Neurotropic Viral Challenge [Cellular Response to Infection]

  • In permissive mouse central nervous system (CNS) neurons, measles virus (MV) spreads in the absence of hallmark viral budding or neuronal death, with transmission occurring efficiently and exclusively via the synapse. MV infection also initiates a robust type I interferon (IFN) response, resulting in the synthesis of a large number of genes, including bone marrow stromal antigen 2 (Bst2)/tetherin/CD317. Bst2 restricts the release of some enveloped viruses, but to date, its role in viral infection of neurons has not been assessed. Consequently, we investigated how Bst2 was induced and what role it played in MV neuronal infection. The magnitude of induction of neuronal Bst2 RNA and protein following IFN exposure and viral infection was notably higher than in similarly treated mouse embryo fibroblasts (MEFs). Bst2 synthesis was both IFN and Stat1 dependent. Although Bst2 prevented MV release from nonneuronal cells, its deletion had no effect on viral pathogenesis in MV-challenged mice. Our findings underscore how cell-type-specific differences impact viral infection and pathogenesis.

    IMPORTANCE Viral infections of the central nervous system can lead to debilitating disease and death. Moreover, it is becoming increasingly clear that nonrenewable cells, including most central nervous system neurons, combat neurotropic viral infections in fundamentally different ways than other rapidly dividing and renewable cell populations. Here we identify type I interferon signaling as a key inducer of a known antiviral protein (Bst2) in neurons. Unexpectedly, the gene is dispensable for clearance of neurotropic viral infection despite its well-defined contribution to limiting the spread of enveloped viruses in proliferating cells. A deeper appreciation of the importance of cell type heterogeneity in antiviral immunity will aid in the identification of unique therapeutic targets for life-threatening viral infections.

  • Characterization of the Interaction between the Matrix Protein of Vesicular Stomatitis Virus and the Immunoproteasome Subunit LMP2 [Virus-Cell Interactions]

  • The matrix protein (M) of vesicular stomatitis virus (VSV) is involved in virus assembly, budding, gene regulation, and cellular pathogenesis. Using a yeast two-hybrid system, the M globular domain was shown to interact with LMP2, a catalytic subunit of the immunoproteasome (which replaces the standard proteasome catalytic subunit PSMB6). The interaction was validated by coimmunoprecipitation of M and LMP2 in VSV-infected cells. The sites of interaction were characterized. A single mutation of M (I96A) which significantly impairs the interaction between M and LMP2 was identified. We also show that M preferentially binds to the inactive precursor of LMP2 (bearing an N-terminal propeptide which is cleaved upon LMP2 maturation). Furthermore, taking advantage of a sequence alignment between LMP2 and its proteasome homolog, PSMB6 (which does not bind to M), we identified a mutation (L45R) in the S1 pocket where the protein substrate binds prior to cleavage and a second one (D17A) of a conserved residue essential for the catalytic activity, resulting in a reduction of the level of binding to M. The combination of both mutations abolishes the interaction. Taken together, our data indicate that M binds to LMP2 before its incorporation into the immunoproteasome. As the immunoproteasome promotes the generation of major histocompatibility complex (MHC) class I-compatible peptides, a feature which favors the recognition and the elimination of infected cells by CD8 T cells, we suggest that M, by interfering with the immunoproteasome assembly, has evolved a mechanism that allows infected cells to escape detection and elimination by the immune system.

    IMPORTANCE The immunoproteasome promotes the generation of MHC class I-compatible peptides, a feature which favors the recognition and the elimination of infected cells by CD8 T cells. Here, we report on the association of vesicular stomatitis virus (VSV) matrix protein (M) with LMP2, one of the immunoproteasome-specific catalytic subunits. M preferentially binds to the LMP2 inactive precursor. The M-binding site on LMP2 is facing inwards in the immunoproteasome and is therefore not accessible to M after its assembly. Hence, M binds to LMP2 before its incorporation into the immunoproteasome. We suggest that VSV M, by interfering with the immunoproteasome assembly, has evolved a mechanism that allows infected cells to escape detection and elimination by the immune system. Modulating this M-induced immunoproteasome impairment might be relevant in order to optimize VSV for oncolytic virotherapy.

  • Differences in Processing Determinants of Nonstructural Polyprotein and in the Sequence of Nonstructural Protein 3 Affect Neurovirulence of Semliki Forest Virus [Pathogenesis and Immunity]

  • The A7(74) strain of Semliki Forest virus (SFV; genus Alphavirus) is avirulent in adult mice, while the L10 strain is virulent in mice of all ages. It has been previously demonstrated that this phenotypic difference is associated with nonstructural protein 3 (nsP3). Consensus clones of L10 (designated SFV6) and A7(74) (designated A774wt) were used to construct a panel of recombinant viruses. The insertion of nsP3 from A774wt into the SFV6 backbone had a minor effect on the virulence of the resulting recombinant virus. Conversely, insertion of nsP3 from SFV6 into the A774wt backbone or replacement of A774wt nsP3 with two copies of nsP3 from SFV6 resulted in virulent viruses. Unexpectedly, duplication of nsP3-encoding sequences also resulted in elevated levels of nsP4, revealing that nsP3 is involved in the stabilization of nsP4. Interestingly, replacement of nsP3 of SFV6 with that of A774wt resulted in a virulent virus; the virulence of this recombinant was strongly reduced by functionally coupled substitutions for amino acid residues 534 (P4 position of the cleavage site between nsP1 and nsP2) and 1052 (S4 subsite residue of nsP2 protease) in the nonstructural polyprotein. Pulse-chase experiments revealed that A774wt and avirulent recombinant virus were characterized by increased processing speed of the cleavage site between nsP1 and nsP2. A His534-to-Arg substitution specifically activated this cleavage, while a Val1052-to-Glu substitution compensated for this effect by reducing the basal protease activity of nsP2. These findings provide a link between nonstructural polyprotein processing and the virulence of SFV.

    IMPORTANCE SFV infection of mice provides a well-characterized model to study viral encephalitis. SFV also serves as a model for studies of alphavirus molecular biology and host-pathogen interactions. Thus far, the genetic basis of different properties of SFV strains has been studied using molecular clones, which often contain mistakes originating from standard cDNA synthesis and cloning procedures. Here, for the first time, consensus clones of SFV strains were used to map virulence determinants. Existing data on the importance of nsP3 for virulent phenotypes were confirmed, another determinant of neurovirulence and its molecular basis was characterized, and a novel function of nsP3 was identified. These findings provide links between the molecular biology of SFV and its biological properties and significantly increase our understanding of the basis of alphavirus-induced pathology. In addition, the usefulness of consensus clones as tools for studies of alphaviruses was demonstrated.

  • L Particles Transmit Viral Proteins from Herpes Simplex Virus 1-Infected Mature Dendritic Cells to Uninfected Bystander Cells, Inducing CD83 Downmodulation [Virus-Cell Interactions]

  • Mature dendritic cells (mDCs) are known as the most potent antigen-presenting cells (APCs) since they are also able to prime/induce naive T cells. Thus, mDCs play a pivotal role during the induction of antiviral immune responses. Remarkably, the cell surface molecule CD83, which was shown to have costimulatory properties, is targeted by herpes simplex virus 1 (HSV-1) for viral immune escape. Infection of mDCs with HSV-1 results in downmodulation of CD83, resulting in reduced T cell stimulation. In this study, we report that not only infected mDCs but also uninfected bystander cells in an infected culture show a significant CD83 reduction. We demonstrate that this effect is independent of phagocytosis and transmissible from infected to uninfected mDCs. The presence of specific viral proteins found in these uninfected bystander cells led to the hypothesis that viral proteins are transferred from infected to uninfected cells via L particles. These L particles are generated during lytic replication in parallel with full virions, called H particles. L particles contain viral proteins but lack the viral capsid and DNA. Therefore, these particles are not infectious but are able to transfer several viral proteins. Incubation of mDCs with L particles indeed reduced CD83 expression on uninfected bystander DCs, providing for the first time evidence that functional viral proteins are transmitted via L particles from infected mDCs to uninfected bystander cells, thereby inducing CD83 downmodulation.

    IMPORTANCE HSV-1 has evolved a number of strategies to evade the host's immune system. Among others, HSV-1 infection of mDCs results in an inhibited T cell activation caused by degradation of CD83. Interestingly, CD83 is lost not only from HSV-1-infected mDCs but also from uninfected bystander cells. The release of so-called L particles, which contain several viral proteins but lack capsid and DNA, during infection is a common phenomenon observed among several viruses, such as human cytomegalovirus (HCMV), Epstein-Barr virus, and HSV-1. However, the detailed function of these particles is poorly understood. Here, we provide for the first time evidence that functional viral proteins can be transferred to uninfected bystander mDCs via L particles, revealing important biological functions of these particles during lytic replication. Therefore, the transfer of viral proteins by L particles to modulate uninfected bystander cells may represent an additional strategy for viral immune escape.

  • Pathogen-Associated Molecular Pattern Recognition of Hepatitis C Virus Transmitted/Founder Variants by RIG-I Is Dependent on U-Core Length [Pathogenesis and Immunity]

  • Despite the introduction of direct-acting antiviral (DAA) drugs against hepatitis C virus (HCV), infection remains a major public health concern because DAA therapeutics do not prevent reinfection and patients can still progress to chronic liver disease. Chronic HCV infection is supported by a variety of viral immune evasion strategies, but, remarkably, 20% to 30% of acute infections spontaneously clear prior to development of adaptive immune responses, thus implicating innate immunity in resolving acute HCV infection. However, the virus-host interactions regulating acute infection are unknown. Transmission of HCV involves one or a few transmitted/founder (T/F) variants. In infected hepatocytes, the retinoic acid-inducible gene I (RIG-I) protein recognizes 5' triphosphate (5'ppp) of the HCV RNA and a pathogen-associated molecular pattern (PAMP) motif located within the 3' untranslated region consisting of poly-U/UC. PAMP binding activates RIG-I to induce innate immune signaling and type 1 interferon antiviral defenses. HCV poly-U/UC sequences can differ in length and complexity, suggesting that PAMP diversity in T/F genomes could regulate innate immune control of acute HCV infection. Using 14 unique poly-U/UC sequences from HCV T/F genomes recovered from acute-infection patients, we tested whether RIG-I recognition and innate immune activation correlate with PAMP sequence characteristics. We show that T/F variants are recognized by RIG-I in a manner dependent on length of the U-core motif of the poly-U/UC PAMP and are recognized by RIG-I to induce innate immune responses that restrict acute infection. PAMP recognition of T/F HCV variants by RIG-I may therefore impart innate immune signaling and HCV restriction to impact acute-phase-to-chronic-phase transition.

    IMPORTANCE Recognition of nonself molecular patterns such as those seen with viral nucleic acids is an essential step in triggering the immune response to virus infection. Innate immunity is induced by hepatitis C virus infection through the recognition of viral RNA by the cellular RIG-I protein, where RIG-I recognizes a poly-uridine/cytosine motif in the viral genome. Variation within this motif may provide an immune evasion strategy for transmitted/founder viruses during acute infection. Using 14 unique poly-U/UC sequences from HCV T/F genomes recovered from acutely infected HCV patients, we demonstrate that RIG-I binding and activation of innate immunity depend primarily on the length of the uridine core within this motif. T/F variants found in acute infection contained longer U cores within the motif and could activate RIG-I and induce innate immune signaling sufficient to restrict viral infection. Thus, recognition of T/F variants by RIG-I could significantly impact the transition from acute to chronic infection.

  • Selective Removal of FG Repeat Domains from the Nuclear Pore Complex by Enterovirus 2Apro [Virus-Cell Interactions]

  • Enteroviruses proteolyze nuclear pore complex (NPC) proteins (Nups) during infection, leading to disruption of host nuclear transport pathways and alterations in nuclear permeability. To better understand how enteroviruses exert these effects on nuclear transport, the mechanisms and consequences of Nup98 proteolysis were examined. The results indicate that Nup98 is rapidly targeted for degradation following enterovirus infection and that this is mediated by the enterovirus 2A protease (2Apro). Incubation of bacterially expressed or in vitro-translated Nup98 with 2Apro results in proteolytic cleavage at multiple sites in vitro, indicating that 2Apro cleaves Nup98 directly. Site-directed mutagenesis of putative cleavage sites identified Gly374 and Gly552 as the sites of 2Apro proteolysis in Nup98 in vitro and in infected cells. Indirect immunofluorescence assays using an antibody that recognizes the N terminus of Nup98 revealed that proteolysis releases the N-terminal FG-rich region from the NPC. In contrast, similar analyses using an antibody to the C terminus indicated that this region is retained at the nuclear rim. Nup88, a core NPC component that serves as a docking site for Nup98, also remains at the NPC in infected cells. These findings support a model whereby the selective removal of Nup FG repeat domains leads to increased NPC permeability and inhibition of certain transport pathways, while retention of structural domains maintains the overall NPC structure and leaves other transport pathways unaffected.

    IMPORTANCE Enteroviruses are dependent upon host nuclear RNA binding proteins for efficient replication. This study examines the mechanisms responsible for alterations in nuclear transport in enterovirus-infected cells that lead to the cytoplasmic accumulation of these proteins. The results demonstrate that the enterovirus 2A protease directly cleaves the nuclear pore complex (NPC) protein, Nup98, at amino acid positions G374 and G552 both in vitro and in infected cells. Cleavage at these positions results in the selective removal of the FG-containing N terminus of Nup98 from the NPC, while the C terminus remains associated. Nup88, a core component of the NPC that serves as a docking site for the C terminus of Nup98, remains associated with the NPC in infected cells. These findings help to explain the alterations in permeability and nuclear transport in enterovirus-infected cells and how NPCs remain functional for certain trafficking pathways despite significant alterations to their compositions.

  • Role of the DNA Sensor STING in Protection from Lethal Infection following Corneal and Intracerebral Challenge with Herpes Simplex Virus 1 [Pathogenesis and Immunity]

  • STING is a protein in the cytosolic DNA and cyclic dinucleotide sensor pathway that is critical for the initiation of innate responses to infection by various pathogens. Consistent with this, herpes simplex virus 1 (HSV-1) causes invariable and rapid lethality in STING-deficient (STINGnndash;/nndash;) mice following intravenous (i.v.) infection. In this study, using real-time bioluminescence imaging and virological assays, as expected, we demonstrated that STINGnndash;/nndash; mice support greater replication and spread in ocular tissues and the nervous system. In contrast, they did not succumb to challenge via the corneal route even with high titers of a virus that was routinely lethal to STINGnndash;/nndash; mice by the i.v. route. Corneally infected STINGnndash;/nndash; mice also showed increased periocular disease and increased corneal and trigeminal ganglia titers, although there was no difference in brain titers. They also showed elevated expression of tumor necrosis factor alpha (TNF-aalpha;) and CXCL9 relative to control mice but surprisingly modest changes in type I interferon expression. Finally, we also showed that HSV strains lacking the ability to counter autophagy and the PKR-driven antiviral state had near-wild-type virulence following intracerebral infection of STINGnndash;/nndash; mice. Together, these data show that while STING is an important component of host resistance to HSV in the cornea, its previously shown immutable role in mediating host survival by the i.v. route was not recapitulated following a mucosal infection route. Furthermore, our data are consistent with the idea that HSV counters STING-mediated induction of the antiviral state and autophagy response, both of which are critical factors for survival following direct infection of the nervous system.

    IMPORTANCE HSV infections represent an incurable source of morbidity and mortality in humans and are especially severe in neonatal and immunocompromised populations. A key step in the development of an immune response is the recognition of microbial components within infected cells. The host protein STING is important in this regard for the recognition of HSV DNA and the subsequent triggering of innate responses. STING was previously shown to be essential for protection against lethal challenge from intravenous HSV-1 infection. In this study, we show that the requirement for STING depends on the infection route. In addition, STING is important for appropriate regulation of the inflammatory response in the cornea, and our data are consistent with the idea that HSV modulates STING activity through inhibition of autophagy. Our results elucidate the importance of STING in host protection from HSV-1 and demonstrate the redundancy of host protective mechanisms, especially following mucosal infection.

  • Inactivation of the Human Cytomegalovirus US20 Gene Hampers Productive Viral Replication in Endothelial Cells [Virus-Cell Interactions]

  • The human cytomegalovirus (HCMV) US12 gene family includes a group of 10 contiguous genes (US12 to US21) encoding predicted seven-transmembrane-domain (7TMD) proteins that are nonessential for replication within cultured fibroblasts. Nevertheless, inactivation of some US12 family members affects virus replication in other cell types; e.g., deletion of US16 or US18 abrogates virus growth in endothelial and epithelial cells or in human gingival tissue, respectively, suggesting a role for some US12 proteins in HCMV cell tropism. Here, we provide evidence that another member, US20, impacts the ability of a clinical strain of HCMV to replicate in endothelial cells. Through the use of recombinant HCMV encoding tagged versions of the US20 protein, we investigated the expression pattern, localization, and topology of the US20-encoded protein (pUS20). We show that pUS20 is expressed as a partially glycosylated 7TMD protein which accumulates late in infection in endoplasmic reticulum-derived peripheral structures localized outside the cytoplasmic virus assembly compartment (cVAC). US20-deficient mutants generated in the TR clinical strain of HCMV exhibited major growth defects in different types of endothelial cells, whereas they replicated normally in fibroblasts and epithelial cells. While the attachment and entry phases in endothelial cells were not significantly affected by the absence of US20 protein, US20-null viruses failed to replicate viral DNA and express representative E and L mRNAs and proteins. Taken together, these results indicate that US20 sustains the HCMV replication cycle at a stage subsequent to entry but prior to E gene expression and viral DNA synthesis in endothelial cells.

    IMPORTANCE Human cytomegalovirus (HCMV) is a major pathogen in newborns and immunocompromised individuals. A hallmark of HCMV pathogenesis is its ability to productively replicate in an exceptionally broad range of target cells, including endothelial cells, which represent a key target for viral dissemination and replication in the host, and to contribute to both viral persistence and associated inflammation and vascular diseases. Replication in endothelial cells depends on the activities of a set of viral proteins that regulate different stages of the HCMV replication cycle in an endothelial cell type-specific manner and thereby act as determinants of viral tropism. Here, we report the requirement of a HCMV protein as a postentry tropism factor in endothelial cells. The identification and characterization of HCMV endotheliotropism-regulating proteins will advance our understanding of the molecular mechanisms of HCMV-related pathogenesis and help lead to the design of new antiviral strategies able to exploit these functions.

  • Remote Activation of Host Cell DNA Synthesis in Uninfected Cells Signaled by Infected Cells in Advance of Virus Transmission [Virus-Cell Interactions]

  • Viruses modulate cellular processes and metabolism in diverse ways, but these are almost universally studied in the infected cell itself. Here, we study spatial organization of DNA synthesis during multiround transmission of herpes simplex virus (HSV) using pulse-labeling with ethynyl nucleotides and cycloaddition of azide fluorophores. We report a hitherto unknown and unexpected outcome of virus-host interaction. Consistent with the current understanding of the single-step growth cycle, HSV suppresses host DNA synthesis and promotes viral DNA synthesis in spatially segregated compartments within the cell. In striking contrast, during progressive rounds of infection initiated at a single cell, we observe that infection induces a clear and pronounced stimulation of cellular DNA replication in remote uninfected cells. This induced DNA synthesis was observed in hundreds of uninfected cells at the extended border, outside the perimeter of the progressing infection. Moreover, using pulse-chase analysis, we show that this activation is maintained, resulting in a propagating wave of host DNA synthesis continually in advance of infection. As the virus reaches and infects these activated cells, host DNA synthesis is then shut off and replaced with virus DNA synthesis. Using nonpropagating viruses or conditioned medium, we demonstrate a paracrine effector of uninfected cell DNA synthesis in remote cells continually in advance of infection. These findings have significant implications, likely with broad applicability, for our understanding of the ways in which virus infection manipulates cell processes not only in the infected cell itself but also now in remote uninfected cells, as well as of mechanisms governing host DNA synthesis.

    IMPORTANCE We show that during infection initiated by a single particle with progressive cell-cell virus transmission (i.e., the normal situation), HSV induces host DNA synthesis in uninfected cells, mediated by a virus-induced paracrine effector. The field has had no conception that this process occurs, and the work changes our interpretation of virus-host interaction during advancing infection and has implications for understanding controls of host DNA synthesis. Our findings demonstrate the utility of chemical biology techniques in analysis of infection processes, reveal distinct processes when infection is examined in multiround transmission versus single-step growth curves, and reveal a hitherto-unknown process in virus infection, likely relevant for other viruses (and other infectious agents) and for remote signaling of other processes, including transcription and protein synthesis.

  • Genome-Wide Screen Reveals Valosin-Containing Protein Requirement for Coronavirus Exit from Endosomes [Virus-Cell Interactions]

  • Coronaviruses are RNA viruses with a large zoonotic reservoir and propensity for host switching, representing a real threat for public health, as evidenced by severe acute respiratory syndrome (SARS) and the emerging Middle East respiratory syndrome (MERS). Cellular factors required for their replication are poorly understood. Using genome-wide small interfering RNA (siRNA) screening, we identified 83 novel genes supporting infectious bronchitis virus (IBV) replication in human cells. Thirty of these hits can be placed in a network of interactions with viral proteins and are involved in RNA splicing, membrane trafficking, and ubiquitin conjugation. In addition, our screen reveals an unexpected role for valosin-containing protein (VCP/p97) in early steps of infection. Loss of VCP inhibits a previously uncharacterized degradation of the nucleocapsid N protein. This inhibition derives from virus accumulation in early endosomes, suggesting a role for VCP in the maturation of virus-loaded endosomes. The several host factors identified in this study may provide avenues for targeted therapeutics.

    IMPORTANCE Coronaviruses are RNA viruses representing a real threat for public health, as evidenced by SARS and the emerging MERS. However, cellular factors required for their replication are poorly understood. Using genome-wide siRNA screening, we identified novel genes supporting infectious bronchitis virus (IBV) replication in human cells. The several host factors identified in this study may provide directions for future research on targeted therapeutics.

  • A Druggable Pocket at the Nucleocapsid/Phosphoprotein Interaction Site of Human Respiratory Syncytial Virus [Structure and Assembly]

  • Presently, respiratory syncytial virus (RSV), the main cause of severe respiratory infections in infants, cannot be treated efficiently with antivirals. However, its RNA-dependent polymerase complex offers potential targets for RSV-specific drugs. This includes the recognition of its template, the ribonucleoprotein complex (RNP), consisting of genomic RNA encapsidated by the RSV nucleoprotein, N. This recognition proceeds via interaction between the phosphoprotein P, which is the main polymerase cofactor, and N. The determinant role of the C terminus of P, and more particularly of the last residue, F241, in RNP binding and viral RNA synthesis has been assessed previously. Here, we provide detailed structural insight into this crucial interaction for RSV polymerase activity. We solved the crystallographic structures of complexes between the N-terminal domain of N (N-NTD) and C-terminal peptides of P and characterized binding by biophysical approaches. Our results provide a rationale for the pivotal role of F241, which inserts into a well-defined N-NTD pocket. This primary binding site is completed by transient contacts with upstream P residues outside the pocket. Based on the structural information of the N-NTD:P complex, we identified inhibitors of this interaction, selected by in silico screening of small compounds, that efficiently bind to N and compete with P in vitro. One of the compounds displayed inhibitory activity on RSV replication, thereby strengthening the relevance of N-NTD for structure-based design of RSV-specific antivirals.

    IMPORTANCE Respiratory syncytial virus (RSV) is a widespread pathogen that is a leading cause of acute lower respiratory infections in infants worldwide. RSV cannot be treated efficiently with antivirals, and no vaccine is presently available, with the development of pediatric vaccines being particularly challenging. Therefore, there is a need for new therapeutic strategies that specifically target RSV. The interaction between the RSV phosphoprotein P and the ribonucleoprotein complex is critical for viral replication. In this study, we identified the main structural determinants of this interaction, and we used them to screen potential inhibitors in silico. We found a family of molecules that were efficient competitors of P in vitro and showed inhibitory activity on RSV replication in cellular assays. These compounds provide a basis for a pharmacophore model that must be improved but that holds promises for the design of new RSV-specific antivirals.

  • Propranolol Decreases Proliferation of Endothelial Cells Transformed by Kaposi's Sarcoma-Associated Herpesvirus and Induces Lytic Viral Gene Expression [Transformation and Oncogenesis]

  • Kaposi's sarcoma (KS) is common in Africa, but economic constraints hinder successful treatment in most patients. Propranolol, a generic bbeta;-adrenergic antagonist, decreased proliferation of KS-associated herpesvirus (KSHV)-infected cells. Downregulation of cyclin A2 and cyclin-dependent kinase 1 (CDK1) recapitulated this phenotype. Additionally, propranolol induced lytic gene expression in association with downregulation of CDK6. Thus, propranolol has diverse effects on KSHV-infected cells, and this generic drug has potential as a therapeutic agent for KS.

  • Adeno-Associated Virus Type 2 Rep68 Can Bind to Consensus Rep-Binding Sites on the Herpes Simplex Virus 1 Genome [Genome Replication and Regulation of Viral Gene Expression]

  • Adeno-associated virus type 2 is known to inhibit replication of herpes simplex virus 1 (HSV-1). This activity has been linked to the helicase- and DNA-binding domains of the Rep68/Rep78 proteins. Here, we show that Rep68 can bind to consensus Rep-binding sites on the HSV-1 genome and that the Rep helicase activity can inhibit replication of any DNA if binding is facilitated. Therefore, we hypothesize that inhibition of HSV-1 replication involves direct binding of Rep68/Rep78 to the HSV-1 genome.

  • Transition toward Human Cytomegalovirus Susceptibility in Early Human Embryonic Stem Cell-Derived Neural Precursors [Virus-Cell Interactions]

  • Congenital human cytomegalovirus (HCMV) infection is associated with neurodevelopmental disabilities. To dissect the earliest events of infection in the developing human brain, we studied HCMV infection during controlled differentiation of human embryonic stem cells (hESC) into neural precursors. We traced a transition from viral restriction in hESC, mediated by a block in viral binding, toward HCMV susceptibility in early hESC-derived neural precursors. We further revealed the role of platelet-derived growth factor receptor alpha (PDGFRaalpha;) as a determinant of the developmentally acquired HCMV susceptibility.

  • Infectious Bursal Disease Virus VP3 Upregulates VP1-Mediated RNA-Dependent RNA Replication [Genome Replication and Regulation of Viral Gene Expression]

  • Genome replication is a critical step in virus life cycles. Here, we analyzed the role of the infectious bursal disease virus (IBDV) VP3, a major component of IBDV ribonucleoprotein complexes, on the regulation of VP1, the virus-encoded RNA-dependent RNA polymerase (RdRp). Data show that VP3, as well as a peptide mimicking its C-terminal domain, efficiently stimulates the ability of VP1 to replicate synthetic single-stranded RNA templates containing the 3' untranslated regions (UTRs) from the IBDV genome segments.

  • Knockout of cGAS and STING Rescues Virus Infection of Plasmid DNA-Transfected Cells [Cellular Response to Infection]

  • It is well known that plasmid DNA transfection, prior to virus infection, negatively affects infection efficiency. Here, we show that cytosolic plasmid DNA activates the cGAS/STING signaling pathway, which ultimately leads to the induction of an antiviral state of the cells. Using a transient one-plasmid clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, we generated cGAS/STING-knockout cells and show that these cells can be infected after plasmid DNA transfection as efficiently as nontransfected cells.

  • Erratum for Gjessing et al., Salmon Gill Poxvirus, the Deepest Representative of the Chordopoxvirinae [Errata]

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    JVI Accepts: Articles Published Ahead of Print

  • Prion infectivity plateaus and conversion to symptomatic disease originate from falling precursor levels and increased oligomeric PrPSc species [PublishAheadOfPrint]

  • In lethal prion neurodegenerative diseases, misfolded prion proteins (PrPSc) replicate by redirecting the folding of the cellular prion glycoprotein (PrPC). Infections of different durations can have a subclinical phase with constant levels of infectious particles, but the mechanisms underlying this plateau and a subsequent exit to overt clinical disease are unknown. Using tandem biophysical techniques, we show that attenuated accumulation of infectious particles in pre-symptomatic disease is preceded by a progressive fall in PrPC level, which constricts replication rate and thereby causes the plateau effect. Furthermore, disease symptoms occurred at the threshold associated with increasing levels of small, relatively less protease-resistant oligomeric prion particles (oPrPSc). While a hypothetical lethal isoform of PrP cannot be excluded, our data argue that diminishing residual PrPC levels and continuously increasing levels of oPrPSc are crucial determinants in the transition from pre-symptomatic to symptomatic prion disease.

    IMPORTANCE Prions are infectious agents that cause lethal brain diseases; they arise from misfolding of a cell-surface protein, PrPC to a form called PrPSc. Prion infections can have long latencies even though there is no protective immune response. Accumulation of infectious prion particles has been suggested to always reach the same plateau in the brain during latent periods, with clinical disease only occurring when hypothetical toxic forms (called PrPL or TPrP) begin to accumulate. We show here that infectivity plateaus arise because PrPC precursor levels become downregulated and that the duration of latent periods can be accounted for by the level of residual PrPC nndash; which transduces a toxic effect nndash; along with the amount of oligomeric forms of PrPSc.

  • Ultrastructural characterization of Turnip mosaic virus induced cellular rearrangements reveal membrane-bound viral particles accumulating in vacuoles [PublishAheadOfPrint]

  • Positive-strand RNA [(+) RNA] viruses remodel cellular membranes to facilitate virus replication and assembly. In the case of Turnip mosaic virus (TuMV), the viral membrane protein 6K2 plays an essential role in endomembrane alterations. Although 6K2-induced membrane dynamics have been widely studied by confocal microscopy, the ultrastructure of this remodeling has not been extensively examined. In this study, we investigated the formation of TuMV-induced membrane changes by chemical fixation and high-pressure freezing/freeze substitution (HPF/FS) for transmission electron microscopy at different times of infection. We observed the formation of convoluted membranes connected to rough endoplasmic reticulum (rER) early in the infection process, followed by the production of single-membrane vesicle-like (SMVL) structures at mid stage of infection. Both SMVL and double-membrane vesicle-like structures with electron-dense cores, as well as electron-dense bodies, were found late in the infection process. Immunogold labeling results showed that the vesicle-like structures were 6K2 tagged, and suggested that only the SMVL structures were viral RNA replication sites. Electron tomography (ET) was used to regenerate a three-dimensional model of these vesicle-like structures, which showed they were in fact tubules. Late in infection, we observed filamentous particle bundles associated with electron-dense bodies, which suggests that these are sites for viral particle assembly. In addition, TuMV particles were observed to accumulate in the central vacuole as membrane-associated linear arrays. Our work thus unravels the sequential appearance of distinct TuMV-induced membrane structures for viral RNA replication, viral particle assembly and accumulation.

    Importance Positive-strand RNA viruses remodel cellular membranes for different stages of the infectious process, such as protein translation and processing, viral RNA synthesis, particle assembly, and virus transmission. The ultrastructure of the Turnip mosaic virus-induced membrane remodeling was investigated over several days of infection. The first change that was observed involved endoplasmic reticulum-connected convoluted membrane accumulation. This was followed by the formation of single-membrane tubules, which were shown to be viral RNA replication sites. Later in the infection process, double-membrane tubular structures were observed and were associated with viral particle bundles. In addition, TuMV particles were observed to accumulate in the central vacuole as membrane-associated linear arrays. This work thus unravels the sequential appearance of distinct TuMV-induced membrane structures for viral RNA replication, viral particle assembly and accumulation.

  • Induction and antagonism of antiviral responses in respiratory syncytial virus-infected pediatric airway epithelium. [PublishAheadOfPrint]

  • Airway epithelium is the primary target of many respiratory viruses. However, virus induction and antagonism of host responses by human airway epithelium remains poorly understood. To address this, we developed a model of respiratory syncytial virus (RSV) infection based on well- differentiated pediatric primary bronchial epithelial cell cultures (WD-PBECs) that mimics hallmarks of RSV disease in infants. RSV is the most important respiratory viral pathogen in young infants worldwide. We found that RSV induces a potent antiviral state in WD-PBECs that was mediated in part by secreted factors, including interferon lambda-1 (IFN1)/IL-29. In contrast, type I interferons were not detected following RSV infection of WD-PBECs., Interferon (IFN) responses in RSV-infected WD-PBECs reflected those in lower airway samples from RSV-hospitalized infants. In view of the prominence of IL-29, we determined whether recombinant IL-29 treatment of WD-PBECs before or after infection abrogated RSV replication. Interestingly, IL-29 demonstrated prophylactic, but not therapeutic, potential against RSV. The absence of therapeutic potential reflected effective RSV antagonism of IFN-mediated antiviral responses in infected cells. Our data are consistent with RSV non-structural proteins 1 and/or 2 perturbing the Jak-STAT signaling pathway, with concomitant reduced expression of antiviral effector molecules, such as MxA/B. Antagonism of Jak-STAT signaling was restricted to RSV-infected cells in WD-PBEC cultures. Importantly, our study provides the rationale to further explore IL-29 as a novel RSV prophylactic.

    IMPORTANCE Most respiratory viruses target airway epithelium for infection and replication, which is central to causing disease. However, for most human viruses we have a poor understanding of their interactions with human airway epithelium. Respiratory syncytial virus (RSV) is the most important viral pathogen of young infants. To help understand RSV interactions with pediatric airway epithelium, we previously developed 3-D primary cell cultures from infant bronchial epithelium that reproduce several hallmarks of RSV infection in infants, indicating that they represent authentic surrogates of RSV infection in infants. We found that RSV induced a potent antiviral state in these cultures and that type III interferon (IL-29) was involved. Indeed, our data suggest that IL-29 has potential to prevent RSV disease. However, we also demonstrated that RSV efficiently circumvents this antiviral immune response and identified mechanisms by which this may occur. Our study provides new insights into RSV interaction with pediatric airway epithelium.

  • A unique 5' translation element discovered in Triticum mosaic virus. [PublishAheadOfPrint]

  • Several plant viruses encode elements at the 5rrsquo; end of their RNAs that, unlike most cellular mRNAs, can initiate translation in the absence of a 5rrsquo; m7GpppG cap. Here, we describe an exceptionally long (739 nt) leader sequence in Triticum mosaic virus (TriMV), a recently-emerged wheat pathogen that belongs to the Potyviridae family of positive-strand RNA viruses. We demonstrate that the TriMV 5rrsquo; leader drives strong cap-independent translation in both wheat germ extract and oat protoplasts through a novel, non-canonical translation mechanism. Translation preferentially initiates at the 13th start codon within the leader sequence independently of eIF4E, but involves eIF4G. We truncated the 5rrsquo; leader to a 300-nucleotide sequence that drives cap-independent translation from the 5rrsquo; end. Within this sequence, we show that the translation activity relies on a stem loop structure identified at nucleotide position 469-490. The disruption of the stem significantly impairs the function of the 5rrsquo; UTR in driving translation and competing against a capped RNA. Additionally, the TriMV 5rrsquo; UTR can direct translation from an internal position of a bicistronic mRNA. And unlike cap-driven translation, it is unimpaired when the 5rrsquo; end is blocked by a strong hairpin in a monocistronic reporter. However, the disruption of the identified stem structure eliminates such translational advantage. Our results reveal a potent and uniquely-controlled translation enhancer that may provide new insights into mechanisms of plant virus translational regulation.

    IMPORTANCE Many members of the Potyviridae family rely on their 5rrsquo; end for translation. Here, we show that the 739 nucleotide-long Triticum mosaic virus 5rrsquo; leader bears a powerful translation element with features distinct from those described for other plant viruses. Despite the presence of twelve AUG start codons within the TriMV 5rrsquo; UTR, translation primarily initiates at the 13th AUG codon. The TriMV 5rrsquo; UTR is capable of driving cap-independent translation in vitro and in vivo, is independent of eIF4E, and can drive internal translation initiation. A hairpin structure at nucleotide position 469-490 is required for cap-independent translation and internal translation initiation abilities of the element, and plays a role in the ability of the TriMV UTR to compete against a capped RNA in vitro. Our results reveal a novel translation enhancer that may provide new insights into the large diversity among plant virus translation mechanisms.

  • Deer Prion Proteins Modulate the Emergence and Adaptation of Chronic Wasting Disease Strains. [PublishAheadOfPrint]

  • Transmission of chronic wasting disease (CWD) between cervids is influenced by the primary structure of the host cellular prion protein (PrPC). In white-tailed deer, PRNP alleles encode polymorphisms Q95G96 (wt), Q95S96 (S96) and H95G96 (H95) that differentially impact CWD progression. We hypothesize that transmission of CWD prions between deer expressing different allotypes of PrPC modifies the contagious agent affecting disease spread. To evaluate the transmission properties of CWD prions derived experimentally from deer of four PRNP genotypes (wt/wt, S96/wt, H95/wt or H95/S96), transgenic (tg) mice expressing wt (tg33) or S96 (tg60) alleles were challenged with these prion agents. Passage of deer CWD into tg33 mice resulted in 100% attack rates, with H95/S96 CWD having significantly longer incubation periods. Disease signs, neuropathological and PrP-res profiles in infected tg33 mice were similar between groups, indicating a prion strain (Wisc-1) common to all CWD inocula was amplified. In contrast, tg60 mice developed prion disease only when inoculated with the H95/wt and H95/S96 CWD allotypes. Serial passage in tg60 mice resulted in adaptation of a novel CWD strain (H95+) with distinct biological properties. Transmission of first-passage tg60CWD-H95+ isolates into tg33 mice, however, elicited two prion disease presentations consistent with a mixture of strains associated with different PrP-res glycotypes. Our data indicates that H95-PRNP heterozygous deer accumulated two CWD strains, whose emergence was dictated by PrPC primary structure of the recipient host. These findings suggest CWD transmission between cervids expressing distinct PrPC molecules results in generation of novel CWD strains.

    IMPORTANCE CWD prions are contagious among wild and captive cervids in North America and in Korea. We present data linking the amino acid variant Q95H in white-tailed deer cellular prion protein (PrPC) to the emergence of a novel CWD strain (H95+). We show that, upon infection, deer expressing H95-PrPC molecules accumulated a mixture of CWD strains selectively propagated depending on the PRNP genotype of the host in which passaged. Our study also demonstrates that mice expressing the deer S96-PRNP allele, previously shown to be resistant to various cervid prions, are susceptible to H95+ CWD prions. The potential for generation of novel strains raises the possibility of an expanded host range for CWD.

  • An RNA Domain Imparts Specificity and Selectivity to a Viral DNA Packaging Motor [PublishAheadOfPrint]

  • During assembly, double-stranded DNA viruses, including bacteriophages and herpesviruses, utilize a powerful molecular motor to package their genomic DNA into a preformed viral capsid. An integral component of the packaging motor in the Bacillus subtilis bacteriophage 29 is a viral-encoded pentameric ring of RNA (pRNA). pRNA is a 174b transcript comprised of two domains, Domains I and II. Early studies initially isolated a 120b form (Domain I only) that retains high biological activity in vitro; hence, no function could be assigned to Domain II. Here we define a role for this domain in the packaging process. DNA packaging using restriction digests of 29 DNA showed that motors with 174b pRNA supported correct polarity of DNA packaging, selectively packaging the DNA left-end. In contrast, motors containing 120b pRNA had compromised specificity, packaging both left- and right- end fragments. The presence of Domain II also provides selectivity in competition assays with genomes from related phages. Furthermore, motors with 174b pRNA were restrictive for only one DNA fragment packaged, whereas motors with 120b pRNA packaged several fragments into the head, indicating multiple initiation events. These results show that Domain II imparts specificity and stringency to the motor during the packaging initiation events that precede DNA translocation. Heteromeric rings of pRNA demonstrated that one or two copies of Domain II were sufficient to impart this selectivity/stringency. Although 29 differs from other dsDNA phages in having an RNA motor component, function provided by pRNA is encoded in the motor protein components in other phages.

    IMPORTANCE During virus assembly, genome packaging involves the delivery of newly synthesized viral nucleic acid into a protein shell. In the double-stranded DNA phages and herpesviruses, this is accomplished by a powerful molecular motor that translocates the viral DNA into a preformed viral shell. A key event in DNA packaging is recognition of the viral DNA amongst other nucleic acids in the host cell. Commonly, a DNA-binding protein mediates the interaction of viral DNA with the motor/head shell. Here we show that for the bacteriophage 29, this essential step of genome recognition is mediated by a viral-encoded RNA, rather than protein. A domain of the prohead RNA (pRNA) imparts specificity and stringency to the motor by ensuring the correct orientation of DNA packaging and restricting initiation to a single event. Since this assembly step is unique to the virus, DNA packaging is a novel target for the development of anti-viral drugs.

  • AP-2 is the crucial clathrin adaptor protein for CD4 downmodulation by HIV-1 Nef in primary infected CD4+ T cells. [PublishAheadOfPrint]

  • HIV-1 Nef-mediated CD4 downmodulation involves various host factors. We investigated the importance of AP-1, AP-2, AP-3, V1H-ATPase, bbeta;-COP and ACOT8 for CD4 downmodulation in HIV-1 infected shRNA-expressing CD4+ T cells and characterized direct interaction with Nef by FRET. Binding of lentiviral Nefs to CD4 and AP-2 was conserved and only AP-2 knockdown impaired Nef-mediated CD4 downmodulation from primary T cells. Altogether, among the factors tested, AP-2 is the most important player for Nef-mediated CD4 downmodulation.

  • Whole-genome sequencing of KSHV from Zambian Kaposi's sarcoma biopsies reveals unique viral diversity. [PublishAheadOfPrint]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent for Kaposi's sarcoma (KS). Both KSHV and KS are endemic in sub-Saharan Africa where approximately 84% of global KS cases occur. Nevertheless, whole-genome sequencing of KSHV has only been completed using isolates from Western countriesmmdash;where KS is not endemic. The lack of whole-genome KSHV sequence data from the most clinically important geographical region, sub-Saharan Africa, represents an important gap as it remains unclear whether genomic diversity has a role on KSHV pathogenesis. We hypothesized that distinct KSHV genotypes might be present in sub-Saharan Africa compared to Western countries. Using a KSHV-targeted enrichment protocol followed by Illumina deep-sequencing, we generated and analyzed sixteen unique Zambian, KS-derived, KSHV genomes. We enriched KSHV DNA over cellular DNA 1,851 to 18,235-fold. Enrichment provided coverage levels up to 24,740-fold; therefore, supporting highly confident polymorphism analysis. Multiple alignment of the sixteen newly sequenced KSHV genomes showed low level variability across the entire central conserved region. This variability resulted in distinct phylogenetic clustering between Zambian KSHV genomic sequences and those derived from Western countries. Importantly, the phylogenetic segregation of Zambian from Western sequences occurred irrespective of inclusion of the highly variable genes K1 and K15. We also show that four genes within the more conserved region of the KSHV genome contained polymorphisms that partially, but not fully, contributed to the unique Zambian KSHV whole-genome phylogenetic structure. Taken together, our data suggest that the whole KSHV genome should be taken into consideration for accurate viral characterization.

    IMPORTANCE Our results represent the largest number of KSHV whole-genomic sequences published to date and the first time that multiple genomes have been sequenced from sub-Saharan Africa, a geographic area where KS is highly endemic. Based on our new sequence data, it is apparent that whole-genome KSHV diversity is greater than previously appreciated and differential phylogenetic clustering exists between viral genomes of Zambia and Western countries. Furthermore, individual genes may be insufficient for KSHV genetic characterization. Continued investigation of the KSHV genetic landscape is necessary in order to effectively understand the role of viral evolution and sequence diversity on KSHV gene functions and pathogenesis.

  • The contribution of epidemiological predictors in unravelling the phylogeographic history of HIV-1 subtype C in Brazil [PublishAheadOfPrint]

  • The phylogeographic history of the Brazilian HIV-1 subtype C (HIV-1C) epidemic is still unclear. Previous studies have mainly focused on the capital cities of Brazilian federal states and the fact that HIV-1C infections increase at a higher rate than subtype B in Brazil calls for a better understanding of the process of spatial spread. A comprehensive sequence dataset sampled across 22 Brazilian locations was assembled and analyzed. A Bayesian phylogeographic generalized linear model approach was used to reconstruct the spatiotemporal history of HIV-1C in Brazil considering several potential explanatory predictors of the viral diffusion process. Analyses were performed on several subsampled datasets in order to mitigate potential sample biases. We reveal a central role for Porto Alegre city, the capital of the southernmost state, in the Brazilian HIV-1C epidemic (HIV-1C_BR), and the northwards expansion of HIV-1C_BR could be linked to source populations with higher HIV-1 burden and larger proportions of HIV-1C infections. The results presented here bring new insights to the continuing discussion about the HIV-1C epidemic in Brazil, and raise an alternative hypothesis for its spatiotemporal history. The current work also highlights how sampling bias can confound phylogeographic analyses and demonstrates the importance of incorporating external information to protect against this.

    Importance: Subtype C is responsible for the largest HIV infection burden worldwide, but our understanding of its transmission dynamics remains incomplete. Brazil witnessed a relatively recent introduction of HIV-1C compared to HIV-1B, but it swiftly spread throughout the South, where it now circulates as the dominant variant. The northward spread is comparatively slower and HIV-1B still prevails in this region. While epidemiological data and viral genetic analyses have both independently shed light on the dynamics of spread in isolation, their combination has not yet been explored. Here, we complement publically available sequences and new genetic data from 13 cities with epidemiological data to reconstruct the history of HIV-1C spread in Brazil. The combined approach results in more robust reconstructions and can protect against sampling bias. We found evidence for an alternative view on the HIV-1C spatiotemporal history in Brazil, which, contrary to previous explanations, integrates seamlessly with other observational data.

  • Differential inhibition of macrophage activation by lymphocytic choriomeningitis virus and Pichinde virus is mediated by the Z protein N-terminal domain [PublishAheadOfPrint]

  • Several arenavirus pathogens, such as Lassa and Junin viruses, inhibit macrophage activation, the molecular mechanism of which is unclear. We show that lymphocytic choriomeningitis virus (LCMV) can also inhibit macrophage activation, in contrast to Pichinde and Tacaribe virus, which are not known to naturally cause human diseases. Using recombinant Pichinde virus system, we show that LCMV Z NTD mediates the inhibition of macrophage activation and immune functions.

  • Comparison of the Life Cycles of Genetically Distant Species C and Species D Human Adenoviruses Ad6 and Ad26 in Human Cells [PublishAheadOfPrint]

  • Our understanding of adenovirus (Ad) biology is largely extrapolated from human species C Ad5. Most humans are immune to Ad5, so lower seroprevalent viruses like human Ad6 and Ad26 are being tested as therapeutic vectors. Ad6 and Ad26 differ by 34% at the DNA level. To better understand how this might impact their biology, we examined the life cycle of the two viruses in human lung cells in vitro. Both viruses infected A549 cells with similar efficiency, both executed DNA replication with identical kinetics within 12 hours, and both begin killing cells within 72 hours. While Ad6 infected cells remained adherent until death, Ad26 infected cells detached within 12 hours of infection, but remained viable. Next generation sequencing (NGS) of mRNA from infected cells demonstrated that viral transcripts constituted 1% of cellular mRNAs within 6 hours and 8 to 16% within 12 hours. Quantitative PCR and NGS revealed activation of key early genes at 6 hours and transition to late gene activation by 12 hours by both viruses. There were marked differences in the balance of E1A and E1B activation by the two viruses and in the expression of E3 immunevasion mRNAs. Ad6 was markedly more effective at suppressing MHC I display on the cell surface and in evading TRAIL-mediated apoptosis than Ad26. These data demonstrate shared as well as divergent life cycles in these genetically distant human adenoviruses. Understanding these differences expands the knowledge of alternative Ad species and may inform selection of related Ads for therapeutic development.

    IMPORTANCE A burgeoning number of adenoviruses (Ads) are being harnessed as therapeutics, yet the biology of these viruses is generally extrapolated from Ad2 and Ad5. Here, we are first to compare the transcriptional programs of two genetically distant Ads by mRNA next generation sequencing (NGS). Species C Ad6 and Ad26 are being pursued as lower seroprevalence Ad vectors, but differ at the DNA level by 34%. Head to head comparison in human lung cells by NGS revealed that the two viruses generally conform to our general understanding of the Ad transcriptional program. However, fine mapping revealed subtle and strong differences in how these two viruses execute these programs including differences in the balance of E1A and B mRNAs and in E3 immunevasion genes. This suggests that not all adenoviruses behave like Ad2 and Ad5 and that they may have unique strategies to infect cells and evade the immune system.

  • Breakthrough virus neutralization resistance as a correlate of protection in a nonhuman primate heterologous SIV vaccine challenge study. [PublishAheadOfPrint]

  • Comprehensive assessments of immune correlates of protection in HIV vaccine trials are essential to vaccine design. Neutralization sieve analysis compares the neutralization sensitivity of the breakthrough transmitted/founder (TF) viruses from vaccinated and control animals to infer the molecular mechanisms of vaccine protection. Here, we report a robust neutralization sieve effect in a nonhuman primate Simian Immunodeficiency Virus (SIV) vaccine trial (DNA-prime/rAd5 boost; VRC-10-332) that demonstrated substantial protective efficacy and revealed a genetic signature of neutralization resistance in the C1 region of env. We found significant enrichment for neutralization resistance in the vaccine compared to control breakthrough TF viruses when tested with plasma from vaccinated study animals, plasma from chronically SIV-infected animals, and a panel of SIV-specific monoclonal antibodies targeting six discrete Env epitopes (pllt;0.008 for all comparisons). Neutralization resistance was significantly associated with the previously identified genetic signature of resistance (pllt;0.0001), and together, the results identify virus neutralization as a correlate of protection. The current findings further demonstrate the in vivo relevance of our previous in vitro analyses of the SIVsmE660 challenge stock, which revealed a broad range in neutralization sensitivity of its component viruses. In sum, the current report demonstrates proof-of-concept that phenotypic sieve analyses can elucidate mechanistic correlates of immune protection following vaccination and raises a cautionary note for SIV and SHIV (Simian-Human Immunodeficiency Virus) vaccine studies that employ challenge strains with envelope glycoproteins that fail to exhibit neutralization resistance profiles typical of TF viruses.

    IMPORTANCE With more than two million new infections annually, the development of an effective vaccine against HIV-1 is a global health priority. Understanding immunologic correlates of protection generated in vaccine trials is critical to advance vaccine development. Here, we assessed the role of vaccine-elicited neutralizing antibodies in a recent nonhuman primate study of a vaccine that showed significant protection against Simian Immunodeficiency Virus (SIV) challenge and suggested a genetic signature of neutralization sensitivity. We found that breakthrough viruses able to establish infection in vaccinated animals were substantially more resistant to antibody-mediated neutralization than were viruses from controls. These findings suggest that vaccine-elicited neutralizing antibodies selectively blocked transmission of more sensitive challenge viruses. Sieve analysis also corroborated the genetic signature of neutralization sensitivity and highlighted the impact of challenge swarm diversity. Our findings suggest an important role for neutralization sieve analyses as an informative component of comprehensive immune correlates analyses.

  • MDA5 is critical to host defense during infection with murine coronavirus [PublishAheadOfPrint]

  • Infection with the murine coronavirus mouse hepatitis virus (MHV) activates the pattern recognition receptors melanoma differentiation-associated gene 5 (MDA5) and toll-like receptor 7 (TLR7) to induce transcription of type 1 interferon. Type 1 interferon is crucial for control of viral replication and spread in the natural host, but the specific contributions of MDA5 signaling to this pathway, as well as to pathogenesis and subsequent immune responses, are largely unknown. In this study, we use MHV infection of the liver as a model to demonstrate that MDA5 signaling is critically important for controlling MHV-induced pathology and regulation of the immune response. Mice deficient in MDA5 expression (MDA5nndash;/nndash; mice) experienced more severe disease following MHV infection, with reduced survival, increased spread of virus to additional sites of infection, and more extensive liver damage. Although type 1 interferon transcription decreased in MDA5nndash;/nndash; mice, the interferon stimulated gene response remained intact. Cytokine production by innate and adaptive immune cells was largely intact in MDA5-/- mice, but perforin induction by natural killer cells, and serum levels of interferon gamma, IL-6, and TNFaalpha; were elevated. These data suggest that MDA5 signaling reduces the severity of MHV-induced disease, at least in part by reducing the intensity of the pro-inflammatory cytokine response.

    IMPORTANCE Multicellular organisms employ a wide range of sensors to detect viruses and other pathogens. One such sensor, MDA5, detects viral RNA and triggers induction of type 1 interferons, chemical messengers that induce inflammation and help regulate the immune responses. In this study we sought to determine the role of MDA5 during infection with mouse hepatitis virus, a murine coronavirus used to model viral hepatitis as well as other human diseases. We found that mice lacking the MDA5 sensor were more susceptible to infection and experienced decreased survival. Viral replication in the liver was similar in mice with and without MDA5, but liver damage was increased in MDA5-/- mice, suggesting that the immune response is causing the damage. Production of several pro-inflammatory cytokines was elevated in MDA5-/- mice, suggesting that MDA5 may be responsible for keeping pathological inflammatory responses in check.

  • Infection of mouse macrophages by seasonal influenza viruses can be restricted at the level of virus entry and at late stage in the virus life cycle [PublishAheadOfPrint]

  • Airway epithelial cells are susceptible to infection with seasonal influenza A viruses (IAV), resulting in productive virus replication and release. Macrophages (M) are also permissive to IAV infection, however virus replication is abortive. Currently, it is unclear how productive infection of M is impaired or the extent to which seasonal IAV replicate in M. Herein, we compared mouse M and epithelial cells for their ability to support genomic replication and transcription, synthesis of viral proteins, assembly of virions and release of infectious progeny following exposure to genetically defined IAV. We confirm that seasonal IAV differ in their ability to utilize cell-surface receptors for infectious entry and that this represents one level of virus restriction. Following virus entry, we demonstrate synthesis of all 8 segments of genomic viral (v)RNA and messenger (m)RNA, as well as 7 distinct IAV proteins, in IAV-infected mouse M. Although newly synthesized hemagglutinin (HA) and neuraminidase (NA) glycoproteins are incorporated into the plasma membrane and expressed at the cell surface, electron microscopy confirmed that virus assembly was defective in IAV-infected M, defining a second level of restriction late in the virus life cycle.

    IMPORTANCE Seasonal influenza A viruses (IAV) and highly pathogenic avian influenza (HPAI) infect macrophages, but only HPAI replicate productively in these cells. Herein, we demonstrate that impaired virus uptake into macrophages represents one level of restriction limiting infection by seasonal IAV. Following uptake, seasonal IAV do not complete productive replication in macrophages, representing a second level of restriction. Using murine macrophages, we demonstrate that productive infection is blocked late in the virus life cycle, such that virus assembly is defective and newly synthesized virions are not released. These studies represent an important step towards identifying host-encoded factors that block replication of seasonal IAV, but not HPAI, in macrophages.

  • Intercellular transmission of viral populations with vesicles. [PublishAheadOfPrint]

  • A common paradigm holds that during cell-to-cell transmission viruses behave as lone soldiers. Recently we discovered that enteroviruses are not only transmitted via vesicles as populations of viral particles, but that this type of transmission enhances their infection efficiency. This mechanism could be advantageous for the overall fitness of the viral population promoting genetic interplay by enabling viral quasispecies to collectively infect a susceptible host cell. Here we discuss these findings in the context of viral pathogenesis and also propose that this novel type of vesicular transmission is likely wide spread among different virus families and includes populations of both viral particles and naked viral genomes.

  • The fusion protein specificity of the parainfluenza virus hemagglutinin-neuraminidase protein is not solely defined by the primary structure of its stalk domain [PublishAheadOfPrint]

  • Virus-specific interaction between the attachment protein (HN) and the fusion protein (F) is prerequisite for the induction of membrane fusion by parainfluenza viruses. This HN-F interaction is presumably mediated by particular amino acids in the HN stalk domain and those in the F head domain. We found in the present study, however, that an SV41 F-specific chimeric HPIV2 HN protein, SCA, whose cytoplasmic, transmembrane, and stalk domains were derived from the SV41 HN protein, could not induce cell-cell fusion of BHK-21 cells when coexpressed with an SV41 HN-specific chimeric PIV5 F protein, no. 36. Similarly, a headless form of the SV41 HN protein failed to induce fusion with chimera no. 36, whereas it was able to induce fusion with the SV41 F protein. Interestingly, substitution of 13 amino acids of the SCA head domain, that are located at or around the dimer interface of the head domain, with the SV41 HN counterparts resulted in a chimeric HN protein, SCA-RII, which induced fusion with chimera no. 36 but not with the SV41 F protein. More interestingly, retrosubstitution of 11 out of the 13 amino acids of SCA-RII with the SCA counterparts resulted in another chimeric HN protein, IM18, which induced fusion either with chimera no. 36 or with the SV41 F protein similarly to the SV41 HN protein. We thus conclude that the F protein specificity of the HN protein that is observed in the fusion event is not solely defined by the primary structure of the HN stalk domain.

    IMPORTANCE It is appreciated that the HN head domain initially conceals the HN stalk domain but exposes it after the head domain has bound to the receptors, which allows particular amino acids in the stalk domain to interact with the F protein and trigger it to induce fusion. However, other regulatory roles of the HN head domain in the fusion event have been ill defined. We have shown in the current study that removal of the head domain or amino acid substitutions in a particular region of the head domain drastically change the F protein specificity of the HN protein, suggesting that the ability of a given HN protein to interact with an F protein is not only defined by the primary structure of the HN stalk domain but also by its conformation. This notion seems to account for the unidirectional substitutability among rubulavirus HN proteins in triggering noncognate F proteins.

  • Flavivirus infection impairs peroxisome biogenesis and early anti-viral signaling [PublishAheadOfPrint]

  • Flaviviruses are significant human pathogens that have an enormous impact on the global health burden. Currently, there are very few vaccines against or therapeutic treatments for flaviviruses and our understanding of how these viruses cause disease is limited. Evidence suggests that the capsid proteins of flaviviruses play critical non-structural roles during infection and therefore, elucidating how these viral proteins affect cellular signaling pathways could lead to novel targets for antiviral therapy. We used affinity purification to identify host cell proteins that interact with the capsid proteins of West Nile and Dengue viruses. One of the cellular proteins that formed a stable complex with flavivirus capsid proteins is the peroxisome biogenesis factor, Pex19. Intriguingly, flavivirus infection resulted in a significant loss of peroxisomes, an effect that may be due in part to capsid expression. We posited that capsid protein-mediated sequestration and/or degradation of Pex19 results in loss of peroxisomes, a situation that could result in reduced early anti-viral signaling. In support of this hypothesis, we observed that induction of the interferon- mRNA in response to a viral RNA mimic, was reduced by more than 80%. Together our findings indicate that inhibition of peroxisome biogenesis may be a novel mechanism by which flaviviruses evade the innate immune system during early stages of infection.

    IMPORTANCE RNA viruses infect hundreds of millions of people each year causing significant morbidity and mortality. Chief among these pathogens are the flaviviruses, which include Dengue virus and West Nile virus. Despite their medical importance, there are very few prophylactic or therapeutic treatments for these viruses. Moreover, the manner in which they subvert the innate immune response in order to establish infection in mammalian cells is not well understood. Recently, peroxisomes were reported to function in early anti-viral signaling but very little is known if or how pathogenic viruses affect these organelles. We report for the first time that flavivirus infection results in significant loss of peroxisomes in mammalian cells which may indicate that targeting of peroxisomes is a key strategy used by viruses to subvert early anti-viral defenses.

  • Differentiation-Coupled Induction of Human Cytomegalovirus Replication by Union of the Major Enhancer Retinoic Acid, cAMP, and NF-{kappa}B Response Elements [PublishAheadOfPrint]

  • Triggers and regulatory pathways that effectively link human cytomegalovirus (HCMV) major immediate-early (MIE) latent-lytic switch activation with progeny production are incompletely understood. In the quiescently infected human NTera2 cell model of primitive neural stem cells, we found that co-stimulation with vasoactive intestinal peptide (V) and phorbol ester (P) synergistically activated viral infection, but this effect waned over time. Coupling retinoic acid (R), an inducer of neuronal differentiation, to VP pulse-stimulation attenuated the decline in viral activity and promoted spread of the active infection through concentric layers of neighboring cells as cellular differentiation progressed. R stimulation alone was unable to activate the infection. The MIE enhancer cis-regulatory mechanisms responsible for this result were characterized by a strategy of combinatorial mutagenesis of five cis-acting element types (retinoic acid receptor binding elements (RARE), cAMP response elements (CRE), NF-B binding sites (kB), serum response element, and ETS/ELK-1 binding site) and multiple methods of assessment. We found that the CRE and kB combination sets pre-induction enhancer tone, is the major initiator and amplifier of RVP-induced MIE gene expression, and cooperates with RARE during cellular differentiation to enhance viral spread. In pre-differentiated NTera2, we also found that the CRE-kB combination functions as initiator and amplifier of unstimulated HCMV MIE gene expression and cooperatively interacts with RARE to enhance viral spread. We conclude that RVP-stimulated signaling cascades and cellular differentiation operate through the enhancer CRE-kB-RARE core in strengthening induction of HCMV MIE gene expression in linkage with viral propagation.

    IMPORTANCE Cytomegalovirus-seropositive persons commonly lack detectable levels of cytomegalovirus replication, even when profoundly immunocompromised. In a human NTera2 cell model of primitive neural stem cells carrying resting cytomegalovirus genomes, we show that co-stimulation of protein kinase A and C-delta signaling cascades in conjunction with retinoic acid-induced neuronal differentiation brings about progeny virus propagation. Iterated DNA binding sites for retinoic acid receptor, CREB, and NF-B family members in the cytomegalovirus major enhancer are at the crux in the pathway to HCMV activation. The stimulated CREB and NF-B binding site combination vigorously initiates and amplifies the active cytomegalovirus infection and cooperates with activated retinoic acid receptor binding sites to further promote viral proliferation and spread between differentiated cells. These results support a paradigm in which a specific combination of stimuli coupled with cellular differentiation satisfies a core cis-activating code that unlocks enhancer silence to re-power the cycle of cytomegalovirus propagation.

  • Antiviral RNAi against Orsay virus is neither systemic nor transgenerational in Caenorhabditis elegans [PublishAheadOfPrint]

  • Antiviral RNA-mediated silencing (RNAi) acts as a powerful innate immunity defence in plants, invertebrates and mammals. In C. elegans RNAi is systemic, i. e. RNAi silencing signals can move between cells and tissues. Furthermore RNAi effects can be inherited transgenerationally and may last for many generations. Neither the biological relevance of systemic RNAi nor transgenerational RNAi are currently understood. Here we examined the role of both pathways to protect C. elegans from viral infection. We studied the Orsay virus, a positive strand RNA virus related to Nodaviridae, and the first and only virus known to infect C. elegans. Immunity to Orsay virus infection requires the RNAi pathway. Surprisingly, we found that genes required for systemic or transgenerational RNAi did not have a role in antiviral defence. Furthermore, we found that Orsay virus infection did not elicit a systemic RNAi response even when a target for RNAi was provided using transgenes. Finally, we show that viral siRNAs, the effectors of RNAi, are not inherited to a level that provides any significant resistance to viral infection in the next generation. We conclude that systemic or transgenerational RNAi does not play a role in the defence to natural Orsay virus infection. Furthermore, our data suggest that there is a qualitative difference between experimental RNAi and antiviral RNAi. Our data are consistent with a model of systemic and transgenerational RNAi that requires a nuclear or germline component which is lacking in almost all RNA virus infections.

    Importance Since its discovery in Caenorhabditis elegans, RNAi has proven a valuable scientific tool in many organisms. In C. elegans exogenous RNAi spreads throughout the entire organism and can be passed between generations, however there has been controversy as to the endogenous role/s that the RNAi pathway plays. One endogenous role for which spreading both within the infected organism and between generations would be advantageous is a role in viral defence. In plants antiviral RNAi is systemic, and the spread of RNAi between cells provides protection against subsequent viral infection. Here we investigate this using the only naturally occurring virus known to infect C. elegans, Orsay virus, and surprisingly find that in contrast to the exogenous RNAi pathway, the antiviral RNAi response targeted against this virus does not spread systemically throughout the organism and cannot be passed between generations. These results suggest that there are differences between the two pathways which remain to be discovered.

  • The interplay between the HSV-1 gB cytodomains and the gH cytotail during cell-cell fusion [PublishAheadOfPrint]

  • Herpesvirus entry into cells is mediated by the viral fusogen gB, which is thought to refold from the prefusion to the postfusion form in a series of large conformational changes that energetically couple refolding to membrane fusion. In contrast to most viral fusogens, gB requires a conserved heterodimer gH/gL as well as other non-conserved proteins. In a further mechanistic twist, gB-mediated cell-cell fusion appears restricted by its intraviral or cytoplasmic domain (cytodomain) because mutations within it result in a hyperfusogenic phenotype. Here, we characterized a panel of hyperfusogenic HSV-1 gB cytodomain mutants and show that they are fully functional in cell-cell fusion at shorter co-incubation times and at lower temperatures than the WT gB, which suggests that these mutations reduce the kinetic energy barrier to fusion. Despite this, the mutants require both gH/gL and gD. We confirm the previous observation that gH cytotail is an essential component of the cell-cell fusion mechanism and show that the N-terminal portion of the gH cytotail is critical for this process. Moreover, the fusion levels achieved by all gB constructs, WT and mutant, were proportionate to the length of the gH cytotail. Putting these results together, we propose that the gH cytotail, in addition to the gH/gL ectodomain, plays an essential role in gB activation, potentially acting as a "wedge" to release the gB cytodomain "clamp" and enable gB activation.

    IMPORTANCE Herpesviruses infect their hosts for life and cause a substantial disease burden. Herpes Simplex viruses cause oral and genital sores as well as rare yet severe encephalitis and a panoply of ocular ailments. Infection initiates when the viral envelope fuses with the host cell membrane in a process orchestrated by the viral fusogen gB assisted by viral glycoproteins gH, gL, gD, and a cellular gD receptor. This process is more complicated than that of most other viruses and is subject to multiple regulatory inputs. Antiviral and vaccine development would benefit from a detailed mechanistic knowledge of this process and how it is regulated.

  • Human cytomegalovirus pUL93 is required for viral genome cleavage and packaging [PublishAheadOfPrint]

  • Human cytomegalovirus (HCMV) pUL93 is essential for virus growth, but its precise function in the virus life cycle is unknown. Here, we characterize a UL93 stop mutant virus (UL93st-TB40/E-BAC) to demonstrate that absence of this protein does not restrict viral gene expression; however, cleavage of viral DNA into unit length genomes as well as genome packaging is abolished. Thus, pUL93 is required for viral genome cleavage and packaging.

  • Single neutralizing monoclonal antibodies targeting the VP1 GH-loop of enterovirus 71 inhibit both virus attachment and internalization during viral entry [PublishAheadOfPrint]

  • Antibodies play a critical role in the immunity against enterovirus 71 (EV71). However, how EV71-specific antibodies neutralize infections remains poorly understood. Here we report the working mechanism for a group of three potently neutralizing monoclonal antibodies (MAbs) against EV71. We found that these three MAbs (termed D5, H7 and C4, respectively) recognized the same conserved neutralizing epitope within the VP1 GH-loop of EV71. Single MAbs in this group, exemplified by D5, could inhibit EV71 infection in cell cultures at both pre- and post-attachment stages in a cell type-independent manner. Specifically, MAb treatment resulted in blockade of multiple steps of EV71 entry, including virus attachment, internalization, and subsequent uncoating and RNA release. Furthermore, we showed that D5 and C4 antibodies could interfere with EV71 binding to its key receptors, including heparan sulfate, SCARB2 and PSGL-1, thus providing a possible explanation for the observed multi-inhibitory function of the MAbs. Collectively, our study unravels the mechanism of neutralization by a unique group of anti-EV71 MAbs targeting the conserved VP1 GH-loop. The findings should enhance our understanding of MAb-mediated immunity against enterovirus infections and accelerate the development of MAb-based anti-EV71 therapeutic drugs.

    IMPORTANCE Enterovirus 71 (EV71) is a major causative agent of hand, foot and mouth disease (HFMD) which has caused significant morbidities and mortalities in young children. Neither vaccine nor antiviral drug is available. Neutralizing antibodies are major protective components in EV71 immunity. Here, we unraveled an unusual mechanism of EV71 neutralization by a group of three neutralizing monoclonal antibodies (MAbs). All of these MAbs bound the same conserved epitope located at the VP1 GH-loop of EV71. Interestingly, mechanistic studies showed single antibodies in this MAb group could block EV71 attachment and internalization during the viral entry process and interfere with EV71 binding to heparan sulfate, SCARB2, and PSGL-1 molecules, which are key receptors involved in different steps of EV71 entry. Our findings greatly enhance the understanding of the interplays among EV71, neutralizing antibodies and host receptors, which in turn should facilitate the development of a MAb-based anti-EV71 therapy.

  • Replication competent influenza B reporter viruses as tools for screening antivirals and antibodies [PublishAheadOfPrint]

  • Influenza B virus is a human pathogen responsible for significant health and economic burden. Research into this pathogen has been limited by the lack of reporter viruses. Here we describe the development of both a replication-competent fluorescent and bioluminescent influenza B reporter virus. Furthermore, we demonstrate these reporter viruses can be used to quickly monitor viral growth and permit the rapid screening of antiviral compounds and neutralizing antibodies.

  • Early Vertebrate Evolution of the Host Restriction Factor Tetherin [PublishAheadOfPrint]

  • Tetherin is an interferon-inducible restriction factor targeting a broad range of enveloped viruses. Its antiviral activity depends on an unusual topology comprising an N-terminal transmembrane domain (TMD), followed by an extracellular coiled-coil region and a C-terminal glycosylphosphatidylinositol (GPI) anchor. One of the two membrane anchors is inserted into assembling virions, while the other remains in the plasma membrane of the infected cell. Thus, tetherin entraps budding viruses by physically bridging viral and cellular membranes. Although tetherin restricts the release of a large variety of diverse human and animal viruses, only mammalian orthologs have been described to date. Here, we examined the evolutionary origin of this protein and demonstrate that tetherin orthologs are also found in fish, reptiles and birds. Notably, alligator tetherin efficiently blocks the release of retroviral particles. Thus, tetherin emerged early during vertebrate evolution and acquired its antiviral activity before the mammal/reptile divergence. Although there is only limited sequence homology, all orthologs share the typical topology. Two unrelated proteins of the slime mold Dictyostelium discoideum also adopt a tetherin-like configuration with an N-terminal TMD and a C-terminal GPI anchor. However, these proteins showed no evidence for convergent evolution and failed to inhibit virion release. In summary, our findings demonstrate that tetherin emerged at least 450 million years ago and is more widespread than previously anticipated. The early evolution of antiviral activity together with the high topology but low sequence conservation suggests that restriction of virus release is the primary function of tetherin.

    IMPORTANCE The continuous arms race with viruses has driven the evolution of a variety of cell-intrinsic immunity factors that inhibit different steps of the viral replication cycle. One of these restriction factors, tetherin, inhibits the release of newly formed progeny virions from infected cells. Although tetherin targets a broad range of enveloped viruses, including retro-, filo-, herpes- and arenaviruses, the evolutionary origin of this restriction factor and its antiviral activity remained obscure. Here, we examined diverse vertebrate genomes for genes encoding cellular proteins that share the highly unusual combination of an N-terminal transmembrane domain and a C-terminal glycosylphosphatidylinositol anchor with tetherin. We show that tetherin orthologs are found in fish, reptiles, and birds and demonstrate that alligator tetherin efficiently inhibits the release of retroviral particles. Our findings identify tetherin as an evolutionarily ancient restriction factor and provide new important insights into the continuous arms race between viruses and their hosts.

  • Vpr enhances TNF production by HIV-1 infected T cells [PublishAheadOfPrint]

  • The HIV-1 accessory protein Vpr displays different activities potentially impacting viral replication, including the arrest of the cell cycle in the G2 phase and the stimulation of apoptosis and DNA damage response pathways. Vpr also modulates cytokine production by infected cells, but this property remains partly characterized. Here, we investigated the effect of Vpr on the production of the pro-inflammatory cytokine TNF. We report that Vpr significantly increases TNF secretion by infected lymphocytes. De novo production of Vpr is required for this effect. Vpr mutants known to be defective for G2 cell cycle arrest induce lower levels of TNF secretion, suggesting a link between these two functions. Silencing experiments and the use of chemical inhibitors further implicate the cellular proteins DDB1 and TAK1 in this activity of Vpr. TNF secreted by HIV-1 infected cells triggers NFB activity in bystander cells and allows viral reactivation in a model of latently infected cells. Thus, the stimulation of the pro-inflammatory pathway by Vpr may impact HIV-1 replication in vivo.

    IMPORTANCE The role of the HIV-1 accessory protein Vpr remains only partially characterized. This protein is important for viral pathogenesis in infected individuals but is dispensable for viral replication in most cell culture systems. Some of the functions described for Vpr remain controversial. In particular, it remains unclear whether Vpr promotes or on the contrary prevents pro-inflammatory and antiviral immune responses. In this study, we show that Vpr promotes the release of TNF, a pro-inflammatory cytokine associated with rapid disease progression. Using Vpr mutants or inhibiting selected cellular genes, we show that the cellular proteins DDB1 and TAK1 are involved in the release of TNF by HIV-infected cells. This study provides novel insights into how Vpr manipulates TNF production and helps clarify the role of Vpr in innate immune responses and inflammation.

  • A General Molecular Strategy For Development Of Arenavirus Live-Attenuated Vaccines [PublishAheadOfPrint]

  • Hemorrhagic fever arenaviruses (HFA) pose important public health problems in their endemic regions. Thus, Lassa virus (LASV) infects several hundred thousand individuals yearly in West Africa causing a high number of Lassa fever cases associated with high morbidity and mortality. Concerns about human pathogenic arenaviruses are exacerbated because of the lack of FDA-licensed arenavirus vaccines and current anti-arenaviral therapy being limited to an off-label use of ribavirin that is only partially effective. The MOPV/LASV reassortant (ML29) is a LASV candidate live-attenuated vaccine (LAV) that has shown promising results in animal models. Nevertheless, the mechanism of ML29 attenuation remains unknown, which raises concerns about the phenotypic stability of ML29 in response to additional mutations. Development of LAV based on well-defined molecular mechanisms of attenuation will represent a major step to combat HFA. We have used the prototypic arenavirus LCMV to develop a general molecular strategy for arenavirus attenuation. Our approach involved replacement of the non-coding intergenic region (IGR) of the L genome segment by the IGR of the S genome segment to generate a recombinant LCM virus, rLCMV(IGR/S-S), that was highly attenuated in vivo but induced protection against a lethal challenge with wild type LCMV. Attenuation of rLCMV(IGR/S-S) was associated with a stable reorganization of the control of viral gene expression. This strategy can facilitate the rapid development of LAV with the same antigenic composition of the parental HFA, and a mechanism of attenuation that minimizes concerns about increased virulence that could be caused by genetic changes in the LAV.

    IMPORTANCE Hemorrhagic fever arenaviruses (HFA) cause high morbidity and mortality, and pose important public health problems in their endemic regions. Implementation of live-attenuated vaccines (LAV) will represent a major step to combat HFA. Here we have used the prototypic arenavirus LCMV to document a general molecular strategy for arenavirus attenuation that can facilitate the rapid development of safe and effective, as well as stable, LAV to combat HFA.

  • The U4 antibody epitope on human papillomavirus 16 identified by cryo-EM [PublishAheadOfPrint]

  • The human papillovavirus (HPV) major structure protein L1 composes capsomers that are linked together through interactions mediated by the L1 C-terminus to constitute a T=7 icosahedral capsid. H16.U4 is a type-specific monoclonal antibody recognizing a conformational-dependent neutralizing epitope of HPV thought to include the L1 protein C-terminus. The structure of HPV16 complexed with H16.U4 fragments of antibody (Fab) was solved by cryo-electron microscopy (cryo-EM) image reconstruction. Atomic structures of virus and Fab were fitted into the corresponding cryo-EM densities to identify the antigenic epitope. The antibody footprint mapped predominately to the L1 C-terminal arm with an additional contact point on the side of the capsomer. This footprint describes an epitope that is presented capsid-wide. However, although the H16.U4 epitope suggests the presence of 360 potential binding sites exposed in the capsid valley between each capsomer, H16.U4 Fab only bound to epitopes located around the icosahedral five-fold vertex of the capsid. Thus the binding characteristics of H16.U4 defined in this study showed a distinctive selectivity for local conformation-dependent interactions with specific L1 invading arms between five-fold related capsomers.

    IMPORTANCE Human papillomavirus 16 (HPV16) is the most prevalent oncogenic genotype in HPV-associated anogenital and oral cancers. Here we use cryo-EM reconstruction techniques to solve the structures of the HPV16 capsid complexes using H16.U4 fragment of antibody (Fab). Different from most other antibodies directed against surface loops, H16.U4 monoclonal antibody is unique in targeting the C-terminal arm of the L1 protein. This Mab is used throughout the HPV research community in HPV serological and vaccine development and to define mechanisms of HPV uptake. The unique binding mode of H16.U4 defined here shows important conformation-dependent interactions within the HPV16 capsid. By targeting an important structural and conformational epitope, H16.U4 may identify subtle conformational changes in different maturation stages of the HPV capsid and provide a key probe to analyze the mechanisms of HPV uptake during the early stages of virus infection. Our analyses precisely define important conformational epitopes on HPV16 capsids that are key targets for successful HPV prophylactic vaccines.

  • Genetic diversity within alphaherpesviruses: characterization of a novel variant of herpes simplex virus type 2 (HSV-2) [PublishAheadOfPrint]

  • Very low levels of variability have been reported for the herpes simplex virus type 2 (HSV-2) genome. We recently described a new genetic variant of HSV-2 (HSV-2v) characterized by a much higher degree of variability for the UL30 gene (DNA polymerase) than observed in the HG52 reference strain. Retrospective screening of 505 clinical isolates of HSV-2 in a specific real-time PCR assay targeting the UL30 gene led to the identification of 13 additional HSV-2v isolates, resulting in an overall prevalence of 2.8%. Phylogenetic analyses on the basis of microsatellite markers and gene sequences showed clear differences between HSV-2v and classical HSV-2. Thirteen of the 14 patients infected with HSV-2v originated from West or Central Africa, and nine of these patients were co-infected with HIV. These results raise questions about the origin of this new virus. Preliminary results suggest that HSV-2v may have acquired genomic segments from the chimpanzee alphaherpesvirus (ChHV) by recombination.

    IMPORTANCE This article deals with the highly topical question of the origin of this new HSV-2v variant identified in patients with HIV co-infection originating mostly from West or Central Africa. HSV-2v clearly differed from classical HSV-2 isolates in phylogenetic analyses and may be linked to simian ChHV. This new HSV-2 variant highlights the possible occurrence of recombination between human and simian herpesviruses in natural conditions, potentially presenting greater challenges for the future.

    Article Summary Line Potential recombination events between human herpes simplex type 2 (HSV-2) and chimpanzee alphaherpesvirus (ChHV) raise questions about the evolutionary origins of a newly described variant of HSV-2.

  • Rotavirus controls the activation of the OAS/RNase L pathway using at least two distinct mechanisms. [PublishAheadOfPrint]

  • The innate immune response is the first defense of the host cell to a viral infection. In turn, viruses have evolved a wide variety of strategies to hide from, and to directly antagonize with, the host innate immune pathways. One of these pathways is the 2rrsquo; -5rrsquo; -oligoadenylate synthetase (OAS)/RNase L pathway. OAS is activated by dsRNA to produce 2rrsquo; -5rrsquo; oligoadenylates, which are the activators of RNase L; this enzyme degrades viral and cellular RNAs restricting viral infection. It has been recently found that the carboxy terminal domain (CTD) of rotavirus VP3 has a 2rrsquo; -5rrsquo; -phosphodiesterase (PDE) activity that is able to functionally substitute the PDE activity of the mouse hepatitis virus ns2 protein. This particular phosphodiesterase cleaves the 2rrsquo; -5rrsquo; phosphodiester bond of the oligoadenylates, antagonizing the OAS/RNase L pathway. However, whether this activity of VP3 is relevant during the replication cycle of rotavirus is not known. Here, we demonstrate that after rotavirus infection the OAS/RNase L complex becomes activated, however, the virus is able to control its activity using at least two distinct mechanisms: A virus-cell interaction that occurs during or previous to rotavirus endocytosis triggers a signal that prevents the early activation of RNase L, while later on the control is taken by the newly synthesized VP3. Co-silencing the expression of VP3 and RNase L in infected cells yields viral infectious particles at levels similar to those obtained in control infected cells, where no genes were silenced, suggesting that the capping activity of VP3 is not essential for the formation of infectious viral particles.

    IMPORTANCE Rotaviruses represent an important cause of severe gastroenteritis in the young of many animal species including humans. In this work we have found that the OAS/RNase L pathway is activated during rotavirus infection, but the virus uses two different strategies to prevent the deleterious effects of this innate immune response of the cell. Early during virus entry, the initial interactions of the viral particle with the cell result in the inhibition of the RNase L activity during the first hours of the infection. Later on, once viral proteins are synthesized, the phosphodiesterase activity of VP3 degrades the cellular 2rrsquo; -5rrsquo; oligoadenylates, which are potent activators of RNase L, preventing its activation. This work demonstrates that the OAS/RNase L pathway plays an important role during the infection, and that the phosphodiesterase activity of VP3 is relevant during the replication cycle of the virus.

  • Modulation of the microRNA cluster miR-183-96-182 expression by the Epstein-Barr virus latent membrane protein 1 [PublishAheadOfPrint]

  • Epstein-Barr virus (EBV) is an oncogenic human herpesvirus involved in the pathogenesis of Burkitt's lymphoma (BL) and various other lymphoproliferative disorders. In BL, EBV protein expression is restricted to the EBNA1, but small non-coding RNAs such as the EBERs and micro (mi)RNAs can also be detected. miRNAs play major roles in crucial processes such as proliferation, differentiation and cell death. It has recently become clear that alteration in the expression profile of miRNAs contribute to the pathogenesis of a number of malignancies. During latent infection, EBV expresses 25 viral pre-miRNAs and modulates the expression of specific cellular miRNAs, such as miR-155 and miR-146, which potentially play a role in oncogenesis. Here, we established the small RNA expression profiles of three BL cell lines. Using large-scale sequencing coupled to northern blot and real-time RT-PCR analysis validation, we demonstrated the differential expression of some cellular and viral miRNAs. High-level expression of the miR-183-96-182 cluster and EBV miR-BART cluster was significantly associated with EBV type I latency. This expression was not affected by viral reactivation since TGF-bbeta;1 stimulation did not significantly change the miRNA profiles. However, using several approaches, including de novo infection with a mutant virus, we present evidence that the expression of latent membrane protein (LMP) 1 triggered down-regulation of the expression of the miR-183-96-182 cluster. We further show that this effect involves the Akt signaling pathway.

    IMPORTANCE In addition to expressing their own miRNAs, herpesviruses also impact the expression levels of cellular miRNAs. This regulation can be either positive or negative and usually results in the perturbation of pathways to create a cellular environment that is more "virus-friendly". For example, EBV induces the expression of miR-155, a well-characterized oncomiR, which leads to increased cell proliferation and decreased cell death. Here, we show that EBV-encoded LMP-1 protein is also involved in the down-regulation of a cluster of three miRNAs, miR-183, -96 and -182, which are known to be also repressed in several cancers. We therefore identify yet another potential player in EBV-induced oncogenesis.

  • A synthetic porcine reproductive and respiratory syndrome virus strain confers unprecedented levels of heterologous protection [PublishAheadOfPrint]

  • Current vaccines do not provide sufficient levels of protection against divergent porcine reproductive and respiratory syndrome virus (PRRSV) strains circulating in the field, mainly due to the substantial variation of the viral genome. We describe here a novel approach to generate a PRRSV vaccine candidate that could confer unprecedented levels of heterologous protection against divergent PRRSV isolates. Using a set of 59 non-redundant, full genome sequences of type-2 PRRSV, a consensus genome (designated as PRRSV-CON) was generated by aligning these 59 PRRSV full genome sequences, followed by selecting the most common nucleotide found at each position of the alignment. Next, the synthetic PRRSV-CON virus was generated through the use of reverse genetics. The PRRSV-CON virus replicates as efficiently as our prototype PRRSV strain FL12, both in vitro and in vivo. Importantly, when inoculated in pigs, the PRRSV-CON virus confers significantly broader levels of heterologous protection than the wild-type PRRSV. Collectively, our data demonstrates that the PRRSV-CON virus can serve as an excellent candidate for the development of a broadly protective PRRS vaccine.

    Importance The extraordinary genetic variation of RNA viruses poses a monumental challenge for the development of broadly protective vaccines against these viruses. To minimize the genetic dissimilarity between vaccine immunogens and the contemporary circulating viruses, computational strategies have been developed for generation of artificial immunogen sequences (so-called "centralized" sequences) that have equal genetic distances to the circulating viruses. Thus far, "centralized" vaccine immunogens have been carried out at the level of individual viral proteins. We expand this concept to for PRRSV, a highly variable RNA virus, by creating a synthetic PRRSV strain based on a "centralized" PRRSV genome sequence. This study provides the first example of "centralizing" the whole genome of an RNA virus to improve vaccine coverage. This concept may be significant for the development of vaccines against genetically variable viruses that require active viral replication in order to achieve complete immune protection.

  • Interaction Between Reverse Transcriptase and Integrase is Required for Reverse Transcription During HIV-1 Replication [PublishAheadOfPrint]

  • Human immunodeficiency virus type 1 (HIV-1) replication requires reverse transcription of its RNA genome into a double-stranded cDNA copy, which is then integrated into the host cell chromosome. The essential steps of reverse transcription and integration are catalyzed by the viral enzymes reverse transcriptase (RT) and integrase (IN), respectively. In vitro, HIV-1 RT can bind with IN, and the C-terminal domain (CTD) of IN is necessary and sufficient for this binding. To better define the RT-IN interaction, we performed nuclear magnetic resonance spectroscopy experiments to map a binding surface on the IN CTD in the presence of RT pre-bound to a duplex DNA construct that mimics the primer-binding site in the HIV-1 genome. To determine the biological significance of the RT-IN interaction during viral replication, we used the NMR chemical shift mapping information as a guide to introduce single amino acid substitutions of nine different residues on the putative RT-binding surface in the IN CTD. We found that six viral clones bearing such IN substitutions (R231E, W243E, G247E, A248E, V250E, and I251E) were non-infectious. Further analyses of the replication-defective IN mutants indicated that the block in replication took place specifically during early reverse transcription. The recombinant INs purified from these mutants, though retaining enzymatic activities, had diminished ability to bind RT in a co-sedimentation assay. The results indicate that the RT-IN interaction is functionally relevant during the reverse transcription step of the HIV-1 life cycle.

    Importance To establish a productive infection, human immunodeficiency virus type I (HIV-1) needs to reverse transcribe its RNA genome to create a double-stranded DNA copy, and then integrate this viral DNA genome into the chromosome of the host cell. These two essential steps are catalyzed by HIV-1 enzymes reverse transcriptase (RT) and integrase (IN), respectively. We have shown previously that IN physically interacts with RT but the importance of this interaction during HIV-1 replication has not been fully characterized. In this study, we have established the biological significance of the HIV-1 RT-IN interaction during the viral life cycle by demonstrating that altering the RT-binding surface on IN disrupts both reverse transcription and viral replication. These findings contribute to our understanding of the RT-IN binding mechanism, as well as indicate that the RT-IN interaction can be exploited as a new anti-viral drug target.

  • Response of mammalian macrophages to challenge with the Chlorovirus ATCV-1 [PublishAheadOfPrint]

  • It was recently reported that 44% of healthy humans in a study cohort had DNA sequences similar to Chlorovirus ATCV-1 (family Phycodnaviridae) in oropharyngeal samples and had decreases in visual processing and visual motor speed compared with individuals in whom no virus was detected. Moreover, mice inoculated orally with ATCV-1 developed immune responses to ATCV-1 proteins and had decreases in certain cognitive domains. Because heightened IL-6, nitric oxide (NO), and ERK MAP-kinase activation from macrophages are linked to cognitive impairments, we evaluated cellular responses and viral plaque forming units in murine RAW264.7 and primary macrophages after exposure to ATCV-1 in vitro for up to 72 h after virus challenge. Approximately 8% of the ATCV-1 inoculum was associated with macrophages after 1 h and increased 2-3 fold over 72 h. Immunoblots using rabbit anti-ATCV-1 detected a 55 kDa protein consistent with viral capsid protein from 1 to 72 h and an increasing de novo synthesis of a previously unidentified 17 kDa protein beginning at 24 h. Emergence of the 17 kDa protein did not occur and persistence of the 55 kDa protein declined over time when cells were exposed to heat-inactivated ATCV-1. Moreover, starting at 24 h, RAW264.7 cells exhibited cytopathic effects, Annexin V staining and cleaved-caspase 3. Activation of ERK MAP-kinases occurred in these cells by 30 min post challenge, which preceded expression of IL-6 and NO. Therefore ATCV-1 persistence in and induction of inflammatory factors by these macrophages may contribute to declines in cognitive abilities of mice and humans.

    IMPORTANCE Virus infections that persist in and stimulate inflammatory factors from macrophages contribute to pathologies in humans. A previous study showed that DNA sequences homologous to Chlorovirus ATCV-1 were found in a significant fraction of oropharyngeal samples from a healthy human cohort. We show here unexpectedly that ATCV-1, whose only known host is an eukaryotic green alga (Chlorella heliozoae) that is an endosymbiont of the heliozoon Acanthocystis turfacea, can persist within murine macrophages and trigger inflammatory responses, including factors that contribute to immunopathologies. The inflammatory factors that are produced in response to ATCV-1 include IL-6 and nitric oxide whose inductions are preceded by activation of ERK MAP-kinases. Other responses of ATCV-1-challenged macrophages include an apoptotic cytopathic effect, an innate anti-viral response, and a metabolic shift towards aerobic glycolysis. Therefore, mammalian encounters with chloroviruses may contribute to chronic inflammatory responses from macrophages.

  • Determinants involved in hepatitis C virus and GB virus B primate host restriction [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) only infects humans and chimpanzees, while GB virus B (GBV-B), another hepatotropic hepacivirus infects small New World primates (tamarins, marmosets). In an effort to develop an immunocompetent small primate model for HCV infection to study HCV pathogenesis and vaccine approaches, we investigated the HCV life cycle step(s) that may be restricted in small primate hepatocytes. First, we found that replication-competent, genome-length HCV chimeric RNAs encoding GBV-B structural proteins in place of HCV equivalent sequences, designed to allow entry into simian hepatocytes, failed to induce viremia in tamarins following intrahepatic inoculation, nor led to progeny virus in permissive, transfected human Huh7.5 hepatoma cells upon serial passage. This likely reflected the disruption of interactions between distantly-related structural and nonstructural proteins that are essential for virion production, whereas such cross-talks could be restored via adaptive mutations in NS3 protease or helicase domain in HCV intergenotypic recombinants designed similarly. Next, HCV entry in small primate hepatocytes was directly examined using HCV pseudo-typed retroviral particles (HCVpp). HCVpp infected efficiently tamarin hepatic cell lines and primary marmoset hepatocyte cultures through the use of simian CD81 ortholog as a co-receptor, indicating that HCV entry is not restricted in small New World primate hepatocytes. Furthermore, we observed efficient genomic replication and modest virus secretion following infection of primary marmoset hepatocyte cultures with a highly cell culture-adapted HCV strain. Thus, HCV life cycle can be successfully completed in primary simian hepatocytes, suggesting the possibility to adapt some HCV strains in small primate hosts.

    IMPORTANCE Hepatitis C virus (HCV) is an important human pathogen that infects over 150 million individuals worldwide and leads to chronic liver disease. The lack of a small animal model for this infection impedes the development of a preventive vaccine and pathogenesis studies. Toward establishing a small primate model for HCV, we first attempted to generate recombinants between HCV and GB virus B (GBV-B), a hepacivirus that infects small New World primates (tamarins, marmosets). This approach revealed that the genetic distance between these hepaciviruses likely prevented virus morphogenesis. We next showed that HCV pseudo-particles were able to infect efficiently tamarin or marmoset hepatocytes, demonstrating that there was no restriction in HCV entry into these simian cells. Furthermore, we found that a highly cell culture-adapted HCV strain was able to achieve a complete viral cycle in primary marmoset hepatocyte cultures, providing promising bases toward further HCV adaptation to small primate hosts.

  • Infectious bronchitis coronavirus inhibits STAT1 signalling and requires accessory proteins for resistance to type I interferon [PublishAheadOfPrint]

  • The innate immune response is the first line of defence against viruses and the type I interferon (IFN) is a critical component of this response. Similar to other viruses, the Gammacoronavirus infectious bronchitis virus (IBV) has evolved under evolutionary pressure to evade and counteract the IFN response to enable its survival. Previously, we reported that IBV induces a delayed activation of the IFN response. In the present work, we describe the resistance of IBV to IFN and the potential role of accessory proteins herein. We show that IBV is fairly resistant to the antiviral state induced by IFN and identify that the viral accessory proteins 3a is involved in resistance to IFN, as its absence renders IBV less resistant to IFN treatment. In addition to this, we find that independently of its accessory proteins, IBV inhibits IFN-mediated phosphorylation and translocation of STAT1. In summary, we show that IBV uses multiple strategies to counteract the IFN response.

    IMPORTANCE In the present study we show that infectious bronchitis virus (IBV) is resistant to IFN treatment and identify a role for the accessory proteins 3a in the resistance against the type I IFN response. We also demonstrated that, in a time-dependent manner, IBV effectively interferes with IFN signalling and that accessory proteins are dispensable for this activity. This study demonstrates that the Gammacoronavirus IBV, similar to its mammalian counterparts, has evolved multiple strategies to efficiently counteract the IFN response of its avian host, and identifies accessory protein 3a as multifaceted antagonist of the avian IFN system.

  • SARS Coronavirus ORF7a inhibits BST-2 virion tethering through a novel mechanism of glycosylation interference [PublishAheadOfPrint]

  • Severe Acute Respiratory Syndrome (SARS) emerged in November 2002 as a case of atypical pneumonia in China and the causative agent of SARS was identified as a novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV). Bone marrow stromal antigen 2 (BST-2; also known as CD317 or tetherin) was initially identified as a pre-B-cell growth promoter but also inhibits the release of the retrovirus human immunodeficiency virus type 1 (HIV-1) virions by tethering budding virions to the host cell membrane. Further work has shown that BST-2 restricts the release of many other viruses, including the human coronavirus hCoV-229E, and many of these viruses encode BST-2 antagonists to overcome BST-2 restriction. Given the previous studies on BST-2, we aimed to determine if BST-2 has the ability to restrict SARS-CoV and if SARS-CoV encodes any proteins that modulate BST-2's anti-viral function. Through an in vitro screen we identified four potential BST-2 modulators encoded by SARS-CoV: PLPro, nsp1, ORF6, and ORF7a. As the function of ORF7a in SARS-CoV replication was previously unknown, we focused our study on ORF7a. We found that BST-2 does restrict SARS-CoV, but the loss of ORF7a leads to a much greater restriction, confirming the role of ORF7a as an inhibitor of BST-2. We further characterized the mechanism of BST-2 inhibition by ORF7a and found that ORF7a localization changes when BST-2 is overexpressed and ORF7a binds directly to BST-2. Finally, we also show that SARS-CoV ORF7a blocks the restriction activity of BST-2 by blocking with the glycosylation of BST-2.

    IMPORTANCE The severe acute respiratory syndrome coronavirus (SARS-CoV) emerged from zoonotic sources in 2002 and caused over 8000 infections and 800 deaths in 32 countries around the world. Identifying host factors that regulate SARS-CoV pathogenesis is critical to understanding how this lethal virus causes disease. We have found that BST-2 is capable of restricting SARS-CoV release from cells, however we also identified a SARS-CoV protein that inhibits BST-2 function. We show that the SARS-CoV protein, ORF7a, inhibits BST-2 glycosylation leading to loss of BST-2's anti-viral function.

  • Characterization of the Drug Resistance Profiles of Integrase Strand Transfer Inhibitors in SIVmac239 [PublishAheadOfPrint]

  • We previously showed that SIVmac239 is susceptible to HIV integrase strand transfer inhibitors (INSTIs) and that the same integrase (IN) drug resistance mutations result in similar phenotypes in both viruses. Now, we wished to determine whether tissue culture drug selection studies with SIV would yield the same resistance mutations as in HIV. Tissue culture selection experiments were performed using rhesus macaque peripheral blood mononuclear cells (PBMCs) infected with SIVmac239 viruses in the presence of increasing concentrations of dolutegravir (DTG), elvitegravir (EVG), and raltegravir (RAL). We now show that 22 weeks of selection pressure with DTG yielded a mutation at position R263K in SIV, similar to what has been observed in HIV, and that selections with EVG led to emergence of the E92Q substitution, which is a primary INSTI-resistance mutation in HIV associated with EVG treatment failure. To study this at a biochemical level, purified recombinant SIVmac239 WT, E92Q, T97A, G118R, Y143R, Q148R, N155H, R263K, E92Q/T97A, E92Q/Y143R, R263K/H51Y and G140S/Q148R-containing IN enzymes were produced and each of strand transfer, 3rrsquo; processing activity, and INSTI inhibitory constants were assessed in cell-free assays. The results show that the G118R and G140S/Q148R substitutions decreased Km' and Vmax'/Km' for strand transfer compared to WT. RAL and EVG showed reduced activity against both viruses and against enzymes containing Q148R, E92Q/Y143R and G140S/Q148R. Both viruses and enzymes containing Q148R and G140S/Q148R showed moderate levels of resistance against DTG. This study further confirms that the same mutations associated with drug resistance in HIV display similar profiles in SIV.

    Importance of the study Our goal was to definitively establish whether HIV and simian immunodeficiency virus (SIV) share similar resistance pathways under tissue culture drug selection pressure with integrase strand transfer inhibitors and to test the effect of HIV-1 integrase resistance-associated mutations on SIV integrase catalytic activity and resistance to integrase strand transfer inhibitors. Clinically relevant HIV-integrase resistance associated mutations were selected in SIV in our tissue culture experiments. Not only do we report on the characterization of SIV recombinant integrase enzyme catalytic activities, we also provide the first research anywhere on the effect of mutations within recombinant integrase SIV enzymes on drug resistance.

  • Monoclonal antibodies directed towards the Hepatitis C virus glycoprotein E2 detect antigenic differences modulated by HVR1, HVR2 and the igVR. [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 form a heterodimer and mediate receptor interactions and viral fusion. Both E1 and E2 are targets of the neutralizing antibody (NAb) response and are candidates for the production of vaccines that generate humoral immunity. Previous studies have demonstrated that the N-terminal hypervariable region 1 (HVR1) can modulate the neutralization potential of MAbs but no information is available for the influence of HVR2 or the intergenotypic variable region (igVR) on antigenicity. In this study, we examined how the variable regions influence the antigenicity of E2661 using a panel of monoclonal antibodies (MAbs) raised to E2661, E2661 lacking HVR1, HVR2 and the igVR (123), and well-characterized MAbs isolated from infected humans. We show for a subset of both neutralizing and non-neutralizing MAbs, that all three variable regions decrease the ability of MAbs to bind E2661 and reduce the ability of MAbs to inhibit E2-CD81 interactions. In addition, we describe a new MAb towards the 411-428 region of E2 (MAb24) that demonstrates broad neutralization against all 7 genotypes of HCV. The ability of MAb24 to inhibit E2-CD81 interactions is strongly influenced by the three variable regions. Our data suggest that HVR1, HVR2 and the igVR modulate exposure of epitopes on the core domain of E2 and their ability to prevent E2-CD81 interactions. These studies suggest that the function of HVR2 and the igVR is to modulate antibody recognition of glycoprotein E2 and may contribute to immune evasion.

    Importance. This study reveals conformational and antigenic differences between the 123 and intact E2661 glycoproteins and provides new structural and functional data about the three variable regions and their role in occluding neutralizing and non-neutralizing epitopes on the E2 core domain. The variable regions may therefore function to reduce the ability of HCV to elicit NAbs directed to the conserved core domain. Future studies aimed at generating a three dimensional structure for intact E2 containing HVR1 and the adjoining major neutralization epitope, and HVR2 will reveal how the variable regions modulate antigenic structure.

  • Mumps virus is released from the apical surface of polarized epithelial cells and the release is facilitated by a Rab11-mediated transport system [PublishAheadOfPrint]

  • Mumps virus (MuV) is an airborne virus that causes a systemic infection in patients. In vivo the epithelium is a major replication site of MuV, and thus the mode of MuV infection of epithelial cells is a subject of interest. Our data in the present study showed that MuV entered cells via both the apical and basolateral surfaces of polarized epithelial cells, while progeny viruses were predominantly released from the apical surface. In polarized cells, intracellular transport of viral ribonucleoprotein (vRNP) complexes was dependent on Rab11-positive endosomes, and vRNP complexes were transported to the apical membrane. Expression of a dominant-negative form of Rab11 (Rab11S25N) reduced the progeny virus release in polarized cells but not in non-polarized cells. Although in this way these effects were correlated with cell polarity, the Rab11S25N did not modulate the direction of virus release from the apical surface. Therefore, our data suggested that Rab11 is not a regulator of selective apical release of MuV, although it acts as an activator of virus release from polarized epithelial cells. In addition, our data and previous studies on Sendai virus, respiratory syncytial virus, and measles virus suggested that selective apical release from epithelial cells is used by many paramyxoviruses even though they cause either a systemic infection or a local respiratory infection.

    Importance Mumps virus (MuV) is the etiological agent of mumps and causes a systemic infection. However, the precise mechanism by which MuV breaks through the epithelial barriers and achieves a systemic infection remains unclear. In the present study, we show that the entry of MuV was bipolar while the release was predominantly from the apical surface in polarized epithelial cells. In addition, the release of progeny virus was facilitated by a Rab11-positive recycling endosome and microtubule network. Our data provide important insights into the mechanism of transmission and pathogenesis of MuV.

  • Mimivirus fibrils are important for viral attachment to microbial world by a diverse glycoside interaction repertoire [PublishAheadOfPrint]

  • Acanthamoeba polyphaga mimivirus (APMV) is a giant virus from the Mimiviridae family. It has many unusual features, such as a pseudo-icosahedral capsid that presents a starfish shape in one of its vertices, through which the ~1.2 Mb dsDNA is released. It also has a dense glycoprotein fibril layer covering the capsid that has not yet been functionally characterized. Here, we verified that although these structures are not essential for viral replication, they are truly necessary for viral adhesion to amoebae, its natural host. In the absence of fibrils, APMV had a significantly lower attachment to the Acanthamoeba castellanii surface. This adhesion is mediated by glycans, specifically mannose and N-acetylglucosamine (a monomer of chitin and peptidoglycan), both of which are largely distributed in nature as structural components of several organisms. Indeed, APMV was able to attach to different organisms, such as Gram-positive bacteria, fungi, and arthropods, but not to Gram-negative bacteria. This prompted us to predict that i) arthropods, mainly insects, might act as mimivirus dispersors and ii) by attaching to other microorganisms, APMV could be ingested by amoebae, leading to the successful production of viral progeny. To date, this mechanism has never been described in the virosphere.

    IMPORTANCE Acanthamoeba polyphaga mimivirus (APMV) is a complex giant virus, both genetically and structurally. Its size is similar to that of small bacteria and it replicates inside amoebae. The viral capsid is covered by a dense glycoprotein fibril layer, but its function has remained unknown until now. We found that the fibrils are not essential for mimivirus replication, but that they are truly necessary for viral adhesion to the cell surface. This interaction is mediated by glycans, mainly N-acetylglucosamine. We also verified that APMV is able to attach to bacteria, fungi and arthropod. This indicates that insects might act as mimivirus dispersors and that adhesion to other microorganisms could facilitate viral ingestion amoebae, a mechanism never before described in the virosphere.

  • A population genetic perspective on geminivirus infection [PublishAheadOfPrint]

  • The selective accumulation of both DNA components of a bipartite geminivirus, Abutilon mosaic virus, was recorded during early systemic infection of Nicotiana benthamiana plants. Purified nuclei were diagnosed for viral DNA using hybridization specific for DNA A or DNA B, either to detect these individual genome components alone, or both simultaneously by dual color staining. Although this virus needs both components for symptomatic infection, DNA A alone was transported to upper leaves, where it was imported into phloem nuclei and replicated autonomously. The coinfection with DNA A and DNA B revealed an independent spread of both molecules, which resulted in a stochastic distribution of DNA A and DNA A/B-infected nuclei. A population genetic evaluation of the respective frequencies was compared to a model computation. This elucidated a surprisingly simple relationship between the initial frequencies of the viral DNA components and the number of susceptible cells during the course of early systemic infection.

    Importance For bipartite begomoviruses, DNA B-independent long-distance spread of DNA A has been described before, but it has never been shown whether viral DNA A alone invades nuclei of systemic tissues and replicates therein. This is demonstrated now for the first time. During infection with DNA A and DNA B, a similar solitary spread of DNA A can be recognized at early stages. A first population genetic model is considered how the hit probabilities of DNA A and DNA B for susceptible cells determine the relative frequencies of either genome component during the course of infection.

  • Resolving the origin of rabbit haemorrhagic disease virus (RHDV): Insights from an investigation of the viral stocks released in Australia [PublishAheadOfPrint]

  • To resolve the evolutionary history of rabbit haemorrhagic disease virus (RHDV) we performed a genomic analysis of the viral stocks imported and released as a biocontrol into Australia, as well as a global phylogenetic analysis. Importantly, conflicts were identified between the sequences determined here and those previously published that may have affected previous estimates of evolutionary rate. By removing likely erroneous sequences we show that RHDV emerged only shortly before its initial description in China.

  • A novel tricyclic polyketide, vanitaracin A, specifically inhibits the entry of hepatitis B and D viruses through targeting NTCP [PublishAheadOfPrint]

  • Anti-hepatitis B virus (HBV) drugs are currently limited to nucleos(t)ide analogs (NAs) and interferons. For a challenge of drug development, it is demanded to identify small molecules that suppress HBV infection from new chemical sources. Here, from a fungal-derived secondary metabolite library, we identify a structurally novel tricyclic polyketide, named vanitaracin A, which specifically inhibits HBV infection. Vanitaracin A inhibited the viral entry process with a submicromolar IC50 (IC50 = 0.61 pplusmn; 0.23 mmu;M) without evident cytotoxicity (CC50 ggt; 256 mmu;M, selectivity index ggt; 419) in primary human hepatocytes. Vanitaracin A did not affect HBV replication process. This compound was found to directly interact with the HBV entry receptor, sodium taurocholate cotransporting polypeptide (NTCP), and impaired its bile acid transport activity. Consistent with this NTCP targeting, antiviral activity of vanitaracin A was observed with hepatitis D virus (HDV), but not hepatitis C virus. Importantly, vanitaracin A inhibited infection of all the HBV genotypes tested (genotype A-D) and clinically-relevant NA-resistant HBV isolates. Thus, we identified a fungal metabolite, vanitaracin A, which was a potent, well-tolerated, and broadly active inhibitor of HBV and HDV entry. This compound, or its related analogs, could be part of an antiviral strategy for preventing reinfection with HBV, including clinically-relevant nucleos(t)ide analog-resistant virus.

    IMPORTANCE For achieving better treatment and prevention against hepatitis B virus (HBV), anti-HBV agents targeting a new molecule have been greatly demanded. Although sodium taurocholate cotransporting polypeptide (NTCP) has recently been reported as an essential host factor for HBV entry, there is limited number of report that identify new compounds targeting NTCP and inhibiting HBV entry. Here, from an uncharacterized chemical library, we isolated a structurally new compound, named vanitaracin A, which inhibited the entry process of HBV and hepatitis D virus (HDV). This compound was suggested to directly interact with NTCP and inhibit its transporter activity. Importantly, vanitaracin A inhibited the entry of all the HBV genotypes examined and that of a clinically-relevant nucleos(t)ide analog-resistant HBV isolate.

  • Genomic analysis, phenotype, and virulence of the historical Brazilian smallpox vaccine strain IOC: Implications for the origins and evolutionary relationships of vaccinia virus [PublishAheadOfPrint]

  • Smallpox was declared eradicated in 1980 after an intensive vaccination program using different strains of vaccinia virus (VACV; Poxviridae). VACV strain IOC (VACV-IOC) was the seed strain of the smallpox vaccine manufactured by the major vaccine producer in Brazil during the smallpox eradication programme. However, little is known about the biological and immunological features, as well as the phylogenetic relationships of this first-generation vaccine. In this work, we present a comprehensive characterization of two clones of VACV-IOC. Both clones had low virulence in infected mice and induced a protective immune response against a lethal infection comparable to the response of the licensed vaccine ACAM2000 and the parental strain VACV-IOC. Full genome sequencing revealed the presence of several fragmented virulence genes that are probably non-functional, e.g., F1L, B13R, C10L, K3L and C3L. Most notably, phylogenetic inference supported by the structural analysis of the genome ends provide evidence of a novel, independent cluster in VACV phylogeny formed by VACV-IOC, the Brazilian field strains Cantagalo (CTGV) and Serro 2 viruses, and horsepox virus, a VACV-like virus supposedly related to an ancestor of the VACV lineage. Our data strongly support the hypothesis that CTGV-like viruses represent feral VACV that evolved in parallel with VACV-IOC after splitting from a most recent common ancestor, probably an ancient smallpox vaccine strain related to horsepox virus. Our data, together with an interesting historical investigation, revisit the origins of VACV and propose new evolutionary relationships between ancient and extant VACV strains, mainly horsepox virus, VACV-IOC/CTGV-like viruses, and Dryvax strain.

    IMPORTANCE First-generation vaccines used to eradicate smallpox had rates of adverse effects that are not acceptable by current healthcare standards. Moreover, these vaccines are genetically heterogeneous and consist of a pool of quasispecies of VACV. Therefore, the search for new-generation smallpox vaccines that combine low pathogenicity, immune protection, and genetic homogeneity is extremely important. In addition, the phylogenetic relationships and origins of VACV strains are quite nebulous. We show the characterization of two clones of VACV-IOC, a unique smallpox vaccine strain that contributed to smallpox eradication in Brazil. The immunogenicity and reduced virulence make the IOC clones good options for alternative second-generation smallpox vaccines. More importantly, this study reveals the phylogenetic relationship between VACV-IOC, feral VACV established in nature, and the ancestor-like horsepox virus. Our data expand the discussion on the origins and evolutionary connections of VACV lineages.

  • Regulation Of Viral RNA Synthesis By The V Protein Of Parainfluenza Virus 5 [PublishAheadOfPrint]

  • Paramyxoviruses include many important animal and human pathogens. The Parainfluenza virus 5 (PIV5), a prototypical paramyxovirus, encodes a V protein that inhibits the viral RNA synthesis. In this work, the mechanism of inhibition was investigated. Using mutational analysis and a mini-genome system, we identified regions in the N- and C- termini protein that inhibit viral RNA synthesis: one at the very N-terminus and the second at the C-terminus of V. Furthermore, we determined that residue L16 and I17 are critical for the inhibitory function of the N-terminal region of the V protein. Both regions interact with the nucleocapsid protein (NP), an essential component of the viral RNA genome complex (RNP). Mutations at L16 and I17 abolish the interaction between NP and the N-terminal domain of V. This suggests that the interaction between NP and the N-terminal domain plays a critical role in V inhibition of viral RNA synthesis by the N-terminal domain. Both the N- and C-terminal regions inhibit viral RNA replication. The C-terminus inhibits viral RNA transcription, while the N-terminal domain enhances viral RNA transcription, suggesting that the two domains affect viral RNA through different mechanisms. Interestingly, V also inhibits viral RNA synthesis of other paramyxoviruses such as Nipah virus (NiV), human parainfluenza virus 3 (HPIV3), measles virus (MeV), mumps virus (MuV) and respiratory syncytial virus (RSV). This suggests that a common host factor may be involved in replication of these paramyxoviruses.

    Significance We identified two regions of V that interact with NP and determined that one of these regions enhances viral RNA transcription via its interaction with NP. Our data suggest a common host factor may be involved in the regulation of paramyxovirus replication and could be a target for broad antiviral drug development. Understanding the regulation of paramyxovirus replication will enable rational design of vaccines and potential antiviral drugs.

  • Role of gB and pUS3 in EHV-1 transfer between PBMC and endothelial cells: a dynamic in vitro model [PublishAheadOfPrint]

  • Infected peripheral blood mononuclear cells (PBMC) effectively transport equine herpesvirus type 1 (EHV-1), but not EHV-4, to endothelial cells (EC) lining the blood vessels of the pregnant uterus or central nervous system, a process that can result in abortion or myeloencephalopathy. We examined, using a dynamic in vitro model, the differences between EHV-1 and EHV-4 infection of PBMC and PBMC-EC interactions. In order to evaluate viral transfer between infected PBMC and EC, co-cultivation assays were performed. Only EHV-1 was transferred from PBMC to EC and viral glycoprotein B (gB) was shown to be mainly responsible for this form of cell-to-cell transfer. For addressing the more dynamic aspects of PBMC-EC interaction, infected PBMC were perfused through a flow channel containing EC in the presence of neutralizing antibodies. By simulating capillary blood flow and analyzing the behavior of infected PBMC through live fluorescence imaging and automated cell tracking, we observed that EHV-1 was able to maintain tethering and rolling of infected PBMC on EC more effectively than EHV-4. Deletion of US3 reduced the ability of infected PBMC to tether and roll compared to parental virus, which resulted in a significant reduction in virus transfer from PBMC to EC. Taken together, we conclude that systemic spread and EC infection of EHV-1, but not EHV-4, is caused by its ability to infect and/or reprogram mononuclear cells with respect to their tethering and rolling behavior on EC and consequent virus transfer.

    IMPORTANCE EHV-1 is widespread throughout the world, and causes substantial economic losses through outbreaks of respiratory disease, abortion and myeloencephalopathy. Despite many years of research, no fully protective vaccines have been developed and several aspects of viral pathogenesis still need to be uncovered. In the current study, we investigated the molecular mechanisms that facilitate the cell-associated viremia, which is arguably the most important aspect of EHV-1 pathogenesis. The newly discovered functions of gB and pUS3 add new facets to their previously reported roles. Due to the conserved nature of cell-associated viremia among numerous herpesviruses, these results are also very relevant for viruses such as varicella-zoster virus, pseudorabies virus, human cytomegalovirus and others. In addition, the constructed mutant and recombinant viruses exhibit potent in vitro replication, but significant defects in certain stages of the disease course. These viruses therefore show much promise as candidates for future live vaccines.


  • The 4E10 antibody recognizes the membrane-proximal external region (MPER) of the HIV-1 Env glycoprotein gp41 transmembrane subunit, exhibiting one of the broadest neutralizing activities known to date. The neutralizing activity of 4E10 requires solvent-exposed hydrophobic residues at the apex of the complementarity-determining region (CDR) H3 loop, but the molecular basis for this requirement has not been clarified. Herein, we report the co-crystal structures and the energetic parameters of binding of a peptide bearing the 4E10-epitope sequence (4E10ep) to non-neutralizing versions of the 4E10 Fab. Non-neutralizing Fabs were obtained by shortening and decreasing the hydrophobicity of the CDR-H3 loop (termed Loop), or by substituting the two tryptophan residues of the CDR-H3 apex with Asp residues (termed WDWD), which also decreases hydrophobicity but preserves the length of the loop. The analysis was complemented by the first crystal structure of the 4E10 Fab in its ligand-free state. Collectively, the data ruled out major conformational changes of CDR-H3 at any stage during the binding process (equilibrium or transition state). Although these mutations did not impact the affinity of wild-type Fab for the 4E10ep in solution, the two non-neutralizing versions of 4E10 were deficient in binding to MPER inserted in the plasma membrane (mimicking the environment faced by the antibody in vivo). The conclusions of our structure-function analysis strengthen the idea that to exert effective neutralization, the hydrophobic apex of the solvent-exposed CDR-H3 loop must recognize an antigenic structure more complex than just the linear aalpha;-helical epitope, and likely constrained by the viral membrane lipids.

    IMPORTANCE The broadly neutralizing anti-HIV-1 4E10 antibody blocks infection caused by nearly all viral strains and isolates examined so far. However, 4E10 (or 4E10-like) antibodies are rarely found in HIV-1 infected individuals or elicited through vaccination. Impediments to the design of successful 4E10 immunogens are partly attributed to an incomplete understanding of the structural and binding characteristics of this class of antibodies. Since the broadly neutralizing activity of 4E10 is abrogated by mutations of the tip of the CDR-H3, we investigated their impact on binding of the MPER-epitope at the atomic and energetic levels. We conclude that the difference between neutralizing and non-neutralizing antibodies of 4E10 is neither structural nor energetic, but related to the capacity to recognize the HIV-1 gp41 epitope inserted in biological membranes. Our findings strengthen the idea that to elicit similar neutralizing antibodies, the suitable MPER vaccine must be "delivered" in a membrane environment.

  • Differential susceptibility of human lung primary cells to H1N1 influenza viruses [PublishAheadOfPrint]

  • Human alveolar epithelial cells (AECs) and alveolar macrophages (AMs) are the first lines of lung defense. In this study we report that AECs are the direct targets for H1N1 viruses that have circulated since the 2009 pandemic (H1N1pdm09). AMs are less susceptible to H1N1pdm09 virus, but they produce significantly more inflammatory cytokines than AECs from the same donor. AECs form an intact epithelial barrier that is destroyed by H1N1pdm09 infection. However, there is significant variation in the cellular permissiveness to H1N1pdm09 infection among different donors. AECs from obese donors appear to be more susceptible to H1N1pdm09 infection, whereas gender, smoking history, and age do not appear to affect AEC susceptibility. There is also a difference in response to different strains of H1N1pdm09 viruses. Compared to A/California04/09 (CA04), A/New York/1682/09 (NY1682) is more infectious and causes more epithelial barrier injury, although it stimulates less cytokine production. We further determined that a single amino acid residue substitution in NY1682 hemagglutinin is responsible for the difference in infectivity. In conclusion, this is the first study of host susceptibility of human lung primary cells and the integrity of the alveolar epithelial barrier to influenza. Further elucidation of the mechanism of increased susceptibility of AECs from obese subjects may facilitate the development of novel protection strategies against influenza infection.

    IMPORTANCE Disease susceptibility of influenza is determined by host and viral factors. Human alveolar epithelial cells (AECs) form the key line of lung defenses against pathogens. Using primary AECs from different donors, we provided cellular level evidence that obesity might be a risk factor for increased susceptibility to influenza. We also compared the infection of two closely related 2009 pandemic H1N1 strains in AECs from the same donor and identified a key viral factor that affected host susceptibility, the dominance of which may be correlated with disease epidemiology. In addition, primary human AECs can serve as a convenient and powerful model to investigate the mechanism of influenza induced lung injury and determine the effect of genetic and epigenetic factors on host susceptibility to pandemic influenza infection.

  • Evidence for an ancestral association of human coronavirus 229E with bats [PublishAheadOfPrint]

  • We previously showed that close relatives of human coronavirus (HCoV)-229E exist in African bats. The small sample and limited genomic characterizations prevented further analyses so far. Here, we tested 2,087 fecal specimens from 11 bat species sampled in Ghana for HCoV-229E-related viruses by RT-PCR. Only hipposiderid bats tested positive. To compare the genetic diversity of bat viruses and HCoV-229E, we tested historical isolates and diagnostic specimens sampled globally over 10 years. Bat viruses were five- to sixfold more diversified than HCoV-229E in RNA-dependent RNA polymerase (RdRp) and Spike genes. In phylogenetic analyses, HCoV-229E strains were monophyletic and not intermixed with animal viruses. Bat viruses formed three large clades in close and more distant sister relationship. A recently described 229E-related alpaca virus occupied an intermediate phylogenetic position between bat and human viruses. According to taxonomic criteria, human, alpaca and bat viruses form a single CoV species showing evidence for multiple recombination events. HCoV-229E and the alpaca virus showed a major deletion in the Spike S1 region compared to all bat viruses. Analyses of four full genomes from 229E-related bat CoVs revealed an eighth open reading frame (ORF8) located at the genomic 3rrsquo; -end. ORF8 also existed in the 229E-related alpaca virus. Re-analysis of HCoV-229E sequences showed a conserved transcription regulatory sequence preceding remnants of this ORF, suggesting its loss after acquisition of a 229E-related CoV by humans. These data suggested an evolutionary origin of 229E-related CoVs in hipposiderid bats, hypothetically with camelids as intermediate hosts preceding the establishment of HCoV-229E.

    IMPORTANCE The ancestral origins of major human coronaviruses (HCoV) likely involve bat hosts. Here, we provide conclusive genetic evidence for an evolutionary origin of the common cold virus HCoV-229E in hipposiderid bats by analyzing a large sample of African bats and characterizing several bat viruses on a full genome level. Our evolutionary analyses show that animal and human viruses are genetically closely related, can exchange genetic material and form a single viral species. We show that the putative host switches leading to the formation of HCoV-229E were accompanied by major genomic changes including deletions in the viral spike glycoprotein gene and loss of an open reading frame. We re-analyze a previously described genetically related alpaca virus and discuss the role of camelids as potential intermediate hosts between bat and human viruses. The evolutionary history of HCoV-229E likely shares important characteristics with that of the recently emerged highly pathogenic MERS-Coronavirus.

  • Deficiencies in cellular processes modulated by the retinoblastoma protein do not account for reduced human cytomegalovirus replication in its absence [PublishAheadOfPrint]

  • Despite encoding multiple viral proteins that modulate the retinoblastoma (Rb) protein in a manner classically defined as inactivation, human cytomegalovirus (HCMV) requires the presence of the Rb protein to replicate efficiently. In uninfected cells, Rb controls numerous pathways that the virus also commandeers during infection. These include cell cycle progression, senescence, mitochondrial biogenesis, apoptosis, and glutaminolysis. We investigated if a potential inability of HCMV to regulate these Rb-controlled pathways in the absence of the Rb protein was the reason for reduced viral productive replication in Rb-knockdown cells. We found that HCMV was equally able to modulate these pathways in the parental Rb-expressing and Rb-depleted cells. Our results suggest that Rb may be required to enhance a specific viral process during HCMV productive replication.

    Importance The retinoblastoma (Rb) tumor suppressor is well established as a repressor of E2F-dependent transcription. Rb hyperphosphorylation, degradation, and binding by viral oncoproteins are also codified. Recent reports indicate Rb can be monophosphorylated, repress the transcription of antiviral genes in association with adenovirus E1A, modulate cellular responses to polycomb-mediated epigenetic methylations in human papillomavirus type 16 E7 expressing cells, and increase the efficiency of human cytomegalovirus (HCMV) productive replication. As Rb function also now extends to regulation of mitochondrial function (apoptosis, metabolism), it's clear that our current understanding of this protein is insufficient to explain its roles in virus-infected cells and tumors. Work here reinforces this concept, showing the known roles of Rb are insufficient to explain its positive impact on HCMV replication. Therefore, HCMV, along with other viral systems, provide valuable tools to probe functions of Rb that might be modulated with therapeutics for cancers with viral or non-viral etiologies.

  • Vaccine-Derived Neutralizing Antibodies to the Human Cytomegalovirus gH/gL Pentamer Potently Block Primary Cytotrophoblast Infection [PublishAheadOfPrint]

  • Human cytomegalovirus (HCMV) elicits neutralizing antibodies (NAb) of varying potency and cell-type specificity to prevent HCMV entry into fibroblasts (FB) and epithelial/endothelial cells (EpC/EnC). NAb targeting the major essential envelope glycoprotein complexes gB and gH/gL inhibit both FB and EpC/EnC entry. In contrast to FB infection, HCMV entry into EpC/EnC is additionally blocked by extremely potent NAb to conformational epitopes of the gH/gL/UL128/UL130/UL131A pentamer complex (PC). We recently developed a vaccine concept based on co-expression of all five PC subunits by a single Modified Vaccinia Ankara (MVA) vector, termed MVA-PC. Vaccination of mice and rhesus macaques with MVA-PC resulted in high titer and sustained NAb that blocked EpC/EnC infection and lower titer NAb that inhibited FB entry. However, antibody function responsible for the neutralizing activity induced by the MVA-PC vaccine is uncharacterized. Here, we demonstrate that MVA-PC elicits NAb with cell-type specific neutralization potency and antigen recognition pattern similar to human NAb targeting conformational and linear epitopes of the UL128/130/131A subunits or gH. In addition, we show that the vaccine-derived PC-specific NAb are significantly more potent than the anti-gH NAb to prevent HCMV spread in EpC and infection of human placental cytotrophoblasts (CTB), cell types thought to be of critical importance for HCMV transmission to the fetus. These findings further validate MVA-PC as a clinical vaccine candidate to elicit NAb that resemble those induced during HCMV infection and provide valuable insights into the potency of PC-specific NAb to interfere with HCMV cell-associated spread and infection of key placental cells.

    IMPORTANCE As a consequence of the leading role of human cytomegalovirus (HCMV) in causing permanent birth defects, developing a vaccine against HCMV has been assigned a major public health priority. We have recently introduced a vaccine strategy based on a widely-used, safe, and well-characterized poxvirus vector platform to elicit potent and durable NAb responses targeting the HCMV envelope pentamer complex (PC), which has been suggested as a critical component for a vaccine to prevent congenital HCMV infection. With this work, we confirm that the NAb elicited by the vaccine vector have properties that are similar to that of human NAb isolated from individuals chronically infected with HCMV. In addition, we show that PC-specific NAb have potent ability to prevent infection of key placental cells that HCMV utilizes to cross the fetal-maternal interface, suggesting that NAb targeting the PC may be essential to prevent HCMV vertical transmission.

  • Differential reovirus-specific and herpesvirus-specific AP-1 activation of secretogranin II leads to altered virus secretion [PublishAheadOfPrint]

  • Viruses utilize host cell machinery for propagation and manage to evade cellular host defense mechanisms in the process. Much remains unknown regarding how the host responds to viral infection. We recently performed global proteomic screens of mammalian reovirus TIL- and T3D-infected, and herpesvirus (HSV-1)-infected, HEK293 cells. The non-enveloped RNA reoviruses caused an up-regulation, whereas the enveloped DNA HSV-1 caused a down-regulation of cellular secretogranin II (SCG2). SCG2, a member of the granin family that functions in hormonal peptide sorting into secretory vesicles, has not been previously linked to virus infections. We confirmed SCG2 up-regulation, and found SCG2 phosphorylation, by 18hpi in reovirus-infected cells. We also found a decrease in the amount of reovirus secretion from SCG2 knockdown cells. Similar analyses of cells infected with HSV-1 showed an increase in the amount of secreted virus. Analysis of the SAPK/JNK pathway indicated that each virus activates different pathways leading to AP-1 activation, which is the known SCG2 transcription activator. We conclude from these experiments that the negative correlation between SCG2 quantity and virus secretion for both viruses indicates a virus-specific role for SCG2 during infection.

    Importance Mammalian reoviruses affect the gastrointestinal system or cause respiratory infections in humans. Recent work has shown that all MRV strains (most specifically T3D) may be useful oncolytic agents. The ubiquitous herpes simplex viruses cause common sores in mucosal areas of their host, and have co-evolved with hosts over many years. Both of these virus species are prototypical representatives of their viral families, and investigation of these viruses can lead to further knowledge of how they and the other more pathogenic members of their respective families interact with the host. Here we show that secretogranin II (SCG2), a protein not previously studied in the context of virus infections, alters virus output in a virus-specific manner, and that the quantity of SCG2 is inversely related to amounts of infectious virus secretion. Herpesviruses may target this protein to facilitate enhanced virus release from the host.

  • Replication and transmission of novel bovine influenza D virus in guinea pig model [PublishAheadOfPrint]

  • Influenza D virus (FLUDV) is a novel influenza virus that infects cattle and swine. The goal of this study was to investigate the replication and transmission of bovine FLUDV in guinea pigs. Following direct intranasal inoculation of animals, the virus was detected in nasal washes of infected animals during the first 7 days post infection. High viral titer was obtained from nasal turbinates and lung tissues of animals directly inoculated. Further, bovine FLUDV was able to transmit from the infected guinea pigs to the sentinel animals by means of contact and not by aerosol under experimental conditions tested in this study. Despite exhibiting no clinical signs, infected guinea pigs developed seroconversion and the viral antigen was detected in lungs of animals by immunohistochemistry. The observation that bovine FLUDV replicated in the respiratory tract of guinea pigs was similar to those described previously in experimentally infected gnotobiotic calves and pigs with bovine FLUDV but different from experimental infection in ferrets and swine with a swine FLUDV, which supported virus replication only in upper respiratory tract, not in lower respiratory tract including lung. Our study established that guinea pigs could be used as an animal model for studying this newly emerging influenza virus.

    Importance Influenza D virus (FLUDV) is a novel emerging pathogen with bovine as its primary host. The epidemiology and pathogenicity of the virus is yet to be known. FLUDV also spreads to swine and presence of FLUDV-specific antibodies in humans could indicate that there is a potential for zoonosis. Our results showed that bovine FLUDV replicated in the nasal turbinate and lungs of guinea pigs at high titers and also was able to transmit from infected animal to sentinel animals by contact. The fact that bovine FLUDV replicated productively in both the upper and lower respiratory tract of guinea pigs, similar to virus infection in its native host, demonstrates that guinea pigs would be a suitable model host to study the replication and transmission potential of bovine FLUDV.

  • CD8lowCD100- T cells identify a novel CD8 T cell subset associated with viral control during human Hantaan virus infection [PublishAheadOfPrint]

  • Hantaan virus (HTNV) infection can cause a severe lethal hemorrhagic fever with renal syndrome (HFRS) in humans. CD8+ T cells play a critical role in combatting HTNV virus infections. However, the contribution of different CD8+ T cell subsets to immune response against viral infection is poorly understood. Here, we identified a novel subset of CD8+ T cells characterized by the CD8lowCD100- phenotype in HFRS patients. The CD8lowCD100- subset accounted for a median of 14.3% of the total CD8+ T cells in early phase of HFRS, and this percentage subsequently declined in late phase of infection, whereas this subset was absent in healthy controls. Furthermore, the CD8lowCD100- cells were associated with high activation and expressed high levels of cytolytic effector molecules and exhibited a distinct expression profile of effector CD8+ T cells (CCR7+/-CD45RA-CD127highCD27intCD28lowCD62L-). When stimulated with specific HTNV-nucleocapsid protein-derived peptide pools, most responding CD8+ cells (IFN-+ and/or TNF-aalpha;+) were CD8lowCD100- cells. The frequency of CD8lowCD100- cells among HTNV-specific CD8+ T cells was higher in milder cases than more severe cases. Importantly, the proportion of the CD8lowCD100- subset among CD8+ T cells in early phase of HFRS was negatively correlated with the HTNV viral load, suggesting that CD8lowCD100- cells may be associated with viral clearance. The contraction of the CD8lowCD100- subset in late phase of infection may be related to the consistently high expression levels of PD-1. These results may provide new insights into our understanding of CD8+ T cell-mediated protective immunity as well as immune homeostasis after HTNV infection in humans.

    IMPORTANCE section CD8+ T cells play important roles in the antiviral immune response. We found that the proportion of CD8lowCD100- cells among CD8+ T cells from HFRS patients was negatively correlated with the HTNV viral load, and the frequency of CD8lowCD100- cells among virus-specific CD8+ T cells was higher in milder HFRS cases than in more severe cases. These results implied a beneficial role for the CD8lowCD100- cell subset in viral control during human HTNV infection.

  • RIG-I Signaling Is Essential For Influenza B Virus-Induced Rapid Interferon Gene Expression [PublishAheadOfPrint]

  • Influenza B virus causes annual epidemics and along with influenza A virus accounts for substantial disease and economic burden throughout the world. Influenza B infects only humans and some marine mammals and is not responsible for pandemics possibly due to a very low frequency of reassortation and a lower evolutionary rate compared to influenza A virus. Influenza B virus has been less studied than influenza A and thus a comparison of influenza A and B virus infection mechanisms may provide new insight into virus-host interactions. Here we analyzed the early events in influenza B virus infection and interferon (IFN) gene expression in human monocyte-derived macrophages and dendritic cells. We show that influenza B virus induces IRF3 activation and IFN-1 gene expression with a faster kinetics compared to influenza A virus without a requirement for viral protein synthesis or replication. Influenza B virus-induced activation of IRF3 required the fusion of viral and endosomal membranes and nuclear accumulation of IRF3 and viral NP occurred concurrently. In comparison, immediate early IRF3 activation was not observed in influenza A virus-infected macrophages. Experiments with RIG-I, MDA5 and RIG-I/MDA5 deficient mouse fibroblasts showed that RIG-I is the critical pattern recognition receptor needed for the influenza B virus-induced activation of IRF3. Our results show that innate immune mechanisms are activated immediately after influenza B virus entry through the endocytic pathway, whereas influenza A virus avoids the early IRF3 activation and IFN gene induction.

    IMPORTANCE Recently, a great deal of interest has been paid on identifying the ligands for RIG-I under a natural infection as many of the previous studies have been based on transfecting the cells with different types of viral or synthetic RNA structures. We shed light on this question by analyzing the earliest step in innate immune recognition of influenza B virus by human macrophages. We show that influenza B virus induces IRF3 activation leading to the IFN gene expression after the viral vRNPs are released to the cytosol and are recognized by RIG-I receptor meaning that already the incoming influenza B virus is able to activate IFN gene expression. In contrast, influenza A (H3N2) virus failed to activate IRF3 at very early times of infection suggesting that there are differences in the innate immune recognition between influenza A and B viruses.

  • Retargeting Oncolytic Vesicular Stomatitis Virus (VSV) to Human T-cell lymphotropic Virus 1 (HTLV1) Associated Adult T-cell leukemia (ATL) [PublishAheadOfPrint]

  • Adult T cell leukemia/lymphoma (ATL) is an aggressive cancer of CD4/CD25+ T lymphocytes, the etiological agent of which is human T cell lymphotropic virus 1 (HTLV1). ATL is highly refractory to presently existing therapies, making the development of new treatments a high priority. Oncolytic viruses such as Vesicular Stomatitis Virus (VSV) are presently being considered as anti-cancer agents since they readily infect transformed cells compared to normal cells, the former appearing to exhibit defective innate immune responses. Here, we have evaluated the efficacy and safety of a recombinant VSV that has been retargeted to specifically infect and replicate in transformed CD4+ cells. This was achieved by replacing the single VSV glycoprotein (G) with human immunodeficiency virus (HIV1) gp160, to create a hybrid fusion protein, gp160G. The resultant virus, VSV-gp160G, was found to only target cells expressing CD4 and retained robust oncolytic activity against HTLV-1 actuated ATL cells. VSV-gp160G was further noted to be highly attenuated and did not replicate efficiently in or induce significant cell death of primary CD4+ T cells. Accordingly, VSV-gp160G did not elicit any evidence of neurotoxicity even in severely immunocompromised animals such as NOD/Shi-scid, IL-2-c null mice (NSG). Importantly, VSV-gp160G effectively exerted potent oncolytic activity in patient-derived ATL transplanted into NSG mice and facilitated a significant survival benefit. Our data indicates that VSV-gp160G exerts potent oncolytic efficacy against CD4+ malignant cells and either alone or in conjunction with established therapies may provide an effective treatment in patients displaying ATL.

    IMPORTANCE Adult T cell leukemia (ATL) is a serious form of cancer with a high mortality rate. Human T cell lymphotropic virus 1 (HTLV1) infection is the etiological agent of ATL and unfortunately, most patients succumb to the disease within a few years. Current treatment options have failed to significantly improve survival rate. In this study, we developed a recombinant strain of Vesicular Stomatitis Virus (VSV) that specifically targets transformed CD4+ T cells through replacement of the G protein of VSV with a hybrid fusion protein, combining domains from gp160 of Human Immunodeficiency Virus (HIV) and VSV-G. This modification eliminated the normally broad tropism of VSV and restricted infection to primarily the transformed CD4+ cell population. This effect greatly reduced neurotoxic risk associated with VSV infection while still allowing VSV to effectively target ATL cells.

  • Dengue virus NS1 protein modulates cellular energy metabolism by increasing glyceraldehyde-3-phosphate dehydrogenase activity [PublishAheadOfPrint]

  • Dengue is one of the main public health concerns worldwide. Recent estimates indicate that over 390 million people are infected annually with the dengue virus (DENV), resulting in thousands of deaths. Among the DENV nonstructural proteins, the NS1 protein is the only one whose function during replication is still unknown. NS1 is a 46-55 kDa glycoprotein commonly found as both a membrane-associated homodimer and a soluble hexameric barrel-shaped lipoprotein. Despite its role in the pathogenic process, NS1 is essential for proper RNA accumulation and virus production. In the present study, we identified that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with intracellular NS1. Molecular docking revealed that this interaction occurred through the hydrophobic protrusion of NS1 and the hydrophobic residues located at the opposite side of the catalytic site. Moreover, addition of purified recombinant NS1 enhanced the glycolytic activity of GAPDH in vitro. Interestingly, we observed that DENV infection promoted the re-localization of GAPDH to the perinuclear region, where NS1 is commonly found. Both DENV infection and expression of NS1 itself resulted in increased GAPDH activity. Our findings indicate that the NS1 protein acts to increase glycolytic flux and, consequently, energy production, which is consistent with the recent finding that DENV induces and requires glycolysis for proper replication. This is the first report to propose that NS1 is an important modulator of cellular energy metabolism. The data presented here provide new insights that may be useful for further drug design and the development of alternative DENV antiviral therapies.

    IMPORTANCE Dengue represents a serious public health problem worldwide and is caused by the infection of dengue virus (DENV). Estimates indicate that half of the global population is at risk of infection, with almost 400 million cases occurring per year. The NS1 glycoprotein is found in both the intracellular and extracellular milieu. Despite the fact that NS1 has been commonly associated with DENV pathogenesis, it plays a pivotal, but unknown role in the replication process. In an effort to understand the role of intracellular NS1, we demonstrated that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with NS1. Our results indicated that NS1 increases the glycolytic activity of GAPDH in vitro. Interestingly, the GAPDH activity was increased during DENV infection, and NS1 expression alone was sufficient to enhance intracellular GAPDH activity in BHK-21 cells. Overall, our findings suggest that NS1 is an important modulator of cellular energy metabolism by increasing glycolytic flux.

  • Role of Substitutions in the Hemagglutinin in the Emergence of the 1968 Pandemic Influenza Virus [PublishAheadOfPrint]

  • Hemagglutinin (HA) of H3N2/1968 pandemic influenza viruses differed from putative avian precursor by seven amino acid substitutions. Substitutions Q226L and G228S are known to be essential for avian-to-mammalian adaptation. We found that introduction of avian-virus-like amino acids at five other HA positions (62, 81, 92, 144 and 193) of A/Hong Kong/1/1968 virus decreased viral replication in human cells and transmission in pigs. Thus, substitutions at some of these positions facilitated emergence of the pandemic virus.

  • Characterization of a Novel Hepadnavirus in the White Sucker (Catostomus commersonii) from the Great Lakes Region of the USA [PublishAheadOfPrint]

  • The white sucker Catostomus commersonii is a freshwater teleost often utilized as a resident sentinel. Here, we sequenced the full genome of a hepatitis B-like virus that infects white suckers from the Great Lakes Region of the USA. Dideoxysequencing confirmed the white sucker hepatitis B virus (WSHBV) has a circular genome (3542 bp) with the prototypical codon organization of hepadnaviruses. Electron microscopy demonstrated that complete virions of approximately 40 nm were present in the plasma of infected fish. Compared to avi- and orthohepadnaviruses, sequence conservation of the core, polymerase and surface proteins was low and ranged from 16-27% at the amino acid level. An X protein homologue common to the orthohepadnaviruses was not present. The WSHBV genome included an atypical, presumptively non-coding region absent in previously described hepadnaviruses. Phylogenetic analyses confirmed WSHBV as distinct from previously documented hepadnaviruses. The level of divergence in protein sequences between WSHBV other hepadnaviruses, and the identification of an HBV-like sequence in an African cichlid provide evidence that a novel genus of the family Hepadnaviridae may need to be established that includes these hepatitis B-like viruses in fishes. Viral transcription was observed in 9.5% (16 of 169) of white suckers evaluated. The prevalence of hepatic tumors in these fish was 4.9%, of which only 2.4% were positive for both virus and hepatic tumors. These results are not sufficient to draw inferences regarding the association of WSHBV and carcinogenesis in white sucker.

    IMPORTANCE We report the first full length genome of a hepadnavirus from fishes. Phylogenetic analysis of this genome indicates divergence from those of previously described hepadnaviruses from mammalian and avian hosts and supports the creation of a novel genus. The discovery of this novel virus may better our understanding of the evolutionary history of hepatitis B-like viruses of other hosts. In fishes, knowledge of this virus may provide insight regarding possible risk factors associated with hepatic neoplasia in the white sucker. This may also offer another model system for mechanistic research.

  • Sulfolobus spindle-shaped virus 1 contains glycosylated capsid proteins, a cellular chromatin protein and host-derived lipids [PublishAheadOfPrint]

  • Geothermal and hypersaline environments are rich in virus-like particles among which spindle-shaped morphotypes dominate. Currently, viruses with spindle- or lemon-shaped virions are exclusive to Archaea and belong to two distinct viral families. The larger of the two families, the Fuselloviridae, comprises tail-less, spindle-shaped viruses, which infect hosts from phylogenetically distant archaeal lineages. Sulfolobus spindle-shaped virus 1 (SSV1) is the best known member of the family and was one of the first hyperthermophilic archaeal virus to be isolated. SSV1 is an attractive model for understanding virus-host interactions in Archaea; however, the constituents and architecture of SSV1 particles remain only partially characterized. Here, we have conducted an extensive biochemical characterization of highly purified SSV1 virions and identified four virus-encoded structural proteins, VP1-VP4, as well as one DNA-binding protein of cellular origin. The virion proteins VP1, VP3 and VP4 undergo post-translational modification by glycosylation seemingly at multiple sites. VP1 is also proteolytically processed. In addition to the viral DNA-binding protein VP2, we show that viral particles contain the Sulfolobus solfataricus chromatin protein Sso7d. Finally, we present evidence indicating that SSV1 virions contain glycerol dibiphytanyl glycerol tetraether (GDGT) lipids, resolving a long-standing debate on SSV1 lipid issue. Comparison of the contents of lipids isolated from the virus and its host cell suggests that GDGTs are acquired by the virus in a selective manner from the host cytoplasmic membrane, likely during the progeny egress.

    IMPORTANCE Although spindle-shaped viruses represent one of the most prominent viral groups in Archaea, structural data on their virion constituents and architecture are still scarce. The comprehensive biochemical characterization of the hyperthermophilic virus SSV1 presented here brings novel and significant insights into the organization and architecture of spindle-shaped virions. The obtained data permit the comparison between spindle-shaped viruses residing in widely different ecological niches, improving our understanding on the adaptation of viruses with unusual morphotypes to extreme environmental conditions. Our findings also pave the way for future research on the unique archaeal virosphere that will further shed light on the structure and biology of viruses.

  • Virus-like particle vaccine containing the F protein of respiratory syncytial virus confers protection without pulmonary disease by modulating specific subsets of dendritic cells and effector T cells [PublishAheadOfPrint]

  • There is no licensed vaccine against respiratory syncytial virus (RSV) since the failure of formalin inactivated RSV (FI-RSV) due to its vaccine-enhanced disease. We investigated immune correlates conferring protection without causing disease after intranasal immunization with virus-like particle vaccine containing the RSV fusion protein (F VLP) in comparison with FI-RSV and live RSV. Upon RSV challenge, FI-RSV immune mice showed severe weight loss, eosinophilia, and histopathology, and RSV re-infection also caused substantial RSV disease despite their viral clearance. In contrast, F VLP immune mice showed least weight loss and no sign of histopathology and eosinophilia. High levels of IL-4+ and TNFaalpha;+ CD4+ T cells were found in FI-RSV immune mice whereas IFN-+ and TNFaalpha;+ CD4+ T cells dominantly detected in live RSV infected mice. More importantly, in contrast to FI-RSV and live RSV that induced higher levels of CD11b+ dendritic cells, F VLP immunization induced CD8aalpha;+ and CD103+ dendritic cells as well as F-specific IFN-+ and TNFaalpha;+ CD8+ T cells. These results suggest that F VLP can induce protection without causing pulmonary RSV disease by inducing RSV neutralizing antibodies as well as modulating specific subsets of dendritic cells and CD8 T cell immunity.

    IMPORTANCE It has been a difficult challenge to develop an effective and safe vaccine against RSV, a leading cause of respiratory disease. Immune correlates conferring protection but preventing vaccine-enhanced disease remain poorly understood. RSV F VLP would be an efficient vaccine platform conferring protection. Here we investigated the protective immune correlates without causing disease after intranasal immunization with RSV F VLP in comparison with FI-RSV and live RSV. In addition to inducing RSV neutralizing antibodies responsible for clearing lung viral loads, we show that modulation of specific subsets of dendritic cells and CD8 T cells producing T helper type 1 cytokines are important immune correlates conferring protection but not causing vaccine-enhanced disease.

  • Successive Respiratory Syncytial Virus Epidemics in Local Populations Arise from Multiple Variant Introductions Providing Insights into Virus Persistence [PublishAheadOfPrint]

  • Respiratory syncytial virus (RSV) is a global respiratory pathogen of humans, occurring characteristically as recurrent seasonal epidemics. Unlike influenza viruses, little attention has been paid to the mechanism underlying worldwide spread and persistence of RSV and how this may be informed through an improved understanding of the introduction and persistence of RSV viruses in local communities. We analyzed 651 attachment (G) glycoprotein nucleotide sequences of RSV B collected over 11 epidemics (2002-12) in Kilifi, Kenya and contemporaneous data collected elsewhere in Kenya and 18 other countries worldwide (2002-12). Based on phylogeny, genetic distance and clustering patterns, we set out pragmatic criteria to designate local viruses into distinct genotypes and variants, identifying those newly introduced and those locally persisting. Three genotypes were identified in the Kilifi dataset: BA (n=500), SAB1 (n=148) and SAB4 (n=3). Recurrent RSV epidemics in the local population were composed of numerous genetic variants most of which have been newly introduced rather than persisting in the location from season to season. Global comparison revealed that (i) most Kilifi variants do not cluster closely with strains from elsewhere beyond Kenya, (ii) some Kilifi variants were closely related to those observed outside Kenya (mostly Western Europe) and (iii) many variants were circulating elsewhere but never sampled in Kilifi. These results are consistent with the hypothesis that year-to-year presence of RSV at the local level (i.e. Kilifi) is primarily, but not exclusively, through introductions from a pool of variants that are geographically restricted (ie Kenya/Regionally) rather than global.

    IMPORTANCE The mechanism by which RSV persists and reinvades local populations is poorly understood. We have investigated this by studying the temporal patterns of RSV variants in a tropical Africa rural setting and compared with contemporaneous variants circulating in other countries. We find that the periodic seasonal RSV transmission at the local level appears to require regular new introductions of variants. But importantly, the evidence suggests that the source of new variants is mostly geographically restricted and we hypothesize that year-to-year RSV persistence is at country level rather than global level. This has implications for control.

  • Consequences of HLA-B*13-associated escape mutations on HIV-1 replication and Nef function [PublishAheadOfPrint]

  • HLA-B*13 is associated with superior in vivo HIV-1 viremia control. Protection is thought to be mediated by sustained targeting of key CTL epitopes and viral fitness costs of CTL escape in Gag, although additional factors may contribute. We assessed the impact of 10 published B*13-associated polymorphisms in Gag, Pol and Nef, in 23 biologically relevant combinations, on HIV-1 replication capacity and Nef-mediated reduction of cell surface CD4 and HLA class I expression. Mutations were engineered into HIV-1NL4.3 and replication capacity was measured using a GFP-reporter T-cell line. Nef-mediated CD4 and HLA-A*02 downregulation was assessed by flow cytometry, and T cell recognition of infected target cells measured via co-culture with an HIV-specific luciferase-reporter cell line. When tested individually, only Gag-I147L and Gag-I437L incurred replicative costs (5% and 17% respectively), consistent with prior reports. The Gag-I437L-mediated replication defect was rescued to wild-type levels by the adjacent K436R mutation. A novel B*13 epitope, terminating at Gag-147, was identified in p24Gag (GQMVHQAIGag140-147). No other single or combination Gag, Pol or Nef mutants impaired viral replication. Single Nef mutations did not affect CD4 or HLA downregulation; however, the double mutant E24Q-Q107R showed 40% impairment in HLA downregulation with no evidence of Nef stability defects. Moreover, target cells infected with HIV-1-NefE24Q-Q107R were recognized nearly two-fold better by HIV-specific T-cells compared to those infected with HIV-1NL4.3 or single Nef mutants. Our results indicate that CTL escape in Gag and Nef can be functionally costly and suggest that these effects may contribute to long-term HIV-1 control by HLA-B*13.

    IMPORTANCE Protective effects of HLA-B*13 on HIV-1 disease progression are mediated in part by fitness costs of CTL escape mutations in conserved Gag epitopes, but other mechanisms remain incompletely known. We extend our knowledge of the impact of B*13-driven escape on HIV-1 replication by identifying Gag-K436R as a compensatory mutation for the fitness-costly Gag-I437L. We also identify Gag-I147L, the most rapidly and commonly selected B*13-driven substitution in HIV-1, as a putative C-terminal anchor residue mutation in a novel B*13 epitope. Most notably, we identify a novel escape-driven fitness defect: B*13-driven substitutions E24Q and Q107R in Nef, when present together, substantially impair this protein's ability to downregulate HLA class I. This in turn increases the visibility of infected cells to HIV-specific T-cells. Our results suggest that B*13-associated escape mutations impair HIV-1 replication by two distinct mechanisms nndash; by reducing Gag fitness and dampening Nef immune evasion function.


  • We have previously reported that hepatitis C virus (HCV) infection of primary human hepatocytes (PHH) induces epithelial mesenchymal transition (EMT) state and extends hepatocyte life span. These hepatocytes displayed sphere formation on ultralow binding plate, and survived for more than 12 weeks. The sphere forming hepatocytes expressed a number of cancer stem-like cell (CSC) markers, including high level of stem cell factor receptor c-Kit. The c-Kit receptor is regarded as one of the CSC markers in HCC. Analysis of c-Kit mRNA displayed a significant increase in the liver biopsies of chronically HCV-infected patients. We also found c-Kit is highly expressed in transformed human hepatocytes (THH) infected in vitro with cell culture grown HCV genotype 2a. Further studies suggested that HCV core protein significantly upregulates c-Kit expression at the transcriptional level. HCV infection of THH led to a significant increase in the number of spheres displayed on ultra-low binding plate, enhanced EMT and CSC markers, and tumor growth in immunodeficient mice. Use of imatinib or dasatinib as a c-Kit inhibitor reduced sphere forming cells in culture. The sphere forming cells were sensitive to treatment with sorafenib, a multikinase inhibitor, and is used for HCC treatment. Further, stattic; an inhibitor of the Stat3 molecule, induced sphere forming cell death. A combination of sorafenib and stattic had a significantly stronger effect leading to cell death. These results suggested that HCV infection potentiates CSC generation, and selected drugs can be targeted to efficiently inhibit cell growth.

    IMPORTANCE HCV infection may develop into HCC as an end stage liver disease. We focused to understand the mechanism for risk of HCC from chronic HCV infection and identify targets for treatment. HCV infected primary and transformed human hepatocytes (PHH or THH) generated CSC. HCV induced spheres were highly sensitive to cell death from sorafenib and static treatment. Thus, our study is highly significant for HCV associated HCC, with the potential for developing target specific strategy for improved therapies.

  • A highly immunogenic and protective MERS-Coronavirus vaccine based on recombinant MV vaccine platform [PublishAheadOfPrint]

  • In 2012, first cases of infection with the Middle East Respiratory Syndrome coronavirus (MERS-CoV) were identified. In the meantime more than one thousand cases of MERS-CoV infection have been confirmed, which typically are associated with considerable morbidity and, in approximately 30% of the cases, mortality. Currently, there is no protective vaccine available. Replication competent recombinant measles virus (MV) expressing foreign antigens constitutes a promising tool to induce protective immunity against respective pathogens. Therefore, we generated MVs expressing the spike glycoprotein of MERS-CoV in its full length (MERS-S) or a truncated, soluble variant of MERS-S (MERS-solS). The genes encoding for MERS-S and MERS-solS were cloned into vaccine strain MVvac2 genome and the respective viruses were rescued (MVvac2-CoV-S and MVvac2-CoV-solS). These recombinant MV were amplified and characterized in passages three and 10. The replication of MVvac2-CoV-S in Vero cells turned out to be comparable with the control virus MVvac2-GFP while titers of MVvac2-CoV-solS were approx. 3-fold impaired. The genomic stability and expression of the inserted antigens was confirmed via sequencing of viral cDNA and immunoblot analysis. In vivo, immunization of IFNAR-/--CD46Ge mice with 2x105 TCID50 MVvac2-CoV-S(H) or MVvac2-CoV-solS(H) in a prime-boost regimen induced robust levels of both MV and MERS-CoV neutralizing antibodies. Additionally, induction of specific T cells could be demonstrated by T cell proliferation, antigen-specific T-cell cytotoxicity, and IFN- secretion after stimulation of splenocytes with MERS-CoV-S presented by murine DCs. MERS-CoV challenge experiments indicate protective capacity of these immune responses in vaccinated mice.

    IMPORTANCE Although MERS-CoV has not yet acquired extensive distribution being mainly confined to the Arabic and Korean peninsulas, it could adapt to spread more readily amongst humans and thereby become pandemic. Therefore, the development of a vaccine is mandatory. The integration of antigen-coding genes into recombinant MV resulting in co-expression of MV and foreign antigens can efficiently be achieved. Thus, in combination with the excellent safety profile of the MV vaccine, recombinant MV seems to constitute an ideal vaccine platform. The present study shows that a recombinant MV expressing MERS-S was genetically stable and induced strong humoral and cellular immunity against MERS-CoV in vaccinated mice. Subsequent challenge experiments indicate protection of vaccinated animals, illustrating the potential of MV as vaccine platform with the potential to target emerging infections such as MERS-CoV.

  • Regulation of PACT-mediated PKR activation by the OV20.0 protein of orf virus [PublishAheadOfPrint]

  • Double-stranded RNA (dsRNA)-activated protein kinase (PKR), a major component of the cellular antiviral system, is activated by the binding of either dsRNA or the cellular PKR activator, the PACT protein. Suppression of PKR activation is one of the main strategies that viruses employ to circumvent interferon signaling. Orf virus (ORFV), a parapoxvirus from the Poxviridae family, causes contagious pustular dermatitis in small ruminants. Previous studies have demonstrated that various OV20.0 isoforms, encoded by the OV20.0L gene, are able to inhibit PKR activation both by sequestering dsRNA and by physically interacting with PKR in vitro. Thus this gene acts as a virulence factor of ORFV when tested using a mouse infection model. In the present study, the regions within OV20.0 that interacts with dsRNA and with PKR have been mapped. Furthermore, this study demonstrates for the first time that OV20.0 is also able to interact with the dsRNA binding domain of PACT and that the presence of dsRNA strengthened the interaction of these two molecules. The presence of OV20.0 diminishes PKR phosphorylation when this is stimulated by PACT. Nevertheless, the association of OV20.0 with PKR, rather than with PACT, was found to be essential for reducing PACT-mediated PKR phosphorylation. These observations elucidate a new strategy whereby innate immunity can be evaded by ORFV.

    IMPORTANCE Our previous study indicated that ORFV's two OV20.0 isoforms act as a PKR antagonist via sequestering the PKR activator, dsRNA, and by interacting with PKR leading to an inhibition of PKR activation. In current study, the possible mechanisms by which OV20.0 protein counteracts PKR activation were studied in depth. OV20.0 is able to binds PKR and its two activators, dsRNA and PACT. In addition, OV20.0 binds directly to the RNA binding domains (RBDs) of PKR, and this interaction does not require dsRNA. Moreover, OV20.0 interacts with or occupies the RBD2 and the kinase domain of PKR, which then prevents PACT binding to PKR. Finally, OV20.0 associates with PACT via the RBDs, which may reduce the ability of PACT to induce PKR activation. The findings in this study provide new concepts in relation to how ORFV modulates PKR activation.


  • Type I interferons (IFNs) are induced upon viral infection and important mediators of innate immunity. While there is 1 IFN-bbeta; protein, there are 12 different IFN-aalpha; subtypes. It has been reported extensively that different viruses induce distinct patterns of IFN subtypes, but it has not been previously shown how viral MOI can affect IFN induction. In this study, we discovered the novel finding that human U937 cells infected with 2 different concentrations of Sendai virus (SeV) induce 2 distinct type I IFN subtype profiles. Cells infected with a lower MOI induced more subtypes than cells infected with the higher MOI. We found that this was due to the extent of signaling through the IFNAR. The cells infected with the lower viral MOI induced the IFNAR2- dependent IFN-aalpha; subtypes 4, 6, 7, 10 and 17, which were not induced in cells infected with higher virus concentrations. IFN-bbeta; and IFN-aalpha; 1, 2 and 8 were induced in an IFNAR-independent manner in cells infected with both virus concentrations. IFN-aalpha; 5, 14, 16 and 21 were induced in an IFNAR-dependent manner in cells infected with lower virus concentrations and in an IFNAR-independent manner in cells infected with higher virus concentrations. These differences in IFN subtype profiles in the 2 virus concentrations also resulted in a distinct interferon stimulated gene induction. These results present the novel finding that different viral MOIs will differentially activate JAK/STAT signaling through the IFNAR, which greatly affects the profile of IFN subtypes that are induced.

    IMPORTANCE Type I IFNs are pleiotropic cytokines that are instrumental in combating viral diseases. Understanding how the individual subtypes are induced is important in developing strategies to block viral replication. Many studies have reported that different viruses induce distinct type I IFN subtype profiles due to differences in the way viruses are sensed in different cell types. However, we report in our study the novel finding that the amount of virus used to infect a system can also affect which type I IFN subtypes are induced due to the extent of activation of certain signaling pathways. These distinct IFN subtype profiles in cells infected with differing MOIs are correlated with differences in interferon stimulated gene induction, indicating that the same virus can induce distinct antiviral responses depending on MOI. Because type I IFNs are used as therapeutic agents to treat viral diseases, understanding their antiviral mechanisms can enhance clinical treatments.

  • Characterization of the determinants of NS2-3-independent virion morphogenesis of pestiviruses [PublishAheadOfPrint]

  • A peculiarity of the Flaviviridae is the critical function of nonstructural (NS) proteins for virus particle formation. For pestiviruses, like bovine viral diarrhea virus (BVDV), uncleaved NS2-3 represents an essential factor for virion morphogenesis while NS3 is an essential component of the viral replicase. Accordingly, in natural pestivirus isolates processing at the NS2-3 cleavage site is incomplete to allow for virion morphogenesis. When compared, virion morphogenesis of the related hepatitis C virus (HCV) shows a major deviation: while RNA replication also requires free NS3, virion formation does not depend on uncleaved NS2-NS3.

    Recently, we described a BVDV-1 chimera based on strain NCP7 encompassing the NS2-4B*-coding region of strain Osloss. This chimera allowed for the production of infectious virus particles in the absence of uncleaved NS2-3. The Osloss sequence deviates in the NS2-4B* part from NCP7 in 48 amino acids, as well as an ubiquitin insertion between NS2 and NS3. The present study demonstrates that in the NCP7 backbone only two amino acid exchanges in NS2 (E1576V) and NS3 (V1721A), respectively, are sufficient and necessary to allow for efficient NS2-3-independent virion morphogenesis. The adaptation of a bicistronic virus encompassing an IRES element between the NS2- and NS3-coding sequences to efficient virion morphogenesis led to the identification of additional amino acids in E2, NS2, and NS5B, critically involved in this process. The surprisingly small requirements for approximating the packaging schemes of pestiviruses and HCV with respect to the NS2-3 region, is in favor of a common mechanism in an ancestral virus.

    IMPORTANCE For positive-strand RNA viruses the processing products of the viral polyprotein serve in RNA replication as well as virion morphogenesis. For bovine viral diarrhea virus nonstructural protein NS2-3 is of critical importance to switch between these processes: While free NS3 is essential for RNA replication, uncleaved NS2-3, which accumulates over time in the infected cell, is required for virion morphogenesis. Surprisingly, the virion morphogenesis of the related hepatitis C virus is independent from uncleaved NS2-NS3. Here we demonstrate that pestiviruses can adapt to virion morphogenesis in the absence of uncleaved NS2-3 by just two amino acid exchanges. While the mechanism behind this gain of function remains elusive the fact that it can be achieved by such minor changes is in line with the assumption that an ancestral virus already used this mechanism but lost it in the course of adapting to a new host/infection strategy.

  • The nucleoprotein is required for lymphocytic choriomeningitis virus-based vaccine vector immunogenicity [PublishAheadOfPrint]

  • Glycoprotein-deficient lymphocytic choriomeningitis virus-based vaccine vectors (rLCMV/GP) are potent CD8+ T cell inducers. To investigate the underlying molecular requirements we generated a nucleoprotein-deficient vector counterpart (rLCMV/NP). NP but not GP is a minimal trans-acting factor for viral transcription and genome replication. We found that unlike rLCMV/GP, rLCMV/NP failed to elicit detectable CD8+ T cell responses unless NP was trans-complemented in a transgenic host. Hence, NP-dependent intracellular gene expression is essential for LCMV vector immunogenicity.

  • Requirement for Host RNA Silencing Components and the Virus Silencing Suppressor when Second-Site Mutations Compensate For Structural Defects in the 3' UTR [PublishAheadOfPrint]

  • Turnip crinkle virus (TCV) contains a structured 3rrsquo; region with hairpins and pseudoknots that form a complex network of non-canonical RNA:RNA interactions supporting higher order structure critical for translation and replication. We investigated several second-site mutations in the p38 coat protein ORF that arose in response to a mutation in the asymmetric loop of a critical 3rrsquo; UTR hairpin that disrupts local higher order structure. All tested second-site mutations improved accumulation of TCV in conjunction with a partial reversion of the primary mutation (TCV-rev1) but had neutral or a negative effect on wt TCV or TCV with the primary mutation. SHAPE structure probing indicated that these second-site mutations reside in an RNA domain that includes most of p38 (Domain 2), and evidence for RNA:RNA interactions between Domain 2 and 3rrsquo; UTR-containing Domain 1 was found. However, second-site mutations were not compensatory in the absence of p38, which is also the TCV silencing suppressor, or in dcl-2/dcl4 or ago1/ago2 backgrounds. One second-site mutation reduced silencing suppressor activity of p38 by altering one of two GW motifs that are required for p38 binding to dsRNAs and interaction with RISC-associated AGO1/AGO2. Another second-site mutation substantially reduced accumulation of TCV-rev1 in the absence of p38 or DCL2/DCL4. We suggest that the second-site mutations in the p38 ORF exert positive effects through a similar downstream mechanism, by either enhancing accumulation of beneficial DCL-produced viral small RNAs that positively regulate the accumulation of TCV-rev1, or by affecting susceptibility of TCV-rev1 to RISC loaded with viral small RNAs.

    IMPORTANCE Genomes of positive-strand RNA viruses fold into high-order RNA structures. Viruses with mutations in regions critical for translation and replication often acquire second-site mutations that exert a positive compensatory effect through re-establishment of canonical base pairing with the altered region. In this report, two distal second-site mutations that individually arose in response to a primary mutation in a critical 3rrsquo; UTR hairpin in the genomic RNA of Turnip crinkle virus did not directly interact with the primary mutation. Although different second-site changes had different attributes, compensation was dependent on the production of the viral p38 silencing suppressor and on the presence of silencing-required DCL and AGO proteins. Our results provide an unexpected connection between a 3rrsquo; UTR primary-site mutation proposed to disrupt higher order structure and the RNA silencing machinery.

  • Ecuador Paraiso Escondido virus, a new flavivirus isolated from New World sandflies in Ecuador is the first representative of a novel clade in the genus Flavivirus [PublishAheadOfPrint]

  • A new flavivirus Ecuador Paraiso Escondido virus (EPEV), named after the village where it was discovered, was isolated from Psathyromyia abonnenci (formerly Lutzomyia abonnenci) that are unique to the New World. This represents the first sandfly-borne flavivirus identified in the New World and, as will be shown, EPEV exhibited a typical flavivirus genome organization. Nevertheless, the maximum pairwise amino acid identity was 52.8% with currently recognised flaviviruses. Phylogenetic analysis of the complete coding sequence showed that EPEV represented a distinct clade which diverged from a lineage that was ancestral to the non-vectored flaviviruses, Entebbe bat virus, Yokose virus, Sokoluk virus and also the Aedes-spp.-associated mosquito-borne flaviviruses which include yellow fever virus, Sepik virus, Saboya virus and others. EPEV replicated in C6/36 mosquito cells yielding high infectious titres but failed to reproduce in either vertebrate cell lines (Vero, BHK, SW13, and XTC) or in suckling mouse brains. This surprising result, which appears to eliminate an association with vertebrate hosts in the life cycle of EPEV, is discussed in the context of the evolutionary origins of EPEV in the New World.

    IMPORTANCE The flaviviruses are rarely (if at all) vectored by sandfly species at least in the Old World. We have identified the first representative of a sandfly-associated flavivirus, Ecuador Paraiso Escondido virus (EPEV) in the New World which constitutes a novel clade according to current knowledge of the flaviviruses. Phylogenetic analysis of the encoded genome showed that EPEV roots the Aedes spp.-associated mosquito-borne flaviviruses including yellow fever virus. In light of this new discovery, the New World origin of EPEV is discussed together with the other flaviviruses.

  • YB-1 Stabilizes Hepatitis C Virus NS5A via Phosphorylation-Mediated Interaction with NS5A to Regulate Viral Propagation [PublishAheadOfPrint]

  • Replication of hepatitis C virus (HCV) is dependent on viral-encoded proteins and numerous cellular factors. DDX3 is a well-known host cofactor of HCV replication. Here, we investigated the role of a DDX3-interacting protein, Y-box binding protein-1 (YB-1), in HCV life cycle. Both YB-1 and DDX3 interacted with the viral nonstructural protein NS5A. During HCV infection, YB-1 partially colocalized with NS5A and the HCV replication intermediate dsRNA in HCV-infected Huh-7.5.1 cells. Despite sharing the same interacting partners, YB-1 participated in HCV RNA replication, but was dispensable in steady-state HCV RNA replication, as different from the action of DDX3. Moreover, knockdown of YB-1 in HCV-infected cells prevented infectious virus production and reduced the ratio of hyperphosphorylated (p58) to hypophosphorylated (p56) forms of NS5A, whereas DDX3 silencing did not affect the ratio of p58 to p56 phosphoforms of NS5A. Interestingly, silencing of YB-1 severely reduced NS5A protein stability in NS5A-ectopically expressing, replicon-containing and HCV-infected cells. Furthermore, mutations of serine 102 of YB-1 affected both YB-1/NS5A interaction and NS5A-stabilizing activity of YB-1, indicating that this Akt phosphorylation site of YB-1 plays an important role in stabilizing NS5A. Collectively, our results support a model in which the event of YB-1 phosphorylation-mediated interaction with NS5A results in stabilizing NS5A to sustain HCV RNA replication and infectious HCV production. Overall, our study may reveal a new aspect for the development of novel anti-HCV drugs.

    IMPORTANCE Chronic hepatitis C virus (HCV) infection induces liver cirrhosis and hepatocellular carcinoma. The viral nonstructural protein NS5A co-opting various cellular signaling pathways and cofactors to support viral genome replication and virion assembly is a new strategy for anti-HCV drug development. NS5A phosphorylation is believed to modulate switches between different stages of HCV life cycle. Here, we identified the cellular protein YB-1 as a novel NS5A-interacting protein. YB-1 is a multifunctional protein participating in oncogenesis, and is an oncomarker of hepatocellular carcinoma (HCC). We found that YB-1 protects NS5A from degradation and likely regulates NS5A phosphorylation through its phosphorylation-dependent interaction with NS5A, which might be controlled by HCV-induced signaling pathways. Our observations suggest a model in which HCV modulates NS5A level and the ratio of p58 to p56 phosphoforms for efficient viral propagation via regulating cellular signaling inducing YB-1 phosphorylation. Our finding may provide new aspects for developing novel anti-HCV drugs.

  • KSHV vIRF1 interacts with a member of the Interferon Stimulated Gene 15 pathway [PublishAheadOfPrint]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus known to establish lifelong latency in the human host. We and others have previously shown that three KSHV homologs of cellular Interferon Regulatory Factors (IRFs), known as viral IRFs (vIRFs), participate in evasion of the host interferon (IFN) response. We report that vIRF1 interacts with the cellular interferon stimulated gene 15 (ISG15) E3 ligase, HERC5 in the context of TLR3 activation and IFN induction. The ISG15 protein is covalently conjugated to target proteins upon activation of the interferon response. Interaction between vIRF1 and HERC5 was confirmed by immunoprecipitation and the region between amino acids 224 and 349 of vIRF1 was required for interaction with HERC5. We further report that expression of vIRF1 in the context of TLR3 activation results in decreased ISG15 conjugation of proteins. Specifically, TLR3-induced ISG15 conjugation and protein levels of cellular IRF3, a known ISG15 target, were decreased in the presence of vIRF1 compared to control. vIRF1 itself was also identified as a target of ISG15 conjugation. KSHV-infected cells exhibited increased ISG15 conjugation upon reactivation from latency in coordination with increased IFN. Furthermore, knockdown of ISG15 in latently infected cells resulted in a higher level of KSHV reactivation and an increase in infectious virus. These data suggest that the KSHV vIRF1 protein affects ISG15 conjugation and interferon responses and may contribute to effective KSHV viral replication.

    IMPORTANCE The KSHV vIRF1 protein can inhibit interferon activation in response to viral infection. We identified a cellular protein named HERC5, which is the major ligase for ISG15, as a vIRF1 binding partner. vIRF1 association with HERC5 altered ISG15 modification of cellular proteins and knockdown of ISG15 augmented reactivation of KSHV from latency.

  • Computational and functional analysis of the virus-receptor interface reveals host range trade-offs in New World arenaviruses [PublishAheadOfPrint]

  • Animal viruses frequently cause zoonotic disease in humans. As these viruses are highly diverse, evaluating the threat they pose remains a major challenge, and efficient approaches are needed to rapidly predict virus-host compatibility. Here, we develop a combined computational and experimental approach to assess the compatibility of New World arenaviruses, endemic in rodents, with the host TfR1 entry receptors of different potential new host species. Using signatures of positive selection, we identify a small motif on rodent TfR1 that conveys species-specificity to the entry of viruses into cells. However, we show that mutations in this region affect the entry of each arenavirus differently. Moreover, a human SNP in this region, L212V, makes human TfR1 a weaker receptor for one arenavirus, Machupo virus, but a stronger receptor for two other arenaviruses, Junin and Sabia viruses. Collectively, these findings set the stage for potential evolutionary trade-offs, where natural selection for resistance to one virus may make humans or rodents susceptible to other arenavirus species. Given the complexity of this host-virus interplay, we propose a computational method to predict these interactions, based on homology modeling and computational docking of the virus-receptor protein-protein interaction. We demonstrate the utility of this model for Machupo virus, for which a suitable co-crystal structural template exists. Our model effectively predicts whether the TfR1 receptors of different species will be functional receptors for Machupo virus entry. Approaches such at this could provide a first step towards computationally predicting the "host jumping" potential of a virus into a new host species.

    IMPORTANCE We demonstrate how evolutionary trade-offs may exist in the dynamic evolutionary interplay between viruses and their hosts, where natural selection for resistance to one virus could make humans or rodents susceptible to other virus species. We present an algorithm that predicts which species have cell surface receptors that make them susceptible to Machupo virus, based on computational docking of protein structures. Few molecular models exist for predicting the risk of spillover of a particular animal virus into humans or new animal populations. Our results suggest that a combination of evolutionary analysis, structural modeling, and experimental verification may provide an efficient approach for screening and assessing potential spillover risks of viruses circulating in animal populations.

  • Exclusive decoration of SIV Env with high-mannose type N-glycans is not compatible with mucosal transmission in rhesus macaques [PublishAheadOfPrint]

  • HIV and SIV envelope proteins (Env) are extensively decorated with N-glycans, predominantly of the high-mannose type. However, it is unclear how high-mannose N-glycans on Env impact viral spread. We show that exclusive modification of SIV Env with these N-glycans reduces viral infectivity and abrogates mucosal transmission, despite increasing viral capture by immune cell lectins. Thus, high-mannose N-glycans have opposed effects on SIV infectivity and lectin-reactivity and a balance might be required for efficient mucosal transmission.

  • Mutational disruption of the CRE(2C) in coxsackievirus B3 leads to 5' terminal genomic deletions. [PublishAheadOfPrint]

  • Following natural human or experimental murine infections, and in cell culture, coxsackie B virus (CVB) RNA can persist for weeks in the absence of CPE yet with detectable viral RNA. Our earlier studies demonstrated that this persistence produced viral RNA with up to 49 nucleotide deletions at the 5rrsquo; genomic terminus which partially degraded the cloverleaf (or domain I), an RNA structure required for efficient viral replication. A cis-acting replication element (CRE) in the 2C protein coding region [CRE(2C)] templates the addition of two uridine residues to the virus-encoded RNA replication primer, VPg, prior to positive strand synthesis. Because our previous work also demonstrated that 5rrsquo; terminally deleted CVB (CVB-TD) genomes have VPg covalently linked despite rarely terminating in the canonical UU donated by CRE(2C) mediated uridylylation of VPg, we hypothesized that a functional (uridylylating) CRE(2C) would be unnecessary for CVB-TD replication. Using the same 16 mutations in the CVB3 CRE(2C) structure that were considered lethal for this virus by others, we demonstrate here that the wild type (wt) and the CVB3-TD virus carrying these mutations are viable with a non-uridylylating CRE(2C) both in infected cell cultures and in mice. While the wt genome with the mutated CRE(2C) displays suppressed replication levels similar to that observed in a CVB3-TD strain, mutation of CRE(2C) function in a CVB3-TD strain does not further decrease replication. Finally, we show that replication of the parental CVB3 strain containing the mutated CRE(2C) drives de novo generation of 5rrsquo; terminal genomic deletions.

    IMPORTANCE In this report, we demonstrate that while CVB can replicate without a uridylylating CRE(2C), replication rate suffers significantly. Further, 5rrsquo; genomic terminal deletions are generated in this virus population, supplanting the wildtype population. This demonstrates that VPg can prime without being specifically uridylylated and that this priming is error prone, resulting in loss of 5rrsquo; terminal sequence information. These findings have significance when considering the replication of HEV, and we believe that these data are unattainable in a cell-free system due to the poor replication of these CRE-deficient viruses.

  • Cooperation between the hepatitis C virus p7 and NS5B proteins enhances virion infectivity [PublishAheadOfPrint]

  • The molecular mechanisms that govern hepatitis C virus (HCV) assembly, release, and infectivity are still not yet fully understood. In the present study, we sequenced a genotype 2A strain of HCV (JFH-1) that had been cell culture adapted in Huh-7.5 cells to produce nearly 100-fold higher viral titers than the parental strain. Sequence analysis identified nine mutations in the genome, present within both the structural and non-structural genes. The infectious clone of this virus containing all nine culture-adapted mutations had 10-fold higher levels of RNA replication and RNA release into the supernatant, but had nearly 1000-fold higher viral titers, resulting in an increased specific infectivity compared to wild-type JFH-1. Two mutations, identified in the p7 polypeptide and NS5B RNA-dependent RNA polymerase, were sufficient to increase the specific infectivity of JFH-1. We found that the culture-adapted mutation in p7 promoted an increase in the size of cellular lipid droplets following transfection of viral RNA. In addition, we found that the culture-adaptive mutations in p7 and NS5B acted synergistically to enhance the specific viral infectivity of JFH-1 by decreasing the level of sphingomyelin in the virion. Overall, these results reveal a genetic interaction between p7 and NS5B that contributes to virion specific infectivity. Furthermore, our results demonstrate a novel role for the RNA-dependent RNA polymerase NS5B in HCV assembly.

    IMPORTANCE Hepatitis C virus assembly and release depends on viral interactions with host lipid metabolic pathways. Here we demonstrate that the viral p7 and NS5B proteins cooperate to promote virion infectivity by decreasing sphingomyelin content in the virion. Our data uncover a new role for the viral RNA-dependent RNA polymerase NS5B and p7 proteins in contributing to virion morphogenesis. Overall, these findings are significant because they reveal a genetic interaction between p7 and NS5B, as well as an interaction with sphingomyelin that regulates virion infectivity. Our data provide new strategies for targeting host lipid-viral interactions as potential targets for therapies against HCV infection.

  • Down-regulation of toll-like receptor 9 expression by beta human papillomavirus type 38 and implications for cell cycle control [PublishAheadOfPrint]

  • Innate immunity is the first line of host defence against infections. Many oncogenic viruses can deregulate several immune-related pathways to guarantee the persistence of the infection. Here, we show that the cutaneous human papillomavirus (HPV) type 38 E6 and E7 oncoproteins suppress the expression of the double-stranded DNA sensor TLR9 in human foreskin keratinocytes (HFK), a key mediator of the anti-viral innate immune host response. In particular, HPV38 E7 induces TLR9 mRNA down regulation by promoting accumulation of Np73aalpha;, an antagonist of p53 and p73. Inhibition of Np73aalpha; expression by antisense oligonucleotide in HPV38 E6/E7 HFK strongly rescues mRNA levels of TLR9, highlighting a key role of Np73aalpha; in this event. Chromatin immunoprecipitation experiments showed that Np73aalpha; is part of a negative transcriptional regulatory complex with IB kinase beta (IKKbbeta;) that binds to a NF-B responsive element within the TLR9 promoter. In addition, the polycomb protein enhancer of zeste homolog 2 (EZH2), responsible for gene expression silencing, is also recruited into the complex leading to histone 3 tri-methylation at lysine 27 (H3K27me3) in the same region of the TLR9 promoter. Ectopic expression of TLR9 in HPV38 E6/E7 cells resulted in an accumulation of the cell cycle inhibitors p21WAF1 and p27Kip1, decreased CDK2-associated kinase activity and inhibition of cellular proliferation. In summary, our data show that HPV38, similarly to other viruses with well-known oncogenic activity can down-regulate TLR9 expression. In addition, they highlight a new role for TLR9 in cell cycle regulation.

    IMPORTANCE The mucosal high-risk HPV types have been clearly associated with human carcinogenesis. Emerging lines of evidence suggest the involvement of certain cutaneous HPV types in development of skin squamous cell carcinoma, although this association is still under debate. Oncogenic viruses have evolved different strategies to hijack the host immune-system in order to guarantee the persistence of the infection. Their capability to evade the immune system is as important as their ability to promote cellular transformation. Therefore, understanding the viral mechanisms involved in viral persistence is a valid tool to evaluate their potential role in human carcinogenesis. Here, we show that E6 and E7 oncoproteins from the cutaneous HPV type 38 down-regulate the expression of the double-stranded DNA sensor TLR9 of innate immunity. We also present evidence that the HPV38-mediated down-regulation of TLR9 expression, in addition to its potential impact on the innate immune response, is linked to cell cycle deregulation.

  • Mapping enterovirus A71 antigenic determinants from viral evolution [PublishAheadOfPrint]

  • Human enterovirus A71 (EV-A71) belongs to the Enterovirus A species in the picornaviridae family. Several vaccines against EV-A71, a disease causing severe neurological complications or even death, are currently under development and tested in clinical trials, and preventative vaccination programs are expected to start soon. To characterize the potential for antigenic change of EV-A71, we compared the sequences of two antigenically diverse genotype B4 and B5 strains of EV-A71 and identified substitutions at residues 98, 145, and 164 in the VP1 capsid protein as antigenic determinants. To examine the effect of these three substitutions on antigenicity, we constructed a series of recombinant viruses containing different mutation combinations at these three residues with a reverse genetics system and then investigated the molecular basis of antigenic changes with antigenic cartography. We found that a novel EV-A71 mutant, containing lysine, glutamine, and glutamic acid at the respective residues 98, 145, and 164 in the VP1 capsid protein, exhibited neutralization reduction against patients' antisera and substantially increased virus binding ability to human cells. These observations indicated that this low neutralization-reactive EV-A71 VP1-98K/145Q/164E mutant potentially increases viral binding ability, and that surveillance studies should look out for these mutants that could compromise vaccine efficacy.

    IMPORTANCE Emerging and re-emerging EV-A71 viruses can cause severe neurological etiology, primarily affecting children, especially around Asia-Pacific countries. We identified a set of mutations in EV-A71 that both reduced neutralization activity against humoral immunity in antisera of patients and healthy adults and greatly increased the viral binding ability to cells. These findings provide important insights for EV-A71 antigenic determinants, and emphasize the importance of continuous surveillance, especially after EV-A71 vaccination programs begin.

  • STAT3 Regulates Lytic Activation of Kaposi's Sarcoma-associated Herpesvirus [PublishAheadOfPrint]

  • Lytic activation of Kaposi's Sarcoma-associated Herpesvirus (KSHV) from latency is a critical contributor to pathogenesis and progression of KSHV-mediated disease. Development of targeted treatment strategies and improvement of lytic phase-directed oncolytic therapies therefore hinge on gaining a better understanding of latency-to-lytic transition. A key observation in that regard, also common to other herpesviruses, is the partial permissiveness of latently-infected cells to lytic cycle inducing agents. Here, we address the molecular basis for why only some KSHV-infected cells respond to lytic stimuli. Since cellular Signal Transducer and Activator of Transcription 3 (STAT3) is overactive in KSHV-associated cancers, KSHV activates STAT3, and STAT3 has been found to regulate lytic activation of Epstein-Barr virus (EBV)-infected cells, we asked if STAT3 contributes similarly to the life cycle of KSHV. We found that high levels of STAT3 correlate with the refractory state at the single cell level under conditions of both spontaneous and induced lytic activation; importantly, STAT3 also regulates lytic susceptibility. Further, knockdown of STAT3 suppresses the cellular transcriptional co-repressor Kruuuml;ppel-associated box domain-associated protein 1 (KAP1), and suppression of KAP1 activates lytic genes including the viral lytic switch RTA, thereby linking STAT3 via KAP1 to regulation of the balance between lytic and latent cells. These findings, taken together with those from EBV-infected, and more recently HSV-1-infected cells, cement the contribution of host STAT3 to persistence of herpesviruses, and simultaneously reveal an important lead to devise strategies to improve lytic phase-directed therapies for herpesviruses.

    IMPORTANCE Lytic activation of the cancer-causing Kaposi's Sarcoma-associated Herpesvirus (KSHV) is vital to its life cycle and causation of disease. Like other herpesviruses, however, a substantial fraction of latently-infected cells are resistant to lytic-inducing stimuli. Investigating the molecular basis for this refractory state is essential for understanding how the virus persists, how it causes disease, and to guide efforts to improve treatment of KSHV-mediated diseases. We find that like two other herpesviruses, EBV and HSV-1, KSHV exploits the cellular transcription factor STAT3 to regulate susceptibility of latently-infected cells to lytic triggers. These findings highlight a common STAT3-centered strategy used by herpesviruses to maintain persistence in their host, while also revealing a key molecule to pursue while devising methods to improve herpesvirus lytic phase-directed therapies.

  • Immune protection against virus challenge in aging mice is not affected by latent herpesviral infections [PublishAheadOfPrint]

  • Latent herpesvirus infections alter the immune homeostasis. To understand if this results in aging-related loss of immune protection against emerging infections, we challenged old mice carrying latent mouse CMV, HSV-1 and/or MHV-68 with Influenza, WNV or VSV. We observed no increase in mortality or weight-loss over herpesvirus-negative counterparts and a relative, but no absolute reduction in CD8 responses against acute infections. Therefore, herpesviruses do not appear to increase susceptibility to emerging infections in aging.

  • The ns12.9 accessory protein of human coronavirus OC43 is a viroporin involved in virion morphogenesis and pathogenesis [PublishAheadOfPrint]

  • An accessory gene between the S and E gene loci is contained in all coronaviruses (CoVs), and its function has been studied in some coronaviruses. This gene locus in human coronavirus OC43 (HCoV-OC43) encodes the ns12.9 accessory protein; however, its function during viral infection remains unknown. Here, we engineered a recombinant mutant virus lacking the ns12.9 protein (HCoV-OC43-ns12.9) to characterize the contributions of ns12.9 in HCoV-OC43 replication. The ns12.9 accessory protein is a transmembrane protein and forms ion channels in both Xenopus oocytes and yeast through homo-oligomerization, suggesting that ns12.9 is a newly recognized viroporin. HCoV-OC43-ns12.9 presented at least 10-fold reduction of viral titer in vitro and in vivo. Intriguingly, exogenous ns12.9 and heterologous viroporins with ion channel activity could compensate the production of HCoV-OC43-ns12.9, indicating that the ion channel activity of ns12.9 plays a significant role in the production of infectious virions. Systematic dissection of single-cycle replication revealed that ns12.9 protein had no measurable effect on virus entry, subgenomic messenger RNA (sgmRNA) synthesis and protein expression. Further characterization revealed that HCoV-OC43-ns12.9 was less efficient in virion morphogenesis than recombinant wild-type virus (HCoV-OC43-WT). Moreover, reduced viral replication, inflammatory response and virulence in HCoV-OC43-ns12.9-infected mice were observed compared with HCoV-OC43-WT-infected mice. Taken together, our results demonstrated that the ns12.9 accessory protein functions as a viroporin, and is involved in virion morphogenesis and the pathogenesis of HCoV-OC43 infection.

    IMPORTANCE HCoV-OC43 was isolated in the 1960s and is a major agent of the common cold. The functions of HCoV-OC43 structural proteins have been well studied, but few studies have focused on its accessory proteins. In the present study, we demonstrated that the ns12.9 protein is a newly recognized viroporin, and the ns12.9 gene knockout virus (HCoV-OC43-ns12.9) presents a growth defect in vitro and in vivo. We identified the important functions of the ns12.9 viroporin in virion morphogenesis during HCoV-OC43 infection. Furthermore, mice infected with HCoV-OC43-ns12.9 exhibited reduced inflammation and virulence accompanied by a lower titer in the brain compared with wild-type infected mice, suggesting the ns12.9 viroporin influences virus pathogenesis. Therefore, our findings revealed that the ns12.9 viroporin facilitates the virion morphogenesis to enhance viral production, and these results provided a deeper understanding of HCoV-OC43 pathogenesis.

  • HSV capsid/organelle association in the absence of the large tegument protein UL36p [PublishAheadOfPrint]

  • UL36p (VP1/2) is the largest protein encoded by HSV-1 and resides in the innermost layer of the viral tegument, lying between the capsid and the envelope. UL36p performs multiple functions in the HSV life cycle, including an essential role in cytoplasmic envelopment. We earlier described the isolation of a virion-associated cytoplasmic membrane fraction from HSV-infected cells. Biochemical and ultrastructural analyses showed that the organelles in this buoyant fraction contain enveloped infectious HSV particles in their lumens, and naked capsids docked to their cytoplasmic surfaces. These organelles can also recruit molecular motors and transport their cargo virions along microtubules in vitro. Here we examine the properties of these HSV-associated organelles in the absence of UL36p. We find that, while capsid envelopment is clearly defective, a subpopulation of capsids nevertheless still associate with the cytoplasmic faces of these organelles. The existence of these capsid/membrane structures was confirmed by subcellular fractionation, immunocytochemistry, lipophilic dye fluorescence microscopy, thin section electron microscopy and correlative light and electron microscopy. We conclude that capsid/membrane binding can occur in the absence of UL36p, and propose that this association may precede the events of UL36p-driven envelopment.

    IMPORTANCE Membrane-association and envelopment of the HSV capsid is essential for the assembly of an infectious virion. Envelopment involves the complex interplay of a large number of viral and cellular proteins, however the function of most of them is unknown. One example of this is the viral protein UL36p, which is clearly essential for envelopment but plays a poorly understood role. Here we demonstrate that organelles utilized for HSV capsid envelopment still accumulate surface-bound capsids in the absence of UL36p. We propose that UL36p-independent binding of capsids to organelles occurs prior to the function of UL36p in capsid envelopment.

  • Structural basis of the inhibition of STAT1 activity by Sendai virus C protein [PublishAheadOfPrint]

  • Sendai virus C protein inhibits the signal transduction pathways of interferon (IFN)-aalpha;/bbeta; and IFN- by binding to the N-terminal domain of STAT1 (STAT1ND), thereby allowing SeV to escape from host innate immunity. Here we determined the crystal structure of STAT1ND associated with the C-terminal half of the C protein (Y3, amino acids 99nndash;204) at a resolution of 2.0 AAring;. This showed that two molecules of Y3 symmetrically bind to each niche created between two molecules of the STAT1ND dimer. Molecular modeling suggested that an anti-parallel form of the full-length STAT1 dimer can bind only one Y3 molecule and that a parallel form can bind two Y3 molecules. Affinity analysis demonstrated anti-cooperative binding of two Y3 molecules with the STAT1 dimer, which is consistent with the hypothetical model that the second Y3 molecule can only target the STAT1 dimer in a parallel form. STAT1 with excess amounts of Y3 was prone to inhibit the dephosphorylation at Tyr701 by a phosphatase. In an electrophoretic mobility shift assay, tyrosine-phosphorylated (pY)-STAT1 with Y3 associated with the -activated sequence probably as high molecular weight complexes (HMWCs), which may account for partial inhibition of a reporter assay from IFN- by Y3. Our study suggests that the full-length C protein interferes with the domain arrangement of the STAT1 dimer, leading to the accumulation of pY-STAT1 and the formation of HMWCs. In addition, we discuss the mechanism by which phosphorylation of STAT2 is inhibited in the presence of the C protein after stimulation by IFN-aalpha;/bbeta;.

    IMPORTANCE Sendai virus, a paramyxovirus that causes respiratory diseases in rodents, possesses the C protein, which inhibits the signal transduction pathways of interferon-aalpha;/bbeta; and interferon- by binding to the transcription factor STAT1. In virus-infected cells, phosphorylation of STAT1 at the Tyr701 residue is potently enhanced, although transcription by STAT1 is inert. Here, we determined the crystal structure of the N-terminal domain of STAT1 associated with the C-terminal half of the C protein. Molecular modeling and experiments suggested that the two C proteins bind to and stabilize the parallel form of the STAT1 dimer, which are likely to be phosphorylated at Tyr701, further inducing high molecular weight complex formation and inhibition of transcription by interferon-. We also discuss the possible mechanism of inhibition of the interferon-aalpha;/bbeta; pathways by the C protein. This is the first structural report of the C protein, suggesting a mechanism of evasion of the paramyxovirus from innate immunity.

  • Molecular determinants of virulence and stability of a reporter-expressing H5N1 influenza A virus [PublishAheadOfPrint]

  • We previously reported that an H5N1 virus carrying the Venus reporter gene, which was inserted into the NS gene segment from A/Puerto Rico/8/1934 (H1N1) virus (Venus-H5N1 virus), became more lethal to mice and the reporter gene was more stably maintained after mouse adaptation compared with the wild-type Venus-H5N1 (WT-Venus-H5N1) virus. However, the basis for this difference in virulence and Venus stability was unclear. Here, we investigated the molecular determinants behind this virulence and reporter stability by comparing WT-Venus-H5N1 virus with a mouse-adapted Venus-H5N1 (MA-Venus-H5N1) virus. To determine the genetic basis for these differences, we used reverse genetics to generate a series of reassortants of these two viruses. We found that reassortants with PB2 from MA-Venus-H5N1 (MA-PB2), MA-PA, or MA-NS expressed Venus more stably than did WT-Venus-H5N1 virus. We also found that a single mutation in PB2 (V25A) or in PA (R443K) increased the virulence of WT-Venus-H5N1 virus in mice, and that the presence of both of these mutations substantially enhanced the pathogenicity of the virus. Our results suggest roles for PB2 and PA in the stable maintenance of a foreign protein as an NS1 fusion protein in influenza A virus.

    IMPORTANCE The ability to visualize influenza viruses has far-reaching benefits in influenza virus research. Previously, we reported that an H5N1 virus bearing the Venus reporter gene became more pathogenic to mice and its reporter gene was more highly expressed and more stably maintained after mouse adaptation. Here, we investigated the molecular determinants behind this enhanced virulence and reporter stability. We found that mutations in PB2 (V25A) and PA (R443K) play crucial roles in the stable maintenance of a foreign protein as an NS1 fusion protein in influenza A virus and in the virulence of influenza virus in mice. Our findings further our knowledge of the pathogenicity of influenza virus in mammals and will help advance influenza virus-related live imaging studies in vitro and in vivo.

  • Epstein-Barr viruses deficient in EBER RNAs give higher LMP2 RNA expression in lymphoblastoid cell lines and efficiently establish persistent infection in humanized mice [PublishAheadOfPrint]

  • Functions of EBER RNAs were tested in lymphoblastoid cell lines containing EBER mutants of Epstein-Barr Virus (EBV). Binding of EBER1 to RPL22 was confirmed. Deletion of EBER1 or EBER2 correlated with increased cytoplasmic EBV LMP2 RNA and with small effects on specific cellular miRNA levels but protein levels of LMP1 and LMP2A were not affected. Wild type and EBER deletion EBV had approximately equal ability to infect immunodeficient mice reconstituted with a human haematopoietic system.

  • Release of VSVG-pseudotyped Lentivirus from the Host Cell is impaired upon Low-density Lipoprotein Receptor Overexpression [PublishAheadOfPrint]

  • Production of a VSVG-pseudotyped lentiviral expression vector in HEK293 cells decreased on overexpression of LDLR but not of ICAM1 or TfR1. RT-qPCR revealed a reduction in vector RNA as a function of LDLR expression. Decreased syncytia formation suggested diminished surface expression of VSVG. Intracellular VSVG granules colocalized with LDLR, ERGIC53, LAMP2, and vimentin, but neither with GM130 nor calnexin suggesting that VSVG interacts with LDLR within the ERGIC resulting in re-routing into the aggresome/autophagosome pathway.

  • The genome of a tortoise herpesvirus (testudinid herpesvirus 3) has a novel structure and contains a large region that is not required for replication in vitro or virulence in vivo [PublishAheadOfPrint]

  • Testudinid herpesvirus 3 (TeHV-3) is the causative agent of a lethal disease affecting several tortoise species. The threat that this virus poses to endangered animals is focusing efforts on characterizing its properties, in order to enable the development of prophylactic methods. We have sequenced the genomes of the two most studied TeHV-3 strains (1976 and 4295). TeHV-3 strain 1976 has a novel genome structure and is most closely related to a turtle herpesvirus, thus supporting its classification into genus Scutavirus, subfamily Alphaherpesvirinae, family Herpesviridae. The sequence of strain 1976 also revealed viral counterparts of cellular interleukin-10 and semaphorin, which have not been described previously in members of subfamily Alphaherpesvirinae. TeHV-3 strain 4295 is a mixture of three forms (m1, m2, and M), in which, in comparison to strain 1976, the genomes exhibit large, partially overlapping deletions of 12.5 to 22.4 kb. Viral subclones representing these forms were isolated by limiting dilution, and each replicated in cell culture comparably to strain 1976. With the goal of testing the potential of the three forms as attenuated vaccine candidates, strain 4295 was inoculated intranasally into Hermann's tortoises (Testudo hermanni). All inoculated subjects died, and PCR analyses demonstrated the ability of the m2 and M forms to spread and invade the brain. In contrast, the m1 form was detected in none of the organs tested, suggesting its potential as the basis of an attenuated vaccine candidate. Our findings represent a major step towards characterizing TeHV-3 and developing prophylactic methods against it.

    IMPORTANCE Testudinid herpesvirus 3 (TeHV-3) causes a lethal disease in tortoises, several species of which are endangered. We have characterized the viral genome, and used this information to take steps towards developing an attenuated vaccine. We have sequenced the genomes of two strains (1976 and 4295), compared their growth in vitro, and investigated the pathogenesis of strain 4295, which consists of three deletion mutants. The major findings are: (i) TeHV-3 has a novel genome structure; (ii) its closest relative is a turtle herpesvirus; (iii) it contains interleukin-10 and semaphorin genes, the first time these have been reported in an alphaherpesvirus; (iv) a sizeable region of the genome is not required for viral replication in vitro or virulence in vivo; and (v) one of the components of strain 4295, which has a deletion of 22.4 kb, exhibits properties indicating that it may serve as the starting point for an attenuated vaccine.

  • Influences on the design and purification of soluble, recombinant native-like HIV-1 envelope glycoprotein trimers [PublishAheadOfPrint]

  • We have investigated factors that influence the production of native-like soluble, recombinant trimers based on the env genes of two isolates of human immunodeficiency virus type 1 (HIV-1), specifically 92UG037.8 (clade A) and CZA97.012 (clade C). When made according to the SOSIP.664 design and purified by affinity chromatography using broadly neutralizing antibodies (bNAbs) against quaternary epitopes (PGT145 and PGT151, respectively), the resulting trimers are highly stable and fully native-like. They also have a native-like (i.e., abundant) oligomannose glycan composition, and display multiple bNAb epitopes while occluding those for non-neutralizing antibodies. In contrast, uncleaved, Foldon (Fd) domain-containing gp140 proteins (gp140UNC-Fd-His), based on the same env genes, very rarely form native-like trimers consistent with their antigenic and biophysical properties and glycan composition. The addition of a 20-residue flexible linker between the gp120 and gp41ECTO subunits to make the uncleaved 92UG037.8 gp140-FL20 construct is not sufficient to create a native-like trimer, but a small percentage of native-like trimers were produced when an I559P substitution in gp41ECTO was also present. The further addition of a disulfide bond (SOS) to link the gp120 and gp41 subunits in the uncleaved gp140-FL20-SOSIP protein increases native-like trimer formation to ~20-30%. Analysis of the disulfide bond content shows that misfolded gp120 subunits are abundant in uncleaved CZA97.012 gp140UNC-Fd-His proteins but very rare in native-like trimer populations. The design and stabilization method and the purification strategy are, therefore, all important influences on the quality of trimeric Env proteins and hence their suitability as vaccine components.

    IMPORTANCE Soluble, recombinant multimeric proteins based on the HIV-1 env gene are current candidate immunogens for vaccine trials in humans. These proteins are generally designed to mimic the native trimeric envelope glycoprotein (Env) that is the target of virus-neutralizing antibodies on the surface of virions. The underlying hypothesis is that an Env-mimetic protein may be able to induce antibodies that can neutralize the virus broadly and potently enough for a vaccine to be protective. Multiple different designs for Env-mimetic trimers have been put forth. Here, we used the CZA97.012 and 92UG037.8 env genes to compare some of these designs and determine which ones best mimic virus-associated Env trimers. We conclude that the most widely used versions of CZA97.012 and 92UG037.8 oligomeric Env proteins do not resemble the trimeric Env glycoprotein on HIV-1 viruses, which has implications for the design and interpretation of ongoing or proposed clinical trials of these proteins.

  • Long-term effect of serial infections with H13 and H16 low pathogenic avian influenza viruses in black-headed gulls [PublishAheadOfPrint]

  • Infections of domestic and wild birds with low pathogenic avian influenza viruses (LPAIV) have been associated with protective immunity to subsequent infection. However, the degree and duration of immunity in wild birds from previous LPAIV infectionmmdash;by the same or a different subtypemmdash;are poorly understood. Therefore, we inoculated H13N2 (A/Black-headed gull/Netherlands/7/2009) and H16N3 (A/Black-headed gull/Netherlands/26/2009) LPAIVs into black-headed gulls (Chroicocephalus ridibundus)mmdash;their natural host speciesmmdash;and measured the long-term immune response and protection against one or two re-infections over a period of more than one year. This is the typical interval between LPAIV epizootics in wild birds. Re-infection with the same virus resulted in progressively less virus excretion, with complete abrogation of virus excretion after two infections for H13 but not H16. However, re-infection with the other virus affected neither level nor duration of virus excretion. Virus excretion by immunologically naiiuml;ve birds did not differ in total level of excreted H13 or H16 virus between first- and second-year birds, but duration of H13 excretion was shorter for second-year birds. Furthermore, serum antibody levels did not correlate with protection against LPAIV infection. LPAIV-infected gulls showed no clinical signs of disease. These results imply that the epidemiological cycles of H13 and H16 in black-headed gulls are relatively independent from each other and depend mainly on infection of first-year birds.

    IMPORTANCE Low pathogenic avian influenza viruses (LPAIV) circulate mainly in wild waterbirds, but are occasionally transmitted to other species including humans, where they cause subclinical to fatal disease. To date, the effect of LPAIV-specific immunity on the epidemiology of LPAIV in wild birds is poorly understood. In this study, we investigated the effect of H13 and H16 LPAIV infection in black-headed gulls on susceptibility and virus excretion of subsequent infection with the same or the other virus within the same breeding season and between breeding seasons. These are the only two LPAIV hemagglutinin subtypes predominating in this species. The findings suggest H13 and H16 LPAIV cycles in black-headed gull populations are independent of each other, indicate the importance of first-year birds in LPAIV epidemiology and emphasize the need for alternatives to AIV-specific serum antibodies as evidence of past LPAIV infection and correlate of protection against LPAIV infection in wild birds.

  • HuR displaces PTB to facilitate La binding to the 3' UTR and enhances HCV replication [PublishAheadOfPrint]

  • HuR is a ubiquitous, RNA binding protein that influences the stability and translation of several cellular mRNAs. Here we report a novel role for HuR, as a regulator of proteins assembling at the 3rrsquo; untranslated region of viral RNA in the context of Hepatitis C virus infection. HuR relocalizes from the nucleus to the cytoplasm upon HCV infection, interacts with the viral polymerase (NS5B) and gets redistributed into compartments of viral RNA synthesis. Depletion in HuR levels leads to significant reduction in viral RNA synthesis. We further demonstrate that the interaction of HuR with the 3rrsquo; UTR of the viral RNA affects the interaction of two host proteins La and PTB at this site. HuR interacts with La and facilitates La binding to the 3rrsquo; UTR, enhancing La mediated circularization of the HCV genome and thus viral replication. In addition, it competes with the polypyrimidine tract binding protein (PTB) for association with the 3rrsquo; UTR, which might stimulate viral replication. Results suggest that HuR influences the formation of a cellular/viral ribonucleoprotein complex, which is important for efficient initiation of viral RNA replication. Our study unravels a novel strategy of regulation of HCV replication through interplay of host and viral proteins, orchestrated by HuR.

    IMPORTANCE Hepatitis C virus (HCV) is highly dependent on various host factors for efficient replication of the viral RNA. Here, we have shown how a host factor (HuR) migrates from the nucleus to the cytoplasm and gets recruited in the protein complex assembling at the 3rrsquo; untranslated region (3rrsquo; UTR) of HCV RNA. At the 3rrsquo; UTR, it facilitates circularization of the viral genome through interaction with another host factor La, which is critical for replication. Also, it competes with the host protein PTB, which is a negative regulator of viral replication. Results demonstrate a unique strategy of regulation of HCV replication by a host protein through alteration of its subcellular localization and interacting partners. The study has advanced our knowledge of the molecular mechanism of HCV replication and unravelled the complex interplay between the host factors and viral RNA that could be targeted for therapeutic interventions.

  • Viral small RNA analysis of Bombyx mori larval midgut during persistent and pathogenic cytoplasmic polyhedrosis virus infection [PublishAheadOfPrint]

  • Lepidopteran innate immune response against RNA viruses remains poorly understood, while in other insects several studies have highlighted an essential role for the exo-RNAi pathway in combating viral infection. Here, by employing deep sequencing technology for viral small RNA (vsRNA) assessment, we provide evidence that exo-RNAi is operative in the silkworm Bombyx mori against both persistent and pathogenic infection of B. mori cytoplasmic polyhedrosis virus (BmCPV) which is characterized by a segmented dsRNA genome. Further, we show that Dicer-2 predominantly targets viral dsRNA and produces 20 nt vsRNAs, whereas an additional pathway is responsive to viral mRNA derived from segment 10. Importantly, vsRNA distributions, which define specific hot- and cold-spot profiles for each viral segment, are to a considerable degree overlapping between Dicer-2-related (19-21 nt) and Dicer-2-unrelated vsRNAs suggesting a common origin for these profiles. We found a degenerate motif significantly enriched at the cut sites of vsRNAs of various lengths which link an unknown ribonuclease to the origins of vsRNAs biogenesis and distribution. Accordingly, the indicated RNase activity may be an important early factor for the host's antiviral defense in Lepidoptera.

    IMPORTANCE This work contributes to the elucidation of the lepidopteran antiviral response against infection of segmented dsRNA virus (CPV; Reoviridae) and highlights the importance of vsRNA analysis for getting insights into host-pathogen interactions. Three vsRNA pathways are implicated in antiviral defense. For dsRNA, two pathways are proposed, either based on Dicer-2 cleavage to generate 20 nt vsRNAs, or based on the activity of an uncharacterized endoRNase that cleaves the viral RNA substrate at a degenerate motif. The analysis indicates also the existence of a degradation pathway that targets the (+)-strand of segment 10.

  • Preferential budding of vesicular stomatitis virus from the basolateral surface of polarized epithelial cells is not solely directed by matrix protein or glycoprotein [PublishAheadOfPrint]

  • VSV has been shown to bud basolaterally, and the matrix protein but not glycoprotein was proposed to mediate this asymmetry. Using polarized T84 monolayers, we demonstrate that no single viral protein is sufficient for polarized budding. Particles are released from the apical and basolateral surfaces and are indistinguishable, indicating that there is no apical assembly defect. We propose that aspects of host cell polarity create a more efficient budding process at the basolateral surface.

  • Th1/17 polarization of CD4 T cells supports HIV-1 DNA persistence during antiretroviral therapy [PublishAheadOfPrint]

  • The ability to persist long term in latently-infected CD4 T cells represents a characteristic feature of HIV-1 infection and the predominant barrier to efforts aiming at viral eradication and cure. Yet, increasing evidence suggest that only small subsets of CD4 T cells with specific developmental and maturational profiles are capable to effectively support HIV-1 long-term persistence. Here, we analyzed how the functional polarization of CD4 T cells shapes and structures the reservoirs of HIV-1 infected cells. We found that CD4 T cells enriched for a Th1/Th17 polarization had elevated susceptibilities to HIV-1 infection in ex-vivo assays, harbored high levels of HIV-1 DNA in persons treated with antiretroviral therapy, and made a disproportionately increased contribution to the viral reservoir relative to their contribution to the CD4 T memory cell pool. Moreover, HIV-1 DNA levels in Th1/Th17 cells remained stable over many years of antiretroviral therapy, resulting in a progressively increasing contribution of these cells to the viral reservoir, and phylogenetic studies suggested preferential long-term persistence of identical viral sequences during prolonged antiretroviral treatment in this cell compartment. Together, these data suggest that Th1/Th17 CD4 T cells represent a preferred site for HIV-1 DNA long-term persistence in patients receiving antiretroviral therapy.

    IMPORTANCE Current antiretroviral therapy is very effective in suppressing active HIV-1 replication, but does not fully eliminate virally infected cells. The ability of HIV-1 to persist long-term despite suppressive antiretroviral combination therapy represents a perplexing aspect of HIV-1 disease pathogenesis, since most HIV-1 target cells are activated, short-lived CD4 T cells. This study suggests that CD4 T helper cells with Th1/Th17 polarization have a preferential role as a long-term reservoir for HIV-1 infection during antiretroviral therapy, possibly because these cells may imitate some of the functional properties traditionally ascribed to stem cells, such as the ability to persist for extremely long periods of time and to repopulate their own pool size through homeostatic self-renewal. These observations support the hypothesis that HIV-1 persistence is driven by small subsets of long-lasting stem cell-like CD4 T cells that may represent particularly promising targets for clinical strategies aiming at HIV-1 eradication and cure.

  • Differential PI3K-Akt-mTOR activation by Semliki Forest and chikungunya virus, dependent on nsP3 and connected to replication complex internalisation [PublishAheadOfPrint]

  • Many viruses affect or exploit the phosphatidylinositol-3-kinase (PI3K)nndash;Aktnndash;mammalian target of Rapamycin (mTOR) pathway, a crucial pro-survival signalling cascade. We report that this pathway was strongly activated in cells upon infection with the Old World alphavirus Semliki Forest virus (SFV), even under conditions of complete nutrient starvation. We mapped this activation to the hyperphosphorylated/acidic domain in the C-terminal tail of SFV non-structural protein nsP3. Viruses with a deletion of this domain (SFV-50), but not other regions in nsP3, displayed a clearly delayed and reduced capacity of Akt stimulation. Ectopic expression of nsP3-wildtype, but not -50, equipped with a membrane anchor was sufficient to activate Akt. We linked PI3Knndash;Aktnndash;mTOR stimulation to the intracellular dynamics of viral replication complexes, which are formed at the plasma membrane and subsequently internalised in a process blocked by the PI3K inhibitor Wortmannin. Replication complex internalisation was observed upon infection of cells with SFV-wt and SFV mutants with deletions in nsP3, but not with SFV-50, where replication complexes were typically accumulated at the cell periphery. In cells infected with the closely related chikungunya virus (CHIKV), the PI3Knndash;Aktnndash;mTOR pathway was only moderately activated. Replication complexes of CHIKV were predominantly located at the cell periphery. Exchanging the hypervariable C-terminal tail of nsP3 between SFV and CHIKV induced the phenotype of strong PI3Knndash;Aktnndash;mTOR activation and replication complex internalisation in CHIKV. In conclusion, infection with SFV but not CHIKV boosts PI3Knndash;Aktnndash;mTOR through the hyperphosphorylated/acidic domain of nsP3 to drive replication complex internalisation.

    Importance SFV and CHIKV are very similar in terms of molecular and cell biology, e.g. regarding replication and molecular interactions, but are strikingly different regarding pathology: CHIKV is a relevant human pathogen, causing high fever and joint pain, while SFV is a low-pathogenic model virus, albeit neuropathogenic in mice. We show that SFV and CHIKV both activate the pro-survival PI3Knndash;Aktnndash;mTOR pathway in cells, but greatly differ in their capacity to do so: Akt is strongly and persistently activated by SFV infection, but only moderately by CHIKV. We mapped this activation capacity to a region in the non-structural protein 3 of SFV and could functionally transfer this region to CHIKV. Akt activation is linked to the subcellular dynamics of replication complexes, which are efficiently internalised from the cell periphery for SFV but not CHIKV. This difference in signal pathway stimulation and replication complex localisation may have implications for pathology.

  • Characterising the diverse mutational pathways associated with R5-tropic maraviroc resistance: HIV-1 that uses the drug-bound CCR5 coreceptor [PublishAheadOfPrint]

  • Entry-inhibitors represent a potent class of antiretroviral drugs that target a host cell protein, CCR5, an HIV-1 entry coreceptor, and not viral protein. Lack of sensitivity can occur due to pre-existing virus that uses the CXCR4 coreceptor, while true resistance occurs through viral adaptation to use drug-bound CCR5 coreceptor. To understand this R5-resistance pathway we analysed ggt;500 envelope protein sequences and phenotypes from viruses of 20 patients from the clinical trials MOTIVATE 1 and 2 in which treatment experienced patients received maraviroc plus optimised background therapy. The resistant viral population was phylogenetically distinct and associated with a genetic bottleneck in each patient, consistent with de novo emergence of resistance. Recombination analysis shows the C2-V3-C3 region tends to genotypically correspond to the recombinant's phenotype, indicating its primary importance in conferring resistance. Between patients, there is a notable lack of commonality in the specific sites conferring resistance confirming the unusual nature of R5-tropic resistance. We use coevolutionary and positive selection analysis to characterise the genotypic determinants of resistance and find: (1) complicated covariation networks indicating frequent coevolutionary/compensatory changes in the context of protein structure; (2) covarying sites under positive selection are enriched in resistant viruses; (3) CD4 binding sites form part of a unique covariation network independent of the V3 loop; (4) the covariation network formed between the V3 loop, and other regions of gp120 and gp41 intersects with sites involved in glycosylation and protein secretion. These results demonstrate that while envelope sequence mutations are the key to conferring maraviroc resistance, the specific changes involved are context-dependent and thus inherently unpredictable.

    Importance The entry-inhibitor drug maraviroc makes the cell coreceptor CCR5 unavailable for use by HIV-1 and is now used in combination antiretroviral therapy. Treatment failure with drug resistant virus is particularly interesting because it tends to be rare with lack of sensitivity usually associated with the presence of CXCR4-using virus (the main alternative coreceptor HIV-1 uses in addition to CD4). We analysed envelope sequences from HIV-1 obtained from 20 patients who enrolled in maraviroc clinical trials and experienced treatment failure without detection of CXCR4-using virus. Evolutionary analysis was employed to identify molecular changes that confer maraviroc resistance. We find that in these individuals, resistant viral populations form a distinct population that evolved once and were successful as a result of the drug pressure. Further evolutionary analysis places the complex network of inter-dependent mutational changes into functional groups that help explain the impediments to the emergence of maraviroc-associated R5 drug resistance.

  • Tracking the fate of genetically distinct vesicular stomatitis virus matrix proteins highlights the role for late domains in assembly [PublishAheadOfPrint]

  • Vesicular stomatitis virus (VSV) assembly requires condensation of the viral ribonucleoprotein (RNP) core with the matrix protein (M) during budding from the plasma membrane. The RNP core comprises the negative-sense genomic RNA completely coated by the nucleocapsid protein (N) and associated by a phosphoprotein (P) to the large polymerase protein (L). To study assembly of single viral particles, we tagged M and P with fluorescent proteins. We selected from a library of insertions in the M gene, a replication competent virus containing a fluorescent M, and combined that with our previously described virus containing fluorescent P. Virus particles containing those fusions maintain the same bullet-shape appearance as wild type VSV with a modest increase in particle length reflecting the increased genome size. Imaging of the released particles reveals a variation in the amount of M and P assembled into the virions, consistent with a flexible packaging mechanism. We used the recombinants to further study the importance of the "late" domains in M, which serve to recruit the endosomal sorting complexes required for transport (ESCRT) machinery during budding. Mutations in late domains result in the accumulation of virions that fail to pinch off from the plasma membrane. Imaging of single virions released from cells that were coinfected with M-eGFP and M-mCherry variants in which the late domains of one virus were inactivated by mutation showed a strong bias against the incorporation of the late domain mutant into the released virions. By contrast, intracellular expression and membrane association of the two variants were unaltered. These studies provide new tools for imaging particle assembly and enhance our resolution of existing models for assembly of VSV.

    Importance Assembly of vesicular stomatitis virus (VSV) particles requires the separate trafficking of the viral replication machinery, a matrix protein (M) and a glycoprotein to the plasma membrane. The matrix protein contains a motif termed a "late domain" that engages the host endosomal sorting complex required for transport (ESCRT) machinery to facilitate release of viral particles. Inactivation of the late domains through mutation results in the accumulation of virions arrested at the point of release. Here we develop new tools to study VSV assembly by fusing fluorescent proteins to M and to a constituent of the replication machinery, the phosphoprotein (P). We use those tools to show that the late domains of M are required for efficient incorporation into viral particles and that the particles contain a variable quantity of M and P.

  • SLAM family receptor homologs in New World monkey cytomegaloviruses [PublishAheadOfPrint]

  • Throughout evolution, large DNA viruses have been usurping genes from their hosts to equip themselves with proteins that restrain host immune defenses. Signalling lymphocytic activation molecule (SLAM) family receptors are involved in the regulation of both innate and adaptive immunity, which occurs upon engagement with their ligands via homotypic or heterotypic interactions. Here we report a total of seven SLAM family (SLAMF) genes encoded by two cytomegalovirus (CMV) species, squirrel monkey CMV (SMCMV) and owl monkey CMV (OMCMV), that infect New World monkeys. Our results indicate that host genes were captured by retrotranscription at different stages of the CMV-host co-evolution. The most recent acquisition led to S1 in SMCMV, which encodes a SLAMF6 homolog with an amino acid identity of 97% in its ligand-binding N-terminal Ig domain. We demonstrate that S1 is a cell-surface glycoprotein capable of binding to host SLAMF6. Furthermore, OMCMV encodes A33, a LY9 (SLAMF3) homolog, and A43, a CD48 (SLAMF2) homolog, two soluble glycoproteins which recognize their respective cellular counter-receptors, and thus are likely to be viral SLAMF decoy receptors. In addition, distinct copies of further divergent CD48 homologs were found in both CMV genomes. Remarkably, all these molecules display a number of unique features, including cytoplasmic tails lacking characteristic SLAMF signalling motifs. Taken together, our findings indicate a novel immune evasion mechanism in which incorporation of host SLAMF receptors that retain their ligand binding properties enables viruses to interfere with SLAMF functions and to supply themselves with convenient structural moulds for expanding their immunomodulatory repertoires.

    IMPORTANCE The way in which viruses shape their genomes, under the continual selective pressure exerted by the host immune system, is central for their survival. Here, we report that New World monkey cytomegaloviruses have broadly captured and duplicated immune-cell receptors of the signalling lymphocyte activation molecule (SLAM) family during host-virus co-evolution. Notably, we demonstrate that several of these viral SLAMs exhibit exceptional preservation of their N-terminal immunoglobulin domains, which results in maintenance of their ligand-binding capacities. At the same time, these molecules present distinctive structural properties, including soluble forms and absence of typical SLAM signalling motifs in their cytoplasmic domains, likely reflecting the evolutionary adaptation undergone to efficiently interfere with host SLAM family activities. The observation that other large DNA viruses might bear SLAM family homologs further underscores the importance of these molecules as a novel class of immune regulators and as convenient scaffolds for viral evolution.

  • Modification of the asparagine-linked glycan density for the design of hepatitis B virus (HBV) virus-like particles with enhanced immunogenicity [PublishAheadOfPrint]

  • The small envelope proteins (HBsAgS) derived from hepatitis B virus (HBV) represent the antigenic components of the HBV vaccine, and are platforms for the delivery of foreign antigenic sequences. To investigate structurenndash;immunogenicity relationships for the design of improved immunization vectors, we have generated biochemically modified VLPs exhibiting glycoengineered HBsAgS. For the generation of hypoglycosylated VLPs, the wildtype (WT) HBsAgS N146 glycosylation site was converted to N146Q; for constructing hyperglycosylated VLPs, potential glycosylation sites were introduced in the HBsAgS external loop region at positions T116 and G130 in addition to the WT site. The introduced sites T116N and G130N were utilized as glycosylation anchors resulting in the formation of hyperglycosylated VLPs. Mass spectroscopic analyses showed that the hyperglycosylated VLPs carry the same types of glycans as WT VLPs with minor variations regarding the degree of fucosylation, bisecting N-acetylglucosamins, and sialylation. Antigenic fingerprints for the WT, hypo-, and hyperglycosylated VLPs using a panel of 19 anti-HBsAgS monoclonal antibodies revealed that 15 antibodies retained their binding ability to the different VLP glyco-analogues suggesting that the additional N-glycans did not shield extensively for the HBsAgS-specific antigenicity. Immunization studies with the different VLPs showed a strong correlation between N-glycan abundance and antibody titres. The T116N VLPs induced earlier and longer lasting antibody responses compared to the hypoglycosylated and WT VLPs. The ability of non-native VLPs to promote immune responses possibly due to differences in their glycosylation-related interaction with the innate immune system illustrates pathways for the design of immunogens for superior preventive applications.

    IMPORTANCE The use of biochemically modified, non-native immunogens represent an attractive strategy for the generation of modulated or enhanced immune responses possibly due to differences in their interaction with immune cells. We have generated virus-like particles (VLPs) composed of hepatitis B virus envelope proteins (HBsAgS) with additional N-glycosylation sites. Hyperglycosylated VLPs were synthesized and characterized demonstrating that they carry the same types of glycans as wildtype VLPs. Comparative immunization studies demonstrated that the VLPs with the highest N-glycan density induce earlier and longer lasting antibody immune responses compared to wildtype or hypoglycosylated VLPs, possibly allowing reduced numbers of vaccine injections. The ability to modulate the immunogenicity of an immunogen will provide opportunities to develop optimized vaccines and VLP delivery platforms for foreign antigenic sequences, possibly in synergy with the use of suitable adjuvanting compounds.

  • Doubly Spliced RNA of Hepatitis B Virus Suppresses Viral Transcription via TATA-Binding Protein and Induces Stress Granule Assembly [PublishAheadOfPrint]

  • The risk of liver cancer in patients infected with the hepatitis B virus (HBV) and their clinical response to interferon-aalpha; therapy vary based on HBV genotype. The mechanisms underlying these differences in HBV pathogenesis remain unclear. In HepG2 cells transfected with a mutant HBVG2335A expression plasmid that does not transcribe the 2.2-kb doubly spliced RNA (2.2DS-RNA) expressed by wild-type HBV genotype A, the level of HBV pgRNA was higher than that in cells transfected with an HBV genotype-A expression plasmid. By using co-transfection with an HBV genotype-D and 2.2DS-RNA plasmids, we found that reduction of pgRNA was observed in the cells even in low amount of the 2.2DS-RNA plasmids. Moreover, ectopic expression of 2.2DS-RNA in the HBV-producing cell line, 1.3ES2, reduced the expression of pgRNA. Further analysis showed that exogenously transcribed 2.2DS-RNA inhibited a reconstituted transcription in vitro. In Huh7 cells ectopically expressing 2.2DS-RNA, RNA immunoprecipitation revealed that 2.2DS-RNA interacted with TATA-binding protein (TBP) and that nucleotides 432 to 832 of 2.2DS-RNA were required for efficient TBP binding. Immunofluorescence experiments showed that 2.2DS-RNA colocalized with cytoplasmic TBP and the stress granule components, G3BP and PABP1, in Huh7 cells. In conclusion, our study reveals that 2.2DS-RNA acts as a repressor of HBV transcription through an interaction with TBP that induces stress granule formation. The expression of 2.2DS-RNA may be one of viral factors involved in viral replication, which may underlie differences in clinical outcomes of liver disease and response to interferon-aalpha; therapy between patients infected with different HBV genotypes.

    IMPORTANCE Patients infected with certain genotypes of HBV have a lower risk of hepatocellular carcinoma, and exhibit a more favorable response to antiviral therapy than patients infected with other HBV genotypes. Using cultured human hepatoma cells as a model of HBV infection, we found that the expression of 2.2DS-RNA caused a decrease in HBV replication. In cultured cells, the ectopic expression of 2.2DS-RNA obviously reduced the intracellular levels of HBV mRNAs. Our analysis of the 2.2DS-RNA-mediated suppression of viral RNA expression showed that 2.2DS-RNA inhibited transcription via binding to TATA-binding protein and stress granule proteins. Our findings suggest that the 2.2DS-RNA acts as a suppressive noncoding RNA that modulates HBV replication, which may in turn influence the development of chronic hepatitis B.

  • Phenotypic Correlates of HIV-1 Macrophage Tropism [PublishAheadOfPrint]

  • HIV-1 is typically CCR5-using (R5) and T cell-tropic (T-tropic), targeting memory CD4+ T cells throughout acute and chronic infection. However, viruses can expand into alternative cells types. Macrophage-tropic (M-tropic) HIV-1 variants have evolved to infect macrophages, which have only low levels of surface CD4. Most M-tropic variants have been isolated from the central nervous system during late-stage chronic infection. We used the HIV-1 env genes of well-defined, subject-matched M-tropic and T-tropic viruses to characterize phenotypic features of the M-tropic Env protein. We found that, compared to T-tropic viruses, M-tropic viruses infect monocyte-derived macrophages (MDMs) on average 28-fold more efficiently, use low-density CD4 more efficiently, have increased sensitivity to soluble CD4 (sCD4), and show trends toward sensitivity to some CD4 binding site antibodies, but no difference in sensitivity to antibodies targeting the CD4-bound conformation. M-tropic viruses also displayed a trend toward resistance to neutralization by monoclonal antibodies targeting the V1/V2 region of Env, suggesting subtle changes in Env protein conformation. The paired M- and T-tropic viruses did not differ in autologous serum neutralization, temperature sensitivity, entry kinetics, intrinsic infectivity, or Env protein incorporation. We also examined viruses with modestly increased CD4 usage. These variants have significant sensitivity to sCD4 and may represent evolutionary intermediates. CD4 usage is strongly correlated with infectivity of MDMs over a wide range of CD4 entry phenotypes. These data suggest that emergence of M-tropic HIV-1 includes multiple steps in which a phenotype of increased sensitivity to sCD4 and enhanced CD4 usage accompany subtle changes in Env conformation.

    IMPORTANCE HIV-1 typically replicates in CD4+ T cells. However, HIV-1 can evolve to infect macrophages, especially within the brain. Understanding how CCR5-using macrophage-tropic viruses evolve and differ from CCR5-using T cell-tropic viruses may provide insights into viral evolution and pathogenesis within the central nervous system. We characterized the HIV-1 env viral entry gene from subject-matched macrophage-tropic and T cell-tropic viruses to identify entry features of macrophage-tropic viruses. We observed several differences between T cell-tropic and macrophage-tropic Env proteins, including functional differences with host CD4 receptor engagement and possible changes in the CD4 binding site and V1/V2 region. We also identified viruses with phenotypes between that of "true" macrophage-tropic and T cell-tropic viruses, which may represent evolutionary intermediates in a multi-step process to macrophage tropism.

  • Spatiotemporal reconstruction of the introduction of hepatitis C virus into Scotland and its subsequent regional transmission [PublishAheadOfPrint]

  • A more comprehensive understanding of hepatitis C virus (HCV) transmission dynamics could facilitate public health initiatives to reduce the prevalence of HCV in people who inject drugs. We aimed to determine how HCV sequences entered and spread throughout Scotland and to identify transmission hotspots. A Scottish dataset with embedded demographic data was created by sequencing the NS5B of 125 genotype (Gt) 1a and 166 Gt3a samples and analysed alongside sequences from public databases. Applying Bayesian inference methods, we reconstructed the global origin and local spatiotemporal dissemination of HCV in Scotland. Scottish sequences mainly formed discrete clusters interspersed between sequences from the rest of the world; the most recent common ancestors of these clusters dated to 1942-1952 (Gt1a) and 1926-1942 (Gt3a), coincident with global diversification and distribution. Extant Scottish sequences originated in Edinburgh (Gt1a) and Glasgow (Gt3a) in the 1970s but both genotypes spread from Glasgow to other regions. The dominant Gt1a strain differed between Edinburgh (cluster [C]2), Glasgow (C3) and Aberdeen (C4) whereas significant Gt3a strain-specificity only occurred in Aberdeen. Specific clusters initially formed separate transmission zones in Glasgow that subsequently overlapped occasioning city-wide co-circulation. Transmission hotspots were detected with 45% of samples from patients residing in just nine of Glasgow's 57 postcode districts. HCV was introduced into Scotland in the 1940s concomitant with its worldwide dispersal likely arising from global-scale historical events. Cluster-specific transmission hubs were identified in Glasgow, the key Scottish city implicated in HCV dissemination. This fine-scale spatiotemporal reconstruction improves understanding of HCV transmission dynamics in Scotland.

    IMPORTANCE HCV is a major health burden and the leading cause of hepatocellular carcinoma. Public health needle exchange and "treatment as prevention" strategies targeting HCV are designed to reduce prevalence of the virus in people who inject drugs (PWID), potentially mitigating the future burden of HCV-associated liver disease. Understanding HCV transmission dynamics could increase the effectiveness of such public health initiatives by identifying and targeting regions playing a central role in virus dispersal. In this study we examined HCV transmission in Scotland by analysing the genetic relatedness of strains from PWID alongside data inferring the year individuals became infected and residential information at a geographically finer-scale resolution than in previous studies. Clusters of Scotland-specific strains were identified with regional specificity and mapping the spread of HCV allowed the identification of key areas central to HCV transmission in Scotland. This research provides a basis for identifying HCV transmission hotspots.

  • The combination of the R263K and T66I resistance substitutions in HIV-1 integrase is incompatible with high level viral replication and the development of high-level drug resistance [PublishAheadOfPrint]

  • Background: The R263K substitution in integrase has been selected in tissue culture with dolutegravir (DTG) and been reported in several treatment-experienced individuals receiving DTG as part of salvage therapy. R263K seems to be incompatible with the presence of common resistance mutations associated with raltegravir (RAL), a different integrase strand transfer inhibitor (INSTI). T66I is a substitution that is common in individuals who have developed resistance against a different INSTI termed elvitegravir (EVG), but it is not known whether these two mutations might be compatible in the context of resistance against DTG or what impact the combination of these substitutions might have on resistance against INSTIs. E138K is a common secondary substitution observed with various primary resistance substitutions in RAL- and EVG- treated individuals.

    Methods: Viral infectivity, replicative capacity, and resistance against INSTIs were measured in cell-based assays. Strand-transfer and 3rrsquo; processing activities were measured biochemically.

    Results: The combination of R263K and T66I decreased HIV-1 infectivity, replicative capacity, and strand-transfer activity. The addition of the E138K substitution partially compensated for these deficits and resulted in high levels of resistance against EVG but not against DTG or RAL.

    Conclusions: These findings suggest that the presence of T66I will not compromise the activity of DTG and may also help to prevent the additional generation of R263K. Our observations support the use of DTG in second-line therapy for individuals who experience treatment failure with EVG due to the T66I substitution.

    Importance The integrase strand transfer inhibitors (INSTIs) elvitegravir and dolutegravir are newly developed inhibitors against human immunodeficiency virus-1 (HIV-1). HIV drug-resistant mutations in integrase that can arise in individuals treated with elvitegravir commonly include the T66I substitution whereas R263K is a signature resistant substitution against dolutegravir. In order to determine how different combinations of resistance integrase mutations can influence the outcome of therapy, we report here the effects of the T66I, E138K, and R263K substitutions, alone and in combination, on viral replicative capacity and resistance to integrase inhibitors. Our results show that the addition of R263K to the T66I substitution diminishes viral replicative capacity and strand-transfer activity while not compromising susceptibility to dolutegravir. This supports the use of dolutegravir in second-line therapy for patients failing elvitegravir who harbor the T66I resistance substitution.

  • Novel reassortant human-like H3N2 and H3N1 influenza A viruses detected in pigs are virulent and antigenically distinct from endemic viruses [PublishAheadOfPrint]

  • Human-like swine H3 influenza A viruses (IAV) were detected by the USDA surveillance system. We characterized two novel swine human-like H3N2 and H3N1 viruses with HA genes similar to human seasonal H3 strains and the internal genes closely related to 2009 H1N1 pandemic viruses. The H3N2 NA was of the contemporary human N2 lineage, while the H3N1 NA was of the classical swine N1 lineage. Both viruses were antigenically distant from swine H3 viruses that circulate in the U.S. and from swine vaccine strains, and also showed antigenic drift from human seasonal H3N2. Their pathogenicity and transmission in pigs were compared to a human H3N2 with common HA ancestry. Both swine human-like H3 viruses efficiently infected pigs and transmitted to indirect contacts, whereas the human H3N2 was much less efficient. To evaluate the role of genes from the swine isolates on their pathogenesis, reverse genetics-generated reassortants between the swine human-like H3N1 and the seasonal human H3N2 were tested in pigs. Gene segment contribution to virulence was complex with the swine HA and internal genes showing effect in vivo. The experimental infections indicate that these novel H3 viruses are virulent and can sustain onward transmission in pigs, and the naturally occurring mutations in the HA were associated with antigenic divergence from H3 IAV from human and swine. Consequently, these viruses could have a significant impact on the swine industry if they cause more widespread outbreaks, and the potential risk of these emerging swine IAV to humans should be considered.

    IMPORTANCE Pigs are important hosts in the evolution of influenza A viruses (IAV). Human-to-swine transmissions of IAV have resulted in the circulation of reassortant viruses containing human-origin genes in pigs, greatly contributing to the diversity of IAV in swine worldwide. New human-like H3N2 and H3N1 viruses that contain a mix of human and swine gene segments were recently detected by the USDA surveillance system. The human-like viruses efficiently infected pigs and resulted in onward airborne transmission, likely due to multiple changes identified between human and swine H3 viruses. The human-like swine viruses are distinct from contemporary U.S. H3 swine viruses and from the strains used in swine vaccines, which could have a significant impact on the swine industry due to lack of population immunity. Additionally, public health experts should consider appropriate risk assessment for these emerging swine H3N1 for the human population.

  • Reorganization of nuclear pore complexes and lamina in late-stage parvovirus infection [PublishAheadOfPrint]

  • Canine parvovirus (CPV) infection induces reorganization of nuclear structures. Our studies indicated that late-stage infection induces accumulation of nuclear pore complexes (NPCs) and lamin B1 concomitantly with decrease of lamin A/C on the apical side of the nucleus. Newly formed CPV capsids are located in close proximity of NPCs on the apical side. These results suggest that parvoviruses cause apical enrichment of NPCs and reorganization of nuclear lamina presumably to facilitate the late-stage infection.

  • Positive selection in CD8+ T-cell epitopes of influenza nucleoprotein revealed by a comparative analysis of human and swine viral lineages [PublishAheadOfPrint]

  • Numerous experimental studies have demonstrated that CD8+ T-cells contribute to immunity against influenza by limiting viral replication. It is therefore surprising that rigorous statistical tests have failed to find evidence of positive selection in the epitopes targeted by CD8+ T-cells. Here we use a novel computational approach to test for selection in CD8+ T-cell epitopes. We define all epitopes in the nucleoprotein (NP) and matrix protein (M1) with experimentally identified human CD8+ T-cell responses, and then compare the evolution of these epitopes in parallel lineages of human and swine influenza that have been diverging since roughly 1918. We find a significant enrichment of substitutions that alter human CD8+ T-cell epitopes in the NP of human versus swine influenza, consistent with the idea that these epitopes are under positive selection. Furthermore, we show that epitope-altering substitutions to human influenza NP are enriched on the trunk versus the branches of the phylogenetic tree, indicating that viruses that acquire these mutations have a selective advantage. However, even in human influenza NP, sites in T-cell epitopes evolve more slowly than non-epitope sites, presumably because these epitopes are under higher inherent functional constraint. Overall, our work demonstrates that there is clear selection from CD8+ T-cells in human influenza NP, and illustrates how comparative analyses of viral lineages from different hosts can identify positive selection that is otherwise obscured by strong functional constraint.

    IMPORTANCE There is a strong interest in correlates of anti-influenza immunity that are protective against diverse viral strains. CD8+ T-cells provide such broad immunity, since they target conserved viral proteins. An important question is whether T-cell immunity is sufficiently strong to drive influenza evolution. Although many studies have shown that T-cells limit viral replication in animal models and are associated with decreased symptoms in humans, no studies have proven with statistical significance that influenza evolves under positive selection to escape T-cells. Here we use comparisons of human and swine influenza to rigorously demonstrate that human influenza evolves under pressure to fix mutations in nucleoprotein that promote escape from T-cells. We further show that viruses with these mutations have a selective advantage since they are preferentially located on the "trunk" of the phylogenetic tree. Overall, our results show that CD8+ T-cells targeting nucleoprotein play an important role in shaping influenza evolution.

  • Viral polymerase-helicase complexes regulate replication fidelity to overcome intracellular nucleotide depletion. [PublishAheadOfPrint]

  • To date, the majority of work on RNA virus replication fidelity has focused on the viral RNA polymerase, while the potential role of other viral replicase proteins in this process is poorly understood. Previous studies used resistance to broad-spectrum RNA mutagens, such as ribavirin, to identify polymerases with increased fidelity that avoid misincorporation of such base analogues. We identified a novel variant in the alphavirus viral helicase/protease, nonstructural protein 2 (nsP2) that operates in concert with the viral polymerase nsP4 to further alter replication complex fidelity, a functional linkage that was conserved among the alphavirus genus. Purified chikungunya virus nsP2 presented delayed helicase activity of the high-fidelity enzyme, yet purified replication complexes manifested stronger RNA polymerization kinetics. Because mutagenic nucleoside analogs such as ribavirin also affect intracellular nucleotide pools, we addressed the link between nucleotide depletion and replication fidelity by using purine and pyrimidine biosynthesis inhibitors. High-fidelity viruses were more resistant to these conditions and viral growth could be rescued by the addition of exogenous nucleosides, suggesting that mutagenesis by base analogues requires nucleotide pool depletion. Taken together this study provides a novel function for nsP2, highlighting the role of other components of the replication complex in regulating viral replication fidelity and suggests that viruses can alter their replication complex fidelity to overcome intracellular nucleotide-depleting conditions.

    IMPORTANCE Previous studies using the RNA mutagen ribavirin to select for drug-resistant variants have highlighted the essential role of the viral RNA-dependent RNA polymerase (RdRp) in regulating replication fidelity. However, the role of other viral replicase components in replication fidelity has not been studied in detail. Here we identified a RNA mutagen-resistant variant of the nsP2 helicase/protease that conferred increased fidelity, yet could not operate in the same manner as high-fidelity polymerases. We show that the alphavirus helicase is a key component of the fidelity-regulating machinery. Our data show that the RNA mutagenic activity of compounds such as ribavirin is coupled to and potentiated by nucleotide depletion, and that RNA viruses can fine-tune their replication fidelity when faced with an intracellular environment depleted of nucleotides.

  • Cross-species infectivity of H3N8 influenza virus in an experimental infection in swine [PublishAheadOfPrint]

  • Avian influenza A viruses have gained increasing attention due to their ability to cross the species barrier and cause severe disease in humans and other mammal species as pigs. H3 and particularly H3N8 viruses, are highly adaptive since they are found in multiple avian and mammal hosts. H3N8 viruses have not been isolated yet from humans; however a recent report showed that equine influenza A viruses (IAV) can be isolated from pigs, although an established infection has not been observed so far in this host. To gain insight into the possibility of H3N8 avian IAV to cross the species barrier into pigs, in vitro experiments and an experimental infection in pigs with four H3N8 viruses from different origins (equine, canine, avian and seal) were performed. As positive control, a H3N2 swine influenza virus A was used. While equine and canine viruses hardly replicated in the respiratory apparatus of pigs, avian and seal viruses replicated substantially and caused detectable lesions in inoculated pigs without previous adaptation. Interestingly, antibodies against HA could not be detected after infection by hemaglutination inhibition test (HAI) with the avian and seal virus. This phenomenon was observed not only in pigs but also in mice immunized with the same virus strains. Our data indicated that H3N8 IAV from wild aquatic birds have the potential to cross the species barrier and establish successful infections in pigs that might spread unnoticed using HAI as diagnostic tool.

    IMPORTANCE SECTION Although natural infection of humans with an avian H3N8 influenza A virus has not yet been reported, this influenza A virus subtype has already crossed the species barrier. Therefore, we have examined the potential of H3N8 from canine, equine, avian and seal origin to productively infect pigs. Our results demonstrated that avian and seal viruses replicated substantially and caused detectable lesions in inoculated pigs without previous adaptation. Surprisingly, we could not detect specific antibodies against HA in any H3N8-infected pigs. Therefore, special attention should be focused towards viruses of the H3N8 subtype as they could behave as stealth viruses in pigs.

  • Host Gene Expression is Regulated by Two Types of Noncoding RNAs Transcribed from the Epstein-Barr Virus BART Region [PublishAheadOfPrint]

  • In Epstein-Barr virus infected epithelial cancers, the alternatively spliced BamHI A rightward transcripts (BARTs) are the most abundant viral polyadenylated RNA. The BART introns form the template for the production of 44 microRNAs and the spliced and polyadenylated exons form nuclear non-protein coding RNAs. Analysis of host cell transcription by RNA-seq during latency in AGS cells identified a large number of reproducibly changed genes. Genes that were downregulated were enriched for BART miRNA targets. Bioinformatics analysis predicted activation of the myc pathway and downregulation of XBP1 as likely mediators of the host transcriptional changes. Effects on XBP1 activity were not detected in these cells, however myc activation was confirmed through use of a myc responsive luciferase reporter. To identify potential regulatory properties of the spliced, polyadenylated BART RNAs, a full length cDNA clone of one of the BART isoforms was obtained and expressed in the EBV negative AGS cells. The BART cDNA transcript remained primarily nuclear yet induced considerable and consistent changes in cellular transcription as profiled by RNA-seq. These transcriptional changes significantly overlapped the transcriptional changes induced during latent EBV infection of these same cells where the BARTs are exclusively nuclear and do not encode for proteins. These data suggest that the nuclear BART RNAs are functional long noncoding RNAs (lncRNAs). The abundant expression of multiple forms of noncoding RNAs that contribute to growth regulation without expression of immunogenic proteins would be an important mechanism for viral oncogenesis in the presence of a functional immune system.

    IMPORTANCE Infection with Epstein-Barr virus is nearly ubiquitous in the human population; however it does contribute to the formation of multiple types of cancer. In immune compromised patients, EBV causes multiple types of lymphomas by expressing viral oncogenes that promote growth and survival of infected B lymphocytes. EBV positive gastric carcinoma does not require immune suppression and the viral oncoproteins that are frequent targets for an immunological response are not expressed. This study demonstrates using transcriptional analysis that the expression of various classes of viral non-protein coding RNAs likely contribute to the considerable changes in the host transcriptional profile in the AGS gastric cancer cell line. This is the first data to show the highly expressed polyadenylated BART viral transcript in gastric carcinoma is in fact a functional viral long noncoding RNA. These studies provide new insight into how EBV can promote transformation in the absence of viral protein expression.

  • A Nucleolar Protein, Ribosomal RNA Processing 1 Homolog B (RRP1B), Enhances the Recruitment of Cellular mRNA in Influenza Virus Transcription [PublishAheadOfPrint]

  • Influenza A virus (IAV) undergoes RNA transcription by a unique capped mRNA-dependent transcription, which is carried out by the viral RNA-dependent RNA polymerase (RdRp), consisting of the viral PA, PB1 and PB2 proteins. But, how the viral RdRp utilizes cellular factors for viral transcription is not clear. Previously, we have conducted a genome-wide pooled shRNA screen to identify host factors important for influenza A virus replication. Ribosomal RNA processing 1 homolog B (RRP1B) was identified as one of the candidates. RRP1B is a nucleolar protein involved in ribosomal biogenesis. Upon IAV infection, part of RRP1B was translocated from the nucleolus to the nucleoplasm, where viral RNA synthesis likely takes place. The depletion of RRP1B significantly reduced IAV mRNA transcription in a mini-replicon assay and in virus-infected cells. Furthermore, we showed that RRP1B interacted with PB1 and PB2 of the RdRp and formed a coimmunoprecipitable complex with RdRp. The depletion of RRP1B reduced the amount of capped mRNA in the RdRp complex. Taken together, these findings indicate that RRP1B is a host factor essential for IAV transcription and provides a target for new antivirals.

    IMPORTANCE Influenza virus is an important human pathogen which causes significant morbidity and mortality and threatens the human population with epidemics and pandemics every year. Due to high mutation rate of the virus, antiviral drugs targeting at viral proteins might ultimately lose their effectiveness. An alternative strategy that explores the genetic stability of host factors indispensable for influenza virus replication would thus be desirable. Here, we characterized the ribosomal RNA processing 1 homolog B (RRP1B) protein as an important cellular factor for influenza A viral transcription. We showed that silencing RRP1B hampered viral RNA-dependent RNA polymerase (RdRp) activity, which is responsible for viral transcription and replication. Furthermore, we reported that RRP1B is crucial for RdRp binding to cellular capped mRNA, which is a critical step of virus transcription. Our study not only provides a deeper understanding of influenza virus-host interplay but also suggests a potential target for antiviral drug development.

  • In vitro Evidence Supports Membrane Alanyl Aminopeptidase N as a Receptor for a Plant Virus in the Pea Aphid Vector [PublishAheadOfPrint]

  • Insect-borne plant viruses cause significant agricultural losses and jeopardize sustainable global food production. While blocking plant virus transmission would allow for crop protection, virus receptors in insect vectors are unknown. Here we identify membrane alanyl aminopeptidase N (APN) as a receptor for Pea enation mosaic virus (PEMV) coat protein (CP) in the gut of the pea aphid, Acyrthosiphon pisum using a far-western blot method. Pull-down and immunofluorescence binding assays, and surface plasmon resonance were used to confirm and characterize CP nndash; APN interaction. PEMV virions and a peptide comprised of PEMV coat protein fused to GFP (CP-P-GFP) specifically bound to APN. Recombinant APN expressed in Sf9 cells resulted in internalization of CP-P-GFP, which was visualized by confocal microscopy; such internalization is an expected hallmark of a functional gut receptor. Finally, in assays with aphid gut-derived brush border membrane vesicles (BBMV), binding of CP-P-GFP competed with binding of GBP3.1, a peptide previously demonstrated to bind to APN in the aphid gut, and to impede PEMV uptake into the hemocoel; this finding supports the hypothesis that GBP3.1 and PEMV bind to and compete for the same APN receptor. These in vitro data combined with previously published in vivo experiments (S. Liu, S. Sivakumar, W.O. Sparks, W.A. Miller, B.C. Bonning, Virology 401:107-16, 2010, doi: 10.1016/j.virol.2010.02.009) support identification of APN as the first receptor in a plant virus vector. Knowledge of this receptor will provide for technologies based on PEMV nndash; APN interaction designed to block plant virus transmission and to suppress aphid populations.

    IMPORTANCE A significant proportion of global food production is lost to insect pests. Aphids, in addition to weakening plants by feeding on their sap, are responsible for transmitting about half of the plant viruses vectored by insects. Growers rely heavily on the application of chemical insecticides to manage both aphids and aphid-vectored plant viral disease. To increase our understanding of plant virus-aphid vector interaction, we provide in vitro evidence supporting earlier in vivo work for identification of a receptor protein in the aphid gut called aminopeptidase N, which is responsible for entry of the plant virus Pea enation mosaic virus into the pea aphid vector. Enrichment of proteins found on the surface of the aphid gut epithelium resulted in identification of this first aphid gut receptor for a plant virus. This discovery is particularly important as disruption of plant virus binding to such a receptor may enable development of a non-chemical strategy for controlling aphid-vectored plant viruses to maximize food production.

  • Structural and functional diversity of nairovirus-encoded nucleoproteins [PublishAheadOfPrint]

  • The nairoviruses include assorted tick-borne bunyaviruses that are emerging as causative infectious diseases among humans and animals. As negative-sense single-stranded RNA (-ssRNA) viruses, nairoviruses encode nucleoprotein (NP) that encapsidates the genomic RNA and further forms ribonucleoprotein complex (RNP) with viral RNA-dependent RNA polymerase (RdRp). We previously revealed that the monomeric NP encoded by Crimean-Congo hemorrhagic fever virus (CCHFV) presents a racket-shaped structure and shows unusual DNA-specific endonuclease activity. To examine the structural and biological variation of nairovirus-encoded NPs, here we systematically solved the crystal structures of NPs encoded by various nairoviruses, including Hazara virus (HAZV), Kupe virus (KUPV), and Erve virus (ERVEV). Combined with biochemical analysis, our results generate a clearer picture to aid in the understanding of the functional diversity of nairovirus-encoded NPs and the formation of nairovirus RNPs.

    Importance Nairoviruses are consisted of several tick-borne bunyaviruses that are emerging as causative infectious diseases among humans and animals, however, little is known of the nairovirus genome assembling and transcription mechanism. Based on the previous study of CCHFV NP reported by different research groups, here we systematically investigate the structural and functional diversity among three different Nairoviruses, these work provide important information on nairovirus nucleoprotein function and the formation of RNPs.

  • Inhibition of HIV expression and integration in macrophages by Methylglyoxal-bis-guanylhydrazone [PublishAheadOfPrint]

  • Macrophages are a target for infection with HIV and represent one of the viral reservoirs that are relatively resistant to current anti-retroviral drugs. Here we demonstrate that methylglyoxal-bis-guanylhydrazone (MGBG), a polyamine analog and potent S-adenosylmethionine decarboxylase inhibitor, decreases HIV expression in monocytes and macrophages. MGBG is selectively concentrated by these cells through a mechanism consistent with active transport by the polyamine transporter. Using a macrophage-tropic reporter virus tagged with the enhanced green fluorescent protein, we demonstrate that MGBG decreases the frequency of HIV infected cells. The effect is dose dependent and correlates with the production of HIV p24 in culture supernatants. This anti-HIV effect was further confirmed using three macrophage tropic primary HIV isolates. Viral life cycle mapping studies show that MGBG inhibits HIV DNA integration into the cellular DNA in both monocytes and macrophages.

    IMPORTANCE Our work demonstrates for the first time the selective concentration of MGBG by monocytes/macrophages, leading to the inhibition of HIV-1 expression and a reduction in proviral load within macrophage cultures. These results suggest that MGBG may be useful in adjunctive macrophage targeted therapy for HIV infection.

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