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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]

  • HIV Vaccine Trial Exploits a Dual and Central Role for Innate Immunity [Commentary]

  • Limited understanding of correlates of protection from HIV transmission hinders development of an efficacious vaccine. D. J. M. Lewis and colleagues (J. Virol. 88:11648nndash;11657, 2014, doi:10.1128/JVI.01621-14) now report that vaginal immunization with an HIVgp140 vaccine linked to the 70-kDa heat shock protein downregulated the human immunodeficiency virus (HIV) coreceptor CCR5 (chemokine [C-C motif] receptor 5) and increased expression of the HIV resistance factor APOBEC3G (apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G), in women. These effects correlated with HIV suppression ex vivo. Thus, vaccine-induced innate responses not only facilitate adaptive immunitynndash;they may prove to be critical for preventing HIV transmission.

  • Reemergence of Chikungunya Virus [Gem]

  • Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes acute fever and acute and chronic musculoskeletal pain in humans. Since 2004, CHIKV has caused millions of cases of disease in the Indian Ocean region and has emerged in new areas, including Europe, the Middle East, and the Pacific region. The mosquito vectors for this virus are globally distributed in tropical and temperate zones, providing the opportunity for CHIKV to continue to expand into new geographic regions. In October 2013, locally acquired cases of CHIKV infection were identified on the Caribbean island of Saint Martin, signaling the arrival of the virus in the Western Hemisphere. In just 9 months, CHIKV has spread to 22 countries in the Caribbean and Central and South America, resulting in hundreds of thousands of cases. CHIKV disease can be highly debilitating, and large epidemics have severe economic consequences. Thus, there is an urgent need for continued research into the epidemiology, pathogenesis, prevention, and treatment of these infections.

  • Effect of Vaginal Immunization with HIVgp140 and HSP70 on HIV-1 Replication and Innate and T Cell Adaptive Immunity in Women [Vaccines and Antiviral Agents]

  • The international effort to prevent HIV-1 infection by vaccination has failed to develop an effective vaccine. The aim of this vaccine trial in women was to administer by the vaginal mucosal route a vaccine consisting of HIV-1 gp140 linked to the chaperone 70-kDa heat shock protein (HSP70). The primary objective was to determine the safety of the vaccine. The secondary objective was to examine HIV-1 infectivity ex vivo and innate and adaptive immunity to HIV-1. Protocol-defined female volunteers were recruited. HIV-1 CN54gp140 linked to HSP70 was administered by the vaginal route. Significant adverse reactions were not detected. HIV-1 was significantly inhibited ex vivo in postimmunization CD4+ T cells compared with preimmunization CD4+ T cells. The innate antiviral restrictive factor APOBEC3G was significantly upregulated, as were CC chemokines which induce downregulation of CCR5 in CD4+ T cells. Indeed, a significant inverse correlation between the proportion of CCR5+ T cells and the concentration of CCL-3 or CCL-5 was found. Importantly, the upregulation of APOBEC3G showed a significant inverse correlation, whereas CCR5 exhibited a trend to correlate with inhibition of HIV-1 infection (r = 0.51). Furthermore, specific CD4+ and CD8+ T cell proliferative responses were significantly increased and CD4+ T cells showed a trend to have an inverse correlation with the viral load (r = nndash;0.60). However, HIVgp140-specific IgG or IgA antibodies were not detected. The results provide proof of concept that an innate mechanism consisting of CC chemokines, APOBEC3G, and adaptive immunity by CD4 and CD8 T cells might be involved in controlling HIV-1 infectivity following vaginal mucosal immunization in women. (This study has been registered at ClinicalTrials.gov under registration no. NCT01285141.)

    IMPORTANCE Vaginal immunization of women with a vaccine consisting of HIVgp140 linked to the 70-kDa heat shock protein (HSP70) elicited ex vivo significant inhibition of HIV-1 replication in postimmunization CD4+ T cells compared with that in preimmunization peripheral blood mononuclear cells. There were no significant adverse events. The vaccine induced the significant upregulation of CC chemokines and the downmodulation of CCR5 expression in CD4+ T cells, as well as an inverse correlation between them. Furthermore, the level of CCR5 expression was directly correlated with the viral load, consistent with the protective mechanism in which a decrease in CCR5 molecules on CD4+ T cells decreases HIV-1 envelope binding. Expression of the antiviral restriction factor APOBEC3G was inversely correlated with the viral load, suggesting that it may inhibit intracellular HIV-1 replication. Both CD4+ and CD8+ T cells showed HIVgp140- and HSP70-specific proliferation. A strong inverse correlation between the proportion of CC chemokine-modulated CCR5-expressing CD4+ T cells and the stimulation of CD4+ or CD8+ T cell proliferation by HIVgp140 was found, demonstrating a significant interaction between innate and adaptive immunity. This is the first clinical trial of vaginal immunization in women using only HIVgp140 and HSP70 administered by the mucosal route (3 times) in which a dual innate protective mechanism was induced and enhanced by significant adaptive CD4+ and CD8+ T cell proliferative responses.

  • Toll-Like Receptor 9-Mediated Protection of Enterovirus 71 Infection in Mice Is Due to the Release of Danger-Associated Molecular Patterns [Pathogenesis and Immunity]

  • Enterovirus 71 (EV71), a positive-stranded RNA virus, is the major cause of hand, foot, and mouth disease (HFMD) with severe neurological symptoms. Antiviral type I interferon (alpha/beta interferon [IFN-aalpha;/bbeta;]) responses initiated from innate receptor signaling are inhibited by EV71-encoded proteases. It is less well understood whether EV71-induced apoptosis provides a signal to activate type I interferon responses as a host defensive mechanism. In this report, we found that EV71 alone cannot activate Toll-like receptor 9 (TLR9) signaling, but supernatant from EV71-infected cells is capable of activating TLR9. We hypothesized that TLR9-activating signaling from plasmacytoid dendritic cells (pDCs) may contribute to host defense mechanisms. To test our hypothesis, Flt3 ligand-cultured DCs (Flt3L-DCs) from both wild-type (WT) and TLR9 knockout (TLR9KO) mice were infected with EV71. More viral particles were produced in TLR9KO mice than by WT mice. In contrast, alpha interferon (IFN-aalpha;), monocyte chemotactic protein 1 (MCP-1), tumor necrosis factor-alpha (TNF-aalpha;), IFN-, interleukin 6 (IL-6), and IL-10 levels were increased in Flt3L-DCs from WT mice infected with EV71 compared with TLR9KO mice. Seven-day-old TLR9KO mice infected with a non-mouse-adapted EV71 strain developed neurological lesion-related symptoms, including hind-limb paralysis, slowness, ataxia, and lethargy, but WT mice did not present with these symptoms. Lung, brain, small intestine, forelimb, and hind-limb tissues collected from TLR9KO mice exhibited significantly higher viral loads than equivalent tissues collected from WT mice. Histopathologic damage was observed in brain, small intestine, forelimb, and hind-limb tissues collected from TLR9KO mice infected with EV71. Our findings demonstrate that TLR9 is an important host defense molecule during EV71 infection.

    IMPORTANCE The host innate immune system is equipped with pattern recognition receptors (PRRs), which are useful for defending the host against invading pathogens. During enterovirus 71 (EV71) infection, the innate immune system is activated by pathogen-associated molecular patterns (PAMPs), which include viral RNA or DNA, and these PAMPs are recognized by PRRs. Toll-like receptor 3 (TLR3) and TLR7/8 recognize viral nucleic acids, and TLR9 senses unmethylated CpG DNA or pathogen-derived DNA. These PRRs stimulate the production of type I interferons (IFNs) to counteract viral infection, and they are the major source of antiviral alpha interferon (IFN-aalpha;) production in pDCs, which can produce 200- to 1,000-fold more IFN-aalpha; than any other immune cell type. In addition to PAMPs, danger-associated molecular patterns (DAMPs) are known to be potent activators of innate immune signaling, including TLR9. We found that EV71 induces cellular apoptosis, resulting in tissue damage; the endogenous DNA from dead cells may activate the innate immune system through TLR9. Therefore, our study provides new insights into EV71-induced apoptosis, which stimulates TLR9 in EV71-associated infections.

  • Human Rhinovirus 16 Causes Golgi Apparatus Fragmentation without Blocking Protein Secretion [Virus-Cell Interactions]

  • The replication of picornaviruses has been described to cause fragmentation of the Golgi apparatus that blocks the secretory pathway. The inhibition of major histocompatibility complex class I upregulation and cytokine, chemokine and interferon secretion may have important implications for host defense. Previous studies have shown that disruption of the secretory pathway can be replicated by expression of individual nonstructural proteins; however the situation with different serotypes of human rhinovirus (HRV) is unclear. The expression of 3A protein from HRV14 or HRV2 did not cause Golgi apparatus disruption or a block in secretion, whereas other studies showed that infection of cells with HRV1A did cause Golgi apparatus disruption which was replicated by the expression of 3A. HRV16 is the serotype most widely used in clinical HRV challenge studies; consequently, to address the issue of Golgi apparatus disruption for HRV16, we have systematically and quantitatively examined the effect of HRV16 on both Golgi apparatus fragmentation and protein secretion in HeLa cells. First, we expressed each individual nonstructural protein and examined their cellular localization and their disruption of endoplasmic reticulum and Golgi apparatus architecture. We quantified their effects on the secretory pathway by measuring secretion of the reporter protein Gaussia luciferase. Finally, we examined the same outcomes following infection of cells with live virus. We demonstrate that expression of HRV16 3A and 3AB and, to a lesser extent, 2B caused dispersal of the Golgi structure, and these three nonstructural proteins also inhibited protein secretion. The infection of cells with HRV16 also caused significant Golgi apparatus dispersal; however, this did not result in the inhibition of protein secretion.

    IMPORTANCE The ability of replicating picornaviruses to influence the function of the secretory pathway has important implications for host defense. However, there appear to be differences between different members of the family and inconsistent results when comparing infection with live virus to expression of individual nonstructural proteins. We demonstrate that individual nonstructural HRV16 proteins, when expressed in HeLa cells, can both fragment the Golgi apparatus and block secretion, whereas viral infection fragments the Golgi apparatus without blocking secretion. This has major implications for how we interpret mechanistic evidence derived from the expression of single viral proteins.

  • The Epstein-Barr Virus (EBV) Glycoprotein B Cytoplasmic C-Terminal Tail Domain Regulates the Energy Requirement for EBV-Induced Membrane Fusion [Virus-Cell Interactions]

  • The entry of enveloped viruses into host cells is preceded by membrane fusion, which in Epstein-Barr virus (EBV) is thought to be mediated by the refolding of glycoprotein B (gB) from a prefusion to a postfusion state. In our current studies, we characterized a gB C-terminal tail domain (CTD) mutant truncated at amino acid 843 (gB843). This truncation mutant is hyperfusogenic as monitored by syncytium formation and in a quantitative fusion assay and is dependent on gH/gL for fusion activity. gB843 can rescue the fusion function of other glycoprotein mutants that have null or decreased fusion activity in epithelial and B cells. In addition, gB843 requires less gp42 and gH/gL for fusion, and can function in fusion at a lower temperature than wild-type gB, indicating a lower energy requirement for fusion activation. Since a key step in fusion is the conversion of gB from a prefusion to an active postfusion state by gH/gL, gB843 may access this activated gB state more readily. Our studies indicate that the gB CTD may participate in the fusion function by maintaining gB in an inactive prefusion form prior to activation by receptor binding.

    IMPORTANCE Diseases resulting from Epstein-Barr virus (EBV) infection in humans range from the fairly benign disease infectious mononucleosis to life-threatening cancer. As an enveloped virus, EBV must fuse with a host cell membrane for entry and infection by using glycoproteins gH/gL, gB, and gp42. Among these glycoproteins, gB is thought to be the protein that executes fusion. To further characterize the function of the EBV gB cytoplasmic C-terminal tail domain (CTD) in fusion, we used a previously constructed CTD truncation mutant and studied its fusion activity in the context of other EBV glycoprotein mutants. From these studies, we find that the gB CTD regulates fusion by altering the energy requirements for the triggering of fusion mediated by gH/gL or gp42. Overall, our studies may lead to a better understanding of EBV fusion and entry, which may result in novel therapies that target the EBV entry step.

  • The 3' Untranslated Region of Pea Enation Mosaic Virus Contains Two T-Shaped, Ribosome-Binding, Cap-Independent Translation Enhancers [Genome Replication and Regulation of Viral Gene Expression]

  • Many plant viruses without 5'caps or 3' poly(A) tails contain 3' proximal, cap-independent translation enhancers (3'CITEs) that bind to ribosomal subunits or translation factors thought to assist in ribosome recruitment. Most 3'CITEs participate in a long-distance kissing-loop interaction with a 5' proximal hairpin to deliver ribosomal subunits to the 5' end for translation initiation. Pea Enation Mosaic Virus (PEMV) contains two adjacent 3'CITEs in the center of its 703-nucleotide 3' untranslated region (3'UTR), the ribosome-binding, kissing-loop T-shaped structure (kl-TSS) and eukaryotic translation initiation factor 4E-binding Panicum mosaic virus-like translation enhance (PTE). We now report that PEMV contains a third, independent 3'CITE located near the 3' terminus. This 3'CITE is composed of three hairpins and two pseudoknots, similar to the TSS 3'CITE of the carmovirus Turnip crinkle virus (TCV). As with the TCV TSS, the PEMV 3'TSS is predicted to fold into a T-shaped structure that binds to 80S ribosomes and 60S ribosomal subunits. A small hairpin (kl-H) upstream of the 3'TSS contains an apical loop capable of forming a kissing-loop interaction with a 5' proximal hairpin and is critical for the accumulation of full-length PEMV in protoplasts. Although the kl-H and 3'TSS are dispensable for the translation of a reporter construct containing the complete PEMV 3'UTR in vitro, deleting the normally required kl-TSS and PTE 3'CITEs and placing the kl-H and 3'TSS proximal to the reporter termination codon restores translation to near wild-type levels. This suggests that PEMV requires three 3'CITEs for proper translation and that additional translation enhancers may have been missed if reporter constructs were used in 3'CITE identification.

    IMPORTANCE The rapid life cycle of viruses requires efficient translation of viral-encoded proteins. Many plant RNA viruses contain 3' cap-independent translation enhancers (3'CITEs) to effectively compete with ongoing host translation. Since only single 3'CITEs have been identified for the vast majority of individual viruses, it is widely accepted that this is sufficient for a virus's translational needs. Pea Enation Mosaic Virus possesses a ribosome-binding 3'CITE that can connect to the 5' end through an RNA-RNA interaction and an adjacent eukaryotic translation initiation factor 4E-binding 3'CITE. We report the identification of a third 3'CITE that binds weakly to ribosomes and requires an upstream hairpin to form a bridge between the 3' and 5' ends. Although both ribosome-binding 3'CITEs are critical for virus accumulation in vivo, only the CITE closest to the termination codon of a reporter open reading frame is active, suggesting that artificial constructs used for 3'CITE identification may underestimate the number of CITEs that participate in translation.

  • Probing the Functions of the Paramyxovirus Glycoproteins F and HN with a Panel of Synthetic Antibodies [Structure and Assembly]

  • Paramyxoviruses are enveloped negative-strand RNA viruses that are significant human and animal pathogens. Most paramyxoviruses infect host cells via the concerted action of a tetrameric attachment protein (variously called HN, H, or G) that binds either sialic acid or protein receptors on target cells and a trimeric fusion protein (F) that merges the viral envelope with the plasma membrane at neutral pH. F initially folds to a metastable prefusion conformation that becomes activated via a cleavage event during cellular trafficking. Upon receptor binding, the attachment protein, which consists of a globular head anchored to the membrane via a helical tetrameric stalk, triggers a major conformation change in F which results in fusion of virus and host cell membranes. We recently proposed a model for F activation in which the attachment protein head domains move following receptor binding to expose HN stalk residues critical for triggering F. To test the model in the context of wild-type viral glycoproteins, we used a restricted-diversity combinatorial Fab library and phage display to rapidly generate synthetic antibodies (sAbs) against multiple domains of the paramyxovirus parainfluenza 5 (PIV5) pre- and postfusion F and HN. As predicted by the model, sAbs that bind to the critical F-triggering region of the HN stalk do not disrupt receptor binding or neuraminidase (NA) activity but are potent inhibitors of fusion. An inhibitory prefusion F-specific sAb recognized a quaternary antigenic site and may inhibit fusion by preventing F refolding or by blocking the F-HN interaction.

    IMPORTANCE The paramyxovirus family of negative-strand RNA viruses cause significant disease in humans and animals. The viruses bind to cells via their receptor binding protein and then enter cells by fusion of their envelope with the host cell plasma membrane, a process mediated by a metastable viral fusion (F) protein. To understand the steps in viral membrane fusion, a library of synthetic antibodies to F protein and the receptor binding protein was generated in bacteriophage. These antibodies bound to different regions of the F protein and the receptor binding protein, and the location of antibody binding affected different processes in viral entry into cells.

  • Combined Effects of the Structural Heterogeneity and Dynamics of Flaviviruses on Antibody Recognition [Structure and Assembly]

  • Flaviviruses are thought to sample an ensemble of structures at equilibrium. One consequence of a structurally dynamic virion is the observed time-dependent increases in neutralization sensitivity that can occur after prolonged incubation with antibody. Differences in how virus strains "breathe" may affect epitope exposure and contribute to the underlying mechanisms of strain-dependent neutralization sensitivity. Beyond the contribution of structural dynamics, flaviviruses exist as a structurally heterogeneous population due to an inefficient virion maturation process. Here, we investigate the interplay between virion maturation and structural dynamics that contributes to antibody-mediated neutralization. Using West Nile (WNV) and dengue (DENV) viruses produced under conditions that modify the extent of virion maturation, we investigated time-dependent changes in neutralization sensitivity associated with structural dynamics. Our results identify distinct patterns of neutralization against viruses that vary markedly with respect to the extent of virion maturation. Reducing the efficiency of virion maturation resulted in greater time-dependent changes in neutralization potency and a marked reduction in the stability of the particle at 37ddeg;C compared to more mature virus. The fact that the neutralization sensitivity of WNV and DENV did not increase after prolonged incubation in the absence of antibody, regardless of virion maturation, suggests that the dynamic processes that govern epitope accessibility on infectious viruses are reversible. Against the backdrop of heterogeneous flavivirus structures, differences in the pathways by which viruses "breathe" represent an additional layer of complexity in understanding maturation state-dependent patterns of antibody recognition.

    IMPORTANCE Flaviviruses exist as a group of related structures at equilibrium that arise from the dynamic motion of E proteins that comprise the antigenic surface of the mature virion. This process has been characterized for numerous viruses and is referred to as viral "breathing." Additionally, flaviviruses are structurally heterogeneous due to an inefficient maturation process responsible for cleaving prM on the virion surface. Both of these mechanisms vary the exposure of antigenic sites available for antibody binding and impact the ability of antibodies to neutralize infection. We demonstrate that virions with inefficient prM cleavage "breathe" differently than their more mature counterparts, resulting in distinct patterns of neutralization sensitivity. Additionally, the maturation state was found to impact virus stability in solution. Our findings provide insight into the complex flavivirus structures that contribute to infection with the potential to impact antibody recognition.

  • Dynamic and Nucleolin-Dependent Localization of Human Cytomegalovirus UL84 to the Periphery of Viral Replication Compartments and Nucleoli [Genome Replication and Regulation of Viral Gene Expression]

  • Protein-protein and protein-nucleic acid interactions within subcellular compartments are required for viral genome replication. To understand the localization of the human cytomegalovirus viral replication factor UL84 relative to other proteins involved in viral DNA synthesis and to replicating viral DNA in infected cells, we created a recombinant virus expressing a FLAG-tagged version of UL84 (UL84FLAG) and used this virus in immunofluorescence assays. UL84FLAG localization differed at early and late times of infection, transitioning from diffuse distribution throughout the nucleus to exclusion from the interior of replication compartments, with some concentration at the periphery of replication compartments with newly labeled DNA and the viral DNA polymerase subunit UL44. Early in infection, UL84FLAG colocalized with the viral single-stranded DNA binding protein UL57, but colocalization became less prominent as infection progressed. A portion of UL84FLAG also colocalized with the host nucleolar protein nucleolin at the peripheries of both replication compartments and nucleoli. Small interfering RNA (siRNA)-mediated knockdown of nucleolin resulted in a dramatic elimination of UL84FLAG from replication compartments and other parts of the nucleus and its accumulation in the cytoplasm. Reciprocal coimmunoprecipitation of viral proteins from infected cell lysates revealed association of UL84, UL44, and nucleolin. These results indicate that UL84 localization during infection is dynamic, which is likely relevant to its functions, and suggest that its nuclear and subnuclear localization is highly dependent on direct or indirect interactions with nucleolin.

    IMPORTANCE The protein-protein interactions among viral and cellular proteins required for replication of the human cytomegalovirus (HCMV) DNA genome are poorly understood. We sought to understand how an enigmatic HCMV protein critical for virus replication, UL84, localizes relative to other viral and cellular proteins required for HCMV genome replication and replicating viral DNA. We found that UL84 localizes with viral proteins, viral DNA, and the cellular nucleolar protein nucleolin in the subnuclear replication compartments in which viral DNA replication occurs. Unexpectedly, we also found localization of UL84 with nucleolin in nucleoli and showed that the presence of nucleolin is involved in localization of UL84 to the nucleus. These results add to previous work showing the importance of nucleolin in replication compartment architecture and viral DNA synthesis and are relevant to understanding UL84 function.

  • Classic Nuclear Localization Signals and a Novel Nuclear Localization Motif Are Required for Nuclear Transport of Porcine Parvovirus Capsid Proteins [Virus-Cell Interactions]

  • Nuclear targeting of capsid proteins (VPs) is important for genome delivery and precedes assembly in the replication cycle of porcine parvovirus (PPV). Clusters of basic amino acids, corresponding to potential nuclear localization signals (NLS), were found only in the unique region of VP1 (VP1up, for VP1 unique part). Of the five identified basic regions (BR), three were important for nuclear localization of VP1up: BR1 was a classic Pat7 NLS, and the combination of BR4 and BR5 was a classic bipartite NLS. These NLS were essential for viral replication. VP2, the major capsid protein, lacked these NLS and contained no region with more than two basic amino acids in proximity. However, three regions of basic clusters were identified in the folded protein, assembled into a trimeric structure. Mutagenesis experiments showed that only one of these three regions was involved in VP2 transport to the nucleus. This structural NLS, termed the nuclear localization motif (NLM), is located inside the assembled capsid and thus can be used to transport trimers to the nucleus in late steps of infection but not for virions in initial infection steps. The two NLS of VP1up are located in the N-terminal part of the protein, externalized from the capsid during endosomal transit, exposing them for nuclear targeting during early steps of infection. Globally, the determinants of nuclear transport of structural proteins of PPV were different from those of closely related parvoviruses.

    IMPORTANCE Most DNA viruses use the nucleus for their replication cycle. Thus, structural proteins need to be targeted to this cellular compartment at two distinct steps of the infection: in early steps to deliver viral genomes to the nucleus and in late steps to assemble new viruses. Nuclear targeting of proteins depends on the recognition of a stretch of basic amino acids by cellular transport proteins. This study reports the identification of two classic nuclear localization signals in the minor capsid protein (VP1) of porcine parvovirus. The major protein (VP2) nuclear localization was shown to depend on a complex structural motif. This motif can be used as a strategy by the virus to avoid transport of incorrectly folded proteins and to selectively import assembled trimers into the nucleus. Structural nuclear localization motifs can also be important for nuclear proteins without a classic basic amino acid stretch, including multimeric cellular proteins.

  • Immunodominant CD4+ T-Cell Responses to Influenza A Virus in Healthy Individuals Focus on Matrix 1 and Nucleoprotein [Pathogenesis and Immunity]

  • Antigen-specific CD4+ T cells are essential for effective virus-specific host responses, with recent human challenge studies (in volunteers) establishing their importance for influenza A virus (IAV)-specific immunity. However, while many IAV CD4+ T cell epitopes have been identified, few are known to stimulate immunodominant CD4+ T cell responses. Moreover, much remains unclear concerning the major antigen(s) responded to by the human CD4+ T cells and the extents and magnitudes of these responses. We initiated a systematic screen of immunodominant CD4+ T cell responses to IAV in healthy individuals. Using in vitro expanded-multispecificity IAV-specific T cell lines and individual IAV protein antigens produced by recombinant vaccinia viruses, we found that the internal matrix protein 1 (M1) and nucleoprotein (NP) were the immunodominant targets of CD4+ T cell responses. Ten epitopes derived from M1 and NP were definitively characterized. Furthermore, epitope sequence conservation analysis established that immunodominance correlated with an increased frequency of mutations, reflecting the fact that these prominent epitopes are under greater selective pressure. Such evidence that particular CD4+ T cells are important for protection/recovery is of value for the development of novel IAV vaccines and for our understanding of different profiles of susceptibility to these major pathogens.

    IMPORTANCE Influenza virus causes half a million deaths annually. CD4+ T cell responses have been shown to be important for protection against influenza and for recovery. CD4+ T cell responses are also critical for efficient CD8+ T cell response and antibody response. As immunodominant T cells generally play a more important role, characterizing these immunodominant responses is critical for influenza vaccine development. We show here that the internal matrix protein 1 (M1) and nucleoprotein (NP), rather than the surface proteins reported previously, are the immunodominant targets of CD4+ T cell responses. Interestingly, these immunodominant epitope regions accumulated many mutations over time, which likely indicates increased immune pressure. These findings have significant implications for the design of T cell-based influenza vaccines.

  • Engineering Recombinant Orsay Virus Directly in the Metazoan Host Caenorhabditis elegans [Genome Replication and Regulation of Viral Gene Expression]

  • The recent identification of Orsay virus, the first virus that is capable of naturally infecting Caenorhabditis elegans, provides a unique opportunity to explore host-virus interaction studies in this invaluable model organism. A key feature of this system is the robust genetic tractability of the host, C. elegans, which would ideally be complemented by the ability to genetically manipulate Orsay virus in parallel. To this end, we developed a plasmid-based reverse genetics system for Orsay virus by creating transgenic C. elegans strains harboring Orsay virus cDNAs. Both wild-type and mutant Orsay viruses, including a FLAG epitope-tagged recombinant Orsay virus, were generated by use of the reverse genetics system. This is the first plasmid-based virus reverse genetics system in the metazoan C. elegans. The Orsay virus reverse genetics we established will serve as a fundamental tool in host-virus interaction studies in the model organism C. elegans.

    IMPORTANCE To date, Orsay virus is the first and the only identified virus capable of naturally infecting Caenorhabditis elegans. C. elegans is a simple multicellular model organism that mimics many fundamental features of human biology and has been used to define many biological properties conserved through evolution. Thus, the Orsay virus-C. elegans infection system provides a unique opportunity to study host-virus interactions. In order to take maximal advantage of this system, the ability to genetically engineer mutant forms of Orsay virus would be highly desirable. Most efforts to engineer viruses have been done with cultured cells. Here we describe the creation of mutant viruses directly in the multicellular organism C. elegans without the use of cell culture. We engineered a virus expressing a genetically tagged protein that could be detected in C. elegans. This provides proof of concept for modifying Orsay virus, which will greatly facilitate studies in this experimental system.

  • The 19S Proteasome Activator Promotes Human Cytomegalovirus Immediate Early Gene Expression through Proteolytic and Nonproteolytic Mechanisms [Genome Replication and Regulation of Viral Gene Expression]

  • Proteasomes are large, multisubunit complexes that support normal cellular activities by executing the bulk of protein turnover. During infection, many viruses have been shown to promote viral replication by using proteasomes to degrade cellular factors that restrict viral replication. For example, the human cytomegalovirus (HCMV) pp71 protein induces the proteasomal degradation of Daxx, a cellular transcriptional repressor that can silence viral immediate early (IE) gene expression. We previously showed that this degradation requires both the proteasome catalytic 20S core particle (CP) and the 19S regulatory particle (RP). The 19S RP associates with the 20S CP to facilitate protein degradation but also plays a 20S CP-independent role promoting transcription. Here, we present a nonproteolytic role of the 19S RP in HCMV IE gene expression. We demonstrate that 19S RP subunits are recruited to the major immediate early promoter (MIEP) that directs IE transcription. Depletion of 19S RP subunits generated a defect in RNA polymerase II elongation through the MIE locus during HCMV infection. Our results reveal that HCMV commandeers proteasome components for both proteolytic and nonproteolytic roles to promote HCMV lytic infection.

    IMPORTANCE Proteasome inhibitors decrease or eliminate 20S CP activity and are garnering increasing interest as chemotherapeutics. However, an increasing body of evidence implicates 19S RP subunits in important proteolytic-independent roles during transcription. Thus, pharmacological inhibition of the 20S CP as a means to modulate proteasome function toward therapeutic effect is an incomplete capitalization on the potential of this approach. Here, we provide an additional example of nonproteolytic 19S RP function in promoting HCMV transcription. These data provide a novel system with which to study the roles of different proteasome components during transcription, a rationale for previously described shifts in 19S RP subunit localization during HCMV infection, and a potential therapeutic intervention point at a pre-immediate early stage for the inhibition of HCMV infection.

  • Conformational Properties of Prion Strains Can Be Transmitted to Recombinant Prion Protein Fibrils in Real-Time Quaking-Induced Conversion [Prions]

  • The phenomenon of prion strains with distinct biological characteristics has been hypothesized to be involved in the structural diversity of abnormal prion protein (PrPSc). However, the molecular basis of the transmission of strain properties remains poorly understood. Real-time quaking-induced conversion (RT-QUIC) is a cell-free system that uses Escherichia coli-derived recombinant PrP (rPrP) for the sensitive detection of PrPSc. To investigate whether the properties of various prion strains can be transmitted to amyloid fibrils consisting of rPrP (rPrP fibrils) using RT-QUIC, we examined the secondary structure, conformational stability, and infectivity of rPrP fibrils seeded with PrPSc derived from either the Chandler or the 22L strain. In the first round of the reaction, there were differences in the secondary structures, especially in bands attributed to bbeta;-sheets, as determined by infrared spectroscopy, and conformational stability between Chandler-seeded (1st-rPrP-fibCh) and 22L-seeded (1st-rPrP-fib22L) rPrP fibrils. Of note, specific identifying characteristics of the two rPrP fibril types seen in the bbeta;-sheets resembled those of the original PrPSc. Furthermore, the conformational stability of 1st-rPrP-fibCh was significantly higher than that of 1st-rPrP-fib22L, as with Chandler and 22L PrPSc. The survival periods of mice inoculated with 1st-rPrP-fibCh or 1st-rPrP-fib22L were significantly shorter than those of mice inoculated with mixtures from the mock 1st-round RT-QUIC procedure. In contrast, these biochemical characteristics were no longer evident in subsequent rounds, suggesting that nonspecific uninfected rPrP fibrils became predominant probably because of their high growth rate. Together, these findings show that at least some strain-specific conformational properties can be transmitted to rPrP fibrils and unknown cofactors or environmental conditions may be required for further conservation.

    IMPORTANCE The phenomenon of prion strains with distinct biological characteristics is assumed to result from the conformational variations in the abnormal prion protein (PrPSc). However, important questions remain about the mechanistic relationship between the conformational differences and the strain diversity, including how strain-specific conformations are transmitted. In this study, we investigated whether the properties of diverse prion strains can be transmitted to amyloid fibrils consisting of E. coli-derived recombinant PrP (rPrP) generated by real-time quaking-induced conversion (RT-QUIC), a recently developed in vitro PrPSc formation method. We demonstrate that at least some of the strain-specific conformational properties can be transmitted to rPrP fibrils in the first round of RT-QUIC by examining the secondary structure, conformational stability, and infectivity of rPrP fibrils seeded with PrPSc derived from either the Chandler or the 22L prion strain. We believe that these findings will advance our understanding of the conformational basis underlying prion strain diversity.

  • A Monomeric Uncleaved Respiratory Syncytial Virus F Antigen Retains Prefusion-Specific Neutralizing Epitopes [Vaccines and Antiviral Agents]

  • Respiratory syncytial virus (RSV) is the leading infectious cause of severe respiratory disease in infants and a major cause of respiratory illness in the elderly. There remains an unmet vaccine need despite decades of research. Insufficient potency, homogeneity, and stability of previous RSV fusion protein (F) subunit vaccine candidates have hampered vaccine development. RSV F and related parainfluenza virus (PIV) F proteins are cleaved by furin during intracellular maturation, producing disulfide-linked F1 and F2 fragments. During cell entry, the cleaved Fs rearrange from prefusion trimers to postfusion trimers. Using RSV F constructs with mutated furin cleavage sites, we isolated an uncleaved RSV F ectodomain that is predominantly monomeric and requires specific cleavage between F1 and F2 for self-association and rearrangement into stable postfusion trimers. The uncleaved RSV F monomer is folded and homogenous and displays at least two key RSV-neutralizing epitopes shared between the prefusion and postfusion conformations. Unlike the cleaved trimer, the uncleaved monomer binds the prefusion-specific monoclonal antibody D25 and human neutralizing immunoglobulins that do not bind to postfusion F. These observations suggest that the uncleaved RSV F monomer has a prefusion-like conformation and is a potential prefusion subunit vaccine candidate.

    IMPORTANCE RSV is the leading infectious cause of severe respiratory disease in infants and a major cause of respiratory illness in the elderly. Development of an RSV vaccine was stymied when a clinical trial using a formalin-inactivated RSV virus made disease, following RSV infection, more severe. Recent studies have defined the structures that the RSV F envelope glycoprotein adopts before and after virus entry (prefusion and postfusion conformations, respectively). Key neutralization epitopes of prefusion and postfusion RSV F have been identified, and a number of current vaccine development efforts are focused on generating easily produced subunit antigens that retain these epitopes. Here we show that a simple modification in the F ectodomain results in a homogeneous protein that retains critical prefusion neutralizing epitopes. These results improve our understanding of RSV F protein folding and structure and can guide further vaccine design efforts.

  • The M33 G Protein-Coupled Receptor Encoded by Murine Cytomegalovirus Is Dispensable for Hematogenous Dissemination but Is Required for Growth within the Salivary Gland [Pathogenesis and Immunity]

  • Human cytomegalovirus (HCMV) is a pathogen found worldwide and is a serious threat to immunocompromised individuals and developing fetuses. Due to the species specificity of cytomegaloviruses, murine cytomegalovirus (MCMV) has been used as a model for in vivo studies of HCMV pathogenesis. The MCMV genome, like the genomes of other beta- and gammaherpesviruses, encodes G protein-coupled receptors (GPCRs) that modulate host signaling pathways presumably to facilitate viral replication and dissemination. Among these viral receptors, the M33 GPCR carried by MCMV is an activator of CREB, NF-B, and phospholipase C-bbeta; signaling pathways and has been implicated in aspects of pathogenesis in vivo, including persistence in the salivary glands of BALB/c mice. In this study, we used immunocompetent nonobese diabetic (NOD) and immunocompromised NOD-scid-gamma (NSG) mice to further investigate the salivary gland defect exhibited by M33 deficiency. Interestingly, we demonstrate that virus with an M33 deletion (M33) can replicate in the salivary gland of immunocompromised animals, albeit with a 400-fold growth defect compared with the growth of wild-type virus. Moreover, we determined that M33 does not have a role in cell-associated hematogenous dissemination but is required for viral amplification once the virus reaches the salivary gland. We conclude that the reduced replicative capacity of the M33 virus is due to a specific defect occurring within the localized environment of the salivary gland. Importantly, since the salivary gland represents a site essential for persistence and horizontal transmission, an understanding of the mechanisms of viral replication within this site could lead to the generation of novel therapeutics useful for the prevention of HCMV spread.

    IMPORTANCE Human cytomegalovirus infects the majority of the American people and can reside silently in infected individuals for the duration of their lives. Under a number of circumstances, the virus can reactivate, leading to a variety of diseases in both adults and developing babies, and therefore, identifying the function of viral proteins is essential to understand how the virus spreads and causes disease. We aim to utilize animal models to study the function of an important class of viral proteins termed G protein-coupled receptors with the ultimate goal of developing inhibitors to these proteins that could one day be used to prevent viral spread.

  • A Chimeric Virus-Mouse Model System for Evaluating the Function and Inhibition of Papain-Like Proteases of Emerging Coronaviruses [Vaccines and Antiviral Agents]

  • To combat emerging coronaviruses, developing safe and efficient platforms to evaluate viral protease activities and the efficacy of protease inhibitors is a high priority. Here, we exploit a biosafety level 2 (BSL-2) chimeric Sindbis virus system to evaluate protease activities and the efficacy of inhibitors directed against the papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus (SARS-CoV), a biosafety level 3 (BSL-3) pathogen. We engineered Sindbis virus to coexpress PLpro and a substrate, murine interferon-stimulated gene 15 (ISG15), and found that PLpro mediates removal of ISG15 (deISGylation) from cellular proteins. Mutation of the catalytic cysteine residue of PLpro or addition of a PLpro inhibitor blocked deISGylation in virus-infected cells. Thus, deISGylation is a marker of PLpro activity. Infection of alpha/beta interferon receptor knockout (IFNARnndash;/nndash;) mice with these chimeric viruses revealed that PLpro deISGylation activity removed ISG15-mediated protection during viral infection. Importantly, administration of a PLpro inhibitor protected these mice from lethal infection, demonstrating the efficacy of a coronavirus protease inhibitor in a mouse model. However, this PLpro inhibitor was not sufficient to protect the mice from lethal infection with SARS-CoV MA15, suggesting that further optimization of the delivery and stability of PLpro inhibitors is needed. We extended the chimeric-virus platform to evaluate the papain-like protease/deISGylating activity of Middle East respiratory syndrome coronavirus (MERS-CoV) to provide a small-animal model to evaluate PLpro inhibitors of this recently emerged pathogen. This platform has the potential to be universally adaptable to other viral and cellular enzymes that have deISGylating activities.

    IMPORTANCE Evaluating viral protease inhibitors in a small-animal model is a critical step in the path toward antiviral drug development. We modified a biosafety level 2 chimeric virus system to facilitate evaluation of inhibitors directed against highly pathogenic coronaviruses. We used this system to demonstrate the in vivo efficacy of an inhibitor of the papain-like protease of severe acute respiratory syndrome coronavirus. Furthermore, we demonstrate that the chimeric-virus system can be adapted to study the proteases of emerging human pathogens, such as Middle East respiratory syndrome coronavirus. This system provides an important tool to rapidly assess the efficacy of protease inhibitors targeting existing and emerging human pathogens, as well as other enzymes capable of removing ISG15 from cellular proteins.

  • An Eriophyid Mite-Transmitted Plant Virus Contains Eight Genomic RNA Segments with Unusual Heterogeneity in the Nucleocapsid Protein [Genetic Diversity and Evolution]

  • Eriophyid mite-transmitted, multipartite, negative-sense RNA plant viruses with membrane-bound spherical virions are classified in the genus Emaravirus. We report here that the eriophyid mite-transmitted Wheat mosaic virus (WMoV), an Emaravirus, contains eight genomic RNA segments, the most in a known negative-sense RNA plant virus. Remarkably, two RNA 3 consensus sequences, encoding the nucleocapsid protein, were found with 12.5% sequence divergence, while no heterogeneity was observed in the consensus sequences of additional genomic RNA segments. The RNA-dependent RNA polymerase, glycoprotein precursor, nucleocapsid, and P4 proteins of WMoV exhibited limited sequence homology with the orthologous proteins of other emaraviruses, while proteins encoded by additional genomic RNA segments displayed no significant homology with proteins reported in GenBank, suggesting that the genus Emaravirus evolved further with a divergent octapartite genome. Phylogenetic analyses revealed that WMoV formed an evolutionary link between members of the Emaravirus genus and the family Bunyaviridae. Furthermore, genomic-length virus- and virus-complementary (vc)-sense strands of all WMoV genomic RNAs accumulated asymmetrically in infected wheat, with 10- to 20-fold more virus-sense genomic RNAs than vc-sense RNAs. These data further confirm the octapartite negative-sense polarity of the WMoV genome. In WMoV-infected wheat, subgenomic-length mRNAs of vc sense were detected for genomic RNAs 3, 4, 7, and 8 but not for other RNA species, suggesting that the open reading frames present in the complementary sense of genomic RNAs are expressed through subgenomic- or near-genomic-length vc-sense mRNAs.

    IMPORTANCE Wheat mosaic virus (WMoV), an Emaravirus, is the causal agent of High Plains disease of wheat and maize. In this study, we demonstrated that the genome of WMoV comprises eight negative-sense RNA segments with an unusual sequence polymorphism in an RNA encoding the nucleocapsid protein but not in the additional genomic RNA segments. WMoV proteins displayed weak or no homology with reported emaraviruses, suggesting that the genus Emaravirus further evolved with a divergent octapartite genome. The current study also examined the profile of WMoV RNA accumulation in wheat and provided evidence for the synthesis of subgenomic-length mRNAs of virus complementary sense. This is the first report to demonstrate that emaraviruses produce subgenomic-length mRNAs that are most likely utilized for genome expression. Importantly, this study facilitates the examination of gene functions and virus diversity and the development of effective diagnostic methods and management strategies for an economically important but poorly understood virus.

  • The Genomes, Proteomes, and Structures of Three Novel Phages That Infect the Bacillus cereus Group and Carry Putative Virulence Factors [Genetic Diversity and Evolution]

  • This article reports the results of studying three novel bacteriophages, JL, Shanette, and Basilisk, which infect the pathogen Bacillus cereus and carry genes that may contribute to its pathogenesis. We analyzed host range and superinfection ability, mapped their genomes, and characterized phage structure by mass spectrometry and transmission electron microscopy (TEM). The JL and Shanette genomes were 96% similar and contained 217 open reading frames (ORFs) and 220 ORFs, respectively, while Basilisk has an unrelated genome containing 138 ORFs. Mass spectrometry revealed 23 phage particle proteins for JL and 15 for Basilisk, while only 11 and 4, respectively, were predicted to be present by sequence analysis. Structural protein homology to well-characterized phages suggested that JL and Shanette were members of the family Myoviridae, which was confirmed by TEM. The third phage, Basilisk, was similar only to uncharacterized phages and is an unrelated siphovirus. Cryogenic electron microscopy of this novel phage revealed a T=9 icosahedral capsid structure with the major capsid protein (MCP) likely having the same fold as bacteriophage HK97 MCP despite the lack of sequence similarity. Several putative virulence factors were encoded by these phage genomes, including TerC and TerD involved in tellurium resistance. Host range analysis of all three phages supports genetic transfer of such factors within the B. cereus group, including B. cereus, B. anthracis, and B. thuringiensis. This study provides a basis for understanding these three phages and other related phages as well as their contributions to the pathogenicity of B. cereus group bacteria.

    IMPORTANCE The Bacillus cereus group of bacteria contains several human and plant pathogens, including B. cereus, B. anthracis, and B. thuringiensis. Phages are intimately linked to the evolution of their bacterial hosts and often provide virulence factors, making the study of B. cereus phages important to understanding the evolution of pathogenic strains. Herein we provide the results of detailed study of three novel B. cereus phages, two highly related myoviruses (JL and Shanette) and an unrelated siphovirus (Basilisk). The detailed characterization of host range and superinfection, together with results of genomic, proteomic, and structural analyses, reveal several putative virulence factors as well as the ability of these phages to infect different pathogenic species.

  • High Eomesodermin Expression among CD57+ CD8+ T Cells Identifies a CD8+ T Cell Subset Associated with Viral Control during Chronic Human Immunodeficiency Virus Infection [Cellular Response to Infection]

  • During HIV infection, increased CD57 expression among CD8+ T cells has been associated with immune senescence and defective immune responses. Interestingly, CD57-expressing CD8+ T cells exhibit a dual profile, being simultaneously highly cytotoxic (terminally differentiated effectors) and poorly proliferative (replicative senescent). Recent publications point toward a positive role of CD57-expressing CD8+ T cell subsets, presumably due to their high cytolytic activity. We further investigated the phenotype of CD57-expressing CD8+ T cells in healthy donors and during HIV infection combining CD57 expression to Eomesodermin (EOMES), a T box transcription factor which determines, coordinately with T-bet, effector and memory CD8+ T cell differentiation. We defined in healthy donors two functionally distinct CD57-expressing CD8+ T cell subsets exhibiting different levels of EOMES expression: EOMEShi CD57+ and EOMESint CD57+ CD8+ T cells. EOMEShi CD57+ cells exhibited low cytotoxic activity but preserved proliferative capacity and interleukin 7 (IL-7) receptor expression, whereas EOMESint CD57+ cells exhibited obvious cytotoxic functions and a more terminally differentiated phenotype. We next performed a similar analysis in different contexts of HIV infection: primary infected patients, long-term viremic patients, aviremic patients treated with antiretroviral therapy, and HIV controllers; we demonstrated a higher percentage of CD57-expressing cells in all HIV-infected patients regardless of virological status. When heterogeneity in EOMES expression among CD57 cells was taken into account, we detected significantly higher proportions of EOMEShi CD57+ cells among HIV-specific and nonspecific CD8+ T cells from HIV controllers than in aviremic antiretroviral-treated patients and viremic patients. Importantly, such a peculiar non-terminally differentiated EOMEShi CD57+ phenotypic profile was associated with viral control.

    IMPORTANCE This study demonstrates that functional heterogeneity exists among CD57-expressing CD8 T cells, which include both terminally differentiated, highly cytotoxic EOMESint CD57+ CD8+ T cells and less differentiated EOMEShi CD57+ CD8 T cells, which do not exhibit immediate cytotoxic functions but present high proliferative capacity. Interestingly, HIV controllers present a high proportion of EOMEShi CD57 cells among CD57-expressing HIV-specific CD8 T cells compared to both long-term viremic and aviremic antiretroviral therapy (ART)-treated patients, suggesting a beneficial role for this cell subset in viral control.

  • Epstein-Barr Virus-Encoded Latent Membrane Protein 2A Promotes the Epithelial-Mesenchymal Transition in Nasopharyngeal Carcinoma via Metastatic Tumor Antigen 1 and Mechanistic Target of Rapamycin Signaling Induction [Transformation and Oncogenesis]

  • Epstein-Barr virus-encoded latent membrane protein 2A (LMP2A) promotes the epithelial-mesenchymal transition (EMT) of nasopharyngeal carcinoma (NPC), thereby increasing tumor invasion. Recently, the dysregulation of metastatic tumor antigen 1 (MTA1) was found to enhance tumor metastasis in a variety of cancers. A molecular connection between these two proteins has been proposed but not firmly established. In this study, we reported the overexpression of MTA1 in 29/60 (48.3%) NPC patients, and the overexpression of MTA1 significantly correlated with tumor metastasis. The overexpression of MTA1 promoted EMT via the Wnt1 pathway and bbeta;-catenin activation. We demonstrated that LMP2A reinforces the expression of MTA1 via the mechanistic target of rapamycin (mTOR) pathway to promote EMT in NPC. Furthermore, by knocking down 4EBP1 in combination with the new mTOR inhibitor INK-128 treatment, we discovered that LMP2A expression activates the 4EBP1-eIF4E axis and increases the expression of MTA1 at the translational level partially independent of c-myc. These findings provided novel insights into the correlation between the LMP2A and MTA1 proteins and reveal a novel function of the 4EBP1-eIF4E axis in EMT of nasopharyngeal carcinoma.

    IMPORTANCE Prevention of the recurrence and metastasis of NPC is critical to achieving a successful NPC treatment. As we all know, EMT has a vital role in metastasis of malignancies. LMP2A, an oncoprotein of Epstein-Barr virus, a well-known NPC activator, induces EMT and has been proved to exert a promoting effect in tumor metastasis. Our study demonstrated that LMP2A could induce EMT by activating MTA1 at the translational level via activating mTOR signaling and the 4EBP1-eIF4E axis. Taken together, our findings bridge the gap between the NPC-specific cell surface molecule and the final phenotype of the NPC cells. Additionally, our findings indicate that LMP2A and mTOR will serve as targets for NPC therapy in the future.

  • Coronaviruses Resistant to a 3C-Like Protease Inhibitor Are Attenuated for Replication and Pathogenesis, Revealing a Low Genetic Barrier but High Fitness Cost of Resistance [Vaccines and Antiviral Agents]

  • Viral protease inhibitors are remarkably effective at blocking the replication of viruses such as human immunodeficiency virus and hepatitis C virus, but they inevitably lead to the selection of inhibitor-resistant mutants, which may contribute to ongoing disease. Protease inhibitors blocking the replication of coronavirus (CoV), including the causative agents of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), provide a promising foundation for the development of anticoronaviral therapeutics. However, the selection and consequences of inhibitor-resistant CoVs are unknown. In this study, we exploited the model coronavirus, mouse hepatitis virus (MHV), to investigate the genotype and phenotype of MHV quasispecies selected for resistance to a broad-spectrum CoV 3C-like protease (3CLpro) inhibitor. Clonal sequencing identified single or double mutations within the 3CLpro coding sequence of inhibitor-resistant virus. Using reverse genetics to generate isogenic viruses with mutant 3CLpros, we found that viruses encoding double-mutant 3CLpros are fully resistant to the inhibitor and exhibit a significant delay in proteolytic processing of the viral replicase polyprotein. The inhibitor-resistant viruses also exhibited postponed and reduced production of infectious virus particles. Biochemical analysis verified double-mutant 3CLpro enzyme as impaired for protease activity and exhibiting reduced sensitivity to the inhibitor and revealed a delayed kinetics of inhibitor hydrolysis and activity restoration. Furthermore, the inhibitor-resistant virus was shown to be highly attenuated in mice. Our study provides the first insight into the pathogenicity and mechanism of 3CLpro inhibitor-resistant CoV mutants, revealing a low genetic barrier but high fitness cost of resistance.

    IMPORTANCE RNA viruses are infamous for their ability to evolve in response to selective pressure, such as the presence of antiviral drugs. For coronaviruses such as the causative agent of Middle East respiratory syndrome (MERS), protease inhibitors have been developed and shown to block virus replication, but the consequences of selection of inhibitor-resistant mutants have not been studied. Here, we report the low genetic barrier and relatively high deleterious consequences of CoV resistance to a 3CLpro protease inhibitor in a coronavirus model system, mouse hepatitis virus (MHV). We found that although mutations that confer resistance arise quickly, the resistant viruses replicate slowly and do not cause lethal disease in mice. Overall, our study provides the first analysis of the low barrier but high cost of resistance to a CoV 3CLpro inhibitor, which will facilitate the further development of protease inhibitors as anti-coronavirus therapeutics.

  • Inhibition of the Human Respiratory Syncytial Virus Small Hydrophobic Protein and Structural Variations in a Bicelle Environment [Structure and Assembly]

  • The small hydrophobic (SH) protein is a 64-amino-acid polypeptide encoded by the human respiratory syncytial virus (hRSV). SH protein has a single aalpha;-helical transmembrane (TM) domain that forms pentameric ion channels. Herein, we report the first inhibitor of the SH protein channel, pyronin B, and we have mapped its binding site to a conserved surface of the RSV SH pentamer, at the C-terminal end of the transmembrane domain. The validity of the SH protein structural model used has been confirmed by using a bicellar membrane-mimicking environment. However, in bicelles the aalpha;-helical stretch of the TM domain extends up to His-51, and by comparison with previous models both His-22 and His-51 adopt an interhelical/lumenal orientation relative to the channel pore. Neither His residue was found to be essential for channel activity although His-51 protonation reduced channel activity at low pH, with His-22 adopting a more structural role. The latter results are in contrast with previous patch clamp data showing channel activation at low pH, which could not be reproduced in the present work. Overall, these results establish a solid ground for future drug development targeting this important viroporin.

    IMPORTANCE The human respiratory syncytial virus (hRSV) is responsible for 64 million reported cases of infection and 160,000 deaths each year. Lack of adequate antivirals fuels the search for new targets for treatment. The small hydrophobic (SH) protein is a 64-amino-acid polypeptide encoded by hRSV and other paramyxoviruses, and its absence leads to viral attenuation in vivo and early apoptosis in infected cells. SH protein forms pentameric ion channels that may constitute novel drug targets, but no inhibitor for this channel activity has been reported so far. A small-molecule inhibitor, pyronin B, can reduce SH channel activity, and its likely binding site on the SH protein channel has been identified. Black lipid membrane (BLM) experiments confirm that protonation of both histidine residues reduces stability and channel activity. These results contrast with previous patch clamp data that showed low-pH activation, which we have not been able to reproduce.

  • Inhibition of Enterovirus 71 by Adenosine Analog NITD008 [Vaccines and Antiviral Agents]

  • Enterovirus 71 (EV71) is a major viral pathogen in China and Southeast Asia. There is no clinically approved vaccine or antiviral therapy for EV71 infection. NITD008, an adenosine analog, is an inhibitor of flavivirus that blocks viral RNA synthesis. Here we report that NITD008 has potent antiviral activity against EV71. In cell culture, the compound inhibits EV71 at a 50% effective concentration of 0.67 mmu;M and a 50% cytotoxic concentration of 119.97 mmu;M. When administered at 5 mg/kg in an EV71 mouse model, the compound reduced viral loads in various organs and completely prevented clinical symptoms and death. To study the antiviral mechanism and drug resistance, we selected escape mutant viruses by culturing EV71 with increasing concentrations of NITD008. Resistance mutations were reproducibly mapped to the viral 3A and 3D polymerase regions. Resistance analysis with recombinant viruses demonstrated that either a 3A or a 3D mutation alone could lead to resistance to NITD008. A combination of both 3A and 3D mutations conferred higher resistance, suggesting a collaborative interplay between the 3A and 3D proteins during viral replication. The resistance results underline the importance of combination therapy required for EV71 treatment.

    IMPORTANCE Human enterovirus 71 (EV71) has emerged as a major cause of viral encephalitis in children worldwide, especially in the Asia-Pacific region. Vaccines and antivirals are urgently needed to prevent and treat EV71 infections. In this study, we report the in vitro and in vivo efficacy of NITD008 (an adenosine analog) as an inhibitor of EV71. The efficacy results validated the potential of nucleoside analogs as antiviral drugs for EV71 infections. Mechanistically, we showed that mutations in the viral 3A and 3D polymerases alone or in combination could confer resistance to NITD008. The resistance results suggest an intrinsic interaction between viral proteins 3A and 3D during replication, as well as the importance of combination therapy for the treatment of EV71 infections.

  • Hepatitis E Virus Inhibits Type I Interferon Induction by ORF1 Products [Virus-Cell Interactions]

  • Hepatitis E virus (HEV) causes both endemic and epidemic human hepatitis by fecal-oral transmission in many parts of the world. Zoonotic transmission of HEV from animals to humans has been reported. Due to the lack of an efficient cell culture system, the molecular mechanisms of HEV infection remain largely unknown. In this study, we found that HEV replication in hepatoma cells inhibited poly(Immiddot;C)-induced beta interferon (IFN-bbeta;) expression and that the HEV open reading frame 1 (ORF1) product was responsible for this inhibition. Two domains, X and the papain-like cysteine protease domain (PCP), of HEV ORF1 were identified as the putative IFN antagonists. When overexpressed in HEK293T cells, the X domain (or macro domain) inhibited poly(Immiddot;C)-induced phosphorylation of interferon regulatory factor 3 (IRF-3), which is the key transcription factor for IFN induction. The PCP domain was shown to have deubiquitinase activity for both RIG-I and TBK-1, whose ubiquitination is a key step in their activation in poly(Immiddot;C)-induced IFN induction. Furthermore, replication of a HEV replicon containing green fluorescent protein (GFP) (E2-GFP) in hepatoma cells led to impaired phosphorylation of IRF-3 and reduced ubiquitination of RIG-I and TBK-1, which confirmed our observations of X and PCP inhibitory effects in HEK293T cells. Altogether, our study identified the IFN antagonists within the HEV ORF1 polyprotein and expanded our understanding of the functions of several of the HEV ORF1 products, as well as the mechanisms of HEV pathogenesis.

    IMPORTANCE Type I interferons (IFNs) are important components of innate immunity and play a crucial role against viral infection. They also serve as key regulators to evoke an adaptive immune response. Virus infection can induce the synthesis of interferons; however, viruses have evolved many strategies to antagonize the induction of interferons. There is little knowledge about how hepatitis E virus (HEV) inhibits induction of host IFNs, though the viral genome was sequenced more than 2 decades ago. This is the first report of identification of the potential IFN antagonists encoded by HEV. By screening all the domains in the open reading frame 1 (ORF1) polyprotein, we identified two IFN antagonists and performed further research to determine how and at which step in the IFN induction pathway they antagonize host IFN induction. Our work provides valuable information about HEV-cell interaction and pathogenesis.

  • Specific Argonautes Selectively Bind Small RNAs Derived from Potato Spindle Tuber Viroid and Attenuate Viroid Accumulation In Vivo [Virus-Cell Interactions]

  • The identification of viroid-derived small RNAs (vd-sRNAs) of 21 to 24 nucleotides (nt) in plants infected by viroids (infectious non-protein-coding RNAs of just 250 to 400 nt) supports their targeting by Dicer-like enzymes, the first host RNA-silencing barrier. However, whether viroids, like RNA viruses, are also targeted by the RNA-induced silencing complex (RISC) remains controversial. At the RISC core is one Argonaute (AGO) protein that, guided by endogenous or viral sRNAs, targets complementary RNAs. To examine whether AGO proteins also load vd-sRNAs, leaves of Nicotiana benthamiana infected by potato spindle tuber viroid (PSTVd) were agroinfiltrated with plasmids expressing epitope-tagged versions of AGO1, AGO2, AGO3, AGO4, AGO5, AGO6, AGO7, AGO9, and AGO10 from Arabidopsis thaliana. Immunoprecipitation analyses of the agroinfiltrated halos revealed that all AGOs except AGO6, AGO7, and AGO10 associated with vd-sRNAs: AGO1, AGO2, and AGO3 preferentially with those of 21 and 22 nt, while AGO4, AGO5, and AGO9 additionally bound those of 24 nt. Deep-sequencing analyses showed that sorting of vd-sRNAs into AGO1, AGO2, AGO4, and AGO5 depended essentially on their 5'-terminal nucleotides, with the profiles of the corresponding AGO-loaded vd-sRNAs adopting specific hot spot distributions along the viroid genome. Furthermore, agroexpression of AGO1, AGO2, AGO4, and AGO5 on PSTVd-infected tissue attenuated the level of the genomic RNAs, suggesting that they, or their precursors, are RISC targeted. In contrast to RNA viruses, PSTVd infection of N. benthamiana did not affect miR168-mediated regulation of the endogenous AGO1, which loaded vd-sRNAs with specificity similar to that of its A. thaliana counterpart.

    IMPORTANCE To contain invaders, particularly RNA viruses, plants have evolved an RNA-silencing mechanism relying on the generation by Dicer-like (DCL) enzymes of virus-derived small RNAs of 21 to 24 nucleotides (nt) that load and guide Argonaute (AGO) proteins to target and repress viral RNA. Viroids, despite their minimal genomes (non-protein-coding RNAs of only 250 to 400 nt), infect and incite disease in plants. The accumulation in these plants of 21- to 24-nt viroid-derived small RNAs (vd-sRNAs) supports the notion that DCLs also target viroids but does not clarify whether vd-sRNAs activate one or more AGOs. Here, we show that in leaves of Nicotiana benthamiana infected by potato spindle tuber viroid, the endogenous AGO1 and distinct AGOs from Arabidopsis thaliana that were overexpressed were associated with vd-sRNAs displaying the same properties (5'-terminal nucleotide and size) previously established for endogenous and viral small RNAs. Overexpression of AGO1, AGO2, AGO4, and AGO5 attenuated viroid accumulation, supporting their role in antiviroid defense.

  • Mutagenesis of the Catalytic and Cleavage Site Residues of the Hypovirus Papain-Like Proteases p29 and p48 Reveals Alternative Processing and Contributions to Optimal Viral RNA Accumulation [Genome Replication and Regulation of Viral Gene Expression]

  • The positive-stranded RNA genome of the prototypic virulence-attenuating hypovirus CHV-1/EP713 contains two open reading frames (ORF), each encoding an autocatalytic papain-like leader protease. Protease p29, derived from the N-terminal portion of ORF A, functions as a suppressor of RNA silencing, while protease p48, derived from the N-terminal portion of ORF B, is required for viral RNA replication. The catalytic and cleavage site residues required for autoproteolytic processing have been functionally mapped in vitro for both proteases but not confirmed in the infected fungal host. We report here the mutagenesis of the CHV-1/EP713 infectious cDNA clone to define the requirements for p29 and p48 cleavage and the role of autoproteolysis in the context of hypovirus replication. Mutation of the catalytic cysteine and histidine residues for either p29 or p48 was tolerated but reduced viral RNA accumulation to ca. 20 to 50% of the wild-type level. Mutation of the p29 catalytic residues caused an accumulation of unprocessed ORF A product p69. Surprisingly, the release of p48 from the ORF B-encoded polyprotein was not prevented by mutation of the p48 catalytic and cleavage site residues and was independent of p29. The results show that, while dispensable for hypovirus replication, the autocatalytic processing of the leader proteases p29 and p48 contributes to optimal virus RNA accumulation. The role of the predicted catalytic residues in autoproteolytic processing of p29 was confirmed in the infected host, while p48 was found to also undergo alternative processing independent of the encoded papain-like protease activities.

    IMPORTANCE Hypoviruses are positive-strand RNA mycoviruses that attenuate virulence of their pathogenic fungal hosts. The prototypic hypovirus CHV-1/EP713, which infects the chestnut bight fungus Cryphonetria parasitica, encodes two papain-like autocatalytic leader proteases, p29 and p48, that also have important functions in suppressing the RNA silencing antiviral defense response and in viral RNA replication, respectively. The mutational analyses of the CHV-1/EP713 infectious cDNA clone, reported here, define the requirements for p29 and p48 cleavage and the functional importance of autoproteolysis in the context of hypovirus replication and exposed an alternative p48 processing pathway independent of the encoded papain-like protease activities. These findings provide additional insights into hypovirus gene expression, replication, and evolution and inform ongoing efforts to engineer hypoviruses for interrogating and modulating fungal virulence.

  • Effect of Formaldehyde Inactivation on Poliovirus [Vaccines and Antiviral Agents]

  • Inactivated polio vaccines, which have been used in many countries for more than 50 years, are produced by treating live poliovirus (PV) with formaldehyde. However, the molecular mechanisms underlying virus inactivation are not well understood. Infection by PV is initiated by virus binding to specific cell receptors, which results in viral particles undergoing sequential conformational changes that generate altered structural forms (135S and 80S particles) and leads to virus cell entry. We have analyzed the ability of inactivated PV to bind to the human poliovirus receptor (hPVR) using various techniques such as ultracentrifugation, fluorescence-activated cell sorting flow cytometry and real-time reverse transcription-PCR (RT-PCR). The results showed that although retaining the ability to bind to hPVR, inactivated PV bound less efficiently in comparison to live PV. We also found that inactivated PV showed resistance to structural conversion in vitro, as judged by measuring changes in antigenicity, the ability to bind to hPVR, and viral RNA release at high temperature. Furthermore, viral RNA from inactivated PV was shown to be modified, since cDNA yields obtained by RT-PCR amplification were severely reduced and no infectious virus was recovered after RNA transfection into susceptible cells.

    IMPORTANCE This study represents a novel insight into the molecular mechanisms responsible for poliovirus inactivation. We show that inactivation with formaldehyde has an effect on early steps of viral replication as it reduces the ability of PV to bind to hPVR, decreases the sensitivity of PV to convert to 135S particles, and abolishes the infectivity of its viral RNA. These changes are likely responsible for the loss of infectivity shown by PV following inactivation. Techniques used in this study represent new approaches for the characterization of inactivated PV products and could be useful in developing improved methods for the production and quality control testing of inactivated polio vaccines. Measuring the antigenicity, capsid stability, and RNA integrity of inactivated PV samples could help establishing the optimal balance between the loss of infectivity and the preservation of virus antigenicity during inactivation.

  • Inactivation of the Human Papillomavirus E6 or E7 Gene in Cervical Carcinoma Cells by Using a Bacterial CRISPR/Cas RNA-Guided Endonuclease [Vaccines and Antiviral Agents]

  • High-risk human papillomaviruses (HPVs), including HPV-16 and HPV-18, are the causative agents of cervical carcinomas and are linked to several other tumors of the anogenital and oropharyngeal regions. The majority of HPV-induced tumors contain integrated copies of the normally episomal HPV genome that invariably retain intact forms of the two HPV oncogenes E6 and E7. E6 induces degradation of the cellular tumor suppressor p53, while E7 destabilizes the retinoblastoma (Rb) protein. Previous work has shown that loss of E6 function in cervical cancer cells induces p53 expression as well as downstream effectors that induce apoptosis and cell cycle arrest. Similarly, loss of E7 allows increased Rb expression, leading to cell cycle arrest and senescence. Here, we demonstrate that expression of a bacterial Cas9 RNA-guided endonuclease, together with single guide RNAs (sgRNAs) specific for E6 or E7, is able to induce cleavage of the HPV genome, resulting in the introduction of inactivating deletion and insertion mutations into the E6 or E7 gene. This results in the induction of p53 or Rb, leading to cell cycle arrest and eventual cell death. Both HPV-16- and HPV-18-transformed cells were found to be responsive to targeted HPV genome-specific DNA cleavage. These data provide a proof of principle for the idea that vector-delivered Cas9/sgRNA combinations could represent effective treatment modalities for HPV-induced cancers.

    IMPORTANCE Human papillomaviruses (HPVs) are the causative agents of almost all cervical carcinomas and many other tumors, including many head and neck cancers. In these cancer cells, the HPV DNA genome is integrated into the cellular genome, where it expresses high levels of two viral oncogenes, called E6 and E7, that are required for cancer cell growth and viability. Here, we demonstrate that the recently described bacterial CRISPR/Cas RNA-guided endonuclease can be reprogrammed to target and destroy the E6 or E7 gene in cervical carcinoma cells transformed by HPV, resulting in cell cycle arrest, leading to cancer cell death. We propose that viral vectors designed to deliver E6- and/or E7-specific CRISPR/Cas to tumor cells could represent a novel and highly effective tool to treat and eliminate HPV-induced cancers.

  • Attachment Protein G of an African Bat Henipavirus Is Differentially Restricted in Chiropteran and Nonchiropteran Cells [Virus-Cell Interactions]

  • Henipaviruses are associated with pteropodid reservoir hosts. The glycoproteins G and F of an African henipavirus (strain M74) have been reported to induce syncytium formation in kidney cells derived from a Hypsignathus monstrosus bat (HypNi/1.1) but not in nonchiropteran BHK-21 and Vero76 cells. Here, we show that syncytia are also induced in two other pteropodid cell lines from Hypsignathus monstrosus and Eidolon helvum bats upon coexpression of the M74 glycoproteins. The G protein was transported to the surface of transfected chiropteran cells, whereas surface expression in the nonchiropteran cells was detectable only in a fraction of cells. In contrast, the G protein of Nipah virus is transported efficiently to the surface of both chiropteran and nonchiropteran cells. Even in chiropteran cells, M74-G was predominantly expressed in the endoplasmic reticulum (ER), as indicated by colocalization with marker proteins. This result is consistent with the finding that all N-glycans of the M74-G proteins are of the mannose-rich type, as indicated by sensitivity to endo H treatment. These data indicate that the surface transport of M74-G is impaired in available cell culture systems, with larger amounts of viral glycoprotein present on chiropteran cells than on nonchiropteran cells. The restricted surface expression of M74-G explains the reduced fusion activity of the glycoproteins of the African henipavirus. Our results suggest strategies for the isolation of infectious viruses, which is necessary to assess the risk of zoonotic virus transmission.

    IMPORTANCE Henipaviruses are highly pathogenic zoonotic viruses associated with pteropodid bat hosts. Whether the recently described African bat henipaviruses have a zoonotic potential as high as that of their Asian and Australian relatives is unknown. We show that surface expression of the attachment protein G of an African henipavirus, M74, is restricted in comparison to the G protein expression of the highly pathogenic Nipah virus. Transport to the cell surface is more restricted in nonchiropteran cells than it is in chiropteran cells, explaining the differential fusion activity of the M74 surface proteins in these cells. Our results imply that surface expression of viral glycoproteins may serve as a major marker to assess the zoonotic risk of emerging henipaviruses.

  • Influenza A Virus Acquires Enhanced Pathogenicity and Transmissibility after Serial Passages in Swine [Pathogenesis and Immunity]

  • Genetic and phylogenetic analyses suggest that the pandemic H1N1/2009 virus was derived from well-established swine influenza lineages; however, there is no convincing evidence that the pandemic virus was generated from a direct precursor in pigs. Furthermore, the evolutionary dynamics of influenza virus in pigs have not been well documented. Here, we subjected a recombinant virus (rH1N1) with the same constellation makeup as the pandemic H1N1/2009 virus to nine serial passages in pigs. The severity of infection sequentially increased with each passage. Deep sequencing of viral quasispecies from the ninth passage found five consensus amino acid mutations: PB1 A469T, PA 1129T, NA N329D, NS1 N205K, and NEP T48N. Mutations in the hemagglutinin (HA) protein, however, differed greatly between the upper and lower respiratory tracts. Three representative viral clones with the five consensus mutations were selected for functional evaluation. Relative to the parental virus, the three viral clones showed enhanced replication and polymerase activity in vitro and enhanced replication, pathogenicity, and transmissibility in pigs, guinea pigs, and ferrets in vivo. Specifically, two mutants of rH1N1 (PB1 A469T and a combination of NS1 N205K and NEP T48N) were identified as determinants of transmissibility in guinea pigs. Crucially, one mutant viral clone with the five consensus mutations, which also carried D187E, K211E, and S289N mutations in its HA, additionally was able to infect ferrets by airborne transmission as effectively as the pandemic virus. Our findings demonstrate that influenza virus can acquire viral characteristics that are similar to those of the pandemic virus after limited serial passages in pigs.

    IMPORTANCE We demonstrate here that an engineered reassortant swine influenza virus, with the same gene constellation pattern as the pandemic H1N1/2009 virus and subjected to only nine serial passages in pigs, acquired greatly enhanced virulence and transmissibility. In particular, one representative pathogenic passaged virus clone, which carried three mutations in the HA gene and five consensus mutations in PB1, PA, NA, NS1, and NEP genes, additionally was able to confer respiratory droplet transmission as effectively as the pandemic H1N1/2009 virus. Our findings suggest that pigs can readily induce adaptive mutational changes to a precursor pandemic-like virus to transform it into a highly virulent and infectious form akin to that of the pandemic H1N1/2009 virus, which underlines the potential direct role of pigs in promoting influenza A virus pathogenicity and transmissibility.

  • Coinfection with Streptococcus pneumoniae Modulates the B Cell Response to Influenza Virus [Cellular Response to Infection]

  • Pathogen-specific antibodies (Abs) protect against respiratory infection with influenza A virus (IAV) and Streptococcus pneumoniae and are the basis of effective vaccines. Sequential or overlapping coinfections with both pathogens are common, yet the impact of coinfection on the generation and maintenance of Ab responses is largely unknown. We report here that the B cell response to IAV is altered in mice coinfected with IAV and S. pneumoniae and that this response differs, depending on the order of pathogen exposure. In mice exposed to S. pneumoniae prior to IAV, the initial virus-specific germinal center (GC) B cell response is significantly enhanced in the lung-draining mediastinal lymph node and spleen, and there is an increase in CD4+ T follicular helper (TFH) cell numbers. In contrast, secondary S. pneumoniae infection exaggerates early antiviral antibody-secreting cell formation, and at later times, levels of GCs, TFH cells, and antiviral serum IgG are elevated. Mice exposed to S. pneumoniae prior to IAV do not maintain the initially robust GC response in secondary lymphoid organs and exhibit reduced antiviral serum IgG with diminished virus neutralization activity a month after infection. Our data suggest that the history of pathogen exposures can critically affect the generation of protective antiviral Abs and may partially explain the differential susceptibility to and disease outcomes from IAV infection in humans.

    IMPORTANCE Respiratory tract coinfections, specifically those involving influenza A viruses and Streptococcus pneumoniae, remain a top global health burden. We sought to determine how S. pneumoniae coinfection modulates the B cell immune response to influenza virus since antibodies are key mediators of protection.

  • Induction of CD8 T Cell Heterologous Protection by a Single Dose of Single-Cycle Infectious Influenza Virus [Pathogenesis and Immunity]

  • The effector functions of specific CD8 T cells are crucial in mediating influenza heterologous protection. However, new approaches for influenza vaccines that can trigger effective CD8 T cell responses have not been extensively explored. We report here the generation of single-cycle infectious influenza virus that lacks a functional hemagglutinin (HA) gene on an X31 genetic background and demonstrate its potential for triggering protective CD8 T cell immunity against heterologous influenza virus challenge. In vitro, X31-sciIV can infect MDCK cells, but infectious virions are not produced unless HA is transcomplemented. In vivo, intranasal immunization with X31-sciIV does not cause any clinical symptoms in mice but generates influenza-specific CD8 T cells in lymphoid (mediastinal lymph nodes and spleen) and nonlymphoid tissues, including lung and bronchoalveolar lavage fluid, as measured by H2-Db NP366 and PA224 tetramer staining. In addition, a significant proportion of X31-sciIV-induced antigen-specific respiratory CD8 T cells expressed VLA-1, a marker that is associated with heterologous influenza protection. Further, these influenza-specific CD8 T cells produce antiviral cytokines when stimulated with NP366 and PA224 peptides, indicating that CD8 T cells triggered by X31-sciIV are functional. When challenged with a lethal dose of heterologous PR8 virus, X31-sciIV-primed mice were fully protected from death. However, when CD8 T cells were depleted after priming or before priming, mice could not effectively control virus replication or survive the lethal challenge, indicating that X31-sciIV-induced memory CD8 T cells mediate the heterologous protection. Thus, our results demonstrate the potential for sciIV as a CD8 T cell-inducing vaccine.

    IMPORTANCE One of the challenges for influenza prevention is the existence of multiple influenza virus subtypes and variants and the fact that new strains can emerge yearly. Numerous studies have indicated that the effector functions of specific CD8 T cells are crucial in mediating influenza heterologous protection. However, influenza vaccines that can trigger effective CD8 T cell responses for heterologous protection have not been developed. We report here the generation of an X31 (H3N2) virus-derived single-cycle infectious influenza virus, X31-sciIV. A one-dose immunization with X31-sciIV is capable of inducing functional influenza virus-specific CD8 T cells that can be recruited into respiratory tissues and provide protection against lethal heterologous challenge. Without these cells, protection against lethal challenge was essentially lost. Our data indicate that an influenza vaccine that primarily relies on CD8 T cells for protection could be developed.

  • Characterization of the Polyproline Region of the Hepatitis E Virus in Immunocompromised Patients [Genetic Diversity and Evolution]

  • Little is known about virus adaptation in immunocompromised patients with chronic genotype 3 hepatitis E virus (HEV3) infections. Virus-host recombinant strains have been isolated recently from chronically infected patients. The nature and incidence of such recombinant events occurring during infections of solid-organ transplant (SOT) recipients are essentially unknown. The polyproline region (PPR) of strains isolated from SOT patients was sequenced during the acute-infection phase (n = 59) and during follow-up of patients whose infections became chronic (n = 27). These 27 HEV strains included 3 (11%) that showed recombinant events 12, 34, 48, or 88 months after infection. In one strain, parts of the PPR and the RNA-dependent RNA polymerase were concomitantly inserted. In the second, a fragment of a human tyrosine aminotransferase (TAT) gene was inserted first, followed by a fragment of PPR. A fragment of the human inter-aalpha;-trypsin inhibitor (ITI) gene was inserted in the third. All the inserted sequences were rich in aliphatic and basic amino acids. In vitro growth experiments suggest that the ITI insertion promoted more vigorous virus growth. In silico studies showed that the inserted sequences could provide potential acetylation, ubiquitination, and phosphorylation sites. We found that recombinant events had occurred in the HEV PPR in approximately 11% of the strains isolated from chronically infected transplant patients followed up in Toulouse University Hospital. These inserted fragments came from the HEV genome or a human gene and could enhance virus replication.

    IMPORTANCE Hepatitis E virus (HEV) can cause chronic infections in immunocompromised patients, including solid-organ transplant (SOT) recipients. Two strains that had undergone recombination with human ribosomal genes were described recently. The strains with inserted sequences replicated better in vitro. Little is known about the frequency of such recombinant events or how such an insertion enhances replication. We therefore investigated 59 SOT patients infected with HEV and found 3 strains with 4 recombinant events in 27 of these patients whose infection became chronic. The 4 inserted sequences were of different origins (human gene or HEV genome), but all were enriched in aliphatic and basic amino acids and provided potential regulation sites. Our data indicate that recombinant events occur in approximately 11% of strains isolated from chronically infected patients. The structures of the inserted sequences provide new clues as to how the inserted sequences could foster virus replication.

  • Cellular Visualization of Macrophage Pyroptosis and Interleukin-1{beta} Release in a Viral Hemorrhagic Infection in Zebrafish Larvae [Virus-Cell Interactions]

  • Hemorrhagic viral diseases are distributed worldwide with important pathogens, such as dengue virus or hantaviruses. The lack of adequate in vivo infection models has limited the research on viral pathogenesis and the current understanding of the underlying infection mechanisms. Although hemorrhages have been associated with the infection of endothelial cells, other cellular types could be the main targets for hemorrhagic viruses. Our objective was to take advantage of the use of zebrafish larvae in the study of viral hemorrhagic diseases, focusing on the interaction between viruses and host cells. Cellular processes, such as transendothelial migration of leukocytes, virus-induced pyroptosis of macrophages. and interleukin-1bbeta; (Il-1bbeta;) release, could be observed in individual cells, providing a deeper knowledge of the immune mechanisms implicated in the disease. Furthermore, the application of these techniques to other pathogens will improve the current knowledge of host-pathogen interactions and increase the potential for the discovery of new therapeutic targets.

    IMPORTANCE Pathogenic mechanisms of hemorrhagic viruses are diverse, and most of the research regarding interactions between viruses and host cells has been performed in cell lines that might not be major targets during natural infections. Thus, viral pathogenesis research has been limited because of the lack of adequate in vivo infection models. The understanding of the relative pathogenic roles of the viral agent and the host response to the infection is crucial. This will be facilitated by the establishment of in vivo infection models using organisms such as zebrafish, which allows the study of the diseases in the context of a complete individual. The use of this animal model with other pathogens could improve the current knowledge on host-pathogen interactions and increase the potential for the discovery of new therapeutic targets against diverse viral diseases.

  • The Composition of West Nile Virus Lipid Envelope Unveils a Role of Sphingolipid Metabolism in Flavivirus Biogenesis [Virus-Cell Interactions]

  • West Nile virus (WNV) is an emerging zoonotic mosquito-borne flavivirus responsible for outbreaks of febrile illness and meningoencephalitis. The replication of WNV takes place on virus-modified membranes from the endoplasmic reticulum of the host cell, and virions acquire their envelope by budding into this organelle. Consistent with this view, the cellular biology of this pathogen is intimately linked to modifications of the intracellular membranes, and the requirement for specific lipids, such as cholesterol and fatty acids, has been documented. In this study, we evaluated the impact of WNV infection on two important components of cellular membranes, glycerophospholipids and sphingolipids, by mass spectrometry of infected cells. A significant increase in the content of several glycerophospholipids (phosphatidylcholine, plasmalogens, and lysophospholipids) and sphingolipids (ceramide, dihydroceramide, and sphingomyelin) was noticed in WNV-infected cells, suggesting that these lipids have functional roles during WNV infection. Furthermore, the analysis of the lipid envelope of WNV virions and recombinant virus-like particles revealed that their envelopes had a unique composition. The envelopes were enriched in sphingolipids (sphingomyelin) and showed reduced levels of phosphatidylcholine, similar to sphingolipid-enriched lipid microdomains. Inhibition of neutral sphingomyelinase (which catalyzes the hydrolysis of sphingomyelin into ceramide) by either pharmacological approaches or small interfering RNA-mediated silencing reduced the release of flavivirus virions as well as virus-like particles, suggesting a role of sphingomyelin-to-ceramide conversion in flavivirus budding and confirming the importance of sphingolipids in the biogenesis of WNV.

    IMPORTANCE West Nile virus (WNV) is a neurotropic flavivirus spread by mosquitoes that can infect multiple vertebrate hosts, including humans. There is no specific vaccine or therapy against this pathogen licensed for human use. Since the multiplication of this virus is associated with rearrangements of host cell membranes, we analyzed the effect of WNV infection on different cellular lipids that constitute important membrane components. The levels of multiple lipid species were increased in infected cells, pointing to the induction of major alterations of cellular lipid metabolism by WNV infection. Interestingly, certain sphingolipids, which were increased in infected cells, were also enriched in the lipid envelope of the virus, thus suggesting a potential role during virus assembly. We further verified the role of sphingolipids in the production of WNV by means of functional analyses. This study provides new insight into the formation of flavivirus infectious particles and the involvement of sphingolipids in the WNV life cycle.

  • Polyomavirus Small t Antigen Interacts with Yes-Associated Protein To Regulate Cell Survival and Differentiation [Virus-Cell Interactions]

  • Murine polyomavirus small t antigen (PyST) regulates cell cycle, cell survival, apoptosis, and differentiation and cooperates with middle T antigen (MT) to transform primary cells in vitro and in vivo. Like all polyomavirus T antigens, PyST functions largely via its interactions with host cell proteins. Here, we show that PyST binds both Yes-associated protein 1 (YAP1) and YAP2, integral parts of the Hippo signaling pathway, which is a subject of increasing interest in human cancer. The transcription factor TEAD, which is a known target of YAP, is also found in PyST complexes. PyST enhanced YAP association with protein phosphatase 2A (PP2A), leading to decreased YAP phosphorylation. PyST increased YAP levels by decreasing its degradation. This effect was mediated by a reduction in YAP association with bbeta;-transducin repeat protein (bbeta;TRCP), which is known to regulate YAP turnover in a phosphorylation-dependent manner. Genetic analysis has identified PyST mutants defective in YAP binding. These mutants demonstrated that YAP binding is important for PyST to block myoblast differentiation and to synergize with the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) to promote cell death in 3T3-L1 preadipocytes placed under differentiation conditions. In addition to YAP binding, both of these phenotypes require PyST binding to PP2A.

    IMPORTANCE The Hippo/YAP pathway is a highly conserved cascade important for tissue development and homeostasis. Defects in this pathway are increasingly being associated with cancer. Polyomavirus small t antigen is a viral oncogene that cooperates with middle T antigen in transformation. On its own, small t antigen controls cell survival and differentiation. By binding YAP, small t antigen brings it together with protein phosphatase 2A. This work shows how this association of small t antigen with YAP is important for its effects on cell phenotype. It also suggests that PyST can be used to characterize cellular processes that are regulated by YAP.

  • Counteracting Effects of Cellular Notch and Epstein-Barr Virus EBNA2: Implications for Stromal Effects on Virus-Host Interactions [Virus-Cell Interactions]

  • A number of diverse environmental cues have been linked to B lymphocyte differentiation and activation. One such cue, Notch-2, may be particularly relevant to the biology of infection with Epstein-Barr virus (EBV), which colonizes the B cell compartment. Activated Notch and EBV nuclear antigen 2 (EBNA2) both function as transcriptional activators by virtue of their interactions with the transcription factor RBP-J. Although EBNA2 and activated Notch appear to have partially overlapping functions, we now report that activated Notch counteracts a crucial EBNA2 function both in newly infected primary B cells and in lymphoblastoid cell lines (LCLs). EBNA2 is directly responsible for the initiation of transcription of the majority of EBV proteins associated with type III latency, leading to the outgrowth of LCLs. One of the key proteins driving this outgrowth is latent membrane protein 1 (LMP1), which is regulated by an EBNA2-responsive element within its ED-L1 promoter. Activation of Notch-2 via Delta-like ligand 1 inhibits EBNA2-mediated initiation of LMP1 transcription. Furthermore, ligated Notch-2 also efficiently turns off LMP1 expression from the ED-L1 promoter in LCLs already expressing LMP1. Modulation of EBV gene expression by Notch was not confined to EBNA2-dependent events. Activated Notch-2 also inhibited EBV entry into the lytic cycle in a B cell non-Hodgkin's lymphoma line by upregulating the cellular transcription factor Zeb2, which represses the transcription of BZLF1. These results support the concept that in vivo, cumulative signals from the microenvironment downregulate EBV gene expression in B cells to the latency 0 gene expression profile observed in B cells entering the peripheral blood.

    IMPORTANCE Experimental infection of resting B cells by Epstein-Barr virus leads to the growth transformation program of virus gene expression and the outgrowth of lymphoblastoid cell lines. Previous studies at the single-cell level revealed complex cellular and viral signaling networks regulating transcription of the viral genome. This study demonstrates that viral gene expression can also be radically altered by molecules expressed on stromal cells in the microenvironment of lymphoid tissue, specifically, Delta-like ligand 1 on stromal cells ligating Notch-2 on infected B cells. Activation of Notch interferes with the transactivation function of EBNA2, downregulates the expression of LMP1 and LMP2a, and inhibits the activation of lytic virus replication in a B cell non-Hodgkin's lymphoma line by preventing expression of BZLF1. The significance of these observations is that they indicate new mechanisms whereby the microenvironment in normal lymphoid tissue may facilitate the repression of viral gene expression, enabling establishment of true latency in memory B cells.

  • Combined Alphavirus Replicon Particle Vaccine Induces Durable and Cross-Protective Immune Responses against Equine Encephalitis Viruses [Vaccines and Antiviral Agents]

  • Alphavirus replicons were evaluated as potential vaccine candidates for Venezuelan equine encephalitis virus (VEEV), western equine encephalitis virus (WEEV), or eastern equine encephalitis virus (EEEV) when given individually or in combination (V/W/E) to mice or cynomolgus macaques. Individual replicon vaccines or the combination V/W/E replicon vaccine elicited strong neutralizing antibodies in mice to their respective alphavirus. Protection from either subcutaneous or aerosol challenge with VEEV, WEEV, or EEEV was demonstrated out to 12 months after vaccination in mice. Individual replicon vaccines or the combination V/W/E replicon vaccine elicited strong neutralizing antibodies in macaques and demonstrated good protection against aerosol challenge with an epizootic VEEV-IAB virus, Trinidad donkey. Similarly, the EEEV replicon and V/W/E combination vaccine elicited neutralizing antibodies against EEEV and protected against aerosol exposure to a North American variety of EEEV. Both the WEEV replicon and combination V/W/E vaccination, however, elicited poor neutralizing antibodies to WEEV in macaques, and the protection conferred was not as strong. These results demonstrate that a combination V/W/E vaccine is possible for protection against aerosol challenge and that cross-interference between the vaccines is minimal.

    IMPORTANCE Three related viruses belonging to the genus Alphavirus cause severe encephalitis in humans: Venezuelan equine encephalitis virus (VEEV), western equine encephalitis virus (WEEV), and eastern equine encephalitis virus (EEEV). Normally transmitted by mosquitoes, these viruses can cause disease when inhaled, so there is concern that these viruses could be used as biological weapons. Prior reports have suggested that vaccines for these three viruses might interfere with one another. We have developed a combined vaccine for Venezuelan equine encephalitis, western equine encephalitis, and eastern equine encephalitis expressing the surface proteins of all three viruses. In this report we demonstrate in both mice and macaques that this combined vaccine is safe, generates a strong immune response, and protects against aerosol challenge with the viruses that cause Venezuelan equine encephalitis, western equine encephalitis, and eastern equine encephalitis.

  • DESC1 and MSPL Activate Influenza A Viruses and Emerging Coronaviruses for Host Cell Entry [Virus-Cell Interactions]

  • The type II transmembrane serine protease (TTSP) TMPRSS2 cleaves and activates the influenza virus and coronavirus surface proteins. Expression of TMPRSS2 is essential for the spread and pathogenesis of H1N1 influenza viruses in mice. In contrast, H3N2 viruses are less dependent on TMPRSS2 for viral amplification, suggesting that these viruses might employ other TTSPs for their activation. Here, we analyzed TTSPs, reported to be expressed in the respiratory system, for the ability to activate influenza viruses and coronaviruses. We found that MSPL and, to a lesser degree, DESC1 are expressed in human lung tissue and cleave and activate the spike proteins of the Middle East respiratory syndrome and severe acute respiratory syndrome coronaviruses for cell-cell and virus-cell fusion. In addition, we show that these proteases support the spread of all influenza virus subtypes previously pandemic in humans. In sum, we identified two host cell proteases that could promote the amplification of influenza viruses and emerging coronaviruses in humans and might constitute targets for antiviral intervention.

    IMPORTANCE Activation of influenza viruses by host cell proteases is essential for viral infectivity and the enzymes responsible are potential targets for antiviral intervention. The present study demonstrates that two cellular serine proteases, DESC1 and MSPL, activate influenza viruses and emerging coronaviruses in cell culture and, because of their expression in human lung tissue, might promote viral spread in the infected host. Antiviral strategies aiming to prevent viral activation might thus need to encompass inhibitors targeting MSPL and DESC1.

  • Increased Replicative Fitness Can Lead to Decreased Drug Sensitivity of Hepatitis C Virus [Genetic Diversity and Evolution]

  • Passage of hepatitis C virus (HCV) in human hepatoma cells resulted in populations that displayed partial resistance to alpha interferon (IFN-aalpha;), telaprevir, daclatasvir, cyclosporine, and ribavirin, despite no prior exposure to these drugs. Mutant spectrum analyses and kinetics of virus production in the absence and presence of drugs indicate that resistance is not due to the presence of drug resistance mutations in the mutant spectrum of the initial or passaged populations but to increased replicative fitness acquired during passage. Fitness increases did not alter host factors that lead to shutoff of general host cell protein synthesis and preferential translation of HCV RNA. The results imply that viral replicative fitness is a mechanism of multidrug resistance in HCV.

    IMPORTANCE Viral drug resistance is usually attributed to the presence of amino acid substitutions in the protein targeted by the drug. In the present study with HCV, we show that high viral replicative fitness can confer a general drug resistance phenotype to the virus. The results exclude the possibility that genomes with drug resistance mutations are responsible for the observed phenotype. The fact that replicative fitness can be a determinant of multidrug resistance may explain why the virus is less sensitive to drug treatments in prolonged chronic HCV infections that favor increases in replicative fitness.

  • Core-Binding Factor Subunit Beta Is Not Required for Non-Primate Lentiviral Vif-Mediated APOBEC3 Degradation [Virus-Cell Interactions]

  • Viral infectivity factor (Vif) is required for lentivirus fitness and pathogenicity, except in equine infectious anemia virus (EIAV). Vif enhances viral infectivity by a Cullin5-Elongin B/C E3 complex to inactivate the host restriction factor APOBEC3. Core-binding factor subunit beta (CBF-bbeta;) is a cell factor that was recently shown to be important for the primate lentiviral Vif function. Non-primate lentiviral Vif also degrades APOBEC3 through the proteasome pathway. However, it is unclear whether CBF-bbeta; is required for the non-primate lentiviral Vif function. In this study, we demonstrated that the Vifs of non-primate lentiviruses, including feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), caprine arthritis encephalitis virus (CAEV), and maedi-visna virus (MVV), do not interact with CBF-bbeta;. In addition, CBF-bbeta; did not promote the stability of FIV, BIV, CAEV, and MVV Vifs. Furthermore, CBF-bbeta; silencing or overexpression did not affect non-primate lentiviral Vif-mediated APOBEC3 degradation. Our results suggest that non-primate lentiviral Vif induces APOBEC3 degradation through a different mechanism than primate lentiviral Vif.

    IMPORTANCE The APOBEC3 protein family members are host restriction factors that block retrovirus replication. Vif, an accessory protein of lentivirus, degrades APOBEC3 to rescue viral infectivity by forming Cullin5-Elongin B/C-based E3 complex. CBF-bbeta; was proved to be a novel regulator of primate lentiviral Vif function. In this study, we found that CBF-bbeta; knockdown or overexpression did not affect FIV Vif's function, which induced polyubiquitination and degradation of APOBEC3 by recruiting the E3 complex in a manner similar to that of HIV-1 Vif. We also showed that other non-primate lentiviral Vifs did not require CBF-bbeta; to degrade APOBEC3. CBF-bbeta; did not interact with non-primate lentiviral Vifs or promote their stability. These results suggest that a different mechanism exists for the Vif-APOBEC interaction and that non-primates are not suitable animal models for exploring pharmacological interventions that disrupt Vifnndash;CBF-bbeta; interaction.

  • Computational Prediction of Vaccine Strains for Human Influenza A (H3N2) Viruses [Vaccines and Antiviral Agents]

  • Human influenza A viruses are rapidly evolving pathogens that cause substantial morbidity and mortality in seasonal epidemics around the globe. To ensure continued protection, the strains used for the production of the seasonal influenza vaccine have to be regularly updated, which involves data collection and analysis by numerous experts worldwide. Computer-guided analysis is becoming increasingly important in this problem due to the vast amounts of generated data. We here describe a computational method for selecting a suitable strain for production of the human influenza A virus vaccine. It interprets available antigenic and genomic sequence data based on measures of antigenic novelty and rate of propagation of the viral strains throughout the population. For viral isolates sampled between 2002 and 2007, we used this method to predict the antigenic evolution of the H3N2 viruses in retrospective testing scenarios. When seasons were scored as true or false predictions, our method returned six true positives, three false negatives, eight true negatives, and one false positive, or 78% accuracy overall. In comparison to the recommendations by the WHO, we identified the correct antigenic variant once at the same time and twice one season ahead. Even though it cannot be ruled out that practical reasons such as lack of a sufficiently well-growing candidate strain may in some cases have prevented recommendation of the best-matching strain by the WHO, our computational decision procedure allows quantitative interpretation of the growing amounts of data and may help to match the vaccine better to predominating strains in seasonal influenza epidemics.

    IMPORTANCE Human influenza A viruses continuously change antigenically to circumvent the immune protection evoked by vaccination or previously circulating viral strains. To maintain vaccine protection and thereby reduce the mortality and morbidity caused by infections, regular updates of the vaccine strains are required. We have developed a data-driven framework for vaccine strain prediction which facilitates the computational analysis of genetic and antigenic data and does not rely on explicit evolutionary models. Our computational decision procedure generated good matches of the vaccine strain to the circulating predominant strain for most seasons and could be used to support the expert-guided prediction made by the WHO; it thus may allow an increase in vaccine efficacy.

  • Regulation of Autophagic Activation by Rta of Epstein-Barr Virus via the Extracellular Signal-Regulated Kinase Pathway [Virus-Cell Interactions]

  • Autophagy is an intracellular degradation pathway that provides a host defense mechanism against intracellular pathogens. However, many viruses exploit this mechanism to promote their replication. This study shows that lytic induction of Epstein-Barr virus (EBV) increases the membrane-bound form of LC3 (LC3-II) and LC3-containing punctate structures in EBV-positive cells. Transfecting 293T cells with a plasmid that expresses Rta also induces autophagy, revealing that Rta is responsible for autophagic activation. The activation involves Atg5, a key component of autophagy, but not the mTOR pathway. The expression of Rta also activates the transcription of the genes that participate in the formation of autophagosomes, including LC3A, LC3B, and ATG9B genes, as well as those that are involved in the regulation of autophagy, including the genes TNF, IRGM, and TRAIL. Additionally, treatment with U0126 inhibits the Rta-induced autophagy and the expression of autophagy genes, indicating that the autophagic activation is caused by the activation of extracellular signal-regulated kinase (ERK) signaling by Rta. Finally, the inhibition of autophagic activity by an autophagy inhibitor, 3-methyladenine, or Atg5 small interfering RNA, reduces the expression of EBV lytic proteins and the production of viral particles, revealing that autophagy is critical to EBV lytic progression. This investigation reveals how an EBV-encoded transcription factor promotes autophagy to affect viral lytic development.

  • A Single Amino Acid Substitution in the Novel H7N9 Influenza A Virus NS1 Protein Increases CPSF30 Binding and Virulence [Pathogenesis and Immunity]

  • Although an effective interferon antagonist in human and avian cells, the novel H7N9 influenza virus NS1 protein is defective at inhibiting CPSF30. An I106M substitution in H7N9 NS1 can restore CPSF30 binding together with the ability to block host gene expression. Furthermore, a recombinant virus expressing H7N9 NS1-I106M replicates to higher titers in vivo, and is subtly more virulent, than the parental virus. Natural polymorphisms in H7N9 NS1 that enhance CPSF30 binding may be cause for concern.

  • Gene Expression of Five Different Iteradensoviruses: Bombyx mori Densovirus, Casphalia extranea Densovirus, Papilio polyxenes Densovirus, Sibine fusca Densovirus, and Danaus plexippus Densovirus [Genome Replication and Regulation of Viral Gene Expression]

  • Iteradensoviruses are 5-kb parvoviruses with typical J-shaped inverted terminal repeats of about 250 nucleotides and terminal hairpins of about 165 nucleotides. The single-stranded DNA genome contains several open reading frames, but their expression strategy is still unknown. Here the transcription maps and expression of the viruses in this genus were explored. As for brevidensoviruses, the two nonstructural (NS) genes were expressed by overlapping promoters with alternate transcription starts at both sides of the NS1 start codon.

  • Parvovirus-Derived Endogenous Viral Elements in Two South American Rodent Genomes [Genetic Diversity and Evolution]

  • We describe endogenous viral elements (EVEs) derived from parvoviruses (family Parvoviridae) in the genomes of the long-tailed chinchilla (Chinchilla lanigera) and the degu (Octodon degus). The novel EVEs include dependovirus-related elements and representatives of a clearly distinct parvovirus lineage that also has endogenous representatives in marsupial genomes. In the degu, one dependovirus-derived EVE was found to carry an intact reading frame and was differentially expressed in vivo, with increased expression in the liver.

  • Herpes Simplex Virus 1 Counteracts Viperin via Its Virion Host Shutoff Protein UL41 [Virus-Cell Interactions]

  • The interferon (IFN)-inducible viperin protein restricts a broad range of viruses. However, whether viperin plays a role during herpes simplex virus 1 (HSV-1) infection is poorly understood. In the present study, it was shown for the first time that wild-type (WT) HSV-1 infection couldn't induce viperin production, and ectopically expressed viperin inhibited the replication of UL41-null HSV-1 but not WT viruses. The underlying molecular mechanism is that UL41 counteracts viperin's antiviral activity by reducing its mRNA accumulation.

  • Human Herpesvirus 8 Viral Interleukin-6 Signaling through gp130 Promotes Virus Replication in Primary Effusion Lymphoma and Endothelial Cells [Virus-Cell Interactions]

  • The contributions of human herpesvirus 8 (HHV-8) viral interleukin-6 (vIL-6) to virus biology remain unclear. Here we examined the role of vIL-6/gp130 signaling in HHV-8 productive replication in primary effusion lymphoma and endothelial cells. Depletion and depletion-complementation experiments revealed that endoplasmic reticulum-localized vIL-6 activity via gp130 and gp130-activated signal transducer and activator of transcription (STAT) signaling, but not extracellular signal-regulated kinase (ERK) activation, was critical for vIL-6 proreplication activity. Our data significantly extend current understanding of vIL-6 function and associated mechanisms in HHV-8 biology.

  • On the Use of 2,5-Dimethyl-Pyrrol-1-yl-Benzoic Acid Derivatives as EPH-Ephrin Antagonists [Letters to the Editor]

  • Reply to "On the Use of 2,5-Dimethyl-Pyrrol-1-yl-Benzoic Acid Derivatives as EPH-Ephrin Antagonists" [Letters to the Editor]

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    JVI Accepts: Articles Published Ahead of Print

  • Hyperglycosylated stable core immunogens designed to present the CD4 binding site preferentially recognized by broadly neutralizing antibodies [PublishAheadOfPrint]

  • The HIV-1 surface envelope glycoprotein (Env) trimer mediates entry into CD4+/CCR5+ host cells. Env possesses conserved antigenic determinants, such as the gp120 primary receptor CD4 binding site (CD4bs), a known neutralization target. Env also contains variable regions and protein surfaces occluded within the trimer that elicit non-neutralizing antibodies. Here, we engineered additional N-linked glycans onto a cysteine-stabilized gp120 core (0G) deleted of its major variable regions to preferentially expose the conformationally fixed CD4bs. Three, 6, 7 and 10 new NXT/S glycan (G) motifs were engineered into 0G to encode 3G, 6G, 7G and 10G cores. Following purification, most glycoproteins were recognized by broadly neutralizing CD4bs-directed antibodies except for 10G. Gel and glycan mass-spectrometry confirmed that additional N-glycans were post-translationally added to the redesigned cores. Binding kinetics revealed high-affinity recognition by seven broadly neutralizing CD4bs-directed antibodies and low-to-no binding by non-broadly-neutralizing CD4bs-directed antibodies. Rabbits inoculated with the hyperglycosylated cores elicited IgM and IgG responses to each given protein that were similar to those elicited by parental 0G. Site-specific glycan masking effects were detected in the elicited sera and the anti-sera competed with b12 for CD4bs-directed binding specificity. However, the core-elicited sera showed very limited neutralization activity. Trimer priming or boosting of the core immunogens elicited tier 1-level neutralization that mapped to both the CD4bs and V3 and appeared solely trimer-dependent. Fine-mapping at the CD4bs indicated that conformational stabilization of the cores and addition of N-glycans altered the molecular surface, suggesting why the elicited neutralization was not improved by this rational-design strategy.

    Importance Major obstacles to develop an effective HIV-1 vaccine are the variability of its surface proteins and its high-density shield generated by incorporation of host (human) glycans (sugars). HIV-1 does harbor highly conserved sites on the exposed envelope protein surface, gp120, one of which is the virus receptor (CD4) binding site. Several broadly neutralizing antibodies elicited from HIV patients do target this gp120 CD4 binding site (CD4bs); however, gp120 immunogens do not elicit broadly neutralizing antibodies. In this study, we targeted the CD4bs by conformational stabilization and additional glycan-masking. We used high-resolution structure to re-engineer gp120 cores to preferentially present the cysteine-stabilized CD4bs and to glycan-mask non-neutralizing determinants. Importantly, glycan masking did successfully focus antibody responses to the CD4bs; however, the elicited CD4bs-directed antibodies did not neutralize HIV or bind to unmodified gp120, presumably due to the structure-guided modifications of this conserved region.

  • Overlapping and distinct molecular determinants dictating the antiviral activities of TRIM56 against flaviviruses and coronavirus [PublishAheadOfPrint]

  • The tripartite motif-containing (TRIM) proteins have emerged as a new class of host antiviral restriction factors, with several demonstrating roles in regulating innate antiviral responses. Of ggt;70 known TRIMs, TRIM56 inhibits replication of bovine viral diarrhea virus, a ruminant pestivirus of the family Flaviviridae, but has no appreciable effect on VSV, a rhabdovirus. Yet, the antiviral spectrum of TRIM56 remains undefined. In particular, how TRIM56 impacts human-pathogenic viruses is unknown. Also unclear are the molecular determinants governing the antiviral activities of TRIM56. Herein, we show that TRIM56 poses a barrier to infections by yellow fever virus (YFV), dengue virus serotype-2 (DENV2), and human coronavirus virus (HCoV)-OC43 but not encephalomyocarditis virus (EMCV). Moreover, by engineering cell lines conditionally expressing various TRIM56 mutants, we demonstrate that TRIM56's anti-flavivirus effects required both the E3 ligase activity that lies in the N-terminal RING domain and the integrity of its C-terminal portion, while the restriction of HCoV-OC43 relied upon the TRIM56 E3 ligase activity alone. Furthermore, TRIM56 was revealed to impair YFV and DENV2 propagation by suppressing intracellular viral RNA accumulation but to compromise HCoV-OC43 infection at a later step in the viral life cycle, suggesting that distinct TRIM56 domains accommodate differing antiviral mechanisms. Altogether, TRIM56 is a versatile antiviral host factor that confers resistance to YFV, DENV2 and HCoV-OC43 through overlapping and distinct molecular determinants.

    Importance We previously reported tripartite-motif protein 56 (TRIM56) as a host restriction factor of bovine viral diarrhea virus, a ruminant pathogen. However, the impact of TRIM56 on human-pathogenic RNA viruses is unknown. Herein, we demonstrate that TRIM56 restricts two medically-important flaviviruses, yellow fever virus (YFV) and dengue virus serotype-2 (DENV2), and a human coronavirus, HCoV-OC43, but not encephalomyocarditis virus, a picornavirus. Further, we show that TRIM56-mediated inhibition of HCoV-OC43 multiplication solely depends on its E3 ligase activity whereas its restriction of YFV and DENV2 requires both the E3 ligase activity and integrity of the C-terminal portion. The differing molecular determinants appear to accommodate distinct antiviral mechanisms TRIM56 adopts to target different families of virusesmmdash; while TRIM56 curbs intracellular YFV/DENV2 RNA replication, it acts at a later step in HCoV-OC43 life cycle. These novel findings illuminate the molecular basis of the versatility and specificity of TRIM56's antiviral activities against positive-strand RNA viruses.

  • DnaJA1/Hsp40 is co-opted by influenza A virus to enhance its viral RNA polymerase activity [PublishAheadOfPrint]

  • The RNA-dependent RNA polymerase (RdRp) of influenza A virus is a heterotrimeric complex composed by PB1, PB2 and PA subunits. The interplay between host factors and the three subunits of the RdRp is critical to enable viral RNA synthesis to occur in the nucleus of infected cells. In this report, we newly identified a host factor DnaJA1, a member of the type I DnaJ/Hsp40 family, acting as a positive regulator for influenza virus replication. We found that DnaJA1 associates with PB2 and PA subunits and enhances viral RNA synthesis both in vivo and in vitro. Moreover, DnaJA1 could be translocated from cytoplasm into the nucleus upon influenza virus infection. The translocation of DnaJA1 is specifically accompanied by the PB1-PA nuclear import. Interestingly, we observed that the effect of DnaJA1 on viral RNA synthesis is mainly dependent on its C-terminal substrate-binding domain but not on its typical J-domain, while the J-domain normally mediates the Hsp70-DnaJ interaction required for regulating Hsp70 ATPase activity. Therefore, we propose that DnaJA1 is co-opted by the influenza A virus to entre nucleus and to enhance its RNA polymerase activity in an Hsp70 cochaperone-independent manner.

    Importance The interplay between host factors and influenza virus RNA polymerase plays a critical role in determining virus pathogenicity and host adaption. In this report, we newly identified a host protein DnaJA1/Hsp40 that is co-opted by influenza A virus RNA polymerase to enhance its viral RNA synthesis in the nucleus of infected cells. We found that DnaJA1 associates with both PB2 and PA subunits and translocates into nucleus along with the nuclear import of PB1-PA dimer during influenza virus replication. Interestingly, the effect of DnaJA1 is mainly dependent on its C-terminal substrate-binding domain but not on its typical J-domain that is required for its Hsp70 cochaperone function. To our knowledge, this is the first report on a member of Hsp40s that is specifically involved in regulating influenza virus RNA polymerase. Targeting the interactions between polymerase subunits and DnaJA1 may provide a novel strategy to develop antiviral drugs.

  • Latency-associated viral IL-10 encoded by human cytomegalovirus modulates cellular IL-10 and CCL8 secretion during latent infection through changes in the cellular miRNA, hsa-miR-92a. [PublishAheadOfPrint]

  • The UL111A gene of human cytomegalovirus encodes a viral homologue of the cellular immunomodulatory cytokine interleukin IL-10 (cIL-10) which, due to alternative splicing, results in expression of two isoforms designated LAcmvIL-10 (expressed during both lytic and latent infection) or cmvIL-10 (identified only during lytic infection).

    We have analysed the functions of LAcmvIL-10 during latent infection of primary myeloid progenitor cells and find that LAcmvIL-10 is responsible, at least in part, for the known increase in secretion of cellular IL-10 and CCL8 in the secretome of latently infected cells. This latency-associated increase in CCL8 expression results from a concomitant LAcmvIL-10-mediated suppression of the expression of cellular miRNA hsa-miR-92a which targets CCL8 directly.

    Taken together, we show that the previously observed downregulation of hsa-miR-92a and upregulation of CCL8 during HCMV latent infection of myeloid cells are intimately linked via the latency-associated expression of LAcmvIL-10.

    Importance HCMV latency causes significant morbidity and mortality in immune compromised individuals yet is carried silently llsquo;latentlyrrsquo; in 50-90% of the population. Understanding of how HCMV maintains infection for the lifetime of an infected individual is critical for the treatment of the immune compromised suffering with disease as a result of HCMV. In this study we analyse one of the proteins which are expressed during the llsquo;latentrrsquo; phase of HCMV, LAcmvIL-10 and find that the expression of this gene modulates the microenvironment of the infected cell leading to evasion of the immune system.

  • Type I interferon signaling enhances CD8+ T cell effector function and differentiation during MHV68 infection [PublishAheadOfPrint]

  • CD8+ T cell responses are critical to the control of replication and reactivation associated with gammaherpesvirus infection. Type I interferons have been shown to have direct and indirect roles in supporting CD8+ T cell development and function during viral infection; however, the role of type I interferons during latent viral infection has not been examined. Mice deficient in type I IFN signaling (IFNAR1nndash;/nndash; mice) have high levels of reactivation during infection with MHV68, a murine gammaherpesvirus model for Epstein-Barr virus. We hypothesized that type I IFNs function to enhance the anti-gammaherpesvirus CD8+ T cell response. To test this, IFNAR1nndash;/nndash; mice were infected with MHV68 and the CD8+ T cell response was analyzed. In the absence of type I IFN signaling there was a marked increase in short-lived effector CD8+ T cells, and MHV68-specific CD8+ T cells had upregulated expression of PD-1 and reduced TNF-aalpha;, IFN-, and IL-2 production. Suppressing MHV68 replication early in infection using the antiviral cidofovir rescued CD8+ T cell cytokine production and reduced PD-1 expression. However, suppressing high levels of reactivation in IFNAR1nndash;/nndash; mice failed to improve CD8+ T cell cytokine production during latency. T cell-specific abrogation of type I IFN signaling showed that the effects of type I IFNs on the CD8+ T cell response during MHV68 infection are independent of direct type I IFN signaling on T cells. Our findings support a model in which type I IFNs likely suppress MHV68 replication, thus limiting viral antigen and facilitating an effective gammaherpesvirus-directed CD8+ T cell response.

    Importance The murine gammaherpesvirus, MHV68, has both genetic and biologic homology to the human gammaherpesvirus Epstein-Barr virus (EBV) which infects over 90% of humans. Latent EBV infection and reactivation are associated with various life-threatening diseases and malignancies. Host suppression of gammaherpesvirus latency and reactivation requires both CD8+ T cells as well as type I interferon signaling. Type I IFNs have been shown to critically support the antiviral CD8+ T cell response in other viral models. Here, we identify an indirect role for type I IFN signaling in enhancing gammaherpesvirus-specific CD8+ T cell cytokine production. Further, this function of type I IFN signaling can be partially rescued by suppressing viral replication during early MHV68 infection. Our data suggest that type I IFN signaling on non-T cells can enhance CD8+ T cell function during gammaherpesvirus infection, potentially through suppressing MHV68 replication.

  • B cell response to herpesvirus infection of the olfactory neuroepithelium [PublishAheadOfPrint]

  • Viruses commonly infect the respiratory tract. Analyses of host defence have focussed on the lungs and the respiratory epithelium. Spontaneously inhaled Murid Herpesvirus-4 (MuHV-4) and Herpes simplex virus type 1 (HSV-1) infect instead the olfactory epithelium, where neuronal cilia are exposed to environmental antigens and provide a route across the epithelial mucus. We used MuHV-4 to define how B cells respond to virus replication in this less well characterized site. Olfactory infection elicited generally weaker acute responses than lung infection, particularly in the spleen, reflecting slower viral replication and spread. Few virus-specific antibody-forming cells (AFCs) were found in the nasal-associated lymphoid tissue (NALT), a prominent response site for respiratory epithelial infection. Instead they appeared first in the superficial cervical lymph nodes. The focus of the AFC response then moved to the spleen, matching the geography of virus dissemination. Little virus-specific IgA response was detected until late on in the bone marrow. Neuroepithelial HSV-1 infection also elicited no significant AFC response in the NALT and a weak IgA response. Thus olfactory herpesvirus infection differed immunologically from an infection of the adjacent respiratory epithelium. Poor IgA induction may help herpesviruses to transmit via long-term mucosal shedding.

    Importance Herpesviruses are widespread, persistent pathogens against which vaccines have had limited success. We need to understand better how they interact with host immunity. MuHV-4 and HSV-1 inhaled by alert mice infect the olfactory neuroepithelium, suggesting that this is a natural entry route. Its immunology is almost completely unknown. The antibody response to neuroepithelial herpesvirus infection started in the cervical lymph nodes, and unlike respiratory influenza virus infection did not involve significantly the nasal-associated lymphoid tissue. MuHV-4 and HSV-1 infections also elicited little virus-specific IgA. Therefore vaccine-induced IgA might provide a defence that herpesviruses are ill-equipped to meet.

  • Hijacking Discs Large 1 for Oncogenic Phosphatidylinositol 3-Kinase Activation in Human Epithelial Cells Is a Conserved Mechanism of Human Adenovirus E4-ORF1 Proteins [PublishAheadOfPrint]

  • The E4-ORF1 gene of human adenoviruses encodes a 14-kDa protein that promotes viral replication as well as cellular metabolic reprogramming, survival, and transformation by constitutively activating cellular phosphatidylinositol 3-kinase (PI3K). We recently reported that the E4-ORF1 protein from subgroup D human adenovirus type 9 upregulates and oncogenically activates PI3K by a novel mechanism involving separate interactions of E4-ORF1 with cellular Discs large 1 (Dlg1) and PI3K to form a ternary complex that translocates to the plasma membrane (K. Kong, M. Kumar, M. Taruishi, and R. T. Javier, PLoS Pathog. 10:e1004102, 2014, doi:10.1371/journal.ppat.1004102). The current study was carried out to investigate whether other human adenovirus E4-ORF1 proteins share this mechanism of action. Results showed that in human MCF10A epithelial cells, stable expression of E4-ORF1 proteins encoded by representative human adenovirus serotypes from subgroups A to D induce ternary complex formation, Dlg1-dependent PI3K activation, PI3K protein elevation, Dlg1 and PI3K membrane recruitment, and PI3K-dependent cellular transformation. The first three of these E4-ORF1 activities were also observed in MCF10A cells infected with each wild-type human adenovirus from subgroups A to D. Our findings indicate that most, if not all, human adenovirus E4-ORF1 proteins share a conserved molecular mechanism of PI3K activation, which confers a common capacity to promote oncogenic transformation in human epithelial cells.

    IMPORTANCE PI3K activation by the adenovirus E4-ORF1 protein mediates oncogenic cellular transformation by human adenovirus type 9, augments viral protein expression and replication by human adenovirus type 5, and dysregulates cellular glucose and lipid metabolism by human adenovirus type 36. For the first time, we report that E4-ORF1 proteins from human adenoviruses in subgroups A to D evolved a conserved molecular mechanism to mediate constitutive PI3K activation that can provoke oncogenic transformation in human epithelial cells. The results raise potential safety concerns about the use of vectors encoding the E4-ORF1 gene in human gene therapy and vaccination. Our findings further suggest that the conserved mechanism revealed here may be targeted for development of therapeutic drugs to treat and prevent adenovirus-associated human diseases.

  • Drosophila C virus systemic infection leads to intestinal obstruction [PublishAheadOfPrint]

  • Drosophila C virus (DCV) is a positive-sense RNA virus belonging to the Dicistroviridae family. This natural pathogen of the model organism Drosophila melanogaster is commonly used to investigate antiviral host-defense in flies, which involves both RNA interference and inducible responses. Although lethality is routinely used as readout for the efficiency of the antiviral immune response in these studies, virus-induced pathologies in flies are still poorly understood. Here, we characterize the pathogenesis associated with systemic DCV infection. Comparison of the transcriptome of flies infected with DCV or two other positive-sense RNA viruses, Flock House virus and Sindbis virus, reveals that DCV infection, unlike those of the other two viruses, represses the expression of a large number of genes. Several of these genes are specifically expressed in the midgut, and are also repressed by starvation. We show that systemic DCV infection triggers a nutritional stress in Drosophila, which result from intestinal obstruction with accumulation of peritrophic matrix at the entry of the midgut, and accumulation of the food ingested in the crop, a blind muscular food storage organ. The related virus Cricket paralysis virus (CrPV), which efficiently grows in Drosophila, does not trigger this pathology. We show that DCV, but not CrPV, infects the smooth muscles surrounding the crop, causing extensive cytopathology and strongly reducing the rate of contractions. We conclude that the pathogenesis associated with systemic DCV infection results from the tropism of the virus for an important organ within the foregut of Dipteran insects, the crop.

    Importance DCV is one of the few identified natural viral pathogens affecting the model organism Drosophila melanogaster. As such, it is an important virus for the deciphering of host-virus interactions in insects. We characterize here the pathogenesis associated with DCV infection in flies, and show that it results from the tropism of the virus for an essential but as yet poorly characterized organ in the digestive tract, the crop. Our results may have relevance for other members of the Dicistroviridae, some of which are pathogenic to beneficial or pest insect species.

  • Activation of RIG-I pathway during influenza vaccination enhances germinal center reactions, T follicular helper cell induction and, provides dose-sparing effect and protective immunity [PublishAheadOfPrint]

  • Pattern Recognition Receptors (PRR) sense certain molecular patterns uniquely expressed by pathogens. Retinoic-acid-inducible gene I (RIG-I) is a cytosolic PRR that senses viral nucleic acids and induces innate immune activation and secretion of type-I IFNs. Here, using influenza vaccine antigens, we investigated the consequences of activating the RIG-I pathway on antigen-specific adaptive immune responses. We found that mice immunized with influenza vaccine antigens co-administered with 5rrsquo; ppp-dsRNA, a RIG-I ligand, developed robust levels of hemagglutination inhibiting antibodies, enhanced germinal center reaction and T follicular helper cell responses. In addition, RIG-I activation enhanced antibody affinity maturation and plasma cell responses in draining lymph nodes, spleen, and bone marrow and conferred protective immunity against virus challenge. Importantly, activation of RIG-I pathway was able to reduce the antigen requirement by 10-100 folds in inducing optimal influenza-specific cellular and humoral responses including protective immunity. The effects induced by 5rrsquo; ppp-dsRNA were significantly dependent on type-I IFN and IPS-1 (adapter protein downstream of RIG-I pathway) signaling, but were independent of MyD88 or TLR3 mediated pathways. Our results show that activation of RIG-I-like receptor pathway programs the innate immunity to achieve qualitatively and quantitatively enhanced protective cellular adaptive immune responses even at low antigen doses and thus, indicate the potential utility of RIG-I ligands as molecular adjuvants for the viral vaccines.

    Study Importance Statement The recently discovered RNA helicase family of RIG-I-like receptors (RLRs) is a critical component of host defense mechanisms responsible for detecting viruses and triggering innate anti-viral cytokines that help control viral replication and dissemination. In this study we show that the RLR-pathway can be effectively exploited for enhancing adaptive immunity and protective immune memory against viral infection. Our results show that activation of RIG-I pathway along with influenza vaccination programs the innate immunity to induce qualitatively and quantitatively superior protective adaptive immunity against pandemic influenza viruses. More importantly, the RIG-I activation at the time of vaccination allows induction of robust adaptive responses even at sparing vaccine antigen doses. These results highlight the potential utility of exploiting RIG-I pathway for enhancing viral vaccine specific immunity and have broader implications for designing better vaccines in general.

  • Vaccinia virus mutations in the L4R gene encoding a virion structural protein produce abnormal mature particles lacking a nucleocapsid [PublishAheadOfPrint]

  • Electron micrographs from the 1960s revealed the presence of an S-shaped tubular structure in the center of the vaccinia virion core. Recently, we showed that packaging of virus transcription enzymes is necessary for the formation of the tubular structure, suggesting that the structure is equivalent to a nucleocapsid. Based on this study and on what is known about nucleocapsids of other viruses, we hypothesized that in addition to transcription enzymes, the tubular structure also contains the viral DNA and a structural protein as a scaffold. The vaccinia virion structural protein L4 stands out as the best candidate for the role of a nucleocapsid structural protein because it is abundant, it is localized in the center of the virion core and it binds DNA. In order to gain more insight into the structure and relevance of the nucleocapsid, we analyzed thermosensitive and inducible mutants in the gene L4R. Using a cryo-fixation method for electron microscopy (high pressure freezing followed by freeze substitution) to preserve labile structures like the nucleocapsid, we were able to demonstrate that in the absence of functional L4, mature particles with defective internal structure are produced under non-permissive conditions. These particles do not contain a nucleocapsid. In addition, the core wall of these virions is abnormal. This suggests that the nucleocapsid interacts with the core wall and that the nucleocapsid structure might be more complex than originally assumed.

    Importance The vaccinia virus nucleocapsid has been neglected since the 1960s due to a lack of electron microscopy techniques to preserve this labile structure. With the advent of cryo-fixation techniques, like High Pressure Freezing/Freeze Substitution, we are now able to consistently preserve and visualize the nucleocapsid. Because vaccinia virus early transcription is coupled to the viral core structure, detailing the structure of the nucleocapsid is indispensable for determining the mechanisms of vaccinia virus core-directed transcription. The present study represents our second attempt to understand the structure and biological significance of the nucleocapsid. We demonstrate the importance of the protein L4 for the formation of the nucleocapsid and reveal in addition that the nucleocapsid and the core wall may be associated, suggesting a higher level of complexity of the nucleocapsid than predicted. In addition, we prove the utility of high pressure freezing in preserving the vaccinia nucleocapsid.

  • Chlorovirus Skp1-binding Ankyrin-Repeat Protein Interplay and Mimicry of Cellular Ubiquitin Ligase Machinery [PublishAheadOfPrint]

  • The ubiquitin-proteasome system is targeted by many viruses that have evolved strategies to redirect host ubiquitination machinery. Members of the genus Chlorovirus are proposed to share a common ancestral lineage with a broader group of related viruses, Nucleo-Cytoplasmic Large DNA Viruses (NCLDV). Chloroviruses encode a Skp1 homolog and ankyrin-repeat (ANK) proteins. Several chlorovirus encoded ANK-repeats contain C-terminal domains characteristic of cellular F-boxes or related NCLDV chordopox PRANC domains. These observations suggested that this unique combination of Skp1 and ANK-repeat proteins might form complexes analogous to the cellular Skp1-Cul1-F-box (SCF) ubiquitin ligase complex. We identified two ANK proteins from the prototypic chlorovirus PBCV-1 that functioned as binding partners for the virus-encoded Skp1, proteins A682L and A607R. These ANK proteins had a C-terminal Skp1 interactional motif that functioned like cellular F-box domains. A C-terminal motif of ANK protein A682L binds Skp1 proteins from widely divergent species. Yeast two-hybrid analyses using serial domain deletion constructs confirmed the C-terminal localization of the Skp1 interactional motif in PBCV-1 A682L. ANK protein A607R represents an ANK family with one member present in all 41 sequenced chloroviruses. A comprehensive phylogenetic analysis of these related ANK and viral Skp1 proteins suggested partnered function tailored to the host alga or common ancestral heritage. Here, we show protein-protein interaction between corresponding family clusters of virus-encoded ANK and Skp1 proteins from three chlorovirus genera. Collectively, our results indicate that chloroviruses have evolved complimenting Skp1 and ANK proteins that mimic cellular SCF-associated proteins.

    Importance Viruses have evolved ways to direct ubiquitination events in order to create environments conducive to their replication. As reported in this manuscript, the large chloroviruses encode several components involved in the SCF ubiquitin ligase complex including a viral Skp1 homolog. Studies on how chloroviruses manipulate their host algal ubiquitination system will provide insights towards viral protein mimicry, substrate recognition, and key interactive domains controlling selective protein degradation. These findings may also help to understand the evolution of other large DNA viruses, like poxviruses, that are reported to share the same monophyly lineage as chloroviruses.

  • Variation of the Specificity of the Human Antibody Responses after Tick-Borne Encephalitis Virus Infection and Vaccination [PublishAheadOfPrint]

  • Tick-borne encephalitis (TBE) virus is an important human-pathogenic flavivirus endemic in large parts of Europe, Central and Eastern Asia. Neutralizing antibodies specific for the viral envelope protein E are believed to mediate long-lasting protection after natural infection and vaccination. To study the specificity and individual variation of human antibody responses we developed immunoassays with recombinant antigens representing viral surface protein domains and domain combinations. These allowed us to dissect and quantify antibody populations of different fine specificities in sera of TBE patients and vaccinees.

    Post-infection and post-vaccination sera both displayed strong individual variation of antibody titers as well as the relative proportions of antibodies to different domains of E, indicating that the immunodominance patterns observed were strongly influenced by individual-specific factors. The contributions of these antibody populations to virus neutralization were quantified by serum depletion analyses and revealed a significantly biased pattern. Antibodies to domain III mmdash; in contrast to what was found in mouse immunization studies with TBE and other flaviviruses mmdash; did not play any role in the human neutralizing antibody response which was dominated by antibodies to domains I and II.

    Importantly, most of the neutralizing activity could be depleted from sera by a dimeric soluble form of the E protein which is the building block of the icosahedral herringbone-like shell of flaviviruses, suggesting that antibodies to more complex quaternary epitopes involving residues from adjacent dimers play only a minor role in the total response to natural infection and vaccination in humans.

    Importance Tick-borne encephalitis (TBE) virus is a close relative of yellow fever, dengue, Japanese encephalitis and West Nile viruses and distributed in large parts of Europe, Central and Eastern Asia. Antibodies to the viral envelope protein E prevent viral attachment and entry into cells and thus mediate virus neutralization and protection from disease. However, the fine specificity and individual variation of neutralizing antibody responses is currently not known. We have therefore developed new in vitro assays for dissecting the antibody populations present in blood serum and determining their contribution to virus neutralization. In our analysis of human post-infection and post-vaccination sera, we found an extensive variation of the antibody populations present in sera, indicating substantial influences of individual-specific factors that control the specificity of the antibody response. Our study provides new insights into the immune response to an important human pathogen that is of relevance for the design of novel vaccines.

  • Evolution of Bluetongue Virus Serotype 1 in Northern Australia over 30 Years [PublishAheadOfPrint]

  • BTV 1 was first isolated in Australia from cattle blood collected in 1979 at Beatrice Hill Farm (BHF), Northern Territory (NT). From long term surveillance programs (1977 to 2011), 2487 isolations of ten BTV serotypes were made. The most frequently isolated serotype was BTV 1 (41%, 1019) followed by BTV 16 (17.5%, 436) and BTV 20 (14%, 348). In three years no BTVs were isolated and in twelve years no BTV 1 was isolated. Seventeen BTV 1 isolates were sequenced and analyzed in comparison with ten Australian prototype serotypes. BTV 1 showed an episodic pattern of evolutionary change characterized by four distinct periods. Each period consisted primarily of slow genetic drift which was punctuated from time to time by genetic shifts generated by segment reassortment and the introduction of new genome segments. Evidence was found for co-evolution of BTV genome segments. Evolutionary dynamics and selection pressures estimates showed strong temporal and clock-like molecular evolutionary dynamics of six Australian BTV genome segments. Bayesian coalescent estimates of mean substitution rates clustered in the range of 3.5 to 5.3 x 10-4 substitutions per site per year. All BTV genome segments evolved under strong purifying (negative) selection with only three sites identified as under pervasive diversifying (positive) selection. The obligate replication in alternate hosts (insect vector and vertebrate host) imposed strong evolutionary constraints. The dominant mechanism generating genetic diversity of BTV 1 at BHF was through the introduction of new viruses and reassortment of genome segments with existing viruses.

    IMPORTANCE Bluetongue virus (BTV) is the causative agent of bluetongue disease in ruminants. It is a disease of concern globally and is transmitted by biting midges (Culicoides species). Analysis of the evolutionary and selection pressures on BTV 1 at a single surveillance site in northern Australia showed strong temporal and clock-like dynamics. Obligate replication in alternate hosts of insect and vertebrate imposed strong evolutionary constraints with all BTV genome segments evolving under strong purifying (negative) selection. Generation of genetic diversity of BTV 1 in northern Australia is through genome segment reassortment and the introduction of new serotypes.

  • The CD8+ memory stem T cell (TSCM) subset is associated with improved prognosis in chronic HIV-1 infection [PublishAheadOfPrint]

  • Memory stem T cells (TSCM) constitute a long-lived, self-renewing lymphocyte population essential for the maintenance functional immunity. The hallmarks of HIV-1 pathogenesis are CD4+ T cell depletion and abnormal cellular activation. We investigated the impact of HIV-1 infection on the TSCM compartment, as well as any protective role these cells may have in disease progression, by characterizing this subset in a cohort of 113 subjects with variable degrees of viral control on and off highly active antiretroviral therapy (HAART). We observed that the frequency of CD8+ TSCM is decreased in all individuals with chronic, untreated HIV-1 infection, and that HAART has a restorative effect on this subset. In contrast, natural controllers of HIV-1 had the highest absolute number of CD4+ TSCM cells among all of the infected groups. The frequency of CD4+ TSCM predicted higher CD8+ TSCM frequencies, consistent with a role for the CD4+ subset in helping to maintain CD8+ memory T cells. In addition, TSCM appeared to be progenitors for effector T cells (TEM), as these two compartments were inversely correlated. Increased frequencies of CD8+ TSCM predicted lower viral loads, higher CD4+ counts and less CD8+ T cell activation. Finally, we found that TSCM express the mucosal homing integrin aalpha;4bbeta;7 and can be identified in the gastrointestinal-associated lymphoid tissue (GALT). The frequency of mucosal CD4+ TSCM was inversely correlated with that in the blood, potentially reflecting the ability of these self-renewing cells to migrate to a crucial site of ongoing viral replication and CD4+ T cell depletion.

    Importance: HIV-1 infection leads to profound impairment of the immune system. TSCM constitute a recently identified lymphocyte subset with stem-cell like qualities including the ability to generate other memory T cell subtypes, and are therefore likely to play an important role in controlling viral infection. We investigated the relationship between the size of the CD8+ TSCM compartment and HIV-1 disease progression in a cohort of chronically infected individuals. Our results suggest that HAART restores a normal frequency of CD8+ TSCM and that the natural preservation of this subset in the setting of untreated HIV-1 infection is associated with improved viral control and immunity. Therefore, the CD8+ TSCM population may represent a correlate of protection in chronic HIV-1 infection that is directly relevant to the design of T cell-based vaccines, adoptive immunotherapy approaches or the pharmacologic induction of TSCM.

  • Exonuclease domain of the Lassa virus nucleoprotein is critical to avoid RIG-I signaling and to inhibit the innate immune response. [PublishAheadOfPrint]

  • Lassa virus (LASV), which causes a viral hemorrhagic fever, inhibits the innate immune response. The exonuclease (ExoN) domain of its nucleoprotein (NP) is implicated in the suppression of RIG-I signaling. We show here that a LASV in which ExoN function has been abolished strongly activates innate immunity and that this effect is dependent on RIG-I signaling. These results highlight the key role of NP ExoN function in the immune evasion that occurs during LASV infection.

  • The exonuclease activity of Lassa virus nucleoprotein is involved in APC-mediated NK cell responses [PublishAheadOfPrint]

  • Lassa virus is an Old World Arenavirus which causes for Lassa hemorrhagic fever in humans mostly in West Africa. Lassa fever is an important public health problem and a safe and effective vaccine is urgently needed. The infection causes immunosuppression, probably due to the absence of activation of antigen-presenting cells (dendritic cells and macrophages), and in low type I IFN production and deficient NK cell function. However, a recombinant Lassa virus carrying D389A and G392A substitutions in the nucleoprotein that abolish the exonuclease activity and IFN activation loses its inhibitory activity and induces strong type I IFN production by dendritic cells and macrophages. We show here that during infection by this mutant Lassa virus, antigen-presenting cells trigger efficient human NK cell responses in vitro including production of IFN- and cytotoxicity. NK cell activation involves close contact with both antigen-presenting cells and soluble factors. We report that infected dendritic cells and macrophages express the NKG2D ligands MHC class I-related chains A and B and they may produce IL-12, IL-15 and IL-18, all involved in NK cell functions. NK cell degranulation is significantly increased in cocultures suggesting that NK cells seem to kill infected dendritic cells and macrophages. This work confirms the inhibitory function of Lassa virus nucleoprotein. Importantly, we demonstrate for the first time that Lassa virus nucleoprotein is involved in the inhibition of antigen presenting cell-mediated NK cell responses.

    Importance Of The Study The pathogenesis and immune responses induced by Lassa virus are poorly known. Recently, an exonuclease domain contained in the viral nucleoprotein has been shown to be able to inhibit type I IFN response by avoiding the recognition of viral RNA by cell sensors. Here, we studied the responses of NK cells to dendritic cells and macrophages infected with a recombinant Lassa virus in which the exonuclease functions have been abolished and demonstrated that NK cells are strongly activated and presented effective functions. These results show that the strategy developed by Lassa virus to evade innate immunity is also effective on NK cells, explaining the weak NK cell activation observed with the wild-type virus. By providing a better understanding of the interactions between Lassa virus and the host immune system, these results are important for the field of arenavirus biology and may be useful for a vaccine approach against Lassa fever.

  • The quantitation of replication-competent HIV-1 in populations of resting CD4+ T cells [PublishAheadOfPrint]

  • Central memory (TCM) CD4+ T cells are the principal reservoir of latent HIV-1 infection that persists despite durable, successful antiretroviral therapy (ART). In a study that measured HIV DNA in 34 patients and replication-competent HIV in four patients, pools of resting and activated transitional memory (TTM) CD4+ T cells were found to be a reservoir for HIV infection. As defective viruses account for the majority of integrated HIV DNA and do not reflect the actual frequency of latent, replication-competent proviral infection, we assessed the specific contribution of resting TTM cells to latent HIV infection. We measured the frequency of replication-competent HIV in purified resting memory cell subpopulations by a limiting dilution, quantitative viral outgrowth assay (QVOA). HIV was routinely detected within the resting central memory compartment, but was infrequently detected within the resting TTM compartment. These observations suggest that prolonged ART may limit persistent latent infection in the TTM compartment. Our results confirm the importance of latent infection within the TCM compartment, and again focus attention on these cells as the most important latent viral reservoir. While proliferation may drive expansion of detectable viral genomes in cells, the frequency of replication-competent HIV must be carefully assessed. Latent infection appears to wane within the transitional memory compartment in patients who have sustained successful viral suppression via ART, or were treated very early in infection.

    IMPORTANCE Antiretroviral therapy (ART) has led to a significant decrease in morbidity and mortality among HIV-infected patients. However, HIV integrates into the genome of CD4+ T cells generating pools of long-lived cells that are reservoirs of latent HIV. Two main subsets of CD4+ T cells, central memory and transitional memory cells were reported to be major reservoirs of HIV infection. However, this study primarily measured the HIV DNA content, which also includes defective proviruses that would not be able to replicate and initiate new rounds of infection. By analyzing the replication-competent virus in both cell subsets we show that transitional memory cells may not be a durable reservoir in patients on successful ART.

  • Structurally Similar Woodchuck and Human Hepadnavirus Core Proteins Have Distinctly Different Temperature Dependences of Assembly [PublishAheadOfPrint]

  • Woodchuck hepatitis virus (WHV), a close relative of human hepatitis B virus (HBV), has been a key model for disease progression and clinical studies. Sequences of the assembly domain of WHV and HBV core proteins (wCp149 and hCp149, respectively) have 65% identity, suggesting similar assembly behavior. We report a cryo-electron microscopy structure of the WHV capsid at sub-nanometer resolution and a characterization of wCp149 assembly. At this resolution, the T=4 capsid structures of WHV and HBV are practically identical. In contrast to their structural similarity, wCp149 demonstrates enhanced assembly kinetics and stronger dimer-dimer interactions compared to hCp149: at 23ddeg;C and 100 mM ionic strength, the pseudo-critical concentrations of assembly of wCp149 and hCp149 are 1.8 mmu;M and 43.3 mmu;M, respectively. Transmission electron microscopy reveals that wCp149 assembles into predominantly T=4 capsids with a sizeable population of larger, non-icosahedral structures. Charge detection mass spectrometry indicates that T=3 particles are extremely rare compared to the ~5% observed in hCp149 reactions. Unlike hCp149, wCp149 capsid assembly is favorable over a temperature range of 4 ddeg;C to 37 ddeg;C; van't Hoff analyses relate the differences in temperature dependence to the large positive values for heat capacity, enthalpy, and entropy of wCp149 assembly. Because the final capsids are so similar, these findings suggest that free wCp149 and hCp149 undergo different structural transitions leading to assembly. The difference in the temperature dependence of wCp149 assembly may be related to the temperature range of its hibernating host.

    IMPORTANCE: In this paper we present a cryo-EM structure of a WHV capsid showing its similarity to HBV. We then observe that assembly properties of the two homologous proteins are very different. Unlike human HBV, the capsid protein of WHV has evolved to function in a non-homeostatic environment. These studies yield insight into the interplay between core protein self-assembly and host environment, which may also be particularly relevant to plant viruses and viruses with zoonotic cycles involving insect vectors.

  • Modeling the timing of anti-latency drug administration during HIV treatment [PublishAheadOfPrint]

  • Latently infected cells are considered a major barrier to cure of HIV infection, as they are long-lived under antiretroviral treatment (ART) and cause viral replication to restart soon after stopping ART. In the last decade different types of anti-latency drugs have been explored with the aim of reactivating and purging this latent reservoir, and the hope of achieving a cure. Because of toxicity and safety considerations, anti-latency drugs can only be given for a short time to patients on long term ART, with small effect.

    We recently investigated the turnover of latently infected cells during active infection, and have found that it was strongly correlated with viral load. This implies that although latently infected cells had long lifespans in the setting of low viral load (as during ART), they turned over fast under high viral load. Possible reasons could be that increased viral load causes increased activation or death of CD4+ T cells, including those that are latently infected. Taking these results into account, we developed a mathematical model to study the most appropriate timing of anti-latency drugs in relationship to the initiation of ART. We found that the best timing of a short-term anti-latency drug would be the start of ART, when viral load, CD4+ T cell activation and latent cell turnover are high. These results have important implications for the design of HIV cure-related clinical trials.

    Importance The antiretroviral therapy of HIV-infected patients currently needs to be life-long, because the cells latently infected with HIV start new rounds of infection as soon as the treatment is stopped. In the last decade, a number of different types of anti-latency drugs have been explored with the aim of "reactivating" and "purging" this latent reservoir, and hoping to achieve a cure. These drugs have so far been tested on patients only after long term ART, and have shown a small or no effect. We use mathematical modeling to argue that the most efficacious timing of a short-term anti-latency treatment may be the start of ART, because of possible interactions of anti-latency drugs with natural activation pathways.

  • p130Cas Scaffolds the Signalosome to Direct Adaptor-Effector Crosstalk During Kaposi's Sarcoma-Associated Herpesvirus Trafficking in Human Microvascular Dermal Endothelial Cells [PublishAheadOfPrint]

  • KSHV interacts with cell surface receptors, such as heparan sulfate, integrins (aalpha;3bbeta;1, aalpha;Vbbeta;3, and aalpha;Vbbeta;5) and EphrinA2 (EphA2), and activates FAK, Src, PI3-K, c-Cbl, and RhoA GTPase signal molecules early during lipid raft (LR) dependent productive macropinocytic entry into human dermal microvascular endothelial cells. Our recent studies have identified CIB1 as a signal amplifier facilitating EphA2 phosphorylation and subsequent cytoskeletal cross-talk during KSHV macropinocytosis. Although CIB1 lacks an enzymatic activity and traditional adaptor domain or known interacting sequence, it associated with the KSHV entry signal complex and the CIB1-KSHV association was sustained over 30 minutes post-infection. To identify factors scaffolding the EphA2-CIB1 signal axis, the role of major cellular scaffold protein p130Cas (Crk associated substrate of Src) was investigated. Inhibitor and siRNA studies demonstrated that KSHV induced p130Cas in an EphA2, CIB1, and Src dependent manner. p130Cas and Crk were associated with KSHV, LRs, EphA2, and CIB1 early during infection. Live cell microscopy and biochemical studies demonstrated that p130Cas knockdown did not affect KSHV entry but significantly reduced productive nuclear trafficking of viral DNA and routed KSHV to lysosomal degradation. p130Cas aided in scaffolding adaptor Crk to downstream guanine nucleotide exchange factor phospho-C3G possibly to coordinate GTPase signaling during KSHV trafficking. Collectively, these studies demonstrate that p130Cas acts as a bridging molecule between the KSHV induced entry signal complex and the downstream trafficking signalosome in endothelial cells, and suggests that simultaneous targeting of KSHV entry receptors with p130Cas would be an attractive potential avenue for therapeutic intervention of KSHV infection.

    IMPORTANCE Eukaryotic cell adaptor molecules, without any intrinsic enzymatic activity, are well known to allow a great diversity of specific and coordinated protein-protein interactions imparting signal amplification to different networks for physiological and pathological signaling. They are involved in integrating signals from growth factors, extra cellular matrix molecules, bacterial pathogens, and apoptotic cells. The present study identifies HMVEC-d cellular scaffold protein p130Cas (Crk associated substrate) as a platform to promote KSHV trafficking. Early during KSHV de novo infection p130Cas associates with lipid rafts and scaffolds EphA2 associated critical adaptor members to downstream effector molecules, promoting successful nuclear delivery of the KSHV genome. Hence, simultaneous targeting of the receptor EphA2 and scaffolding action of p130Cas can potentially uncouple the signal crosstalk between the KSHV entry associated upstream signal complex from the immediate downstream trafficking associated signalosome, consequently, routing KSHV towards lysosomal degradation and eventually blocking KSHV infection and associated malignancies.

  • HIV-1 entry in SupT1-R5, CEM-ss and primary CD4+ T-cells occurs at the plasma membrane and does not require endocytosis [PublishAheadOfPrint]

  • Cytoplasmic entry of HIV-1 requires binding of the viral glycoproteins to the cellular receptor and co-receptor leading to fusion of viral and cellular membranes. Early studies suggested that productive HIV-1 infection occurs by direct fusion at the plasma membrane. Endocytotic uptake of HIV-1 was frequently observed, but was considered to constitute an unspecific dead-end pathway. More recent evidence suggested that endocytosis contributes to productive HIV-1 entry and may even represent the predominant or exclusive route of infection. We have analyzed HIV-1 binding, endocytosis, cytoplasmic entry and infection in T-cell lines and in primary CD4+ T-cells. Efficient cell binding and endocytosis required viral glycoproteins and CD4, but not the co-receptor. The contribution of endocytosis to cytoplasmic entry and infection was assessed by two strategies: (i) Expression of dominant-negative dynamin-2 efficiently blocked HIV-1 endocytosis, but did not affect fusion or productive infection. (ii) Making use of the fact that HIV-1 fusion is blocked at temperatures below 23ddeg;C, cells were incubated with HIV-1 at 22ddeg;C for varying times, and endocytosis was quantified by parallel analysis of transferrin and fluorescent HIV-1 uptake. Subsequently, plasma membrane entry was blocked by high concentrations of the peptidic fusion inhibitor T-20, which does not reach previously endocytosed particles. HIV-1 infection was scored after shifting to 37ddeg;C in the presence of T-20. These experiments revealed that productive HIV-1 entry occurs predominantly at the plasma membrane in SupT1-R5, CEM-ss and primary CD4+ T-cells, with little if any contribution from endocytosed virions.

    Importance HIV-1, like all enveloped viruses, reaches the cytoplasm by fusion of the viral and cellular membranes. Many viruses enter the cytoplasm by endosomal uptake and fusion from the endosome, while cell entry can also occur by direct fusion at the plasma membrane in some cases. Conflicting evidence has been reported regarding the site of HIV-1 fusion with some studies claiming fusion to occur predominantly at the plasma membrane, while others suggested predominant or even exclusive fusion from the endosome. We have revisited HIV-1 entry using a T-cell line that exhibits HIV-1 endocytosis dependent on the viral glycoproteins and the cellular CD4 receptor; results with this cell line were confirmed for another T-cell line and primary CD4+ T-cells. Our studies show that fusion and productive entry occur predominantly at the plasma membrane, and we conclude that endocytosis is dispensable for HIV-1 infectivity in these T-cell lines and in primary CD4+ T-cells.

  • Suppressors of cytokine signaling 1 and 3 are up-regulated in brain resident cells in response to virus induced inflammation of the CNS via at least two distinctive pathways [PublishAheadOfPrint]

  • Suppressors of cytokine signaling (SOCS) proteins are intracellular proteins that inhibit cytokine signaling in a variety of cell types. A number of viral infections have been associated with SOCS up-regulation; however, not much is known about the mechanisms regulating SOCS expression during viral infection.

    In this study, we have used two pathologically distinct intracerebral (i.c.) infection models to characterize temporal and spatial aspects of SOCS expression in the virus-infected CNS, and by employing various knockout mouse models, we have sought to identify regulatory mechanisms that may underlie a virus induced up-regulation of SOCS in the CNS. We found that i.c. infection with either lymphocytic choriomeningitis virus (LCMV) or yellow fever virus (YF) results in gradual up-regulation of SOCS1/3 mRNA expression peaking at day 7 post infection (p.i.). In the LCMV model, SOCS mRNA was expressed in brain resident cells including astrocytes and some neurons, and for SOCS1 in particular this up-regulation was almost entirely mediated by IFN- produced by infiltrating T cells. Following infection with YF, we also found SOCS expression to be up-regulated in brain resident cells with a peak on day 7 p.i., but in this model, the up-regulation was only partially dependent on IFN- and T cells, indicating that at least one other mediator was involved in the up-regulation of SOCS following YF infection. We conclude that virus induced inflammation of the CNS is associated with up-regulation of SOCS1/3 mRNA expression in brain resident cells, and that at least two distinctive pathways can lead to this up-regulation.

    Importance In the present report, we have studied the induction of SOCS1 and SOCS3 expression in the context of virus-induced CNS infection. We find that both a non-cytolytic and a cytolytic virus induce marked up-regulation of SOCS1 and-3 expression. Notably, the kinetics of the observed up-regulation follows that of activity within pro-inflammatory signalling pathways and, interestingly, type II IFN, which is also a key inducer of inflammatory mediators, seems to be essential in initiating this counter-inflammatory response. Another key observation is that not only cells of the immune system, but also CNS resident cells are actively involved in both the pro- and counter-inflammatory immune circuits; thus e. g. astrocytes up-regulates both CXCL10 and SOCS when exposed to type II IFN in vivo.

  • Trunkloads of viruses [PublishAheadOfPrint]

  • Elephant populations are under intense pressure internationally from habitat destruction and poaching for ivory and meat. They also face pressure from infectious agents, including elephant endotheliotropic herpesvirus 1 (EEHV-1), which kills ~20% of Asian elephants (Elephas maximus) born in zoos and causes disease in the wild. EEHV-1 is one of at least 6 distinct EEHV in a phylogenetic lineage that appears to represent a new subfamily (the Deltaherpesvirinae) in family Herpesviridae.

  • Vaginal memory T cells induced by intranasal vaccination are critical for protective T cell recruitment and prevention of genital HSV-2 disease [PublishAheadOfPrint]

  • Protective immunity against genital pathogens causing chronic infections such as herpes simplex virus type 2 (HSV-2) or human immunodeficiency virus requires the induction of cell-mediated immune responses locally in the genital tract. Intranasal immunization with a thymidine kinasemmdash;deficient (TKmmdash;) mutant of HSV-2 effectively induces HSV-2-specific IFN-secreting memory T-cell production and protective immunity against intravaginal challenge with wild-type HSV-2. However, the precise mechanism by which intranasal immunization induces protective immunity in the distant genital mucosa more effectively than does systemic immunization is unknown. Here, we showed that intranasal immunization with live HSV-2 TKmmdash; induced the production of effector T cells and their migration to, and retention in, the vaginal mucosa, whereas systemic vaccination barely established a local effector T cell pool, even when it induced the production of circulating memory T cells in the systemic compartment. The long-lasting HSV-2-specific local effector T cells induced by intranasal vaccination provided superior protection against intravaginal wild-type HSV-2 challenge by starting viral clearance at the entry site earlier than with intraperitoneal immunization. Intranasal immunization is an effective strategy for eliciting high levels of cell-mediated protection of the genital tract by providing long-lasting Ag-specific local effector T cells without introducing topical infection or inflammation.

    Importance Intranasal (IN) vaccines against sexually transmitted diseases, which are caused by viruses, such as herpes simplex virus type 2 (HSV-2), have long been in development, but no vaccine candidate is currently available. Understanding the cellular mechanisms of immune responses in a distant vaginal mucosa induced by IN immunization with HSV-2 will contribute to designing the vaccine. Our study demonstrated that IN immunization with an attenuated strain of HSV-2 generated long-lasting IFN- secreting T cells in vaginal mucosa more effectively than systemic immunization. We found that these vaginal effector memory T cells are critical for the early stage of viral clearance at natural infection sites, and prevent severe vaginal inflammation and herpes encephalitis.

  • The Conserved Disulfide Bond within Domain II (D-II) of Epstein-Barr Virus (EBV) gH has Divergent Roles in Membrane Fusion with Epithelial Cells and B Cells [PublishAheadOfPrint]

  • Epstein-Barr virus (EBV) infects target cells via fusion with cellular membranes. For entry into epithelial cells, EBV requires the herpesvirus conserved core fusion machinery composed of glycoprotein B (gB) and gH/gL. In contrast, for B cell fusion it requires gB and gH/gL with gp42 serving as a cell tropism switch. The available crystal structures for gH/gL allow the targeted analysis of structural determinants of gH to identify functional regions critical for membrane fusion. Domain II of EBV gH contains two disulfide bonds (DB), the first is unique for EBV and closely related -herpesviruses. The second is conserved across the bbeta;- and -herpesviruses and is positioned to stabilize a putative syntaxin-like bundle motif. To analyze the role of these DBs in membrane fusion, gH was mutated by amino acid substitution of the DB cysteines. Mutation of the EBV-specific DB resulted in diminished gH/gL cell surface expression that correlated with diminished B cell and epithelial cell fusion. In contrast, mutation of the conserved DB resulted in wild-type-like B cell fusion whereas epithelial cell fusion was greatly reduced. The gH mutants bound well to gp42 but had diminished binding to epithelial cells. Tyrosine 336, located adjacent to cysteine 335 of the conserved DB, was also found to be important for DB stabilization and gH/gL function. We conclude that the conserved DB has a cell type specific function, since it is important for the binding of gH to epithelial cells initiating epithelial cell fusion but not for fusion with B cells and gp42 binding.

    Importance EBV predominantly infects epithelial and B cells in humans, which can result in EBV-associated cancers such as Burkitt and Hodgkin lymphoma as well as nasopharyngeal carcinoma. EBV is also associated with a variety of lymphoproliferative disorders, typically of B cell origin, observed in immunosuppressed individuals such as post-transplant or HIV/AIDS patients. The gH/gL complex plays an essential but still poorly characterized role as an important determinant for EBV cell tropism. In our current studies, we found that mutants in the DB C278/C335 and a neighboring tyrosine 336 have cell type specific functional deficits with selective decreases in epithelial cell, but not B cell, binding and fusion. The present study brings new insights into the gH function as determinant for epithelial cell tropism during herpesvirus induced membrane fusion and highlights a specific gH motif required for epithelial cell fusion.

  • Induced maturation of human immunodeficiency virus [PublishAheadOfPrint]

  • HIV-1 assembles at the plasma membrane of virus producing cells as an immature, non-infectious particle. Processing of the Gag and Gag-Pol polyproteins by the viral protease (PR) activates the viral enzymes and results in dramatic structural rearrangements within the virion mmdash; termed maturation mmdash; that are a prerequisite for infectivity. Despite its fundamental importance for viral replication, little is currently known about the regulation of proteolysis and about the dynamics and structural intermediates of maturation. This is mainly due to the fact that HIV-1 release and maturation occur asynchronously both at the level of individual cells and at the level of particle release from a single cell. Here, we report a method to synchronize HIV-1 proteolysis in vitro based on protease inhibitor (PI) wash-out from purified immature virions, thereby temporally uncoupling virus assembly and maturation. Drug wash-out resulted in the induction of proteolysis with cleavage efficiencies correlating with koff of the respective PR-PI complex. Proteolysis of Gag was nearly complete and yielded the correct products with an optimal t1/2 of ~5 h, but viral infectivity was not recovered. Failure to gain infectivity following PI wash-out may be explained by the observed formation of aberrant viral capsids and/or by pronounced defects in processing of the RT heterodimer associated with a lack of RT activity. Based on our results, we hypothesize that both the polyprotein processing dynamics and the tight temporal coupling of immature particle assembly and PR activation are essential for correct polyprotein processing and morphological maturation, and thus for HIV-1 infectivity.

    Importance Cleavage of the HIV-1 polyproteins Gag and Gag-Pol into their functional subunits by the viral protease activates the viral enzymes and causes major structural rearrangements essential for HIV-1 infectivity. This proteolytic maturation occurs concomitant with virus release and investigation of its dynamics is hampered by the fact that virus populations in tissue culture contain particles at all stages of assembly and maturation. Here we developed an inhibitor wash-out strategy to synchronize activation of protease in wild-type virus. We demonstrated that nearly complete Gag processing and resolution of the immature virus architecture is accomplished under optimized conditions. Nevertheless, most of the resulting particles displayed irregular morphologies, Gag-Pol processing was not faithfully reconstituted and infectivity was not recovered. These data show that HIV-1 maturation is sensitive to the dynamics of processing, and also that a tight temporal link between virus assembly and PR activation is required for correct polyprotein processing.

  • Transcriptome responses of the host, Trichoplusia ni, to infection by the baculovirus, Autographa californica Multiple Nucleopolyhedrovirus (AcMNPV) [PublishAheadOfPrint]

  • Productive infection of insect Trichoplusia ni cells by the baculovirus AcMNPV leads to expression of ~156 viral genes and results in dramatic cell remodeling. How the cell transcriptome responds to viral infection was unknown due to the lack of a reference genome and transcriptome for T. ni. We used an ~60 Gb RNA-seq dataset from infected and uninfected T. ni cells, to generate and annotate a de novo transcriptome assembly of approximately 70,322 T. ni unigenes, representing the 48 hour infection cycle. Using differential gene expression analysis, we found that the majority of host transcripts were down-regulated after 6 hours post infection (h p.i.) and throughout the remainder of the infection. In contrast, 5.7% (4028) of the T. ni unigenes were up-regulated during the early period (0-6 h p.i.), followed by a decrease through the remainder of the infection cycle. Also, a small subset of genes related to metabolism and stress response showed a significant elevation of transcript levels at 18 and 24 h p.i., but a decrease thereafter. We also examined the responses of genes belonging to a number of specific pathways of interest, including stress responses, apoptosis, immunity, and protein trafficking. We identified specific pathways members that were up-regulated during the early phase of the infection. Combined with the parallel analysis of AcMNPV expression, these results provide both a broad and a detailed view of how baculovirus infection impacts the host cell transcriptome to evade cellular defensive responses, to modify cellular biosynthetic pathways, and to remodel cell structure.

    IMPORTANCE Baculoviruses are insect-specific DNA viruses that are highly pathogenic to their insect hosts. In addition to their use for biological control of certain insects, baculoviruses also serve as viral vectors for numerous biotechnological applications, such as mammalian cell transduction and protein expression for vaccine production. While there is considerable information regarding viral gene expression in infected cells, little is known regarding responses of the host cell to baculovirus infection. In these studies, we assembled a cell transcriptome from the host Trichoplusia ni and used that transcriptome to analyze changes in host cell gene expression throughout the infection cycle. The study was performed in parallel with a prior study of changes in viral gene expression. Combined, these studies provide an unprecedented new level of detail and an overview of events in the infection cycle, and will stimulate new experimental approaches to understand, modify, and utilize baculoviruses for a variety of applications.

  • Effects of Human Anti-Spike Protein Receptor Binding Domain Antibodies on SARS-Coronavirus Neutralization Escape and Fitness [PublishAheadOfPrint]

  • The receptor binding domain (RBD) of the spike (S) glycoprotein of SARS-CoV is a major target of protective immunity in vivo. Although a large number of neutralizing antibodies (nAbs) have been developed, it remains unclear if a single RBD-targeting nAb or two in combination can prevent neutralization escape and if not, attenuate viral virulence in vivo. In this study, we used a large panel of human nAbs against an epitope which overlaps the interface between the RBD and its receptor, ACE2, to assess their cross-neutralization activities against a panel of human and zoonotic SARS-CoVs and neutralization escape mutants. We also investigated the neutralization escape profiles of these nAbs, and evaluated their effects on receptor binding and virus fitness in vitro and in mice. We found that some nAbs had great potency and breadth in neutralizing multiple viral stains including neutralization escape viruses derived from other nAbs, however no single nAb nor a combination of two blocked neutralization escape. Interestingly, in mice the neutralization escape mutant viruses showed either attenuation (Urbani-background) or increased virulence (GD03-background) consistent with the different binding affinities between their RBDs and the mouse ACE2. We conclude that using either single or dual nAb combinations to target a SARS-CoV RBD epitope that shows plasticity may have limitations for preventing neutralization escape during in vivo immunotherapy. However, RBD-directed nAbs may be useful for providing broad neutralization and prevention of escape variants when combined with other nAbs that target a second conserved epitope with less plasticity and more structural constraint.

    Importance The emergence of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) in 2002 and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in 2012 have resulted in severe human respiratory disease with high death rates. Their zoonotic origins highlight the likelihood of reemergence or further evolution into novel human coronavirus pathogens. Broadly neutralizing antibodies (nAbs) that prevent infection of related viruses represent an important immuno-strategy for combating coronaviruses infections, however for this strategy to succeed, it is essential to uncover nAb-mediated escape pathways and to pioneer strategies that prevent escape. Here, we used SARS-CoV as a research model and examined the escaping pathways of broadly nAbs that target the receptor binding domain (RBD) of the virus. We found that neither single nor two nAbs in combination blocked escape. Our results suggest that targeting conserved regions with less plasticity and more structural constraint rather than SARS-CoV RBD-like region(s) should have broader utility for antibody-based immunotherapy.

  • Structural Plasticity of the Coiled-coil Domain of Rotavirus NSP4 [PublishAheadOfPrint]

  • Rotavirus (RV) nonstructural protein NSP4 is a virulence factor that disrupts cellular Ca2+ homeostasis and plays multiple roles regulating RV replication and the pathophysiology of RV-induced diarrhea. Although its native oligomeric state is unclear, crystallographic studies of the coiled-coil domain (CCD) of NSP4 from two different strains suggest that it functions as a tetramer or a pentamer. While the CCD of simian SA11 NSP4 forms a tetramer that binds Ca2+ at its core, the CCD of a human strain ST3 forms a pentamer lacking the bound Ca2+ despite the residues (E120 and Q123) that coordinate Ca2+-binding being conserved. In these previous studies, while the tetramer crystallized at neutral pH, the pentamer crystallized at low pH suggesting that preference for a particular oligomeric state is pH-dependent and that pH could influence Ca2+-binding. Here, we sought to examine if the CCD of NSP4 from a single RV strain can exist in two oligomeric states regulated by Ca2+ or pH. Biochemical, biophysical and crystallographic studies show that while the CCD of SA11 NSP4 exhibits high affinity binding to Ca2+ at neutral pH and forms a tetramer, at low pH, it does not bind Ca2+ and forms a pentamer, and the transition from tetramer to pentamer is reversible with pH. Mutational analysis shows that Ca2+ binding is necessary for the tetramer formation as an E120A mutant forms a pentamer. We propose that the structural plasticity of NSP4 regulated by pH and Ca2+ may form a basis for its pleiotropic functions during RV replication.

    Importance The non-structural protein NSP4 of rotavirus is a multifunctional protein that plays an important role in virus replication, morphogenesis and pathogenesis. Previous crystallography studies of the coiled-coil domain (CCD) of NSP4 from two different rotavirus strains showed two distinct oligomeric states - a Ca2+-bound tetrameric state and a Ca2+-free pentameric state. Whether NSP4 CCD from the same strain can exist in different oligomeric states, and what factors might regulate its oligomeric preferences are not known. This study used a combination of biochemical, biophysical and crystallography techniques and found that the NSP4 CCD can undergo a reversible transition from a Ca2+-bound tetramer to a Ca2+-free pentamer in response to changes in pH. From these studies, we hypothesize that this remarkable structural adaptability of the CCD forms a basis for the pleiotropic functional properties of NSP4.

  • HIV-1 Uncoating is Facilitated by Dynein and Kinesin-1 [PublishAheadOfPrint]

  • Following entry into the target cell, HIV-1 must reverse transcribe its RNA genome to DNA and traffic to the nuclear envelope, where the viral genome is translocated into the nucleus for subsequent integration into the host cell chromosome. During this time, the viral core, which houses the genome, undergoes a poorly understood process of disassembly, known as uncoating. Collectively, many studies suggest that uncoating is tightly regulated to allow nuclear import of the genome while minimizing the exposure of the newly synthesized DNA to cytosolic DNA sensors. However, whether host cellular proteins facilitate this process remain poorly understood. Here, we report that intact microtubules facilitate HIV-1 uncoating in target cells. Disruption of microtubules with Nocodazole substantially delays HIV-1 uncoating, as revealed with three different assay systems. This defect in uncoating did not correlate with defective reverse transcription at early times post-infection, demonstrating that microtubule facilitated uncoating is distinct from the previously reported role for viral reverse transcription on the uncoating process. . We also find that pharmacological or siRNA mediated inhibition of cytoplasmic dynein and the kinesin-1 heavy chain KIF5B delay uncoating, providing detailed insight into how microtubules facilitate the uncoating process. These studies reveal a previously unappreciated role for microtubules and microtubule motor function in HIV-1 uncoating, establishing a functional link between viral trafficking and uncoating. Targeted disruption of capsid/motor interaction may reveal novel mechanisms to inhibit viral infection or provide opportunities to activate cytoplasmic antiviral responses directed against capsid or viral DNA.

    Importance During HIV-1 infection, fusion of viral and target cell membranes dispenses the viral ribonucleoprotein complex into the cytoplasm of target cells. During this time, the virus must reverse transcribe its RNA genome, traffic from the location of fusion to the nuclear membrane, and undergo the process of uncoating, whereby the viral capsid core disassembles to allow the subsequent nuclear import of the viral genome. Numerous cellular restriction factors target the viral capsid, suggesting that perturbation of the uncoating process represents an excellent antiviral target. However, this uncoating process, and cellular factors which facilitate uncoating, remain poorly understood. The main observation from this study is that normal uncoating requires intact microtubules and is facilitated by dynein and kinesin motors. These factors may either directly inhibit infection or delays them enough to trigger mediators of intrinsic immunity which recognize cytoplasmic capsid or DNA and subsequently induce an antiviral state in these cells.

  • Sendai virus pathogenesis in mice is prevented by Ifit2 and exacerbated by interferon [PublishAheadOfPrint]

  • The Type I/III interferon (IFN) system has major roles in regulating viral pathogenesis, usually ameliorating pathogenesis by impairing virus replication through the antiviral actions of one or more IFN-induced proteins. Ifit2 is one such protein which can be induced by IFN or virus infection and is responsible for protecting mice from neuropathogenesis caused by vesicular stomatitis virus. Here, we show that Ifit2 also protects mice from pathogenesis caused by the respirovirus, Sendai virus (SeV). Mice lacking Ifit2 (Ifit2-/-) suffered severe weight loss and succumbed to intranasal infection with SeV strain 52, at a dose that killed only few wild-type mice. Viral RNA was detectable only in lungs and SeV titers were higher in Ifit2-/- mice, compared to wild-type mice. Similar infiltration of immune cells was found in the lungs of both mouse lines, corresponding to similar levels of many induced cytokines and chemokines. In contrast, IFN-bbeta; and IFN-3 expression were considerably higher in the lungs of Ifit2-/- mice. Surprisingly, type I IFN receptor knock-out (IFNAR-/-) mice were less susceptible to SeV than Ifit2-/- mice, although their pulmonary virus titers were similarly high. To test the intriguing possibility that type I IFN-action enhances pathogenesis in the context of elevated SeV replication in lungs, we generated Ifit2/IFNAR-/- double knock-out mice. These mice were less susceptible to SeV than Ifit2-/- mice, although viral titers in their lungs were even higher. Our results indicate that high SeV replication in the lungs of infected Ifit2-/- mice cooperates with elevated IFN-bbeta; induction to cause disease.

    IMPORTANCE The IFN system is an innate defense against virus infections. It is triggered quickly in infected cells, which then secrete IFN. Via their cell surface receptors on surrounding cells, they induce transcription of numerous IFN-stimulated genes (ISG), which in turn protect these cells by inhibiting virus life cycles. Hence, IFNs are commonly considered as beneficial during virus infections. Here, we report two key findings. Firstly, lack of a single ISG in mice, Ifit2, resulted in high mortality after SeV infection of the respiratory tract, following higher virus loads and higher IFN production in Ifit2-/- lungs. Secondly, mortality of Ifit2-/- mice was reduced when mice also lacked the type I IFN receptor, while SeV loads in lungs were still high. This indicates that type I IFN exacerbates pathogenesis in the SeV model, and that limitation of both viral replication and IFN production is needed for effective prevention of disease.

  • Molecular mimicry between dengue virus and coagulation factors induces antibodies to inhibit thrombin activity and enhance fibrinolysis [PublishAheadOfPrint]

  • Dengue virus (DENV) is the most common cause of viral hemorrhagic fever, and it may lead to life-threating dengue hemorrhagic fever and shock syndrome (DHF/DSS). Because most cases of DHF/DSS occur in patients with secondary DENV infection, anti-DENV antibodies are generally considered to play a role in the pathogenesis of DHF/DSS. Previously, we have found that anti-thrombin antibodies (ATAs) with both anti-thrombotic and profibrinolytic activities are present in the sera of dengue patients. However, the mechanism by which these autoantibodies are induced is unclear. In this study, we demonstrated that antibodies induced by DENV immunization in mice and rabbits could bind to DENV antigens as well as to human thrombin and plasminogen (Plg). The binding of anti-DENV antibodies to thrombin and Plg was inhibited by pre-adsorption with DENV nonstructural protein 1. In addition, affinity-purified ATAs from DENV-immunized rabbit sera could inhibit thrombin activity and enhance Plg activation both in vitro and in vivo. Taken together, our results suggest that molecular mimicry between DENV and coagulation factors can induce the production of autoantibodies with biological effects similar to those of ATAs found in dengue patients. These coagulation factor cross-reactive anti-DENV antibodies can interfere with the balance of coagulation and fibrinolysis, which may lead to the tendency of DHF/DSS patients to bleed.

    Importance Dengue virus (DENV) infection is the most common mosquito-borne viral disease in tropical and sub-tropical areas. Over 50 million DENV infection cases develop each year, and more than 2.5 billion people are at risk of dengue-induced hemorrhagic fever and shock syndrome. Currently, there is no vaccine or drug treatment for DENV. In the present study, we demonstrated that DENV immunization could induce thrombin and plasminogen (Plg) cross-reactive antibodies, which were able to inhibit thrombin activity and enhance Plg activation. These results suggest that molecular mimicry between DENV antigens, thrombin, and Plg may elicit antibodies that disturb hemostasis. The selection of appropriate candidate antigens for use in DENV vaccines should prevent these potentially dangerous autoimmune responses.

  • Establishment and characterization of an air-liquid canine corneal organ culture model to study acute herpes keratitis [PublishAheadOfPrint]

  • Despite the clinical importance of herpes simplex virus (HSV)-induced ocular disease, the underlying pathophysiology of this disease remains poorly understood, in part due to the lack of adequate virus-natural host models in which to study the cellular and viral factors involved in acute corneal infection. We developed an air-liquid canine corneal organ culture model and evaluated its susceptibility to canine herpesvirus type 1 (CHV-1) in order to study ocular herpes in a physiologically relevant natural host model. Canine corneas were maintained in culture at an air-liquid interface for up to 25 days and no degenerative changes were observed in the corneal epithelium during cultivation using histology for morphometric analyses, TUNEL assays and transmission electron microscopy (TEM). Next, canine corneas were inoculated with CHV-1 for 48 hours and at that time point post infection, viral plaques could be visualized in the corneal epithelium and viral DNA copies were detected in both the infected corneas as well as in culture supernatants. In addition, we found that canine corneas produced proinflammatory cytokines in response to CHV-1 infection similarly to what has been described for HSV-1. This emphasizes the value of our model as a virus-natural host model to study ocular herpesvirus infections.

    Importance This study is the first to describe the establishment of an air-liquid canine corneal organ culture model as a useful model to study ocular herpesvirus infections. Advantages of this physiologically relevant model include that it (i) provides a system in which ocular herpes can be studied in a virus-natural host setting and (ii) reduces the number of experimental animals needed. In addition, this long-term explant culture model may facilitate research in other fields also, where non-infectious and infectious ocular diseases of dogs and men are being studied.

  • Drift of the HIV-1 envelope glycoprotein gp120 toward increased neutralization resistance over the course of the epidemic: a comprehensive study using the most potent and broadly neutralizing monoclonal antibodies [PublishAheadOfPrint]

  • Extending our previous analyses to the most recently described broadly neutralizing monoclonal antibodies (bNAbs) we confirm a drift of HIV-1 clade B variants over two decades toward higher resistance to bNAbs targeting almost all the identified gp120 neutralizing epitopes. In contrast, the sensitivity to bNAbs targeting the gp41 MPER remained stable, suggesting a selective pressure on gp120 preferentially. Despite this evolution, selected combinations of bNAbs remain capable to neutralize efficiently most of the circulating variants.

  • Characterization of an alphamesonivirus 3C-like protease defines a special group of nidovirus main proteases [PublishAheadOfPrint]

  • Cavally virus (CavV) and related viruses in the family Mesoniviridae diverged profoundly from other nidovirus lineages but largely retained the characteristic set of replicative enzymes conserved in the Corona- and Roniviridae. Expression of these enzymes in virus-infected cells requires extensive proteolytic processing of two large replicase polyproteins, pp1a and pp1ab, by the viral 3C-like protease (3CLpro). Here, we show that CavV 3CLpro autoproteolytic cleavage occurs at two N-terminal (N1 and N2) and one C-terminal (C1) processing site(s). The mature form of 3CLpro was revealed to be a 314-residue protein produced by cleavage at FKNK1386|SAAS (N2) and YYNQ1700|SATI (C1). Site-directed mutagenesis data suggest that the mesonivirus 3CLpro employs a catalytic Cys-His dyad comprised of CavV pp1a/pp1ab residues Cys-1539 and His-1434. The study further suggests that mesonivirus 3CLpro substrate specificities differ from those of related nidovirus proteases. The presence of Gln (or Glu) at the P1 position was not required for cleavage although residues that control Gln/Glu specificity in related viral proteases are retained in the CavV 3CLpro sequence. Asn at the P2 position was identified as a key determinant for mesonivirus 3CLpro substrate specificity. Other positions, including P4 and P1', are each occupied by structurally related amino acids, indicating a supportive role in substrate binding. Together, the data identify a new subgroup of nidovirus main proteases and support previous conclusions on phylogenetic relationships between the main nidovirus lineages.

    Importance Mesoniviruses have been suggested to provide an evolutionary link between nidovirus lineages with small (13-16 kb) and large (26-32 kb) RNA genome sizes and it has been proposed that a specific set of enzymes, including a proofreading exoribonuclease and other replicase-gene encoded proteins, play a key role in the major genome expansion leading to the currently known lineages of "large nidoviruses". Despite their smaller genome size (20 kb), mesoniviruses retained most of the replicative domains conserved in large nidoviruses and are thus considered interesting models for studying possible key events in the evolution of RNA genomes of exceptional size and complexity. Our study provides the first characterization of a mesonivirus replicase gene-encoded nonstructural protein. The data confirm and extend previous phylogenetic studies of mesoni- and related viruses and pave the way for studies into the formation of the mesonivirus replication complex and functional and structural studies of its functional subunits.

  • DDX3 DEAD-box RNA Helicase is a Host Factor that Restricts Hepatitis B Virus Replication at the Transcriptional Level [PublishAheadOfPrint]

  • DDX3 is a member of the DEAD-box RNA helicase family involved in mRNA metabolism including transcription, splicing, and translation. We previously identified DDX3 as a HBV Pol binding protein, and by using a transient transfection, we found that DDX3 inhibits HBV replication at post-transcriptional level, perhaps following encapsidation. To determine the exact mechanism of the inhibition, we here employed diverse HBV experimental system. Inconsistently, we found that DDX3-mediated inhibition occurs at the level of transcription. By using tetracycline-inducible HBV-producing cells, we observed that lentivirus-mediated DDX3 expression led to a reduced level of HBV RNAs. Importantly, knockdown of DDX3 by short hairpin RNA resulted in augmentation of HBV RNAs in two distinct HBV replication systems: (i) tetracycline-inducible HBV producing cells and (ii) constitutive HBV-producing HepG2.2.15 cells. Moreover, DDX3 knockdown in HBV-susceptible HepG2-NTCP cells, where covalently closed circular DNA (cccDNA) serves as the template for viral transcription, resulted in increased HBV RNAs, validating that transcription regulation by DDX3 occurs on a physiological template. Overall, our results demonstrate that DDX3 represents an intrinsic host antiviral factor that restricts HBV transcription.

    Importance Upon entry into host cells, viruses encounter host factors that restrict viral infection. During evolution viruses have acquired the ability to subvert cellular factors that adversely affect their replication. Such host factors include TRIM5aalpha; and APOBEC3G, which were discovered in retroviruses. The discovery of host restriction factors provided deeper insight into the innate immune response and viral pathogenesis, leading to better understanding of host-virus interactions. In contrast to retroviruses, little is known about host factors that restrict hepatitis B virus (HBV), a virus distantly related to retroviruses. DDX3 DEAD-box RNA helicase is best characterized as an RNA helicase involved in RNA metabolism such as RNA processing and translation. Here, we show that DDX3 inhibits HBV infection at the level of viral transcription.

  • Identification of Nucleotide-Level Changes Impacting Gene Content and Genome Evolution in Orthopoxviruses. [PublishAheadOfPrint]

  • Poxviruses are composed of large dsDNA genomes coding for several hundred genes whose variation has supported virus adaptation to a wide variety of hosts over their long evolutionary history. Comparative genomics has suggested that the Orthopoxvirus genus in particular has undergone reductive evolution, with the most recent common ancestor likely possessing a gene complement consisting of all genes present in any existing modern-day orthopoxvirus species, similar to the current Cowpox virus species. As orthopoxviruses adapt to new environments, the selection pressure on individual genes may be altered, driving sequence divergence and possible loss of function. This is evidenced by accumulation of mutations and loss of protein-coding open reading frames (ORFs) that progress from individual missense mutations, to gene truncation through the introduction of early stop mutations (ESMs), gene fragmentation, and in some cases, a total loss of the ORF. In this study, we have constructed a whole-genome alignment for representative isolates from each Orthopoxvirus species and used it to identify the nucleotide-level changes that have led to gene content variation. By identifying the changes that have led to ESMs, we were able to determine that short indels were the major cause of gene truncations, and that the genome length is proportional to the number of ESMs present. We also identified the number and types of protein functional motifs still present in truncated genes to assess their functional significance.

    IMPORTANCE This work contributes to our understanding of reductive evolution in poxviruses by identifying genomic remnants such as SNPs and indels left behind by evolutionary processes. Our comprehensive analysis of the genomic changes leading to gene truncation and fragmentation was able to detect some of the remnants of these evolutionary processes still present in orthopoxvirus genomes, and suggest that these viruses are under continual adaptation due to changes in their environment. These results further our understanding of the evolutionary mechanisms that drive virus variation, allowing orthopoxviruses to adapt to particular environmental niches. Understanding the past evolutionary history of these virus pathogens may help predict their future evolutionary potential.

  • Expanded regulatory T cells in chronically Friend retrovirus infected mice suppress immunity to a mCMV superinfection [PublishAheadOfPrint]

  • It is still unclear whether expanded and activated regulatory T cells (Tregs) in chronic viral infections can influence primary immune responses against superinfections with unrelated viruses. Expanded Tregs found in the spleens of chronically FV-infected mice decreased mCMV-specific CD8+ T cell responses during acute mCMV superinfection. This suppression of mCMV-specific T cell immunity was only found in organs with FV-induced Treg expansion. Surprisingly, acute mCMV infection itself did not expand or activate Tregs.

  • Crystal structure of HSV-2 gD bound to nectin-1 reveals a conserved mode of receptor recognition [PublishAheadOfPrint]

  • Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are among the most prevalent human pathogens. Both viruses can recognize, via the surface envelope glycoprotein D (gD), human nectin-1 as a functional receptor. Previous studies have successfully elucidated the molecular basis of the binding between HSV-1 gD and nectin-1 by co-crystallography. Despite a high sequence identity between HSV-1 and -2 gDs, the atomic inter-molecule details for HSV-2-gD/nectin-1 interaction remain elusive. Here, we reported the crystal structures of both the unbound and the nectin-1-bound HSV-2 gD. The free gD structure expectedly comprises an IgV-like core and the surface-exposed terminal extensions as observed in its HSV-1 counterpart, but lacks traceable electron densities for a large portion of the terminal elements. These terminal residues were clearly traced in the complex structure as a definitive loop in the N-terminus and an aalpha;mmdash;helix in the C-terminus, thereby showing a conserved nectin-1-binding mode as reported for HSV-1 gD. The interface residues in nectin-1 were further mutated and tested for the gD-interaction by surface plasmon resonance. The resultant binding patterns were similar between HSV-1 and -2 gDs, further supporting a homologous receptor-binding basis by the two viruses for nectin-1. These data, together with a cell-based fusion assay showing a cross-inhibition of the gD/nectin-1 mediated cell-cell fusion by soluble HSV-1 and -2 gDs, provided solid structural and functional evidence that HSV-1/-2 recognizes nectin-1 via the same binding mode. Finally, we also demonstrated that nectin-1 I80 is an important residue involved in gD interaction.

    Importance Despite intensified studies, a detailed picture of the molecular features in the HSV-2-gD/nectin-1 interaction remains unavailable. Previous work focused on HSV-1 gD, which folds into an IgV-like core with large terminal extensions and utilizes the extension elements to engage nectin-1. Here, we reported the crystal structures of HSV-2 gD in both the free and the nectin-1-bound forms. The atomic inter-molecule details for HSV-2-gD/nectin-1 interaction were clearly presented. The observed binding mode is identical to that reported for its HSV-1 counterpart. This structural observation was further supported by our comparative functional assays showing that nectin-1 mutations similarly affect the ligand/receptor interaction of both virus gDs. Taken together, we provided comprehensive structural and functional data demonstrating a conserved receptor-binding mode between HSV-1 and -2 for nectin-1. Our results also indicate that the tropism-difference between the two viruses likely arises from other aspects rather than the gD/nectin-1 binding-features.

  • Development of Dose-Response Models of Creutzfeldt-Jakob Disease Infection in Nonhuman Primates for Assessing the Risk of Transfusion-Transmitted variant Creutzfeldt-Jakob Disease [PublishAheadOfPrint]

  • Estimates for the risk of transmitting variant Creutzfeldt-Jakob disease (vCJD) via blood transfusion have largely relied on data from rodent experiments, but the relationship between dose (amount of infected blood) and response (vCJD infection) has never been well quantified. The goal of this study was to develop a dose-response model based on nonhuman primate data to better estimate the likelihood of transfusion-transmitted vCJD (TTvCJD) in humans. Our model used dose-response data from nonhuman primates inoculated intracerebrally (IC) with brain tissues of patients with sporadic and familial CJD. We analyzed the data statistically using a beta-Poisson dose-response model. We further adjusted model parameters to account for the differences in infectivity between blood and brain tissue and in transmission efficiency between intravenous (IV) and IC routes to estimate dose-dependent TTvCJD infection. The model estimates a mean infection rate of 76% among recipients who receive one unit of whole blood collected from an infected donor near the end of the incubation period. The nonhuman primate model provides estimates that are more consistent with those derived from a risk analysis of transfused non-leukoreduced red blood cells in United Kingdom compared to prior estimates based on rodent models.

    IMPORTANCE TTvCJD was recently identified as one of three emerging infectious diseases posing the greatest immediate threat to the safety of the blood supply. Cases of TTvCJD were reported in recipients of non-leukoreduced red blood cells and coagulation Factor VIII manufactured from blood of UK donors. As the quantity of abnormal prions (the causative agent of TTvCJD) varies significantly in different blood components and products, it is necessary to quantify the dose-response relationship for a wide range of doses for the vCJD agent in transfused blood and plasma derivatives. In this paper we suggest the first mechanistic dose-response model for TTvCJD infection based on data from experiments with nonhuman primates. This new model may improve estimates of the possible risk to humans.

  • Analysis of the evolution and structure of a complex intra-host viral population in chronic Hepatitis C mapped by ultra-deep pyrosequencing. [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) causes chronic infection in up to 50-80% of infected individuals. Hypervariable region 1 (HVR1) variability is frequently studied to gain an insight into the mechanisms of HCV adaptation during chronic infection, but the changes to and persistence of HCV subpopulations during intra-host evolution are poorly understood. In this study, we have used ultra-deep pyrosequencing (UDPS) to map viral heterogeneity of a single patient over 9.6 years of chronic HCV genotype 4a infection. Informed error correction of the raw UDPS data was performed using a temporally matched clonal data set. The resultant data set reported the detection of low frequency recombinants throughout the study period implying that recombination is an active mechanism through which HCV can explore novel sequence space. The data indicates that poly-virus infection of hepatocytes has occurred but that the fitness quotients of recombinant daughter virions are too low to compete against the parental genomes. The subpopulations of parental genomes contributing to the recombination events highlighted a dynamic virome where subpopulations of variants are in competition. In addition, we provide direct evidence that demonstrates the growth of subdominant populations to dominance in the absence of a detectable humoral response.

    IMPORTANCE Analysis of ultra-deep pyrosequencing data sets derived from virus amplicons frequently relies on software tools that are not optimised for amplicon analysis, assume random incorporation of sequencing errors and are focused on achieving higher specificity at the expense of sensitivity. Such analysis is further complicated by the presence of hypervariable regions. In this study, we make use of a temporally matched reference sequence data set to inform error correction algorithms. Using this methodology we were able to (1) detect multiple instances of Hepatitis C virus intra-subtype recombination at the E1/E2 junction (a phenomenon rarely reported in the literature) and (2) interrogate the longitudinal quasispecies complexity of the virome. Parallel to the UDPS, isolation of IgG-bound virions was found to be co-incident with the collapse of specific viral subpopulations.

  • Comparative Genome Analysis of Four Elephant Endotheliotropic Herpesviruses EEHV3, EEHV4, EEHV5 and EEHV6 from Cases of Hemorrhagic Disease or Viremia. [PublishAheadOfPrint]

  • Summary: The genomes of three types of novel endotheliotropic herpesviruses (EEHV1A, EEHV1B and EEHV2) associated with lethal hemorrhagic disease in Asian elephants have been previously well characterized and assigned to a new Proboscivirus genus. Here we have generated 112-kb of DNA sequence data from segments of four more types of EEHV by direct targeted PCR from blood or necropsy tissue of six viremic elephants. Comparative phylogenetic analysis of nearly 30 protein-encoding genes of EEHV5 and EEHV6 show that they diverge uniformly by nearly 20% from their closest relatives, EEHV2 and EEHV1A respectively, and are likely to have similar overall gene content and genome organization. In contrast, seven EEHV3 and EEHV4 genes analysed differ from those of all other EEHVs by 37% and have a (G-plus-C)-content of 63% compared to just 42% for the others. Three strains of EEHV5 analyzed clustered into two partially chimeric subgroups EEHV5A and EEHV5B that diverge by 19% within three small non-contiguous segments totalling 6.2-kb. We conclude that all six EEHV types should be designated as independent species within a proposed new fourth Deltaherpesvirinae subfamily of mammalian herpesviruses. These likely initially diverged close to 100 million years ago when the ancestors of modern elephants split from all other placental mammals, then evolved into two major branches with high or low (G-plus-C)-content about 35 million years ago. Later additional branching events subsequently generated three paired sister taxon lineages of which EEHV1 plus EEHV6, EEHV5 plus EEHV2 and EEHV4 plus EEHV3 may represent Asian and African elephant versions, respectively.

    Importance: One of the factors threatening the long-term survival of endangered Asian elephants in both wild range countries and in captive breeding populations in zoos is a highly lethal hemorrhagic herpesvirus disease that has killed at least 70 young Asian elephants worldwide. The genomes of the first three types of EEHVs (or Probosciviruses) identified have been partially characterized in the preceding paper. Here we have used PCR DNA sequence analysis from multiple segments of DNA amplified directly from blood or necropsy tissue of six more selected cases of hemorrhagic disease to partially characterize four other types of EEHVs from either Asian or African elephants. We propose that all six types and two chimeric subtypes of EEHV belong to multiple lineages of both AT-rich and GC-rich branches within a new subfamily to be named the Deltaherpesvirinae, which evolved separately from all other mammalian herpesviruses about100 million years ago.

  • Elephant Endotheliotropic Herpesviruses EEHV1A, EEHV1B and EEHV2 from Cases of Hemorrhagic Disease are Highly Diverged from Other Mammalian Herpesviruses and May Form a New Subfamily. [PublishAheadOfPrint]

  • A family of novel endotheliotropic herpesviruses (EEHV) assigned to the genus Proboscivirus have been identified as the cause of fatal hemorrhagic disease in 70 young Asian elephants worldwide. Although EEHV cannot be grown in cell culture, we have determined a total of 378-kb of viral genomic DNA sequence directly from clinical tissue of six lethal cases and two survivors. Overall, the data obtained encompasses 57 genes, including orthologues of 32 core genes common to all herpesviruses, 14 genes found in some other herpesviruses, plus ten novel genes including a single-large putative transcriptional regulatory protein (ORF-L). Based on differences in gene content and organization plus phylogenetic analyses of conserved core proteins that have just 20% to 50% or less identity to orthologues in other herpesviruses, we propose that EEHV1A, EEHV1B and EEHV2 could be considered a new Deltaherpesvirinae subfamily of mammalian herpesviruses that evolved as an intermediate branch between the Betaherpesvirinae and Gammaherpesvirinae. Unlike cytomegaloviruses, EEHV genomes encode RRB, TK and UL9-like OBP proteins and have an alphaherpesvirus-like dyad-symmetry ori-Lyt domain. They also differ from all known betaherpesviruses by having a 40-kb large-scale inversion of core gene blocks I, II and III. EEHV1 and EEHV2 DNA differ uniformly by more than 25%, but EEHV1 clusters into two major sub-groups designated EEHV1A and EEHV1B with ancient partially chimeric features. Whereas large segments are nearly identical, three non-adjacent loci totalling 15-kb diverge by between 21 and 37%. One strain of EEHV1B analyzed is interpreted to be a modern partial recombinant with EEHV1A.

    Importance: Asian elephants are an endangered species whose survival is under extreme pressure in wild range countries and whose captive breeding populations in zoos are not self-sustaining. In 1999, a novel class of herpesviruses called EEHVs was discovered that have caused a rapidly lethal hemorrhagic disease in 20% of all captive Asian elephant calves born in zoos in the USA and Europe since 1980. The disease is increasingly being recognized in Asian range countries as well. These viruses cannot be grown in cell culture, but by direct PCR DNA sequence analysis from segments totalling 15-30% of the genomes from blood or necropsy tissue of eight different cases, we have determined that they not only fall into multiple types and chimeric subtypes of a novel Proboscivirus genus, but propose that they should also be classified as the first examples of a new mammalian herpesvirus subfamily named the Deltaherpesvirinae.

  • Molecular requirement for sterols in herpes simplex virus entry and infectivity [PublishAheadOfPrint]

  • Herpes simplex virus 1 (HSV-1) required cholesterol for virion-induced membrane fusion. HSV successfully entered DHCR24-/- cells, which lack a desmosterol-to-cholesterol conversion enzyme, indicating entry can occur independently of cholesterol. Depletion of desmosterol from these cells resulted in diminished HSV-1 entry, suggesting a general sterol requirement for HSV-1 entry and that desmosterol can operate in virus entry. Cholesterol functioned more effectively than desmosterol, suggesting that the hydrocarbon tail of cholesterol influences viral entry.

  • Amino Acid Changes in the Influenza A Virus PA Protein that Attenuate Avian H5N1 Viruses in Mammals [PublishAheadOfPrint]

  • The influenza viral polymerase complex affects host tropism and pathogenicity. In particular, several amino acids in the PB2 polymerase subunit are essential for the efficient replication of avian influenza viruses in mammals. The PA polymerase subunit also contributes to host range and pathogenicity. Here, we report that the PA proteins of several highly pathogenic avian H5N1 viruses have attenuating properties in mammalian cells, and that the attenuating phenotype is conferred by strain-specific amino acid changes. Specifically, lysine at position 185 of A/duck/Vietnam/TY165/2010 (TY165; H5N1) PA induced strongly attenuating effects in vitro and in vivo. More importantly, the introduction of the arginine residue commonly found at this position in PA significantly increased the viral polymerase activity of TY165 in mammalian cells, and its virulence and pathogenicity in mice. These findings demonstrate that the PA protein plays an important role in influenza virulence and pathogenicity.

    IMPORTANCE Highly pathogenic influenza viruses of the H5N1 subtype cause severe respiratory infections in humans, which have resulted in death in nearly two-thirds of the laboratory-confirmed cases. We found that the viral PA polymerase subunit of several H5N1 viruses possesses amino acid changes that attenuate virus replication in mammalian cells (yet the H5N1 viruses possessing these mutations are highly pathogenic in mice). Specifically, we found that an arginine-to-lysine substitution at position 185 of an H5N1 virus PA protein significantly affected that virus' virulence and pathogenicity in mice. The PA protein thus plays a role in the pathogenicity of highly pathogenic H5N1 influenza viruses.

  • M27 expressed by cytomegalovirus counteracts effective type I IFN induction of myeloid cells, but not of plasmacytoid dendritic cells [PublishAheadOfPrint]

  • In healthy individuals the functional immune system effectively confines human cytomegalovirus (CMV) replication, while viral immune evasion and persistence preclude sterile immunity. Mouse CMV (MCMV) is a well-established model to study the delicate CMV-host balance. Effective control of MCMV infection depends on the induction of protective type I interferon (IFN-I) responses. Nevertheless, it is unclear whether also in professional antigen presenting cell subsets MCMV-encoded evasins inhibit the induction of IFN-I responses. Upon MCMV treatment, enhanced expression of MCMV immediate early and early proteins was detected in bone marrow cultures of macrophages and myeloid dendritic cells when compared with plasmacytoid dendritic cell cultures, whereas plasmacytoid dendritic cells mounted more vigorous IFN-I responses. Experiments with Toll-like receptor (TLR) and/or RIG-I like helicase (RLH) deficient cell subsets revealed that upon MCMV treatment of myeloid cells IFN-I responses were triggered independent of TLR- and RLH-signaling, whereas in plasmacytoid dendritic cells IFN-I induction was strictly TLR-dependent. Macrophages and myeloid dendritic cells treated either with UV-inactivated MCMV or live MCMV that lacked the STAT2 antagonist pM27 mounted significantly higher IFN-I responses than cells treated with live wild-type MCMV. In contrast, plasmacytoid dendritic cells responded similarly to UV-inactivated and live MCMV. These experiments illustrated that pM27 not only inhibited IFN-I mediated receptor signaling but also evaded the induction of IFN responses in myeloid dendritic cells. Furthermore, we found that additional MCMV encoded evasin(s) were needed to efficiently shut off IFN-I responses of macrophages, but not of myeloid dendritic cells, thus further elucidating the subtle adjustment of the host-pathogen balance.

    Importance MCMV may IFN-I responses in fibroblasts and epithelial as well as in antigen presenting cell subsets. We focused on the analysis of IFN-I responses of antigen presenting cell subsets, including plasmacytoid dendritic cells, myeloid dendritic cells, and macrophages, which all are triggered by MCMV to mount IFN-I responses. Interestingly, myeloid dendritic cells and macrophages, but not plasmacytoid dendritic cells, are readily MCMV infected and support viral gene expression. As expected from previous studies, plasmacytoid dendritic cells sense MCMV Toll-like receptor (TLR)9 dependently, whereas in myeloid cells IFN-I induction is entirely TLR and RLH independent. MCMV encoded pM27 does not impair IFN-I induction of plasmacytoid dendritic cells, while in myeloid dendritic cells it reduces IFN-I responses. In macrophages, pM27 plus other not yet identified evasins profoundly inhibit the induction of IFN-I responses. Collectively, these results illustrate that MCMV evolved diverse mechanisms to differentially modulate IFN-I responses in single immune cell subsets.

  • Herpes Simplex Virus Internalization into Epithelial Cells Requires Na+/H+ Exchangers and p21-activated Kinases but Neither Clathrin- nor Caveolin-Mediated Endocytosis [PublishAheadOfPrint]

  • Herpes simplex virus type 1 (HSV1) is an alphaherpesvirus that has been reported to infect some epithelial cell types by fusion at the plasma membrane but others by endocytosis. To determine the molecular mechanisms of productive HSV1 cell entry, we perturbed key endocytosis host factors using specific inhibitors, RNAi, or overexpression of dominant negative proteins and investigated their effects on HSV1 infection in the permissive epithelial cell lines Vero, HeLa, HEp-2 and PtK2. HSV1 internalization neither required endosomal acidification nor clathrin- or caveolin-mediated endocytosis. In contrast, HSV1 gene expression and internalization were significantly reduced after treatment with EIPA. EIPA blocks the activity of Na+/H+ exchangers which are plasma membrane proteins implicated in all forms of macropinocytosis. HSV1 internalization furthermore required the function of p21-activated kinases that contribute to macropinosome formation. However, in contrast to some forms of macropinocytosis, HSV1 did not enlist the activities of PKC, tyrosine kinases, C-terminal binding protein 1, or dynamin to activate its internalization. These data suggest that HSV1 depends on Na+/H+ exchangers and p21-activated kinases either for macropinocytosis or for local actin rearrangements required for fusion at the plasma membrane or subsequent passage through the actin cortex underneath the plasma membrane.

    Importance After initial replication in epithelial cells, herpes simplex viruses (HSVs) establish latent infections in neurons innervating these regions. Upon primary infection and reactivation from latency, HSVs cause many human skin and neurological diseases, particularly in immunocompromised hosts despite the availability of effective antiviral drugs. Many viruses use macropinocytosis for virus internalization, and many host factors mediating this entry route have been identified although the specific perturbation profiles vary for different host and viral cargo. In addition to an established entry pathway via acidic endosomes, we show here that HSV1 internalization depended on sodium-proton exchangers at the plasma membrane and p21-activated kinases. These results suggest that HSV1 requires a reorganization of the cortical actin cytoskeleton, either for productive cell entry via pH-independent fusion from macropinosomes or for fusion at the plasma membrane, and subsequent cytosolic passage to microtubules that mediate capsid transport to the nucleus for genome uncoating and replication.

  • Adenovirus E1A targets the DREF nuclear factor to regulate virus gene expression, DNA replication and growth [PublishAheadOfPrint]

  • The adenovirus E1A gene is the first gene expressed upon viral infection. E1A re-models the cellular environment to maximize permissivity for viral replication. E1A is also the major transactivator of viral early gene expression and a co-regulator of a large number of cellular genes. E1A carries out its functions predominantly by binding to cellular regulatory proteins and altering their activities. The unstructured nature of E1A enables it to bind to a large variety of cellular proteins and form new molecular complexes with novel functions. The C-terminus of E1A is the least characterized region of the protein with few known binding partners. Here we report the identification of a cellular factor DREF (ZBED1) as a novel, and direct binding partner of E1A. Our studies identify a dual role for DREF in the viral life cycle. DREF contributes to activation of gene expression from all viral promoters early in infection. Unexpectedly, it also functions as a growth restriction factor for adenovirus as knockdown of DREF enhances virus growth and increases viral genome copy number late in the infection. We also identify DREF as a component of viral replication centers. E1A affects the sub-cellular distribution of DREF within PML bodies and enhances DREF SUMOylation. Our findings identify DREF as a novel E1A C-terminus binding partner and provide evidence supporting a role for DREF in viral replication.

    IMPORTANCE This work identifies the putative transcription factor DREF as a new target of the E1A oncoproteins of human adenovirus. DREF was found to primarily localize with PML nuclear bodies in uninfected cells and was re-localized into virus replication centers during infection. DREF was also found to be SUMOylated and this was enhanced in the presence of E1A. Knockdown of DREF reduced the levels of viral transcripts detected at 20 hours, but not at 40 hours post-infection, increased overall virus yield and enhanced viral DNA replication. DREF was also found to localize to viral promoters during infection together with E1A. These results suggest that DREF contributes to activation of viral gene expression. However, like several other PML associated proteins, DREF also appears to function as a growth restriction factor for adenovirus infection.

  • Characterization of a broadly neutralizing monoclonal antibody that targets the fusion domain of group 2 influenza A virus hemagglutinin [PublishAheadOfPrint]

  • Due to continuous changes to its antigenic regions, influenza viruses can evade immune detection and cause a significant amount of morbidity and mortality around the world. Influenza vaccinations can protect against disease, but must be annually reformulated to match the current circulating strains. Towards the development of a broad-spectrum influenza vaccine, the elucidation of conserved epitopes is paramount. To this end, we designed an immunization strategy in mice to boost the humoral response against conserved regions of the hemagglutinin (HA) glycoprotein. Of note, generation and identification of broadly neutralizing antibodies that target group 2 HAs are rare and thus far have yielded only a handful of monoclonal antibodies Here, we demonstrate that mouse mAb, 9H10, has broad and potent in vitro neutralizing activity against H3 and H10 group 2 influenza A subtypes. In the mouse model, mAb 9H10 protects mice against two divergent mouse-adapted H3N2 strains, in both pre- and post-exposure administration regimens. In vitro and cell-free assays suggest that mAb 9H10 inhibits viral replication by blocking HA-dependent fusion of the viral and endosomal membranes early in the replication cycle and by disrupting viral particle egress in the late stage of infection. Interestingly, electron microscopy reconstructions of mAb 9H10 bound to the HA reveal that it binds a similar binding footprint to mAbs CR8020 and CR8043.

    IMPORTANCE The influenza hemagglutinin is the major antigenic target of the humoral immune response. However, due to continuous antigenic changes that occur on the surface of this glycoprotein, influenza viruses can escape the immune system and cause significant disease to the host. Towards the development of broad-spectrum therapeutics and vaccines against influenza virus, elucidation of conserved regions of influenza viruses is crucial. Thus, defining these types of epitopes through the generation and characterization of broadly neutralizing monoclonal antibodies can greatly assist others in highlighting conserved regions of hemagglutinin. Here, we demonstrate that mAb 9H10 that targets the hemagglutinin stalk has broadly neutralizing activity against group 2 influenza A viruses in vitro and in vivo.

  • HBZ stimulates BDNF/TrkB autocrine/paracrine signaling to promote survival of HTLV-1-infected T-cells [PublishAheadOfPrint]

  • Brain-Derived Neurotrophic Factor (BDNF) is a neurotrophin that promotes neuronal proliferation, survival and plasticity. These effects occur through autocrine and paracrine signaling events initiated by interactions between secreted BDNF and its high-affinity receptor, TrkB. A BDNF/TrkB autocrine/paracrine signaling loop has additionally been implicated in augmenting survival of cells representing several human cancers, and is associated with poor patient prognosis. Adult T-cell leukemia (ATL) is a fatal malignancy caused by infection with the complex retrovirus, Human T-cell Leukemia Virus type 1 (HTLV-1). In this study, we found that the HTLV-1-encoded protein, HBZ, activates expression of BDNF, and consistent with this effect, BDNF expression is elevated in HTLV-1-infected T-cell lines compared to uninfected T-cells. Expression of TrkB is also higher in HTLV-1-infected T-cell lines than in uninfected T-cells. Furthermore, levels of both BDNF and TrkB mRNA are elevated in PBMC from ATL patients, and ATL patient sera contain higher concentrations of BDNF than sera from non-infected individuals. Finally, chemically inhibition of TrkB signaling increases apoptosis in HTLV-1-infected T-cells and reduces phosphorylation of GSK-3bbeta;, a downstream target in the signaling pathway. These results suggest that HBZ contributes to an active BDNF/TrkB autocrine/paracrine signaling loop in HTLV-1-infected T-cells that enhances the survival of these cells.

    Importance Infection with Human T-cell Leukemia Virus type 1 (HTLV-1) can cause a rare form of leukemia designated Adult T-cell Leukemia (ATL). Because ATL patients are unresponsive to chemotherapy, this malignancy is fatal. As a retrovirus, HTLV-1 integrates its genome into a host-cell chromosome in order to utilize host factors for replication and expression of viral proteins. However, in infected cells from ATL patients, the viral genome is frequently modified to block expression of all but a single viral protein. This protein, known as HBZ, is therefore believed to modulate cellular pathways necessary for the leukemic state and the chemotherapeutic resistance of the cell. Here we provide evidence to support this hypothesis. We found that HBZ promotes a BDNF/TrkB autocrine/paracrine signaling pathway that is known to enhance the survival and chemotherapeutic resistance of other types of cancer cells. It is possible that inhibition of this pathway may improve treatments for ATL.

  • Hallmarks of hepatitis C virus in equine hepacivirus [PublishAheadOfPrint]

  • Equine hepacivirus (EHcV) has been identified as a closely-related homologue of hepatitis C virus (HCV) in the USA, UK, and Germany, but not in Asian countries. In this study, we genetically and serologically screened 31 sera obtained from Japanese-born domestic horses for EHcV infection and subsequently identified 11 PCR-positive and 7 seropositive sera. We determined the full sequence of the EHcV genome including the 3rrsquo; untranslated region (UTR), which had previously not been completely revealed. The polyprotein of a Japanese EHcV strain showed approximately 95% homology to those of the reported strains. HCV-like cis-acting RNA elements including the stem-loop structures of 3rrsquo; UTR and kissing-loop interaction were deduced from regions around both UTRs of the EHcV genome. A comparison of the EHcV and HCV core proteins revealed that Ile190 and Phe191 of the EHcV core protein could be important for cleavage of the core protein by signal peptide peptidase (SPP), and were substituted with Ala and Leu, respectively, which inhibited intramembrane cleavage of the EHcV core protein. The loss-of-function mutant of SPP abrogated intramembrane cleavage of the EHcV core protein and bound EHcV core protein, suggesting that the EHcV core protein may be cleaved by SPP to become a mature form. The wild-type EHcV core protein, but not the SPP-resistant mutant, was localized on lipid droplets and partially on the lipid raft-like membrane in a similar manner to the HCV core protein. These results suggested that EHcV may conserve the genetic and biological properties of HCV.

    Importance EHcV, which shows the highest amino acid or nucleotide homology to HCV among hepaciviruses, was previously reported to infect horses from Western, but not Asian countries. We herein reported EHcV infection in Japanese-born horses. In this study, HCV-like RNA secondary structures around both UTRs were predicted by determining the whole genome sequence of EHcV. Our results also suggest that the EHcV core protein is cleaved by SPP to become a mature form and then is localized on lipid droplets and partially on lipid raft-like membranes in a similar manner to the HCV core protein. Hence, EHcV was identified as a closely-related homologue of HCV based on its genetic structure as well as biological properties. A clearer understanding of the epidemiology, genetic structure, and infection mechanism of EHcV will assist in elucidating the evolution of hepacivirus as well as the development of surrogate models for the study of HCV.

  • The HERV-K human endogenous retrovirus envelope protein antagonises Tetherin antiviral activity [PublishAheadOfPrint]

  • Endogenous retroviruses are the remnants of past retroviral infections that are scattered within mammalian genomes. In humans, most of these elements are old degenerate sequences that have lost their coding properties. The HERV-K(HML2) family is an exception: it recently amplified in the human genome and corresponds to the most active proviruses, with some intact open reading frames and the potential to encode viral particles. Here, using a reconstructed consensus element, we show that HERV-K(HML2) proviruses are able to inhibit Tetherin, a cellular restriction factor that is active against most enveloped viruses and acts by keeping the viral particles attached to the cell surface. More precisely, we identify the Envelope protein (Env) as the viral effector active against Tetherin. Through immunoprecipitation experiments, we show that recognition of Tetherin is mediated by the surface subunit of Env. Similar to Ebola glycoprotein, HERV-K(HML2) Env does not mediate Tetherin degradation or cell surface removal, and therefore uses a yet undescribed mechanism to inactivate Tetherin. We also assessed all natural complete "alleles" of endogenous HERV-K(HML2) Env described to date for their ability to inhibit Tetherin, and found that two of them (out of six) can block Tetherin restriction. However, due to their recent amplification, HERV-K(HML2) elements are extremely polymorphic in the human population and it is likely that individuals will not all possess the same "anti-Tetherin" potential. Because of Tetherin role as a restriction factor capable of inducing innate immune responses, this could have functional consequences for individual responses to infection.

    IMPORTANCE Tetherin, a cellular protein initially characterised for its role against HIV-1, has been proven to counteract numerous enveloped viruses. It blocks the release of viral particles from producer cells, keeping them tethered to the cell surface. Several viruses have developed strategies to inhibit Tetherin activity, allowing them to efficiently infect and replicate in their host. Here we show that human HERV-K (HML2) elements, the remnants of an ancient retroviral infection, possess an anti-Tetherin activity which is mediated by the envelope protein. It is likely that this activity was an important factor that contributed to the recent, human-specific amplification of this family of elements. Also, due to their recent amplification, HERV-K(HML2) elements are highly polymorphic in the human population. Since Tetherin is a mediator of innate immunity, inter-individual variations among HERV-K(HML2) Env genes may result in differences in immune responses to infection.

  • Early preservation of CXCR5+ PD-1+ helper T cells and B cell activation predict the breadth of neutralizing antibody responses in chronic HIV-1 infection [PublishAheadOfPrint]

  • Much is known about the characteristics of broadly neutralizing antibodies (bNAbs) generated during HIV-1 infection, but little is known about immunological mechanisms responsible for their development in only a minority of those infected by HIV-1. By monitoring longitudinally a cohort of HIV-1 infected subjects, we observed that the preservation of CXCR5+ CD4+ T helper cell frequencies and activation status of B cells during the first year of infection correlates with the maximum breadth of plasma neutralizing antibody responses during chronic infection, independently of viral load. Although, during the first year of infection, no differences were observed in the abilities of peripheral CXCR5+ CD4+ T helper cells to induce antibody secretion by autologous naiiuml;ve B cells, higher frequencies of class-switched antibodies were detected in co-cultures of CXCR5+ CD4+ T and B cells from the subjects who later developed broadly neutralizing antibody responses than those who did not. Furthermore, B cells from the former subjects had higher expression of AICDA than B cells from the latter subjects and transcript levels correlated with the frequency of CXCR5+ CD4+ T cells. Thus, the early preservation of CXCR5+ CD4+ T cells and B cell function are central to the development of bNAbs. Our study provides a possible explanation for their infrequent generation during HIV-1 infection.

    IMPORTANCE Broadly neutralizing antibodies (bNAbs) are developed by HIV-1-infected subjects, but so far (and despite intensive efforts over the past three decades) have not been elicited by immunization. Understanding how bNAbs are generated during natural HIV-1 infection and why only some HIV-1-infected subjects generate such antibodies, will assist our efforts to elicit bNAbs by immunization. CXCR5+ PD-1+ CD4+ T cells are critical for the development of high affinity antigen-specific antibody responses. In our study, we found that not only did the HIV-1-infected subjects who develop bNAbs have a higher frequency of peripheral CXCR5+ PD-1+ CD4+ T cells in early infection, but also that this frequency mirrored what was observed in uninfected subjects and correlated with the level of B cell activation across subjects. Our study highlight the critical role helper T cell function has in the elicitation of broadly neutralizing antibody responses in the context of HIV- infection.

  • Specific cells in the primary salivary glands of the whitefly Bemisia tabaci control retention and transmission of begomoviruses [PublishAheadOfPrint]

  • The majority of plant viruses are vectored by arthropods via persistent-circulative or noncirculative transmission. Previous studies have shown that specific binding sites for noncirculative viruses reside within the stylet or foregut of insect vectors, whereas the transmission mechanisms of circulative viruses remain ambiguous. Here we report the critical roles of whitefly primary salivary glands (PSGs) in the circulative transmission of two begomoviruses. The Middle East Asia Minor 1 (MEAM1) species of the whitefly Bemisia tabaci complex efficiently transmits both Tomato yellow leaf curl China virus (TYLCCNV) and Tomato yellow leaf curl virus (TYLCV), whereas the Mediterranean (MED) species transmits TYLCV but not TYLCCNV. PCR and fluorescence in situ hybridization experiments showed that TYLCCNV efficiently penetrates the PSGs of MEAM1 but not those of MED. When a fragment of the coat protein of TYLCCNV was exchanged with that of TYLCV, mutated TYLCCNV accumulated in the PSGs of MED while mutant TYLCV was nearly undetectable. Confocal microscopy revealed that virion transport in PSGs follows specific paths to reach secretory cells in the central region and, the accumulation of virions in the secretory region of PSGs was correlated with successful virus transmission. Our findings demonstrate that whitefly PSGs, in particular the cells around the secretory region control specificity of begomovirus transmission.

    IMPORTANCE Over 75% of plant viruses are transmitted by insects. However, the mechanisms of virus transmission by insect vectors remain largely unknown. Begomoviruses and whiteflies are a complex of viruses and vectors, which threaten many crops worldwide. We investigated the transmission of two begomoviruses by two whitefly species. We show that specific cells of the whitefly primary salivary glands control viral transmission specificity, and virion transport in the glands follows specific paths to reach secretory cells in the central region and then to the salivary duct. Our results indicate that the secretory cells in the central region of primary salivary glands determine the recognition and transmission of begomoviruses. These findings set a foundation not only for future research on circulative plant virus transmission, but also for other human and animal viruses transmitted by arthropod vectors.

  • CHOLESTEROL-DEPENDENT MEMBRANE FUSION INDUCED BY THE GP41 MPER-TMD CONNECTION SUGGESTS A MECHANISM FOR BROAD HIV-1 NEUTRALIZATION. [PublishAheadOfPrint]

  • The HIV-1 glycoprotein 41 promotes fusion of the viral membrane with that of the target cell. Structural, biochemical and biophysical studies suggest that its membrane-proximal external region (MPER) may interact with the HIV-1 membrane and induce its disruption and/or deformation during the process. However, the high cholesterol content of the envelope (ca. 40-50 mol %) imparts high rigidity, thereby acting against lipid bilayer restructuring. Here, based on the outcome of vesicle stability assays, all-atom molecular dynamics simulations and atomic force microscopy observations, we propose that the conserved sequence connecting MPER with the N-terminal residues of the transmembrane domain (TMD) is involved in HIV-1 fusion. This junction would function by inducing phospholipid protrusion and acyl-chain splay in the cholesterol-enriched rigid envelope. Supporting the functional relevance of such mechanism, membrane fusion was inhibited by the broadly neutralizing 4E10 antibody, but not by a non-neutralizing variant with the CDR-H3 loop deleted. We conclude that the MPER-TMD junction embodies an envelope-disrupting C-terminal fusion peptide that can be targeted by broadly neutralizing antibodies.

    Importance Fusion of the cholesterol-enriched viral envelope with the cell membrane marks the beginning of the infectious HIV-1 replicative cycle. Consequently, the Env glycoprotein-mediated fusion function constitutes an important clinical target for inhibitors and preventive vaccines. Antibodies 4E10 and 10E8 bind to one Env vulnerability site located at the gp41 membrane proximal external region (MPER)-transmembrane domain (TMD) junction and block infection. These antibodies display broad viral neutralization, which underscores the conservation and functionality of the MPER-TMD region. In this work, we combine biochemical assays with molecular dynamics simulations and microscopy observations to describe the unprecedented fusogenic activity of the MPER-TMD junction. The fact that such activity is dependent on cholesterol, and inhibited by the broadly neutralizing 4E10 antibody emphasizes its physiological relevance. Discovery of this functional element adds to our understanding of the mechanisms underlying HIV-1 infection and its blocking by antibodies.

  • Binding Interactions between the Encephalomyocarditis Virus Leader and Protein 2A [PublishAheadOfPrint]

  • The Leader (L) and 2A proteins of cardioviruses are the primary anti-host agents produced during infection. For encephalomyocarditis virus (EMCV), the prototype of this genus, these proteins interact independently with key cellular partners to bring about inhibition of active nucleocytoplasmic trafficking and cap-dependent translation, respectively. L and 2A also bind each other and require this cooperation to achieve their effects during infection. Recombinant L and 2A interact with 1:1 stoichiometry at a KD of 1.5 mmu;M. The mapped contact domains include the amino-proximal third of 2A (first 50 amino acids) and the central hinge region of L. This contact partially overlaps the L segment that makes subsequent contact with RanGTPase in the nucleus, and Ran can displace 2A from L. The equivalent proteins from TMEV (BeAn) and Saffold virus interact similarly in any subtype combination, with varying affinities. The data suggest a mechanism whereby L takes advantage of the nuclear localization signal in the COOH-region of 2A to enhance its trafficking to the nucleus. Once there, it exchanges partners in favor of Ran. This required cooperation during infection explains many observed co-dependent phenotypes of L and 2A mutations.

    IMPORTANCE Cardiovirus pathogenesis phenotypes vary dramatically, from asymptomatic, to mild GI distress, to persistent demyelination and even encephalitic death. Leader and 2A are the primary viral determinants of pathogenesis, so understanding how these proteins cooperate to induce such a wide variety of outcomes for the host is of great important and interest to the field of virology, especially those who use TMEV as a murine model for multiple sclerosis.

  • A unique Nodavirus with novel features: Mosinovirus expresses two subgenomic RNAs, a capsid gene of unknown origin, and a suppressor of the antiviral RNAi pathway [PublishAheadOfPrint]

  • Insects are a reservoir for many known and novel viruses. We discovered an unknown virus, tentatively named Mosinovirus (MoNV), in mosquitoes from a tropical rainforest region in Coocirc;te d'Ivoire. The MoNV genome consists of two segments of positive-sense RNA of 2,972 nt (RNA 1) and 1,801 nt (RNA 2) in length. Its putative RNA-dependent RNA polymerase shares 43% amino acid identity with its closest relative Pariacoto virus (family Nodaviridae). Unexpectedly, for the putative capsid protein maximal pairwise identity of 16% was found to Lake Sinai virus 2, an unclassified virus with a non-segmented RNA genome. Moreover, MoNV virions are non-enveloped and about 50 nm in diameter, larger than any of the known nodaviruses. Mature MoNV virions contain capsid proteins of ~56 kDa, which do not seem to be cleaved from a longer precursor. Northern blot analyses revealed that MoNV expresses two subgenomic RNAs of 580 nt (RNA 3) and 292 nt (RNA 4). RNA 4 encodes a viral suppressor of RNAi that shares its mechanism with the B2 RNAi suppressor protein of other nodaviruses despite lacking recognizable similarity to these proteins. MoNV B2 binds long dsRNA and, accordingly, inhibits Dicer-2-mediated processing of dsRNA into siRNAs. Phylogenetic analyses indicate that MoNV is a novel member of the Nodaviridae family that acquired its capsid gene via reassortment from an unknown, distantly related virus beyond the family level.

    Importance The identification of novel viruses provides important information about virus evolution and diversity. Here, we describe an unknown unique nodavirus in mosquitoes, named Mosinovirus (MoNV). MoNV was classified as a nodavirus based on its genome organization and on phylogenetic analyses of the RNA-dependent RNA polymerase. Notably, its capsid gene was acquired from an unknown virus in distant relationship to nodaviruses. Another remarkable feature of MoNV was that, unlike other nodaviruses, it expresses two sgRNAs. One of the sgRNAs expresses a protein that counteracts antiviral defense of its mosquito host, whereas the function of the other sgRNA remains unknown. Our results show that complete genome segments can be exchanged beyond the species level and suggest that insects harbor a large repertoire of exceptional viruses.

  • Induction of broadly-reactive anti-hemagglutinin stalk antibodies by an H5N1 vaccine in humans [PublishAheadOfPrint]

  • Influenza virus infections are a major public health concern and cause significant morbidity and mortality worldwide. Current vaccines are effective but strain specific due to their focus on the immunodominant globular head domain of the hemagglutinin. It has been hypothesized that sequential exposure of humans to hemagglutinins with divergent globular head domains but conserved stalk domains could refocus the immune response to broadly neutralizing epitopes in the stalk. Humans have pre-existing immunity against H1 (group 1 hemagglutinin) and vaccination with H5 HA (also group 1) nndash; which has a divergent globular head domain but a similar stalk domain nndash; represents one such sequential exposure scenario. To test this hypothesis, we used novel reagents based on chimeric hemagglutinins to screen sera from an H5N1 clinical trial for induction of stalk-specific antibodies by quantitative ELISA and neutralization assays. Importantly, we also investigated the biological activity of these antibodies in a passive transfer in a mouse challenge model. We found that the H5N1 vaccine induced high titers of stalk-reactive antibodies which were biologically active and protective in the passive transfer experiment. The induced response showed exceptional breadth towards divergent group 1 hemagglutinins but did not extend to group 2 hemagglutinins. This data provides evidence for the hypothesis that sequential exposure to hemagglutinins with divergent globular head domains but conserved stalk domains can refocus the immune response towards the conserved stalk domain. Furthermore, the results support the concept of a chimeric hemagglutinin universal influenza virus vaccine strategy that is based on the same principle.

    Significance Influenza virus vaccines have to be re-formulated and re-administered on an annual basis. The development of a universal influenza virus vaccine could abolish the need for this cumbersome and costly process and would also enhance our pandemic preparedness. This study addressed the following questions that are essential for the development of a hemagglutinin stalk based universal influenza virus vaccine: 1) Can stalk-reactive antibodies be boosted by vaccination with divergent HAs that share conserved epitopes? 2) How long-lived are these vaccine induced stalk-reactive antibody responses? 3) What is the breadth of this reactivity. 4) Are these antibodies functional and protective? Our results further strengthen the concept of induction of stalk-reactive antibodies by sequential exposure to hemagglutinin immunogens with conserved stalk and divergent head domains. A universal influenza virus vaccine based on the same principles seems possible and might have a significant impact on global human health.

  • Matrix Proteins of Nipah and Hendra Viruses Interact with Beta Subunits of AP-3 Complexes [PublishAheadOfPrint]

  • Paramyxoviruses and other negative-strand RNA viruses encode matrix proteins that coordinate the virus assembly process. The matrix proteins link together the viral glycoproteins and the viral ribonucleoproteins at virus assembly sites, and often recruit host machinery that facilitates the budding process. Using a co-affinity purification strategy, we have identified the Beta subunit of the AP-3 adapter protein complex, AP3B1, as a binding partner for the M proteins of the zoonotic paramyxoviruses, Nipah virus and Hendra virus. Binding function was localized to the serine-rich and acidic Hinge domain of AP3B1, and a 29-amino acid long Hinge-derived polypeptide was sufficient for M protein binding in co-immunoprecipitation assays. Virus-like particle (VLP) production assays were used to assess the relationship between AP3B1 binding and M protein function. We found that for both Nipah virus and Hendra virus, M protein expression in the absence of any other viral proteins led to the efficient production of VLPs in transfected cells, and this VLP production was potently inhibited upon overexpression of short M-binding polypeptides derived from the Hinge region of AP3B1. Both human and bat (P. alecto) AP3B1-derived polypeptides were highly effective at inhibiting the production of VLPs. VLP production was also impaired through siRNA-mediated depletion of AP3B1 from cells. These findings suggest that AP-3 directed trafficking processes are important for henipavirus particle production, and identify a new host-protein:virus protein binding interface that could become a useful target in future efforts to develop small molecule inhibitors to combat paramyxoviral infections.

    IMPORTANCE Henipaviruses cause deadly infections in humans, with mortality rate of about 40%. Hendra virus outbreaks in Australia, all involving horses and some involving transmission to humans, have been a continuing problem. Nipah virus caused a large outbreak in Malaysia in 1998, killing 109 people, and smaller outbreaks have since occurred in Bangladesh and India. In this study, we have defined, for the first time, host factors that interact with henipavirus M proteins and contribute to viral particle assembly. We have also defined a new host protein:viral protein binding interface that can potentially be targeted for the inhibition of paramyxovirus infections.

  • Common Features of Mucosal and Peripheral Vaccine-Elicited Antibody Responses in Rhesus Monkeys [PublishAheadOfPrint]

  • HIV-1 vaccines that elicit protective antibody responses at mucosal sites would be highly desirable. Here we report that intramuscular immunization of candidate HIV-1 vaccine vectors and purified Env proteins elicited potent and durable humoral immune responses in colorectal mucosa in rhesus monkeys. These mucosal antibody responses shared similar kinetics, isotypes, functionality and specificity to peripheral responses in serum. These data suggest a close immunological relationship between mucosal and systemic antibody responses following vaccination in primates.

  • Circulating clinical strains of human parainfluenza virus reveal the viral entry requirements for in vivo infection [PublishAheadOfPrint]

  • Human parainfluenza viruses (HPIV) cause widespread respiratory infections, with no vaccines or effective treatments. We show that the molecular determinants for HPIV3 growth in vitro are fundamentally different from those required in vivo, and that these differences impact inhibitor susceptibility. HPIV infects its target cells by coordinated action of the receptor binding protein hemagglutinin-neuraminidase (HN) and the fusion (F) envelope glycoprotein which together comprise the molecular fusion machinery; upon receptor engagement by HN, the pre-fusion F undergoes a structural transition, extending and inserting into the target cell membrane and then re-folding into a post-fusion structure that fuses the viral and cell membranes. Peptides derived from key regions of F can potently inhibit HPIV infection at the entry stage, by interfering with the structural transition of F. We show that clinically circulating viruses have fusion machinery that is more stable and less readily activated than viruses adapted to growth in culture. Fusion machinery that is advantageous for growth in human airway epithelia and in vivo confers susceptibility to peptide fusion inhibitors in the host lung tissue or animal, but the same fusion inhibitors have no effect on viruses whose fusion glycoproteins are suited for growth in vitro. We propose that for potential clinical efficacy, antivirals should be evaluated using clinical isolates in natural host tissue, rather than lab strains of virus in cultured cells. The unique susceptibility of clinical strains in human tissues reflects viral inhibition in vivo.

    Importance Acute respiratory infection is the leading cause of mortality in young children under 5 years of age, causing nearly 20% of childhood deaths worldwide each year. The paramyxoviruses, including human parainfluenza viruses (HPIVs), cause a large share of these illnesses. There are no vaccines or drugs for the HPIVs. Inhibiting entry of viruses into the human cell is a promising drug strategy that blocks the first step in infection. To develop antivirals that inhibit entry it is critical to understand the first steps of infection. We found that clinical viruses isolated from patients have very different entry properties than the viruses generally studied in laboratories. The viral entry mechanism is less active and more sensitive to fusion inhibitory molecules. We propose that to interfere with viral infection we test clinically circulating viruses in natural tissues, to develop antivirals against respiratory disease caused by HPIVs.

  • Characterization of pH sensitive molecular switches triggering the structural transition of VSV Glycoprotein from the post- toward the pre-fusion state [PublishAheadOfPrint]

  • Vesicular stomatitis virus (VSV, the prototype rhabdovirus) fusion is triggered at low pH and mediated by glycoprotein G which undergoes a low-pH-induced structural transition. A unique feature of rhabdovirus G is that its conformational change is reversible. This allows G to recover its native pre-fusion state at the viral surface after its transport through the acidic Golgi compartments. The crystal structures of G pre- and post-fusion states have been elucidated, leading to the identification of several acidic amino acid residues, clustered in the post-fusion trimer, as potential pH sensitive switches controlling the transition back toward the pre-fusion state. We mutated these residues and produced a panel of single and double mutants of which the fusion properties, the conformational change characteristics and the ability to pseudotype a virus lacking the glycoprotein gene were assayed. Some of these mutations were also introduced in the genome of recombinant viruses which were further characterized. We show that D268, located in segment [264-273] which refolds into post-fusion helix F during G structural transition, is the major pH sensor while D274, D395 and D393 have additional contributions. Furthermore, a single passage of recombinant virus bearing mutation D268L (which was demonstrated to stabilize G post-fusion state) resulted in a pseudo-revertant with a compensatory second mutation L271P. This revealed that the propensity of segment [264-273] to refold into helix F has to be finely tuned since either an increase (mutation D268L alone) or a decrease (mutation L271P alone) of this propensity is detrimental to the virus.

    Importance Vesicular stomatitis virus enters cells via endocytosis. Endosome acidification induces a structural transition of its unique glycoprotein (G), which mediates fusion between viral and endosomal membranes. G conformational change is reversible upon increasing the pH. This allows G to recover its native pre-fusion state at the viral surface after its transport through the acidic Golgi compartments. We mutated five acidic residues, proposed to be pH sensitive switches controlling the structural transition back toward the pre-fusion state. Our results indicate that residue D268 is the major pH sensor while other acidic residues have additional contributions and reveal that the propensity of segment [264-273] to adopt a helical conformation is finely regulated. This segment might be a good target for antiviral compounds.

  • PRIME-BOOST IMMUNIZATION STRATEGIES AGAINST CHIKUNGUNYA VIRUS [PublishAheadOfPrint]

  • Chikungunya virus (CHIKV) is a reemerging mosquito-borne alphavirus that causes debilitating arthralgia in humans. Here we describe the development and testing of novel DNA replicon and protein CHIKV vaccine candidates and evaluate their ability to induce antigen-specific immune response against CHIKV. We also describe homologous and heterologous prime-boost immunization strategies using the novel as well as previously developed CHIKV vaccine candidates. Immunogenicity and efficacy were studied in a CHIKV infection mouse model and showed that the DNA replicon and protein antigen were potent vaccine candidates, in particular when used for priming and boosting, respectively. Several prime-boost immunization strategies eliciting unmatched humoral and cellular immune responses were identified. Further characterization by antibody epitope mapping revealed differences in the qualitative immune response induced by the different vaccine candidates and immunization strategies. Most vaccine modalities resulted in complete protection against wild type CHIKV infection however we did identify circumstances where certain immunization regimes may lead to enhancement of inflammation upon challenge. These results should help design CHIKV vaccine studies and will form the basis for further pre-clinical and clinical evaluation of these vaccine candidates.

    IMPORTANCE As of today, there is no licensed vaccine available to prevent CHIKV infection. Considering potential new vaccine candidates the preferable situation would be a vaccine that is able to raise long-term protective immunity after a single immunization. While humoral immunity seems to be central for protection against CHIKV infection we do not yet fully understand the correlates of protection. Therefore, in the absence of a functional vaccine there is a need to evaluate a number of different candidates to assess their merits either when used in a single immunization or when used in a homologous or heterologous prime-boost modality. Here we show that while single immunization with various vaccine candidates results in potent responses combined approaches significantly enhances responses suggesting that such approaches need to be consider in the further development of a efficacious CHIKV vaccine.

  • Expression library immunization can confer protection against African swine fever virus lethal challenge [PublishAheadOfPrint]

  • African swine fever is one of the most devastating pig diseases against which there is no vaccine available. Recent work from our laboratory has demonstrated the protective potential of DNA vaccines encoding three African swine fever viral antigens (p54, p30 and the hemagglutinin extracellular domain) fused to ubiquitin. Partial protection was afforded in the absence of detectable antibodies prior to virus challenge and survival correlated with the presence of a large number of hemagglutinin-specific CD8+ T-cells in blood. Aiming to demonstrate the presence of additional CD8+ T-cell determinants with protective potential, an expression library containing more than 4,000 individual plasmid clones was constructed, each one randomly containing a Sau3AI restriction fragment of the viral genome (p54, p30 and hemagglutinin ORFs excluded), fused to ubiquitin. Immunization of farm pigs with the expression library yielded 60% protection against lethal challenge with the virulent E75 strain. These results were further confirmed by using specific pathogen free pigs after challenging with 104 HAU of the cell culture adapted strain E75CV1. On this occasion, 50% of the vaccinated pigs survived the lethal challenge and 2 out of the 8 immunized pigs showed no viremia, or viral excretion at any time post-infection. In all cases, protection was afforded in the absence of detectable specific antibodies prior to challenge and correlated with the detection of specific T-cell responses at time of sacrifice. In summary, our results clearly demonstrate the presence of additional protective determinants within the ASFV genome and open up the possibility for their future identification.

    Importance African swine fever is a highly contagious disease of domestic and wild pigs that is endemic in many Sub-Saharan countries where it causes important economic losses and is currently in continuous expansion across Europe. Unfortunately, there is no treatment, nor an available vaccine. Early attempts using attenuated vaccines demonstrated their potential to protect pigs against experimental infection. However, their use in the field remains controversial due to safety issues. Although inactive and subunit vaccines did not confer solid protection against experimental ASFV infection, our DNA vaccination results have generated new expectations confirming the key role of T-cell responses in protection and the existence of multiple ASFV antigens with protective potential; more of which are currently being identified. Thus, the future might bring complex and safe formulations containing more than one single viral determinant to obtain broader protective vaccines. We believe that obtaining the optimal vaccine formulation it is just a matter of time, investment and willingness.

  • Identification of molecular markers associated with alteration of receptor-binding specificity in a novel genotype of highly pathogenic avian influenza A(H5N1) viruses detected in Cambodia in 2013. [PublishAheadOfPrint]

  • Human infections with influenza A(H5N1) virus in Cambodia increased sharply during 2013. Molecular characterization of viruses detected in clinical specimens from human cases revealed the presence of mutations associated with alteration of receptor-binding specificity (K189R, Q222L) and respiratory droplet transmission in ferrets (N220K with Q222L). Discovery of quasispecies at position 222 (Q/L), in addition to absence of the mutations in poultry/environmental samples, suggested the mutations occurred during human infection and did not transmit further.

  • Envelope Variants Circulating as Initial Neutralization Breadth Developed in Two HIV-Infected Subjects Stimulate Multiclade Neutralizing Antibodies in Rabbits [PublishAheadOfPrint]

  • Identifying characteristics of the HIV-1 Envelope that are effective in generating broad, protective antibodies remains a hurdle to HIV vaccine design. Emerging evidence of the development of broad and potent neutralizing antibodies in HIV-infected subjects suggests that founder and subsequent progeny viruses may express unique antigenic motifs that contribute to this developmental pathway. We hypothesize that over the course of natural infection, B cells are programmed to develop broad antibodies by exposure to select populations of emerging Envelope quasispecies variants. To test this hypothesis, we identified two unrelated subjects whose antibodies demonstrated increasing breadth against a panel of HIV-1 isolates over time. Full-length functional env genes were cloned longitudinally from these subjects from months after infection through 2.6 to 5.8 years of infection. Motifs associated with the development of breadth in published, cross-sectional studies were found in both subjects. We compared the immunogenicity of Envelope vaccines derived from timepoints obtained during and after broadening of neutralization activity within these subjects. Rabbits were co-immunized four times with selected multiple gp160 DNAs and gp140-trimeric Envelope proteins. Affinity of the polyclonal response increased as a function of boosting. The most rapid and persistent neutralization of multi-clade Tier 1 viruses was elicited by Envelopes that were circulating in plasma at timepoints prior to the development of 50% neutralization breadth in both human subjects. The breadth elicited in rabbits was not improved by exposure to later Envelope variants. These data have implications for vaccine development in describing a target timepoint to identify optimal Envelope immunogens.

    IMPORTANCE Vaccine protection against viral infections correlates with the presence of neutralizing antibodies, thus vaccine components capable of generating potent neutralization are likely to be critical constituents in an effective HIV vaccine. However, vaccines tested to date have elicited only weak antibody responses and very modest, waning protection. We hypothesized that B cells develop broad antibodies by exposure to the evolving viral Envelope population and tested this concept using multiple Envelopes from two subjects who developed neutralization breadth within a few years of infection. We compared different combinations of Envelopes from each subject to identify the most effective immunogens and regimens. In each subject, use of HIV Envelopes circulating during the early development and maturation of breadth generated more potent antibodies that were modestly cross neutralizing. These data suggest a new approach to identifying Envelope immunogens that may be more effective in generating protective antibodies in humans.

  • In vivo cellular tropism of gorilla simian foamy virus in blood of infected humans [PublishAheadOfPrint]

  • Simian foamy viruses (SFV) are retroviruses that are widespread among non-human primates. SFV can be transmitted to humans, giving rise to a persistent infection. Only few data are available concerning the distribution of SFV in human blood cells. Here we purified blood mononuclear cell subsets from 11 individuals infected with a Gorilla gorilla SFV strain and quantified SFV DNA levels by quantitative PCR. SFV DNA was detected in the majority of the CD8+, CD4+ and CD19+ lymphocyte samples, and in rare CD14+ monocyte and CD56+ NK lymphocyte samples. The median (Interquartile range, IQR) SFV DNA were 16.0 (11.0-49.8), 11.3 (5.9-28.3) and 17.2 (2.0-25.2) copies/105 cells in CD8+ T lymphocytes, CD4+ T lymphocytes and CD19+ B lymphocytes, respectively. Looking at the CD4 compartment, SFV DNA was detected in both memory and naive CD4+ T lymphocytes. SFV DNA levels in CD4+ T cells were positively correlated with the duration of the infection.

    Our study shows with a quantitative method that CD8+, CD4+ and B lymphocytes are major cellular targets of SFV in the blood of infected humans.

    Importance Investigation of simian foamy virus (SFV) infections in humans is important due to the origin of human immunodeficiency viruses (HIV) and human T-cell lymphotropic viruses (HTLV) from cross-species transmission of their simian counterpart to humans. Surprisingly little is known about many aspects of the biology of SFV in infected humans, including quantitative data concerning the cellular targets of SFV in vivo. Here we show that the distribution of SFV DNA among the different leukocyte populations is not homogeneous, and that viral load in CD4+ T lymphocytes is correlated with the duration of infection. These new data will help in understanding the biology of retroviral infections in humans and can be useful in the growing field of SFV-based gene therapy.

  • Occurrence and reassortment of avian influenza A (H7N9) viruses derived from coinfected birds in China [PublishAheadOfPrint]

  • Over the course of two waves of infection, H7N9 avian influenza A virus has caused 436 human infections and claimed 170 lives in China as of July 2014. To investigate the prevalence and genetic diversity of H7N9 we surveyed avian influenza viruses in poultry in Jiangsu province within the outbreak epicenter. We found frequent occurrence of H7N9/H9N2 co-infection in chickens. Molecular clock phylogenetic analysis confirms co-infection by H7N9/H9N2 viruses and also reveals that the identity of the H7N9 outbreak lineage is confounded by ongoing reassortment between outbreak viruses and diverse H9N2 viruses in domestic birds. Experimental inoculation of a co-infected sample in cell culture yielded two reassortant H7N9 strains with polymerase segments from the original H9N2 strain. Ongoing reassortment between the H7N9 outbreak lineage and diverse H9N2 viruses may generate new strains with the potential to infect humans, highlighting the need for continued viral surveillance in poultry and humans.

    Importance We found frequent occurrence of H7N9/H9N2 co-infection in chickens. The H7N9 outbreak lineage is confounded by ongoing reassortment between H7N9 and H9N2 viruses. The importance of H9N2 viruses as the source of novel avian influenza virus infections in humans requires continuous attention.

  • Accumulation of human-adapting mutations during circulation of A(H1N1)pdm09 influenza in humans in the UK [PublishAheadOfPrint]

  • The influenza pandemic that emerged in 2009 provided an unprecedented opportunity to study adaptation of a virus recently acquired from an animal source during human transmission. In the UK, the novel virus spread in three temporally distinct waves between 2009 and 2011. Phylogenetic analysis of complete viral genomes showed that mutations accumulated over time. Second and third wave viruses replicated more rapidly in human airway epithelial (HAE) cells than first wave virus. In infected mice, weight loss varied between viral isolates from the same wave but showed no distinct pattern with wave, and did not correlate with viral load in the mouse lungs or severity of disease in the human donor. However, second and third wave viruses induced less interferon-aalpha; in the infected mouse lungs. NS1 protein, an interferon antagonist, had accumulated several mutations in second and third wave viruses. Recombinant viruses with third wave NS gene induced less interferon in human cells but this alone was did not account for increased virus fitness in HAE cells. Mutations in HA and NA genes in third wave viruses caused increased binding to aalpha;-2,6 sialic acid, and enhanced infectivity in human mucus. A recombinant virus with these two segments replicated more efficiently in HAE cells. A mutation in PA (N321K) enhanced polymerase activity of third wave viruses and also provided a replicative advantage in HAE cells. Therefore, multiple mutations allowed incremental changes in viral fitness which together may have contributed to the apparent increase in severity of A(H1N1)pdm09 influenza during successive waves.

    Importance Although most people infected with the 2009 pandemic influenza virus had mild or unapparent symptoms, some suffered severe and devastating disease. The reasons for this variability were unknown but the numbers of severe cases increased during successive waves of human infection in the UK. To determine the causes of this variation, we studied genetic changes in virus isolates from individual hospitalized patients. There were no consistent differences between these viruses and those circulating in the community, but we found multiple evolutionary changes that in combination over time increased the virus's ability to infect human cells. These adaptations may explain the remarkable ability of A(H1N1)pdm09 virus to continue to circulate despite widespread immunity, and the apparent increase in severity of influenza over successive waves of infection.

  • Human H7N9 and H5N1 influenza viruses differ in induction of cytokines and tissue tropism [PublishAheadOfPrint]

  • Since emerging in 2013, the avian-origin H7N9 influenza viruses have resulted in over 400 human infections leading to 115 deaths to date. Although the epidemiology differs from human highly pathogenic avian H5N1 influenza infections, there is a similar rapid progression to acute respiratory distress syndrome (ARDS). The aim of these studies was to compare the pathological and immunological characteristics of a panel of human H7N9 and H5N1 viruses in vitro and in vivo. Although there were similarities between particular H5N1 and H7N9 viruses, including association between lethal disease and spread to the alveolar spaces and kidney, there were also strain-specific differences. Both H5N1 and H7N9 viruses are capable of causing lethal infections, with mortality correlating most strongly with wider distribution of viral antigen in the lungs, rather than with traditional measures of viral titer and host responses. Strain-specific differences included hypercytokinemia in H5N1 infections that was not seen with the H7N9 infections regardless of lethality. Conversely, H7N9 viruses showed a greater tropism for respiratory epithelium covering nasal passages and NALT than H5N1 viruses, which may explain the enhanced transmission in ferret models. Overall these studies highlight some distinctive properties of H5N1 and H7N9 viruses in different in vitro and in vivo models.

    Importance The novel avian-origin H7N9 pandemic represents a serious threat to public health. The ability of H7N9 to cause serious lung pathology leading in some cases to the development of acute respiratory distress syndrome is of particular concern. Initial reports of H7N9 infection compared them to infections caused by highly pathogenic avian (HPAI) H5N1 viruses. Thus, it is of critical importance to understand the pathology and immunological response to infection with H7N9 as compared to HPAI H5N1 viruses. We compared these responses in both in vitro and in vivo models, and found that H5N1 and H7N9 infections exhibit distinct pathological, immunological and tissue tropism differences that could explain differences in clinical disease and viral transmission.

  • Broad Protection against Avian Influenza using a Modified Vaccinia Ankara Virus Expressing a Mosaic Hemagglutinin gene [PublishAheadOfPrint]

  • A critical failure in our preparedness for an influenza pandemic is the lack of a universal vaccine. Influenza strains diverge by 1-2% per year, and commercially available vaccines often do not elicit protection from one year to the next, necessitating frequent formulation changes. This represents a major challenge to the development of a cross-protective vaccine that can protect against circulating viral antigenic diversity. We have constructed a recombinant Modified Vaccinia Ankara (MVA) virus that expresses an H5N1 mosaic hemagglutinin (H5M). This mosaic was generated in silico using 2,145 field-sourced H5N1 isolates. A single dose of MVA-H5M provided 100% protection in mice against clades 0, 1 and 2 avian influenza viruses and also protected against seasonal H1N1 virus (PR8). It also provided short- (10 days) and long-term (6 months) protection post-vaccination. Both neutralizing antibodies and antigen-specific CD4+ and CD8+ T cells were still detected after 5 months post-vaccination suggesting long-lasting immunity.

    Importance Influenza viruses infect a billion people every year and cause up to 500,000 deaths. A major problem in combating influenza is the lack of broadly effective vaccines. One solution from the field of human immunodeficiency virus vaccinology involves a novel in silico mosaic approach that has been shown to provide broad and robust protection against highly variable viruses. Unlike a consensus algorithm which picks the most frequent residue at each position, the mosaic method chooses the most frequent T-cell epitopes and combines them to form a synthetic antigen. These studies demonstrated that a mosaic influenza H5 hemagglutinin expressed by a viral vector can elicit full protection against diverse H5N1 challenges as well as inducing broader immunity than a wild-type hemagglutinin.

  • Modified vaccinia Ankara encoding influenza virus hemagglutinin induces heterosubtypic immunity in macaques [PublishAheadOfPrint]

  • Current influenza vaccines primarily aim to induce neutralizing antibodies (NAbs). Modified vaccinia Ankara (MVA) is a safe and well-characterized vector for inducing both antibody and cellular immunity. We evaluated the immunogenicity and protective efficacy of MVA encoding influenza virus hemagglutinin (HA) and/or nucleoprotein (NP) in cynomolgus macaques. Animals were given 2 doses of MVA based vaccines 4 weeks apart, and were challenged with a 2009 pandemic H1N1 isolate (H1N1pdm) 8 weeks after the last vaccination. MVA-based vaccines encoding HA induced potent serum antibody responses against homologous H1 or H5 HAs, but did not stimulate strong T cell responses prior to challenge. However, animals that received MVA encoding influenza HA and/or NP had high frequencies of virus-specific CD4+ and CD8+ T cell responses within the first 7 days of H1N1pdm infection, while animals vaccinated with MVA encoding irrelevant antigens did not. We detected little or no H1N1pdm replication in animals that received vaccines encoding H1 (homologous) HA, while a vaccine encoding NP from an H5N1 isolate afforded no protection. Surprisingly, H1N1pdm viral shedding was reduced in animals vaccinated with MVA encoding HA and NP from an H5N1 isolate. This reduced shedding was associated with cross-reactive antibodies capable of mediating antibody-dependent cellular cytotoxicity (ADCC) effector functions. Our results suggest that ADCC may play a role in cross-protective immunity against influenza. Vaccines optimized to stimulate cross-reactive antibodies with ADCC function may provide an important measure of protection against emerging influenza viruses when NAbs are ineffective.

    Importance Current influenza vaccines are designed to elicit neutralizing antibodies (NAbs). Vaccine-induced NAbs are typically effective, but highly specific for particular virus strains. Consequently, current vaccines are poorly suited for preventing the spread of newly emerging pandemic viruses. We therefore evaluated a vaccine strategy designed to induce both antibody and T cell responses, which may provide more broadly cross-protective immunity against influenza. Here we show, in a translational primate model, that vaccination with a modified vaccinia Ankara (MVA) encoding hemagglutinin from a "heterosubtypic" H5N1 virus was associated with reduced shedding of a pandemic H1N1 virus challenge, while vaccination with MVA encoding nucleoprotein, an internal viral protein, was not. Unexpectedly, this reduced shedding was associated with non-neutralizing antibodies that bound H1 hemagglutinin and activated natural killer cells. Antibody-dependent cellular cytotoxicity (ADCC) may therefore play a role in cross-protective immunity to influenza. Vaccines that stimulate "ADCC antibodies" may enhance protection against pandemic influenza.

  • A bivalent vaccine based on a replication-incompetent influenza virus protects against Streptococcus pneumoniae and influenza virus infection [PublishAheadOfPrint]

  • Streptococcus pneumoniae (S. pneumoniae) is a major causative pathogen in community-acquired pneumonia; together with influenza virus, it represents an important public health burden. Although vaccination is the most effective prophylaxis against these infectious agents, no single vaccine simultaneously provides protective immunity against both S. pneumoniae and influenza virus. Previously, we demonstrated that several replication-incompetent influenza viruses efficiently elicit IgG in serum and IgA in the upper and lower respiratory tracts. Here, we generated a replication-incompetent hemagglutinin-knockout (HA-KO) influenza virus possessing the sequence for the antigenic region of pneumococcal surface protein A (PspA). Although this virus (HA-KO/PspA virus) could replicate only in an HA-expressing cell line, it infected wild-type cells and expressed both viral proteins and PspA. PspA- and influenza virus-specific antibodies were detected in nasal wash, bronchoalveolar lavage, and serum from mice intranasally inoculated with HA-KO/PspA virus, and mice inoculated with HA-KO/PspA virus were completely protected from lethal challenge with either S. pneumoniae or influenza virus. Further, bacterial colonization of the nasopharynx was prevented in mice immunized with HA-KO/PspA virus. These results indicate that HA-KO/PspA virus is a promising bivalent vaccine candidate that simultaneously confers protective immunity against both S. pneumoniae and influenza virus. We believe that this strategy offers a platform for the development of bivalent vaccines, based on replication-incompetent influenza virus, against pathogens that cause respiratory infectious diseases.

    Importance Streptococcus pneumoniae and influenza viruses cause contagious diseases, but no single vaccine can simultaneously provide protective immunity against both pathogens. Here, we used reverse genetics to generate a replication-incompetent influenza virus carrying the sequence for the antigenic region of pneumococcal surface protein A and demonstrated that mice immunized with this virus were completely protected from lethal doses of infection with either influenza virus or Streptococcus pneumoniae. We believe that this strategy, which is based on a replication-incompetent influenza virus possessing the antigenic region of other respiratory pathogens, offers a platform of for the development of bivalent vaccines.

  • Viruses and RNA Interference: Issues and Controversies [PublishAheadOfPrint]

  • The question of whether any mammalian cells are able to mount an effective RNA interference-mediated antiviral innate immune response has remained highly controversial. In this Gem, I review recent data addressing this important issue and propose a testable hypothesis that can explain many of the apparently contradictory results published in this area of research.

  • Mutations to PB2 and NP proteins of an avian influenza virus combine to confer efficient growth in primary human respiratory cells [PublishAheadOfPrint]

  • Influenza pandemics occur when influenza A viruses (IAV) adapted to other host species enter humans and spread through the population. Pandemics are relatively rare due to host restriction of IAV: strains adapted to non-human species do not readily infect, replicate in or transmit among humans. IAV can overcome host restriction through reassortment or adaptive evolution, and these are mechanisms by which pandemic strains arise in nature. To identify mutations that facilitate growth of avian IAV in humans, we have adapted influenza A/duck/Alberta/35/1976 (H1N1) [dk/AB/76] virus to a high growth phenotype in differentiated human tracheo-bronchial epithelial (HTBE) cells. Following ten serial passages of three independent lineages, the bulk populations showed similar growth in HTBE cells to that of a human seasonal virus. The coding changes present in six clonal isolates were determined. The majority of changes were located in the polymerase complex and nucleoprotein (NP) and all isolates carried mutations in the PB2 627 domain and regions of NP thought to interact with PB2. Using reverse genetics, the impact on growth and polymerase activity of individual and paired mutations in PB2 and NP was evaluated. The results indicate that coupling of the mammalian-adaptive mutations, PB2 E627K or Q591K, to selected mutations in NP further augments the growth of the corresponding viruses. In addition, minimal combinations of three (PB2 Q236H, E627K and NP N309K), or two (PB2 Q591K and NP S50G), mutations were sufficient to recapitulate the efficient growth in HTBE cells of dk/AB/76 viruses isolated after ten passages in this substrate.

    Importance Influenza A viruses adapted to birds do not typically grow well in humans. However, as has been seen recently with H5N1 and H7N9 subtype viruses, productive and virulent infection of humans with avian influenza viruses can occur. The ability of avian influenza viruses to adapt to new host species is a consequence of their high mutation rate that supports their zoonotic potential. Understanding of the adaptation of avian viruses to mammals strengthens public health efforts aimed at controlling influenza. In particular, it is critical to know how readily and through mutation to which functional components avian influenza viruses gain the ability to grow efficiently in humans. Our data show that as few as three mutations, in the PB2 and NP proteins, support robust growth of a low pathogenic, H1N1 duck isolate in primary human respiratory cells.

  • Interaction of Adenovirus type 5 E4orf4 with the nuclear pore subunit Nup205 is required for proper viral gene expression. [PublishAheadOfPrint]

  • Adenovirus type 5 E4orf4 is a multifunctional protein that regulates viral gene expression. The activities of E4orf4 are mainly mediated through binding to protein phosphatase 2A (PP2A). E4orf4 recruits target phosphoproteins into complexes with PP2A resulting in dephosphorylation of host factors, such as SR splicing factors. In the current study, we utilized immunoprecipitation followed by mass spectrometry to identify novel E4orf4 interacting proteins. In this manner we identified Nup205, a component of the nuclear pore complex (NPC) as an E4orf4 interacting partner. The Arginine Rich Motif (ARM) of E4orf4 was required for interaction with Nup205 and for nuclear localization of E4orf4. ARMs are commonly found on viral nuclear proteins and we observed that Nup205 interacts with three different nuclear viral proteins containing ARMs. E4orf4 formed a trimolecular complex containing both Nup205 and PP2A. Furthermore, Nup205 complexed with E4orf4 was hypophosphorylated suggesting the protein is specifically targeted for dephosphorylation. An adenovirus mutant that does not express E4orf4 (Orf4-) displayed elevated early and reduced late gene expression relative to wild-type. We observed that knockdown of Nup205 resulted in the same phenotype as the Orf4- virus suggesting that the proteins function as a complex to regulate viral gene expression. Furthermore, knockdown of Nup205 resulted in a more than a four-fold reduction in the replication of wild-type adenovirus. Our data show for first time that Ad5 E4orf4 interacts with and modifies the NPC and that Nup205-E4orf4 binding is required for normal regulation of viral gene expression and viral replication.

    Importance Nuclear pore complexes (NPCs) are highly regulated conduits in the nuclear membrane that control transport of macromolecules between the nucleus and cytoplasm. Viruses that replicate in the nucleus must negotiate the NPC during nuclear entry, and viral DNA, mRNA, and proteins must then be exported from the nucleus. Several types of viruses restructure the NPC to facilitate replication and the current study shows that adenovirus type 5 (Ad5) utilizes a novel mechanism to modify NPC function. We demonstrate that a subunit of the NPC, Nup205, is a phosphoprotein that is actively dephosphorylated by the Ad5 encoded protein E4orf4. Moreover, Nup205 is required by Ad5 to regulate viral gene expression and efficient viral replication. Nup205 is a non-structural subunit that is responsible for the gating functions of the NPC and this study suggests for the first time that the NPC is regulated by phosphorylation both during normal physiology and viral infection.

  • Severity of clinical disease and pathology in ferrets experimentally infected with influenza viruses is influenced by inoculum volume [PublishAheadOfPrint]

  • Ferrets are a valuable model for influenza pathogenesis, virus transmission and antiviral therapy studies. However, the contributions of volume of inoculum administered and the ferret's respiratory tract anatomy to disease outcome have not been explored. We noted variation in clinical disease outcomes and the volume of inoculum administered and investigated these differences by administering two influenza viruses (A/California/07/2009 (H1N1pdm) and A/Minnesota/11/2010 (H3N2v) to ferrets intranasally at a dose of 106 TCID50 in a range of inoculum volumes (0.2, 0.5 or 1.0ml), and followed viral replication, clinical disease and pathology over 6 days. Clinical illness and respiratory tract pathology were most severe and most consistent when the viruses were administered in a volume of 1.0 ml. Using a modified micro-CT imaging method and examining gross specimens, we found that the right main stem bronchus was consistently larger in diameter than the left main stem bronchus though the latter was longer and straighter. These anatomic features likely influence the distribution of inoculum in the lower respiratory tract. A 1.0 ml volume of inoculum is optimal for delivery of virus to the lower respiratory tract of ferrets, particularly when evaluation of clinical disease is desired. Furthermore, we highlight important anatomical features of the ferret lung that influence the kinetics of viral replication, clinical disease severity and lung pathology.

    IMPORTANCE Ferrets are a valuable model for influenza pathogenesis, virus transmission and antiviral therapy studies. Clinical disease in ferrets is an important parameter in evaluating the virulence of novel influenza viruses and findings are extrapolated to virulence in humans. Therefore, the accuracy and reproducibility of data between laboratories is highly desirable. We have found that, even when the same virus was administered at similar doses, a range of clinical disease outcomes were reported by different investigators, from asymptomatic infection to severe weight loss, ocular and nasal discharge, sneezing and lethargy. We found that a wide range of inoculum volumes were used to experimentally infect ferrets and we sought to determine whether the variations in disease outcome were the result of the volume of inoculum administered. These data highlight some less explored features of the model, methods of experimental infection, and clinical disease outcomes in a research setting.

  • Two Novel Simian Arteriviruses in Captive and Wild Baboons (Papio spp.) [PublishAheadOfPrint]

  • Since the 1960s, simian hemorrhagic fever virus (SHFV; Nidovirales; Arteriviridae) has caused highly fatal outbreaks of viral hemorrhagic fever in captive Asian macaque colonies. However, the source(s) of these outbreaks and the natural reservoir(s) of this virus remain obscure. Here we report the identification of two novel, highly divergent simian arteriviruses related to SHFV - Mikumi yellow baboon virus 1 (MYBV-1) and Southwest baboon virus 1 (SWBV-1) - in wild and captive baboons, respectively, and demonstrate recent transmission of SWBV-1 among captive baboons. These findings extend our knowledge of the genetic and geographic diversity of the simian arteriviruses, identify baboons as a natural host of these viruses, and provide further evidence that baboons may have played a role in previous outbreaks of simian hemorrhagic fever in macaques, as has long been suspected. This knowledge should aid in the prevention of disease outbreaks in captive macaques and supports the growing body of evidence that suggests simian arterivirus infections are common in Old World monkeys of many different species throughout Africa.

    IMPORTANCE Historically, the emergence of primate viruses - both in humans and in other primate species - has caused devastating outbreaks of disease. One strategy for preventing the emergence of novel primate pathogens is to identify microbes with the potential for cross-species transmission in their natural state, within reservoir species from which they might emerge. Here, we detail the discovery and characterization of two related simian members of the Arteriviridae family; viruses with a history of disease emergence and host-switching. Our results expand the phylogenetic and geographic range of the simian arteriviruses and define baboons as a natural host for these viruses. Our findings also identify a potential threat to captive macaque colonies by showing that simian arteriviruses are actively circulating in captive baboons.

  • Chemical Induction of Unfolded Protein Response Enhances Cancer Cell Killing through Lytic Virus Infection [PublishAheadOfPrint]

  • Cancer cells are susceptible to oncolytic viruses, albeit variably. Human adenoviruses (HAdVs) are widely used oncolytic agents, engineered to produce progeny within the tumor, and elicit bystander effects. We searched for host factors enhancing bystander effects, and conducted a targeted RNA-interference screen against guanine-nucleotide exchange factors (GEFs) of small GTPases. We show that unfolded protein response (UPR), which is readily inducible in aggressive tumor cells, enhances melanoma or epithelial cancer cell killing upon HAdV infection. UPR was triggered by knock-down of Golgi Brefeldin-A resistant guanine-nucleotide-exchange factor-1 (GBF-1), or the GBF-1 inhibitor Golgicide A (GCA), and stimulated HAdV infection. GBF-1 is a GEF for ADP-ribosylation factors (Arfs) regulating ER to Golgi and intra-Golgi transport. Cells treated with GCA enhanced HAdV-induced cytopathic effects in epithelial and melanoma cancer but not normal cells, if the drug was applied several hours prior to HAdV inoculation. This was shown by real-time label-free impedance measurements using xCELLigencettrade;. GCA-treated cells contained fewer incoming HAdV than control cells, but boosted HAdV titers and spreading in cancer cells. GCA enhanced viral gene expression, or transgene expression from the cytomegalovirus promoter of B- or C-species HAdVs, but did not enhance viral E1A expression in uninfected cell lines, or cells transfected with plasmid reporter DNA. The UPR-enhanced cell killing required the nuclease activity of the UPR-sensor inositol-requiring enzyme 1 (IRE-1), and X-box binding protein 1 (XBP-1), which alleviate ER stress. The collective results show that chemical UPR induction and viruses boost tumor cell killing by enhancing oncolytic viral efficacy.

    IMPORTANCE Cancer is difficult to combat. A wide range of oncolytic viruses show promise for killing cancer cells. Yet, the efficacy of oncolytic killing is low. We searched for host factors enhancing adenovirus cancer cell killing, and found that the knock-down of GBF-1 (Golgi Brefeldin-A resistant guanine-nucleotide-exchange factor-1) or chemical inhibition of GBF-1 enhanced adenovirus infection by triggering the IRE- 1/XBP-1 branch of the unfolded protein response (UPR). IRE-1/XBP-1 promote cell survival, and enhanced the levels of the adenoviral immediate early gene product E1A, virus spreading, and killing of cancer cells. Aggressive tumor cells depend on a readily inducible UPR, and hence present prime targets for a combined strategy involving adenoviruses and small chemicals inducing UPR.

  • On the laws of virus Spread through cell populations [PublishAheadOfPrint]

  • The dynamics of viral infections have been investigated extensively, often with a combination of experimental and mathematical approaches. Mathematical descriptions of virus spread through cell populations are well-established in the literature, and have yielded important insights. Yet, the formulation of certain fundamental aspects of virus dynamics models remains uncertain and untested. Here, we investigate the process of infection, and in particular, the effect of varying the target cell population size on the number of productively infected cells generated. Using an in vitro single-round HIV-1 infection system, we find that the established modeling framework cannot accurately fit the data. If the model is fit to data with the lowest number of cells and is used to predict data generated with larger cell populations, the model significantly overestimates the number of productively infected cells generated. Interestingly, this deviation becomes stronger under experimental conditions that promote mixing of cells and viruses. The reason for the deviation is that the standard model makes certain over-simplifying assumptions about the fate of viruses that fail to find a cell in their immediate proximity. We derive a different model from stochastic processes that assumes simultaneous access of the virus to multiple target cells. In this scenario, if no cell is available to the virus at its location, it has a chance to interact with other cells, a process that can be promoted by mixing of the populations. This model can accurately fit the experimental data, and suggests a new interpretation of mass-action in virus dynamics models.

    Importance: Understanding the principles of virus growth through cell populations is of fundamental importance to virology. It helps us make informed decisions about intervention strategies aimed at preventing virus growth, such as drug treatment or vaccination approaches e.g. in HIV infection. Yet, considerable uncertainty remains in this respect. An important variable in this context is the number of susceptible cells available for virus replication. How does the number of susceptible cells influence the growth potential of the virus? Besides the importance of such information for clinical responses, a thorough understanding of this is also important for the prediction of virus levels in patients and the estimation of crucial patient parameters through the use of mathematical models. This paper investigates the relationship between target cell availability and the virus growth potential with a combination of experimental and mathematical approaches, and provides significant new insights.

  • Human coronavirus NL63 utilize heparan sulfate proteoglycans for attachment to target cells [PublishAheadOfPrint]

  • Human coronavirus NL63 (HCoV-NL63) is an alphacoronavirus that was first identified in 2004 in the nasopharyngeal aspirate from a 7-month-old patient with a respiratory tract infection. Previous studies showed that HCoV-NL63 and the genetically distant SARS-CoV employ the same receptor for host cell entry, angiotensin converting enzyme 2 (ACE2), but it is largely unclear whether ACE2 interactions are sufficient to allow HCoV-NL63 binding to cells. The present study showed that directed expression of angiotensin-converting enzyme 2 (ACE2) on cells previously resistant to HCoV-NL63 renders them susceptible, showing that ACE2 protein acts as a functional receptor and its expression is required for infection. However, comparative analysis showed that directed expression or selective scission of the ACE2 protein had no measurable effect on virus adhesion. In contrast, binding of HCoV-NL63 to heparan sulfates was required for viral attachment and infection of target cells, showing that these molecules serve as attachment receptors for HCoV-NL63.

    Importance ACE2 protein has been proposed as a receptor for HCoV-NL63 already in 2005, but the in-depth analysis of early events during virus infection was not performed thus far. Here, we show that the ACE2 protein is required for viral entry, but it is not the primary binding site on the cell surface. Conducted research showed that heparan sulfate proteoglycans function as adhesion molecules, increasing the virus density on cell surface and possibly facilitating interaction between HCoV-NL63 and its receptor. Obtained results show that the initial events during HCoV-NL63 infection are more complex than anticipated and newly described interaction may be essential for understanding the infection process and, possibly, also assist in the drug design.

  • Characterization of SIV that Induces SIV Encephalitis in Rhesus Macaques with High Frequency: Role of TRIM5 and MHC Genotypes and Early Entry to the Brain [PublishAheadOfPrint]

  • Although non-human primate models of neuroAIDS have made tremendous contributions to our understanding of disease progression in the central nervous system (CNS) of HIV-1 infected individuals, each model holds advantages and limitations. In this study, in vivo passage of SIVsmE543 was conducted to obtain a viral isolate that can induce neuropathology in rhesus macaques. After a series of four in vivo passages in rhesus macaques, we have successfully isolated SIVsm804E. SIVsm804E shows efficient replication in PBMCs and MDMs in vitro and induces neuroAIDS in high frequencies in vivo. Analysis of the acute phase of infection revealed that SIVsm804E establishes infection in the CNS during the early phase of the infection, which was not observed in the animals infected with the parental SIVsmE543-3. Comprehensive analysis of disease progression in the animals used in the study suggested that host MHC-I and TRIM5aalpha; genotypes influence the disease progression in the CNS. Taken together, we have successfully isolated a new strain of SIV that is capable of establishing infection in the CNS at early stage of infection and causes neuropathology in infected rhesus macaques at a high frequency (83%) using a single inoculum, when animals with restrictive MHC-I or TRIM5aalpha; genotypes are excluded. SIVsm804E has the potential to augment some of the limitations of existing non-human primate neuroAIDS models.

    Importance Human immunodeficiency virus (HIV) is associated with a high frequency of neurologic complications due to infection of the central nervous system (CNS). Although the use of antiviral treatment has reduced the incidence of severe complications, milder disease of the CNS continues to be a significant problem. Animal models to study development of neurologic disease are needed. This manuscript describes the development of a novel virus isolate that induces neurologic disease in a high proportion of rhesus macaques infected without the need for prior immune modulation as is required for some other models.

  • MAPK-interacting kinase regulates mTOR/AKT signaling and controls SRPK-responsive type 1 IRES-mediated translation and viral oncolysis [PublishAheadOfPrint]

  • Translation machinery is a major recipient of principal mitogenic signaling networks involving Raf-ERK1/2 and PI3K-mTOR. Picornavirus internal ribosomal entry site (IRES)-mediated translation and cytopathogenic effects are susceptible to the status of such signaling cascades in host cells. We determined that tumor-specific cytotoxicity of the polio-/rhinovirus chimera PVSRIPO is facilitated by Raf-ERK1/2 signals to the mitogen activated protein kinase (MAPK)-interacting kinase, MNK, and its effects on partitioning/activity of the Ser-Arg rich protein kinase, SRPK [Brown et al. (2014) Induction of viral, m7G-cap-independent translation and oncolysis by MAPK interacting kinase (MNK)-mediated effects on the Ser-Arg rich protein kinase, SRPK. J Virol, in press]. Here we show that MNK regulates SRPK via the mechanistic target of rapamycin (mTOR) and AKT. Our investigations revealed a MNK-controlled mechanism acting on mTORC2-AKT. The resulting suppression of AKT signaling attenuates SRPK activity to enhance picornavirus type 1 IRES translation and favor PVSRIPO tumor cell toxicity and killing.

    Importance. Oncolytic immunotherapy with PVSRIPO, the type-1 live attenuated poliovirus (PV) (Sabin) vaccine containing a human rhinovirus type 2 (HRV2) IRES, is demonstrating early promise in clinical trials with intratumoral infusion in recurrent glioblastoma (GBM). Our investigations demonstrate that the core mechanistic principle of PVSRIPO, tumor selective translation and cytotoxicity, relies on constitutive ERK1/2-MNK signals that counteract the deleterious effects of runaway AKT-SRPK activity in malignancy.

  • Induction of viral, m7G-cap-independent translation and oncolysis by MAPK interacting kinase (MNK)-mediated effects on the Ser-Arg rich protein kinase, SRPK [PublishAheadOfPrint]

  • Protein synthesis, the most energy-consuming process in cells, responds to changing physiologic priorities, e.g. upon mitogen- or stress-induced adaptations signaled through the mitogen-activated protein kinases (MAPKs). The prevailing status of protein synthesis machinery is a viral pathogenesis factor, particularly for +strand RNA viruses, where immediate translation of incoming viral RNAs shapes host:virus interactions. In this study, we unraveled signaling pathways centered on the ERK1/2 and p38aalpha; MAPK-interacting kinases, MNK1/2, and their role in controlling 7-methyl-guanosine (m7G) llsquo;cap'-independent translation at enterovirus type 1 internal ribosomal entry sites (IRESs). Activation of Raf-MEK-ERK1/2 signals induced viral IRES-mediated translation in a manner dependent on MNK1/2. This effect was not due to MNK's known functions as eukaryotic translation initiation factor (eIF) 4G binding partner or eIF4E(S209) kinase. Rather, MNK catalytic activity enabled viral IRES-mediated translation/host cell cytotoxicity through negative regulation of the Ser-Arg (SR)-rich protein kinase (SRPK). Our investigations suggest that SRPK activity is a major determinant of type 1 IRES competency, host cell cytotoxicity and viral proliferation in infected cells.

    Importance. We are targeting unfettered enterovirus IRES activity in cancer with PVSRIPO, the type-1 live attenuated poliovirus (PV) (Sabin) vaccine containing a human rhinovirus type 2 (HRV2) IRES. A Phase-I clinical trial of PVSRIPO with intratumoral inoculation in patients with recurrent glioblastoma (GBM) is showing early promise. Viral translation proficiency in infected GBM cells is a core requirement for the anti-neoplastic efficacy of PVSRIPO. Therefore, it is critically important to understand the mechanisms controlling viral, cap-independent translation in infected host cells.

  • Molecular characterization of a novel temperate Sinorhizobium bacteriophage {Phi}LM21, encoding DNA methyltransferase with CcrM-like specificity [PublishAheadOfPrint]

  • LM21 is a temperate phage isolated from Sinorhizobium sp. LM21 (Alphaproteobacteria). Genomic analysis and electron microscopy suggested that LM21 is a member of the family Siphoviridae. The phage has an isometric head and a long non-contractile tail. The genome of LM21 has 50,827 bp of linear double-stranded DNA encoding 72 putative proteins, including proteins responsible for the assembly of the phage particles, DNA packaging, transcription, replication and lysis. Virion proteins were characterized using mass spectrometry, leading to the identification of the major capsid and tail components, tape measure and a putative portal protein. We have confirmed the activity of two gene products, lytic enzyme nndash; a putative chitinase and a DNA methyltransferase, sharing sequence specificity with the cell cycle-regulating methyltransferase (CcrM) of the bacterial host. Interestingly, the genome of Sinorhizobium phage LM21 shows very limited similarity to other known phage genome sequences, and is thus considered unique.

    IMPORTANCE Prophages are known to play an important role in the genomic diversification of bacteria via horizontal gene transfer. The influence of prophages on patogenic bacteria is very well documented. However, our knowledge of the overall impact of prophages on the survival of their lysogenic, nonpathogenic bacterial hosts is still limited. In particular, information on prophages of the agronomically-important Sinorhizobium species, is scarce. In this study we describe the isolation and molecular characterization of a novel temperate bacteriophage LM21 of Sinorhizobium sp. LM21. Since we have not found any similar sequences, we propose that this bacteriophage is a novel species. We conducted a functional analysis of selected proteins. We have demonstrated that the phage DNA methyltransferase has the same sequence specificity as the cell cycle-regulating methyltransferase CcrM of its host. We also pointed out that this phenomenon of mimicking the host regulatory mechanisms by viruses is quite common in bacteriophages.

  • Increased replicative fitness of a dengue virus 2 clade in native mosquitoes: Potential contribution to a clade replacement event in Nicaragua [PublishAheadOfPrint]

  • The four dengue virus serotypes (DENV-1 to -4) are transmitted by Aedes aegypti and Ae. albopictus mosquitoes, causing up to 390 million DENV infections worldwide each year. We previously reported a clade replacement of the DENV-2 Asian-American genotype NI-1 clade by the NI-2B clade in Managua, Nicaragua. Here, we describe our studies of the replicative ability of NI-1 and NI-2B viruses in an Ae. aegypti cell line (Aag2) and Ae. aegypti mosquitoes reared from eggs collected in Managua. In co-infection experiments, several different pairs of NI-I and NI-2B clinical isolates were used to infect Aag2 cells or blood-fed Ae. aegypti mosquitoes; results consistently showed a significant replicative advantage of NI-2B over NI-1 viruses early after infection, and in mosquitoes NI-2B viruses attained a higher Replicative Index than NI-1 isolates on 3-7 days post-infection (dpi). At 7 dpi, NI-2B viruses displayed a significantly higher Replicative Index in legs and salivary glands; however, this advantage was lost by 14 and 21 dpi. We also found that the percent of mosquitoes in which NI-2B viruses was dominant was significantly higher than NI-1 viruses on day 7 but not at later time-points. Taken together, these data demonstrate that clade NI-2B holds a replicative advantage over clade NI-1 early in infection that wanes at later time-points. This early fitness advantage of NI-2B over NI-I viruses in the native vector, Ae. aegypti, suggests a shorter extrinsic incubation period for NI-2B viruses, which could have contributed to the clade replacement event in Managua.

    IMPORTANCE Dengue virus (DENV), one of the most medically important arthropod-borne viruses, is transmitted to humans by Aedes aegypti and Ae. albopictus mosquitoes in tropical and sub-tropical regions worldwide. Dengue epidemics continue to increase in frequency, geographic range, and severity and are a major public health concern. This is due to globalization, unplanned urbanization, and climate change, as well as host genetics and immune responses and viral genetic changes. DENV consists of four serotypes, in turn composed of genotypes and genetically distinct clades. What drives the frequent replacement of a previously-circulating DENV clade by another is unclear. Here, we investigate the replicative fitness of two clades of DENV serotype 2 in Aedes aegypti cells and mosquitoes collected from the region where the viruses circulated and conclude that increased replicative fitness could have contributed to a DENV clade replacement event in Nicaragua. These findings provide insight into vector-driven evolution of DENV epidemics.

  • Interactome analysis of the influenza A virus transcription/replication machinery identifies protein phosphatase 6 as a cellular factor required for efficient virus replication [PublishAheadOfPrint]

  • The negative-sense RNA genome of influenza A virus is transcribed and replicated by the viral RNA-dependent RNA polymerase (RdRP). The viral RdRP is an important host range determinant, indicating that its function is affected by interactions with cellular factors. However, the identities and the roles of most of these factors remain unknown. Here, we employed affinity-purification followed by mass spectrometry to identify cellular proteins that interact with the influenza A virus RdRP in infected human cells. We purified RdRPs using a recombinant influenza virus in which the PB2 subunit of the RdRP is fused to a Strep-tag. When this tagged subunit was purified from infected cells, co-purifying proteins included the other RdRP subunits, PB1 and PA, the viral nucleoprotein and neuraminidase, as well as 171 cellular proteins. Label-free quantitative mass spectrometry revealed that the most abundant of these host proteins were chaperones, cytoskeletal proteins, importins, proteins involved in ubiquitination, kinases and phosphatases, mitochondrial and ribosomal proteins. Among the phosphatases, we identified three subunits of the cellular serine/threonine-protein phosphatase 6 (PP6), including the catalytic subunit PPP6C and regulatory subunits PPP6R1 and PPP6R3. PP6 was found to interact directly with the PB1 and PB2 subunits of the viral RdRP, and siRNA-mediated knockdown of the catalytic subunit of PP6 in infected cells resulted in the reduction of viral RNA accumulation and the attenuation of virus growth. These results suggest that PP6 interacts with and positively regulates the activity of the influenza virus RdRP.

    IMPORTANCE Influenza A viruses are serious clinical and veterinary pathogens, causing substantial health and economic impacts. In addition to annual seasonal epidemics, occasional global pandemics occur when viral strains adapt to humans from other species. To replicate efficiently and cause disease, influenza viruses must interact with a large number of host factors. The reliance of the viral RNA-dependent RNA polymerase (RdRP) on host factors makes it a major host range determinant. This study describes and quantifies host proteins that interact, directly or indirectly, with a subunit of the RdRP. It increases our understanding of the role of host proteins in viral replication and identifies a large number of potential barriers to pandemic emergence. Identifying host factors allows their importance for viral replication to be tested. Here, we demonstrate a role for the cellular phosphatase PP6 in promoting viral replication, contributing to our emerging knowledge of regulatory phosphorylation in influenza virus biology.

  • Activation of NF-{kappa}B by the Kaposi's sarcoma herpesvirus K15 protein involves recruitment of the NF-{kappa}B-inducing kinase, I{kappa}B kinases and phosphorylation of p65 [PublishAheadOfPrint]

  • Kaposi's sarcoma herpesvirus (KSHV) (or human herpesvirus 8) is the cause of Kaposi's sarcoma, primary effusion lymphoma (PEL), and the plasma cell variant of multicentric Castleman's disease (MCD). The transmembrane K15 protein, encoded by KSHV, has been shown to activate NF-B, the mitogen-activated protein kinases (MAPKs) c-jun-N-terminal kinase (JNK) and extracellular signal-regulated kinase (Erk), as well as phospholipase C gamma (PLC) and to contribute to KSHV-induced angiogenesis. Here we investigate how the K15 protein activates the NF-B pathway. We show that activation of NF-B involves the recruitment of NF-B-inducing kinase (NIK) and IKK aalpha;/bbeta; to result in the phosphorylation of p65/RelA on Ser536. A K15 mutant devoid in NIK/IKK recruitment fails to activate NF-B, but remains proficient in the stimulation of both NFAT- and AP1-dependent promoters, showing that the structural integrity of the mutant K15 protein has not been altered dramatically. Direct recruitment of NIK represents a novel way for a viral protein to activate and manipulate the NF-B pathway.

    Importance statement KSHV K15 is a viral protein involved in the activation of pro-inflammatory and angiogenic pathways. Previous studies reported that K15 can activate the NF-B pathway. Here we show the molecular mechanism underlying the activation of this signaling pathway by K15, which involves direct recruitment of the NF-B-inducing kinase NIK to K15 as well as NIK-mediated NF-B p65 phosphorylation on Ser536. K15 is the first viral protein shown to activate NF-B through direct recruitment of NIK. These results indicate a new mechanism whereby a viral protein can manipulate the NF-B pathway.

  • Identification of a high efficiency baculovirus DNA replication origin that functions in insect and mammalian cells [PublishAheadOfPrint]

  • The p143 gene from Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) have been found to increase the expression of luciferase, which is driven by the polyhedrin promoter, in a plasmid with virus co-infection. Further study indicated that this is due to the presence of a replication origin (ori) in the coding region of this gene. Transient DNA replication assays showed that a specific fragment of p143 coding sequence, p143-3, underwent a virus-dependent DNA replication in Sf-21 cells. Deletion analysis of the p143-3 fragment showed that sub-fragment p143-3.2a contained the essential sequence of this putative ori. Sequence analysis of this region revealed a unique distribution of imperfect palindromes with high AT-content. No sequence homology or similarity between p143-3.2a and any other known oris were detected, suggesting that it is a novel baculovirus ori. Further study showed that p143-3.2a ori can replicate more efficiently in the infected Sf-21 cells than baculovirus homologous regions (hrs), the major baculovirus ori, or non-hr oris during virus replication. Previously, hr on its own was unable to replicate in mammalian cells, and for mammalian viral oris, viral proteins are generally required for their proper replication in host cells. However, p143-3.2a ori was surprisingly found to function as an efficient ori in mammalian cells without a need of any viral proteins. We conclude that p143 contains a unique sequence that can function as an ori to enhance gene expression in not only insect but also mammalian cells.

    IMPORTANCE Baculovirus DNA replication relies on both hr and non-hr oris, however, so far very little is known on the latter oris. Here we have identified a new non-hr ori, the p143 ori, which resides in the coding region of p143. By developing a novel DNA replication-enhanced reporter system we have identified and located the core region required for p143 ori. This ori contains large number of imperfect inverted repeats and is the most active ori in the viral genome during virus infection in insect cells. We also found that it is a unique ori that can replicate in mammalian cells without the assistance of baculovirus gene products. The identification of this ori should contribute to a better understanding of baculovirus DNA replication. Also, this ori is very useful in assisting gene expression in mammalian cells.

  • A histidine residue of the influenza virus hemagglutinin controls the pH dependence of the conformational change mediating membrane fusion [PublishAheadOfPrint]

  • The conformational change of the influenza virus hemagglutinin (HA) protein mediating the fusion between the virus envelope and the endosomal membrane was hypothesized to be induced by protonation of specific histidine residues since their pKa matches the pH of late endosomes (pKa~6.0). However, such critical key histidine residues remain to be identified. We investigated the highly conserved His184 at the HA1-HA1 interface and His110 at the HA1-HA2 interface of highly pathogenic H5N1 HA as potential pH sensors. By replacing both histidines by different amino acids and analyzing the effect of these mutations on conformational change and fusion we found that His184, but not His110, plays an essential role for the pH dependence of conformational change of HA. Computational modeling of the protonated His184 revealed that His184 is central of a conserved interaction network possibly regulating the pH dependence of conformational change via its pKa. As the propensity of histidine to get protonated largely depends on its local environment, mutation of residues in vicinity of histidine may affect its pKa. HA of highly pathogenic H5N1 viruses carries a Glu-to-Arg mutation at position 216 close to His184. By mutation of residue 216 in the highly as well as the low pathogenic H5 HA we observed a significant influence on the pH dependence of conformational change and fusion. These results are in support of a pKa modulating effect by neighboring residues.

    Importance The main pathogenic determinant of influenza viruses, the hemagglutinin (HA) protein, triggers a key step of the infection process: the fusion of the virus envelope with the endosomal membrane releasing the viral genome. Whereas essential aspects of the fusion inducing mechanism of HA at low pH are well understood, the molecular trigger of the pH dependent conformational change inducing fusion has been unclear. We provide evidence that His184 regulates the pH dependence of the HA conformational change via its pKa. Mutations of neighboring residues which may affect the pKa of His184 could play an important role of virus adaptation to a specific host. We suggest that mutation of neighboring residue 216, which is present in all highly pathogenic phenotypes of H5N1 influenza strains, contributed to the adaptation of these viruses to the human host via its effect on the pKa of His184.

  • Recent Population Expansions of Hepatitis B Virus in the United States [PublishAheadOfPrint]

  • The recent epidemic history of hepatitis B virus (HBV) infections in the United States is complex, as indicated by current disparity in HBV genotype distribution between acute and chronic hepatitis B cases and rapid decline in hepatitis B incidence since the 1990s. We report temporal changes in genetic composition of the HBV population using whole-genome sequences (n=179) from acute hepatitis B cases (n=1206) identified through the Sentinel County Surveillance for Acute Hepatitis (1998-2006). HBV belonged mainly to subtypes A2 (75%) and D3 (18%), with times of their most recent common ancestors being, respectively, 1979 and 1987, respectively. A2 underwent rapid population expansions in ca. 1995 and ca. 2002, coinciding with transient rises in acute hepatitis B notification rates among adults; D3 underwent expansion in ca. 1998. A2 strains from cases identified after 2002, compared to those before 2002, tended to cluster phylogenetically, indicating selective expansion of specific strains, and were significantly reduced in genetic diversity (p = 0.001) and frequency of drug-resistance mutations (p = 0.001). The expansion of genetically close HBV A2 strains was associated with risk of infection among male homosexuals (p = 0.03). Incident HBV strains circulating in the US were recent in origin, and restricted in genetic diversity. Disparate transmission dynamics among phylogenetic lineages affected the genetic composition of HBV populations and their capacity to maintain drug-resistance mutations. The tendency of selectively expanding HBV strains to be transmitted among male homosexuals highlights the need to improve hepatitis B vaccination coverage among at-risk adults.

    IMPORTANCE Hepatitis B virus (HBV) remains an important cause of acute and chronic liver disease globally, and in the United States. Genetic analysis of HBV whole genomes from cases of acute hepatitis B identified from 1998-2006 in the United States showed dominance of genotype A2 (75%), followed by D3 (18%). Strains of both subtypes were recent in origin and underwent rapid population expansions from 1995-2000, indicating increase in transmission rate for certain HBV strains during a period of decline in the reported incidence of acute hepatitis B in the US. HBV A2 strains from a particular cluster that experienced the most recent population expansion were more commonly detected among men who have sex with men. Vaccination needs to be stepped up to protect persons who remain at risk of HBV infection.

  • African great apes are naturally infected with roseoloviruses closely related to Human herpesvirus 7 [PublishAheadOfPrint]

  • Primates are naturally infected with herpesviruses. During the last 15 years, the search for homologues of human herpesviruses in non-human primates allowed the identification of numerous viruses belonging to the different herpesvirus subfamilies and genera. No simian homologue of human herpesvirus 7 has been reported to date. To investigate the putative existence of HHV7-like viruses in African great apes, we applied the CODEHOP-mediated PCR strategy to blood DNA samples from the four common chimpanzee subspecies (Pan troglodytes verus, P. t. ellioti, P. t. troglodytes and P. t. schweinfurthii), pygmy chimpanzees (Pan paniscus) as well as lowland gorillas (Gorilla gorilla gorilla). This study led to the discovery of a novel roseolovirus close to HHV7 in each of these non-human primate species and subspecies. Generation of the partial glycoprotein B (1111-bp) and full-length DNA polymerase (3036/3042-bp) gene sequences allowed the deciphering of their evolutionary relationships. Phylogenetic analyses revealed that HHV7 and its African great ape homologues formed well-supported monophyletic lineages whose topological resemblance with the host phylogeny is suggestive of virusmmdash;host co-divergence. Notably, the evolutionary branching points that separate HHV7 from African great ape HV7 are remarkably congruent with the dates of divergence of their hosts. Our study shows that African great apes are hosts of human herpesvirus homologues, including HHV7 homologues, and that the latter, like other DNA viruses that establish persistent infections, have co-speciated with their hosts.

    IMPORTANCE Human herpesviruses are known to possess simian homologues. However, surprisingly, none has been identified to date for human herpesvirus 7 (HHV7). This study is the first to describe simian homologues of HHV7. The extensive search performed on almost all African great ape species and subspecies, i.e., common chimpanzees of the four subspecies, bonobos and lowland gorillas, has allowed characterizing a specific virus in each. Genetic characterization of the partial glycoprotein B and full-length DNA polymerase gene sequences followed by their phylogenetic analysis and estimation of divergence times have shed light on the evolutionary relationships of these viruses. In this respect, we conclusively demonstrate the co-speciation between these new viruses and their hosts and report cases of cross-species transmission between two common chimpanzee subspecies in both directions.

  • A Leucine Residue in C-terminus of Human Parainfluenza Virus Type 3 Matrix Protein Is Essential for Efficient Virus-Like Particle and Virion Release [PublishAheadOfPrint]

  • Paramyxovirus particles, like other enveloped virus particles, are formed by budding from membranes of infected cells, and matrix (M) proteins are critical for this process. To indentify the M protein important for this process, we have characterized the budding of the human parainfluenza virus type 3 (HPIV3) M protein. Our results showed that expression of the HPIV3 M protein alone is sufficient to initiate the release of virus-like particles (VLPs). Electron microscopy analysis confirmed that VLPs are morphologically similar to HPIV3 virions. We identified a leucine (L302) residue within the C-terminus of the HPIV3 M protein that is critical for M protein-mediated VLP production by regulating the ubiquitination of the M protein. When L302 was mutated into L302A, ubiquitination of M protein was defective, the release of VLPs was abolished, and the membrane-binding and budding ability of M protein was greatly weakened, but the ML302A retained oligomerization activity and had a dominant negative effect on M protein-mediate VLP production. Furthermore, treatment with a proteasome inhibitor also inhibited M protein-mediated VLP production and viral budding. Finally, recombinant HPIV3 containing ML302A mutant could not be rescued. These results suggest that L302 acts as a critical regulating signal for the ubiquitination of the HPIV3 M protein and virion release.

    Importance Human parainfluenza virus type 3 (HPIV3) is an enveloped virus with a nonsegmented negative-strand RNA genome. It can cause severe respiratory tract diseases such as bronchiolitis, pneumonia, and croup in infants and young children. However, no valid antiviral therapy or vaccine is currently available. Thus, further elucidation of its assembly and budding will be helpful in the development of novel therapeutic approaches. Here, we show that a leucine residue (L302) located at the C-terminus of the HPIV3 M protein is essential for efficient production of virus-like particles (VLPs). Furthermore, we found L302 regulated M protein mediated VLP production via regulation of M protein ubiquitination. Recombinant HPIV3 containing ML302A mutant is growth-defective. These findings provide new insight into the critical role of M protein-mediated VLP production and virion release of a residue that does not belong to L-domain and may advance our understanding of HPIV3 viral assembly and budding.

  • The highly conserved proline at position 438 in Pseudorabies Virus gH is important for regulation of membrane fusion [PublishAheadOfPrint]

  • Membrane fusion in herpesviruses requires viral glycoproteins (g)B and gH/gL. While gB is considered the actual fusion protein but is non-fusogenic per se, the function of gH/gL remains enigmatic. Crystal structures for different gH homologs are strikingly similar despite only moderate amino acid sequence conservation. A highly conserved sequence motif comprises the residues serine-proline-cysteine corresponding to position 437-439 in Pseudorabies Virus (PrV) gH. The structures show that proline438 induces bending at the end of an alpha-helix, thereby juxtaposing cysteine404 and cysteine439 to allow formation of a strictly conserved disulfide bond. However, PrV vaccine strain Bartha unexpectedly carries a serine at this conserved position. To test the influence of this substitution we constructed different gH-chimeras carrying proline or serine at position 438 in gH either derived from PrV strain Kaplan or Bartha. Mutants expressing gH with serine438 showed reduced fusion activity in transient fusion assays and during infection, with delayed penetration kinetics and a small plaque phenotype which indicates that proline438 is important for efficient fusion. A more drastic effect was observed when disulfide bond formation was completely blocked by mutation of cysteine404 to serine. Although PrV expressing gHC404S was viable, plaque size and penetration kinetics were drastically reduced. Alteration of serine438 to proline in gH of strain Bartha enhanced cell-to cell spread and penetration kinetics but restoration of full activity required additional alteration of aspartic acid to valine at position 59.

    Importance: The role of the gH/gL complex in herpesvirus membrane fusion is still unclear. Structural studies predicted a critical role for proline438 in PrV gH to allow formation of a conserved disulfide bond and correct protein folding. Functional analyses within this study corroborated these structural predictions: mutation of this residue resulted in a drastic impairment of membrane fusion kinetics not only in vitro in transient transfection-fusion assays but also during virus infection. Elimination of formation of the disulfide bond yielded the same phenotype in transient assays but had a more drastic effect on virus replication. Thus, our studies add important information to the structure-function analyses of herpesvirus gH.

  • PROBING THE SITES OF INTERACTIONS OF ROTAVIRAL PROTEINS INVOLVED IN REPLICATION [PublishAheadOfPrint]

  • Replication and packaging of the rotavirus genome occur in cytoplasmic compartments called viroplasms that form during virus infection. These processes are orchestrated by yet to be understood complex networks of interactions involving non-structural proteins (NSPs) 2,5,6 and structural proteins (VPs) 1,2,3,6. The multifunctional enzyme NSP2, an octamer with RNA binding activity, is critical for viroplasm formation with its binding partner NSP5, and for genome replication/packaging through its interactions with replicating RNA, the viral polymerase VP1 and the inner core protein VP2. Using isothermal calorimetry, bio-layer interferometry and peptide-array screening, we examined the interactions between NSP2, VP1, VP2, NSP5 and NSP6. These studies provide the first evidence that NSP2 can directly bind to VP1, VP2, and NSP6 in addition to the previously known binding to NSP5. The interacting sites identified from reciprocal peptide arrays were found to be in close proximity of the RNA template entry and dsRNA exit tunnels of VP1 and near the catalytic cleft and RNA-binding grooves of NSP2; these sites are consistent with the proposed role of NSP2 in facilitating dsRNA synthesis by VP1. Peptide screening of VP2 identified NSP2-binding sites in the regions close to the inter-subunit junctions suggesting that NSP2 binding could be a regulatory mechanism for preventing the premature self-assembly of VP2. The binding sites on NSP2 for NSP6 were found to overlap with that of VP1, and the NSP5 binding sites overlap with those of VP2 and VP1 suggesting that interaction of these proteins with NSP2 is likely spatially and/or temporally regulated.

    IMPORTANCE Replication and packaging of the rotavirus genome occur in cytoplasmic compartments called viroplasms that form during virus infection and are orchestrated by complex networks of interactions involving non-structural proteins (NSPs) and structural proteins (VPs). A multifunctional RNA-binding NSP2 octamer with nucleotidyl phosphatase activity is central to viroplasm formation and RNA replication. Here we provide the first evidence that NSP2 can directly bind to VP1, VP2, and NSP6, in addition to the previously known binding to NSP5. The interacting sites identified from peptide arrays are consistent with the proposed role of NSP2 in facilitating dsRNA synthesis by VP1 and also point to NSP2's possible role in preventing the premature self-assembly of VP2 cores. Our findings lead us to propose that the NSP2 octamer with multiple enzymatic activities is a principal regulator of viroplasm formation, recruitment of viral proteins into the viroplasms and possibly genome replication.

  • Structural Definition of an Antibody-Dependent Cellular Cytotoxicity (ADCC) Response Implicated in Reduced Risk for HIV-1 Infection [PublishAheadOfPrint]

  • The RV144 vaccine trial implicated epitopes in the C1 region of gp120 (A32-like epitopes) as targets of potentially protective antibody-dependent cellular cytotoxicity (ADCC) responses. A32-like epitopes are highly immunogenic as infected or vaccinated individuals frequently elicit antibodies specific for these determinants. Antibody-binding titers as measured by ELISA against these epitopes, however, do not consistently correlate with protection. Here, we report crystal structures of CD4-stabilized gp120 cores complexed with the Fab fragments of two non-neutralizing, A32-like monoclonal antibodies (mAbs), named N5-i5 and 2.2c, that compete for antigen binding and exhibit similar binding affinities, yet mediate a 75-fold difference in ADCC potency. We find that these mAbs recognize overlapping epitopes formed by mobile layers 1 and 2 of the gp120 inner domain including the C1-C2 regions, but bind gp120 at different angles via juxtaposed VH and VL contact surfaces. A comparison of structural and immunological data further showed that antibody orientation on bound antigen and the capacity to form multivalent antigen-antibody complexes on target cells were key determinants for ADCC potency, with the latter process having the greater impact. These studies provide atomic level definition of A32-like epitopes implicated as targets of protective antibodies in RV144. Moreover, these studies establish that epitope structure and mode of antibody binding can dramatically affect the potency of Fc-mediated effector function against HIV-1. These results provide key insights for understanding, refining, and improving the outcome of HIV-vaccine trials, in which relevant immune responses are facilitated by A32-like elicited responses.

    Importance: HIV-1 Env is a primary target for antibodies elicited during infection. Although a small number of infected individuals elicit broadly neutralizing antibodies, the bulk of humoral response consists of antibodies that do not neutralize or do so with limited breadth but may effect protection through Fc receptor-dependent processes, such as antibody-dependent cellular cytotoxicity (ADCC). Understanding these non-neutralizing responses is an important aspect in elucidating the complete spectrum of immune response against HIV-1 infection. With this report we provide the first atomic level definition of non-neutralizing CD4-induced epitopes in the N-terminal region of the HIV-1 gp120 (A32-like epitopes). Further, our studies point toward the dominant role of precise epitope targeting and mode of antibody attachment in ADCC responses even when largely overlapping epitopes are involved. Such information provides key insights into the mechanisms of Fc-mediated antibody function to HIV-1 and helps understand the outcome of vaccine trials based on humoral immunity.

  • Epstein Barr Virus (EBV) Late Gene Transcription Depends on the Assembly of a Viral Specific Pre-Initiation Complex [PublishAheadOfPrint]

  • During their productive cycle, herpesviruses exhibit a strictly regulated temporal cascade of gene expression that has three general stages: immediate early (IE), early (E), and late (L). Promoter complexity differs strikingly between IE/E genes and L genes. IE and E promoters contain cis-regulating sequences upstream of a TATA box whereas L promoters comprise a unique cis element. In the case of the -herpesviruses, this element is usually a TATT motif found in the position where the consensus TATA box of eukaryotic promoters typically localizes. Epstein-Barr virus encodes a protein, called BcRF1, which has structural homology with the TATA-binding protein and interacts specifically with the TATT box. However, although necessary for the expression of the L genes, BcRF1 is not sufficient, suggesting that other viral proteins are also required. Here, we present the identification and characterization of a viral protein complex necessary and sufficient for the expression of the late viral genes. This viral complex is composed of five different proteins in addition to BcRF1 and interacts with cellular RNA polymerase II. During the viral productive cycle, this complex, that we call vPIC (for viral Pre-Initiation Complex), works in concert with the viral DNA replication machinery to activate expression of the late viral genes. The EBV vPIC components have homologs in bbeta;- and -herpesviruses but not in aalpha;-herpesviruses. Our results not only reveal that bbeta;- and -herpesviruses encode their own transcription pre-initiation complex responsible for the expression of the late viral genes but also indicate their close evolutionary history.

    IMPORTANCE Control of late gene transcription in DNA viruses is a major unsolved question in virology. In eukaryotes, the first step in transcriptional activation is the formation of a permissive chromatin which allows assembly of the pre-initiation complex (PIC) at the core promoter. Fixation of the TATA-binding protein is a key rate-limiting step in this process. This study provides evidence that EBV encodes a complex composed of six proteins necessary for the expression of the late viral genes. This complex is formed around a viral TBP-like protein and interacts with cellular RNA polymerase II, suggesting that it is directly involved in the assembly of a viral-specific PIC (vPIC).

  • Distinct Mechanisms Regulate Exposure of Neutralizing Epitopes in the V2 and V3 Loops of HIV-1 Envelope [PublishAheadOfPrint]

  • Broadly neutralizing antibodies targeting the HIV-1 envelope (Env) are key components for protection against HIV-1. However, many cross-reactive epitopes are often occluded. This study investigates the mechanisms contributing to the masking of V2i (V2-integrin) epitopes compared to V3 epitopes. V2i are conformation-dependent epitopes encompassing the integrin aalpha;4bbeta;7-binding motif on the V1V2 loop of HIV-1 Env gp120. The V2i monoclonal antibodies (mAbs) display extensive cross-reactivity with gp120 monomers from many subtypes, but neutralize only few viruses, indicating V2i's cryptic nature. First, we asked whether CD4-induced Env conformational changes affect V2i epitopes similar to V3. CD4 treatment of BaL and JRFL pseudoviruses increased their neutralization sensitivity to V3 mAbs, but not to the V2i mAbs. Second, contribution of N-glycans on masking V2i versus V3 epitopes was evaluated by testing neutralization of pseudoviruses produced in the presence of a glycosidase-inhibitor, kifunensine. Viruses grown in kifunensine were more sensitive to neutralization by V3 but not V2i mAbs. Finally, we evaluated the time-dependent dynamics of the V2i and V3 epitopes. Extending the time of virus-mAb interaction to 18 hours, before adding target cells, increased virus neutralization by some V2i mAbs and all V3 mAbs tested. Consistent with this, V2i mAb binding to Env on the surface of transfected cells also increased in time-dependent manner. Hence, V2i and V3 epitopes are highly dynamic but distinct factors modulate antibody accessibility of these epitopes. The study reveals the importance of the structural dynamics of V2i and V3 epitopes in determining HIV-1 neutralization by antibodies targeting these sites.

    Importance Conserved neutralizing epitopes are present in the V1V2 and V3 regions of HIV-1 Env, but these epitopes are often occluded from Abs. This study reveals that distinct mechanisms contribute to the masking of V3 epitopes and V2i epitopes in the V1V2 domain. Importantly, V3 mAbs and some V2i mAbs display greater neutralization against relatively resistant HIV-1 isolates when the mAbs interact with the virus for a prolonged period of time. Given their highly immunogenic nature, V3 and V2i epitopes are valuable targets that would augment the efficacy of HIV vaccines.

  • MHC class I downregulation induced by equine herpesvirus type 1 (EHV-1) pUL56 is through dynamin-dependent endocytosis [PublishAheadOfPrint]

  • Equine herpesvirus type 1 (EHV-1) downregulates cell surface expression of major histocompatibility complex class I (MHC-I) in infected cells. We have previously shown that pUL56 encoded by the EHV-1 ORF1 gene regulates the process (G. Ma, S. Feineis, N. Osterrieder, and G. R. Van de Walle, J. Virol. 86:3554-3563, 2012, doi:10.1128/JVI.06994-11). Here, we report that cell surface MHC-I in EHV-1-infected cells is internalized and degraded in the lysosomal compartment in a pUL56-dependent fashion. pUL56-induced MHC-I endocytosis required dynamin and tyrosine kinase, but was independent of clathrin or caveolin-1, the main constituents of clathrin- and raft/caveolae-mediated endocytosis pathway, respectively. Downregulation of cell surface MHC-I was significantly inhibited by the ubiquitin-activating enzyme E1 inhibitor PYR41, indicating that ubiquitination is essential for the process. Finally, we show that downregulation is not specific for MHC-I and that cell surface expression of other molecules including CD46 and CD63 is also removed from the cell surface in a pUL56-dependent fashion.

    IMPORTANCE We show that alphaherpesvirus induces MHC-I downregulation through endocytosis, which is mediated by pUL56. The dynamin-dependent endocytic pathway is responsible for MHC-I internalization in infected cells. Furthermore, we discovered that this endocytic process can be disrupted by inhibiting ubiquitin-activating E1 enzyme, which is indispensible for ubiquitination. Finally, pUL56 action extends to a number of cell surface molecules that are significant for host immunity. Therefore, the protein may exert a more general immunomodulatory effect.

  • Emergence of individual HIV-specific CD8 T cell responses during primary HIV-1 infection can determine long-term disease outcome [PublishAheadOfPrint]

  • Background. Events during primary HIV-1 infection have been shown to be critical for the subsequent rate of disease progression. Early control of viral replication, resolution of clinical symptoms and development of a viral setpoint have been associated with the emergence of HIV-specific CD8 T cell responses. Here we assessed which particular HIV-specific CD8 T cell responses contribute to long-term control of HIV-1.

    Methods. A total of 620 individuals with primary HIV-1 infection were screened by IFN Elispot for HLA class I-restricted, epitope-specific CD8 T cell responses using optimally defined epitopes approximately 2 months post initial presentation. The cohort was predominantly male(97%)Caucasian(83%) (Fiebig: II/III:[n=152], IV:[n=61], V:[n=277], VI:[n=87], nd:[n=23]). Longitudinal viral loads, CD4 count and time to ART were collected for all patients.

    Results. We observed strong associations between viral load at baseline (initial viremia) and the established early viral set points (pllt;0.0001). Both were significantly associated with HLA class I genotypes (p=0.0009). While neither the breadth nor magnitude of HIV-specific CD8 T cell responses showed an influence on the early viral set point, a broader HIV-specific CD8 T cell response targeting epitopes within HIV-1 Gag during primary HIV-1 infection was associated with slower disease progression. Moreover, the induction of certain HIV-specific CD8 T cell responses - but not others mmdash;significantly influenced the time to ART initiation.

    Conclusions. Individual epitope-specific CD8 T cell responses are significantly contributing to HIV-1 disease control, demonstrating that the specificity of the initial HIV-specific CD8 T cell response rather than the restricting HLA class I molecule alone is a critical determinant of their antiviral function.

    IMPORTANCE Understanding which factors are involved in the control of HIV infection is critical for the design of therapeutic strategies for patients living with HIV/AIDS. Here we assessed in a cohort of over 600 individuals with acute and early HIV infection in unprecedented detail the individual contribution of epitope-specific CD8 T cell responses directed against HIV to control of viremia and their impact on the overall course of disease progression.

  • Activation of Intrinsic Immune Responses and Microglial Phagocytosis in an Ex Vivo Spinal Cord Slice Culture Model of West Nile Virus Infection [PublishAheadOfPrint]

  • West Nile virus (WNV) is a neurotropic flavivirus that causes significant neuroinvasive disease involving the brain and/or spinal cord. Experimental mouse models of WNV infection have established the importance of innate and adaptive immune responses in controlling the extent and severity of central nervous system (CNS) disease. However, differentiating between immune responses that are intrinsic to the CNS and those dependent on infiltrating inflammatory cells or resulting from immune events caused by extraneural systemic infection has proven difficult. We used a murine ex vivo spinal cord slice culture (SCSC) model to determine the innate immune processes specific to the CNS during WNV infections. By 7 days post ex vivo infection of SCSCs, the majority of neurons and a substantial percentage of astrocytes were infected with WNV resulting in apoptotic cell death and astrogliosis. Microglia, the resident immune cells of the CNS, were activated by WNV infection as exemplified by their amoeboid morphology, development of filopodia and lamellipodia, and phagocytosis of WNV-infected cells and debris. Microglial cell activation was concomitant with increased expression of pro-inflammatory cytokines and chemokines, including CXCL10, CXCL1, CCL5, CCL3, CCL2, TNFaalpha;, TRAIL, and IL-6. The application of minocycline, an inhibitor of neuroinflammation, altered the WNV-induced pro-inflammatory cytokine/chemokine expression profile with inhibited production of CCL5, CCL2, and IL-6 but not of CXCL10, TRAIL, and TNFaalpha;. Our findings establish that CNS resident cells have the capacity to initiate a robust innate immune response against WNV infection in the absence of infiltrating inflammatory cells and systemic immune responses.

    IMPORTANCE There are no specific treatments of proven efficacy available for WNV neuroinvasive disease. A better understanding of the pathogenesis of WNV CNS infection is crucial for the rational development of novel therapies. Development of a spinal cord slice culture (SCSC) model facilitates study of WNV pathogenesis and allows investigation of the intrinsic immune responses of the CNS. Our studies demonstrate that robust CNS innate immune responses including microglial activation and pro-inflammatory cytokine/chemokine production develop independent of contributions from the peripheral immune system and CNS-infiltrating inflammatory cells.

  • Direct Interrogation of Viral Peptides Presented by the Class I HLA of HIV Infected T Cells [PublishAheadOfPrint]

  • Identification of CD8+ cytotoxic T lymphocyte (CTL) epitopes has traditionally relied upon testing overlapping peptide libraries for their reactivity with T cells in vitro. Here, we pursued Deep Ligand Sequencing (DLS) as an alternative method of directly identifying those ligands that are epitopes presented to CTL by the class I human leukocyte antigens (HLA) of infected cells. Soluble class I HLA-A*11:01 (sHLA) was gathered from HIV-1 NL4-3-infected human CD4+ SUP-T1 cells. HLA-A*11:01 harvested from infected cells was immunoaffinity purified and acid boiled to release heavy and light chains from peptide ligands that were then recovered by size exclusion filtration. The ligands were fractionated first by high pH HPLC and then subjected to separation by nano LCMS at low pH. Approximately 10 million ions were selected for sequencing by tandem mass spectrometry (MS/MS). HLA-A*11:01 ligand sequences were determined with PEAKS software and confirmed by comparison to spectra generated from synthetic peptides. DLS identified 42 viral ligands presented by HLA-A*11:01, 37 of which were previously undetected. These data demonstrate that (1) HIV-1 Gag and Nef are extensively sampled, (2) ligand length variants are prevalent, particularly within Gag and Nef "hot spots" where ligand sequences overlap, (3) non-canonical ligands are T cell reactive, and (4) HIV-1 ligands are derived from de novo synthesis rather than endocytic sampling. Next generation immunotherapies must factor these nascent HIV-1 ligand length variants, and the finding that CTL reactive epitopes may be absent during infection of CD4+ T cells, into strategies designed to enhance T cell immunity.

    Importance HIV-1 epitopes catalogued by the Los Alamos National Laboratory (LANL) have yielded limited success in vaccine trials. Because the HLA of infected cells have not previously been assessed for HIV-1 ligands, the objective here was to directly characterize the viral ligands that mark infected cells. Recovery of HLA-presented peptides from HIV-1 infected CD4+ T cells and interrogation of the peptide cargo by mass spectrometric DLS shows that typical and atypical viral ligands are efficiently presented by HLA and targeted by human CTL. Nef and Gag ligands dominate the infected cell's antigenic profile, largely due to extensive ligand sampling from select "hot spots" within these viral proteins. Also, HIV-1 ligands are often longer than expected, and these length variants are quite antigenic. These findings emphasize that an HLA-based view of HIV-1 ligand presentation to CTL provides previously unrealized information that may enhance the development of immune therapies and vaccines.

  • The Raccoon Polyomavirus Genome and Tumor Antigen Transcription are Stable and Abundant in Neuroglial Tumors [PublishAheadOfPrint]

  • Raccoon polyomavirus (RacPyV) is associated with 100% of neuroglial tumors in free-ranging raccoons. Other tumor-associated polyomaviruses (PyVs), including SV40, murine PyV, and Merkel cell PyV, are found integrated in the host genome in neoplastic cells, where they constitutively express splice variants of the tumor antigen (TAg) gene. We have previously reported that RacPyV exists only as an episome (non-integrated) in neuroglial tumors. Here we have investigated TAg transcription in primary tumor tissue by transcriptome analysis, and we identified the alternatively spliced TAg transcripts for RacPyV. We also determined that TAg was highly transcribed relative to host cellular genes. We further co-localized TAg DNA and mRNA by in situ hybridization, and found that the majority of tumor cells showed positive staining. Lastly, we examined stability of the viral genome and TAg transcription by quantitative reverse-transcriptase PCR in cultured tumor cells in vitro and in a mouse xenograft model. When tumor cells were cultured in vitro, TAg transcription increased nearly two log-fold over that of parental tumor tissue by passage 17. Both episomal viral genome and TAg transcription were faithfully maintained in culture and in tumors arising from xenotransplant of cultured cells in mice. This study represents a minimal criterion for RacPyV's association with neuroglial tumors, and a novel mechanism of stability for a polyomavirus in cancer.

    Importance: The natural cycle of polyomaviruses in mammals is to persist in the host without causing disease, but can cause cancer in humans or in other animals. Because this is an unpredictable and rare event, the oncogenic potential of polyomavirus is primarily evaluated in laboratory animal models. Recently, raccoon polyomavirus (RacPyV) was identified in neuroglial tumors of free-ranging raccoons. Viral copy number was consistently high in these tumors, but was low or undetectable in non-tumor tissue or in unaffected raccoons. Unlike other oncogenic polyomaviruses, RacPyV was episomal, not integrated, in these tumors. To determine the stability of the viral genome and sustained transcription of the oncogenic tumor antigen proteins, we cultured primary raccoon tumor cells and passaged them in mice, confirming the non-integrated state of the virus and the maintenance of viral protein transcription throughout. RacPyV provides a naturally occurring and tractable model for a novel mechanism of polyomavirus-mediated oncogenesis.

  • Lack of interleukin-10-mediated anti-inflammatory signals and upregulated interferon gamma production are linked to increased intestinal epithelial cell apoptosis in pathogenic simian immunodeficiency virus infection [PublishAheadOfPrint]

  • Interleukin (IL)-10 is an immunomodulatory cytokine that is important for maintenance of epithelial cell (EC) survival and anti-inflammatory responses (AIR). The majority of HIV infections occur through the mucosal route despite mucosal epithelium acting as a barrier to HIV. Therefore understanding the role of IL-10 in maintenance of intestinal homeostasis during HIV infection is of interest for better characterization of the pathogenesis of HIV-mediated enteropathy. Here we demonstrated changes in mucosal IL-10 signaling during SIV infection in rhesus macaques. Disruption of the epithelial barrier was manifested by EC apoptosis and loss of tight junction protein ZO-1. Multiple cell types, including a limited number of ECs, produced IL-10. SIV infection resulted in increased levels of IL-10, however this was associated with increased production of mucosal IFN and TNFaalpha;, suggesting that IL-10 was not able to regulate AIR. This observation was supported by downregulation of STAT3, which is necessary to inhibit production of IFN and TNFaalpha;, and upregulation of SOCS1 and SOCS3, which are important regulatory molecules in the IL-10-mediated AIR. We also observed internalization of the IL-10 receptor (IL-10R) in mucosal lymphocytes, which could limit cellular availability of IL-10 for signaling and contribute to the loss of a functional AIR. Collectively, these findings demonstrate that internalization of IL-10R with the resultant impact on IL-10 signaling and dysregulation of the IL-10-mediated AIR might play a crucial role in EC damage and subsequent SIV/HIV pathogenesis.

    IMPORTANCE Interleukin-10 (IL-10), an important immunomodulatory cytokine plays a key role to control inflammatory function and homeostasis of the gastro-intestinal mucosal immune system. Despite recent advancements in the study of IL-10 and its role in HIV infection, the role of mucosal IL-10 in SIV/HIV infection in inducing enteropathy is not well understood. Here we demonstrated changes in mucosal IL-10 signaling during SIV infection in rhesus macaques. Disruption of the intestinal epithelial barrier was evident along with the increased levels of mucosal IL-10 production. Increased production of mucosal IFN and TNFaalpha; during SIV infection suggested that the increased level of mucosal IL-10 was not able to regulate anti-inflammatory responses. Our findings demonstrate that internalization of IL-10R with the resultant impact on IL-10 signaling and dysregulation of the IL-10-mediated anti-inflammatory responses might play a crucial role in epithelial cell damage and subsequent SIV/HIV pathogenesis.

  • A novel marine DNA virus (Singapore grouper iridovirus, SGIV) entry into host cells occurs via clathrin-mediated endocytosis and macropinocytosis in a pH-dependent manner [PublishAheadOfPrint]

  • Iridoviruses are nucleocytoplasmic DNA viruses which cause great economic losses in aquaculture industry, but also show significant threat to global biodiversity. However, a lack of host cells results in poor progress in clarifying iridovirus behaviors. We investigated the crucial events during virus entry using a combination of single-virus particle tracking and biochemical assays, based on the established virus-cell infection model for Singapore grouper iridovirus (SGIV). SGIV infection in host cells was strongly inhibited when cells were pretreated with drugs blocking clathrin-mediated endocytosis, including sucrose and chlorpromazine. Inhibition of key regulators of macropinocytosis, including Na+/H+ exchanger, Rac1 GTPase, p21-activated kinase 1 (PAK1), protein kinase C (PKC) and myosin II significantly reduced SGIV uptake. Cy5-labeled SGIV particles were observed to co-localize with clathrin and macropinosomes. In contrast, disruption of cellular cholesterol by methyl-bbeta;-cyclodextrin and nystatin had no effect on virus infection, suggesting that SGIV entered grouper cells via the clathrin-mediated endocytic pathway and macropinocytosis, but not via caveolae-dependent endocytosis. Furthermore, inhibitors of endosome acidification such as chloroquine and bafilomycin A1 blocked virus infection, indicating that SGIV entered cells in a pH-dependent manner. In addition, SGIV particles were observed to transport along both microtubules and actin filaments, and intracellular SGIV motility was remarkably impaired by depolymerization of microtubules or actin filaments. The results of this study for the first time demonstrate that not only clathrin-dependent pathway, but also macropinocytosis is involved in fish DNA enveloped virus entry, thus providing a convenient tactic for exploring the life cycle of DNA viruses.

    Importance Statement Virus entry into host cells is critically important for initiating infections and usually recognized as an ideal target for the design of antiviral strategies. Iridoviruses are large DNA viruses which cause serious threat to ecological diversity and aquaculture industry worldwide. However, the current understanding of iridovirus entry is limited and controversial. Singapore grouper iridovirus (SGIV) is a novel marine fish DNA virus which belongs to genus Ranavirus, family Iridovirdae. Here, using single-virus particle tracking technology in combination with biochemical assays, we investigated the crucial events during SGIV entry, and demonstrated that SGIV entered grouper cells via clathrin-mediated endocytic pathway in a pH-dependent manner, but not caveolae-dependent endocytosis. Furthermore, we proposed for the first time that macropinocytosis was involved in iridovirus entry. Together, this work not only contributes greatly to understating iridovirus pathogenesis, but also provides an ideal model for exploring the behaviors of DNA viruses in the living cells.

  • Possible footprints of APOBEC3F and/or other APOBEC3 deaminases -- but not APOBEC3G -- on HIV-1 from patients during acute/early and chronic infection [PublishAheadOfPrint]

  • Members of the apolipo-protein-B mRNA-editing-enzyme-catalytic polypeptide-like-3 (APOBEC3) innate cellular cytidine deaminase family, particularly APOBEC3F and APOBEC3G, can cause extensive and lethal G-to-A mutations in HIV-1 plus-strand DNA (termed hypermutation). It is unclear if APOBEC3-induced mutations in vivo are always lethal or can occur at sub-lethal levels that increase HIV-1 diversification and viral adaptation to the host. The viral accessory protein Vif counteracts APOBEC3-activity by binding to APOBEC3 and promoting proteasome degradation; however, the efficiency of this interaction varies as a range of hypermutation frequencies are observed in HIV-1 patient DNA. Therefore, we examined llsquo;footprintsrrsquo; of APOBEC3G- and APOBEC3F-activity in longitudinal HIV-1 RNA pol sequences from approximately 3,000 chronically infected patients by determining whether G-to-A mutations occurred in motifs that were favored or disfavored by these deaminases. G-to-A mutations were more frequent in APOBEC3G-disfavored than in APOBEC3G-favored contexts. By contrast, mutations in APOBEC3F-disfavored contexts were relatively rare, whereas mutations in contexts favoring APOBEC3F (and possibly other deaminases) occurred 16% more often than average G-to-A mutations. These results were supported by analyses of ggt;500 HIV-1 env sequences from acute/early infection.

    Importance (63) Collectively, our results suggest that APOBEC3G-induced mutagenesis is lethal to HIV-1, whereas mutagenesis caused by APOBEC3F and/or other deaminases may result in sub-lethal mutations that might facilitate viral diversification. Therefore, Vif-specific CTL-responses and drugs that manipulate the interplay between Vif and APOBEC3 may have beneficial or detrimental clinical effects depending on how they affect the binding of Vif to various members of the APOBEC3-family.

  • Stepwise priming by acidic pH and high K+ is required for efficient uncoating of influenza A virus cores after penetration [PublishAheadOfPrint]

  • Influenza A virus uses the low pH in late endocytic vacuoles as a cue for penetration by membrane fusion. Here, we analyzed the pre-fusion reactions that prepare the core for uncoating after it has been delivered to the cytosol. We found that this priming process occurs in two steps that are mediated by the envelope-embedded M2 ion channel. The first weakens the interactions between the matrix protein, M1, and the viral ribonucleoprotein bundle. It involves a conformational change in a linker sequence and the C-terminal domain of M1 after exposure to pH below 6.5. The second step is triggered by pH llt; 6.0 and by the influx of K+ ions. It causes additional changes in M1 as well as a loss of stability in the vRNP bundle. Our results indicated that the switch from Na+ to K+ in maturing endosomes together with the decreasing pH are both needed to prime IAV cores for efficient uncoating and infection of the host cell.

    Importance: The entry of IAV involves several steps including endocytosis and fusion at late endosomes. Entry also includes disassembly of the viral core which is composed of the viral ribonucleoproteins and the RNA genome. We have found that the uncoating process of IAV is initiated long before the core is delivered into the cytosol. M2, an ion channel in the viral membrane is activated when the virus passes through early endosomes. Here, we show that protons entering the virus through M2 cause a conformational change in the matrix protein, M1. This weakens interactions between M1 and the viral ribonucleoproteins. A second change was found to occur when the virus enters late endosomes. The pre-acidified core is now exposed to a high concentration of K+, which affects the interactions between the ribonucleoproteins. Thus, when cores are finally delivered to the cytosol they are already partially destabilized and therefore uncoating-competent and infectious.

  • Fluorescent Tagging and Cellular Distribution of the Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Open Reading Frame 45 (ORF45) Tegument Protein [PublishAheadOfPrint]

  • Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), is a cancer-related human virus, classified as a member of the gammaherpesvirinae subfamily. We report here the construction of a dual fluorescent-tagged KSHV genome (BAC16-mCherry-ORF45), which constitutively expresses GFP and contains the tegument multifunctional ORF45 protein as a fusion protein with monomeric Cherry fluorescent protein (mCherry). We confirmed that this virus is properly expressed and correctly replicates, and that mCherry-ORF45 protein is incorporated into the virions. Using this labeled virus, we describe the dynamics of mCherry-ORF45 expression and localization in newly infected cells as well as in latently infected cells undergoing lytic induction, and show that mCherry can be used to monitor cells undergoing the lytic viral cycle. This virus is likely to enable future studies monitoring the dynamics of viral trafficking and tegumentation during viral ingress and egress.

    Importance: The present study describes the construction and characterization of a new recombinant KSHV genome BAC16 clone which expresses mCherry-tagged ORF45. This virus enables the tracking of cells undergoing lytic infection and can be used to address issues related to the trafficking and maturation pathways of KSHV virions.

  • HIV control is mediated in part by CD8+ T-cell targeting of specific epitopes [PublishAheadOfPrint]

  • We investigate the hypothesis that the correlation between the class I HLA types of an individual and whether that individual spontaneously controls HIV-1 is mediated by the targeting of specific epitopes by CD8+ T cells. Measuring interferon- ELISPOT responses to a panel of 257 optimally defined epitopes in 341 untreated HIV- infected persons, including persons who spontaneously control viremia, we find that the correlation between HLA types and control is mediated by the targeting of specific epitopes. Moreover, we perform a graphical model-based analysis suggesting that the targeting of specific epitopes is a cause of such controlmmdash;that is, some epitopes are protective rather than merely associated with controlmmdash;and identify eight epitopes that are significantly protective. In addition, we use an in silico analysis to identify protein regions where mutations are likely to affect the stability of a protein, and find that the protective epitopes identified by the ELISPOT analysis correspond almost perfectly to such regions. This in silico analysis thus suggests a possible mechanism for control, and could be used to identify protective epitopes that are not often targeted in natural infection but that may be potentially useful in a vaccine. Our analyses thus argue for the inclusion (and exclusion) of specific epitopes in an HIV vaccine.

    Importance Some individuals naturally control HIV replication in the absence of anti-retroviral therapy, and this ability to control is strongly correlated with the HLA class I alleles that they express. Here, in a large-scale experimental study, we provide evidence that this correlation is largely mediated by the targeting of specific CD8+ T-cell epitopes, and identify eight epitopes that are likely to cause control. In addition, we provide an in silico analysis indicating that control occurs because mutations within these epitopes change the stability of the protein structures. This in silico analysis also identifies additional epitopes that are not typically targeted in natural infection, but may lead to control when included in a vaccine, provided other epitopes that would otherwise distract the immune system from targeting them are excluded from the vaccine.

  • Investigating a Mystery Disease: Tales from a Viral Detective [PublishAheadOfPrint]

  • Viral outbreak investigation is challenging logistically as well as scientifically. In the context of addressing a fictional emerging viral disease I describe the process of discovery, from the initial report of a problem through discussions of intellectual property and sample management, study design, management, experimental execution and reporting of results.

  • APOBEC3F Determinants of HIV-1 Vif Sensitivity [PublishAheadOfPrint]

  • HIV-1 Vif counteracts restrictive APOBEC3 proteins by targeting them for proteasomal degradation. To determine the regions mediating sensitivity to Vif, we compared human APOBEC3F, which is HIV-1 Vif sensitive, with rhesus APOBEC3F, which is HIV-1 Vif resistant. Rhesus/human APOBEC3F chimeras and amino acid substitution mutants were tested for sensitivity to HIV-1 Vif. This approach identified the alpha-3 and alpha-4 helices of human APOBEC3F as important determinants of the interaction with HIV-1 Vif.

  • Enhanced potency of a broadly neutralizing HIV-1 antibody in vitro improves protection against lentiviral infection in vivo [PublishAheadOfPrint]

  • Over the past five years, a new generation of highly potent and broadly neutralizing HIV-1 antibodies has been identified. These antibodies can protect against lentiviral infection in non-human primates, suggesting that passive antibody transfer would prevent HIV-1 transmission in humans. To increase the protective efficacy of such monoclonal antibodies, we employed next-generation sequencing, computational bioinformatics, and structure-guided design to enhance the neutralization potency and breadth of VRC01, an antibody that targets the CD4 binding site of the HIV-1 envelope. One variant, VRC07-523, was 5- to 8-fold more potent than VRC01, neutralized 96% of viruses tested, and displayed minimal autoreactivity. To compare its protective efficacy to VRC01 in vivo, we performed a series of simian-HIV (SHIV) challenge experiments in non-human primates and calculated the doses of VRC07-523 and VRC01 that provide 50% protection (EC50). VRC07-523 prevented infection in NHPs at a 5-fold lower concentration than VRC01. These results suggest that increased neutralization potency in vitro correlates with improved protection against infection in vivo, documenting the improved functional efficacy of VRC07-523 and its potential clinical relevance for protecting against HIV-1 infection in humans.

    IMPORTANCE In the absence of an effective HIV-1 vaccine, alternative strategies are needed to block HIV-1 transmission. Direct administration of HIV-1-neutralizing antibodies may be able to prevent HIV-1 infections in humans. This approach could be especially useful in individuals at high risk for contracting HIV-1 and could be used together with antiretroviral drugs to prevent infection. To optimize the chance of success, such antibodies can be modified to improve their potency, breadth, and in vivo half-life. Here, knowledge of the structure of a potent neutralizing antibody VRC01, that targets the CD4-binding site of the HIV-1 envelope protein, was used to engineer a next-generation antibody with 5-8 fold increased potency in vitro. When administered to non-human primates, this antibody conferred protection at a five-fold lower concentration than the original antibody. Our studies demonstrate an important correlation between in vitro assays used to evaluate therapeutic potential of antibodies and their in vivo effectiveness.

  • EBV blocks the autophagic flux and appropriates the autophagic machinery to enhance viral replication [PublishAheadOfPrint]

  • Autophagy is a catabolic pathway that helps cells to survive in stressful conditions. Cells also use autophagy to clear microbiological infections but, on the other hand, microbes have learned how to manipulate the autophagic pathway for their own benefit. The experimental evidence obtained in this study suggests that the autophagic flux is blocked at the final steps during the reactivation of EBV from latency. This is indicated by the level of LC3II that does not increase in the presence of bafilomycin and by the lack of colocalization of autophagosomes with lysosomes, which correlates with a reduced Rab7 expression.. Since the inhibition of the early phases of autophagy impaired EBV replication and viral particles were observed in autophagic vescicles in the cytoplasm of producing cells, we suggest that EBV exploits the autophagic machinery for its transportation, in order to enhance viral production. The autophagic block was not mediated by ZEBRA, an immediate early EBV lytic gene, whose transfection in Ramos, Akata and 293 cells promoted a complete autophagic flux, The block occurred only when the complete set of EBV lytic genes was expressed. We suggest that the inhibition of the early autophagic steps or finding strategies to overcome the autophagic block, allowing viral degradation into the lysosomes, could be potentially exploited to manipulate EBV replication.

    IMPORTANCE This study shows for the first time that autophagy is blocked at the final degradative steps during EBV replication, in several cell types. Through this block, EBV hijacks the autophagic vescicles for its intracellular transportation and enhances viral production. A better understanding of virus/host interactions could help to design new therapeutic approaches against EBV-associated malignancies.

  • A Synthetic Biology Approach For a Vaccine Platform Against Known and Newly Emerging Serotypes of Bluetongue Virus [PublishAheadOfPrint]

  • Bluetongue is one of the major infectious diseases of ruminants and is caused by Bluetongue virus (BTV), an arbovirus existing in nature in at least 26 distinct serotypes. Here, we describe the development of a vaccine platform for BTV. The advent of synthetic biology approaches and the development of reverse genetics systems, has allowed the rapid and reliable design and production of pathogen genomes which can be subsequently manipulated for vaccine production. We describe BTV vaccines based on "synthetic" viruses in which the outer core proteins of different BTV serotypes are incorporated into a common tissue-culture adapted backbone. As a means of validation for this approach, we selected two BTV-8 synthetic reassortants and demonstrated their ability to protect sheep against virulent BTV-8 challenge. In addition, to further highlight the possibilities of genome manipulation for vaccine production, we also designed and rescued a synthetic BTV chimera containing a VP2 protein including regions derived from both BTV-1 and BTV-8. Interestingly, while the parental viruses were neutralized only by homologous antisera, the chimeric proteins could be neutralized by both BTV-1 and BTV-8 antisera. These data suggest that neutralizing epitopes are present in different areas of the BTV VP2 and likely "bivalent" strains eliciting neutralizing antibodies for multiple strains can be obtained.

    Importance Overall, this vaccine platform can significantly reduce the time taken from the identification of new BTV strains to the development and production of new vaccines, as the viral genomes of these viruses can be entirely synthesised in vitro. In addition, these vaccines can be brought quickly in the market as they alter the approach, but not the final product, of existing commercial products.

  • Molecular Basis for Ebola Virus VP35 Suppression of Human Dendritic Cell Maturation [PublishAheadOfPrint]

  • Zaire ebolavirus (EBOV) VP35 is a double-stranded RNA (dsRNA) binding protein that inhibits RIG-I signaling and interferon (IFN)-aalpha;/bbeta; responses by both dsRNA-binding dependent and independent mechanisms. VP35 also suppresses DC maturation. Here, we define the pathways and mechanisms through which VP35 impairs DC maturation. Wild-type VP35 (VP35-WT) and two well-characterized VP35 mutants (F239A and R322A) which independently ablate dsRNA-binding and RIG-I inhibition were delivered to primary human monocyte-derived DCs using a lentivirus-based expression system. VP35-WT suppressed not only IFN-aalpha;/bbeta; but also proinflammatory responses following stimulation of MDDCs with activators of RIG-I-like receptor (RLR) signaling, including RIG-I activators such as Sendai virus (SeV) or 5'-triphosphate RNA or MDA5 activators such as encephalomyocarditis virus (EMCV) or poly(I:C). F239A and R322A exhibited greatly reduced suppression of IFN-aalpha;/bbeta; and proinflammatory cytokine production following treatment of DCs with RLR agonists. VP35-WT also blocked the upregulation of DC maturation markers and the stimulation of allogeneic T cell responses upon SeV infection, whereas the mutants did not. In contrast to the RLR activators, VP35-WT and the mutant VP35s impaired IFN-bbeta; production induced by Toll-Like Receptor (TLR) 3 or TLR4 agonists but failed to inhibit proinflammatory cytokine production induced by TLR2, TLR3, or TLR4 agonists. Further, VP35 did not prevent LPS-induced upregulation of surface markers of MDDC maturation and did not prevent LPS-triggered allogeneic T cell stimulation. Therefore, VP35 is a general antagonist of DC responses to RLR activation. However, TLR agonists can circumvent many of the inhibitory effects of VP35. This suggests strategies to counteract VP35 immune evasion functions.

    Importance. The VP35 protein, which is an inhibitor of RIG-I signaling and interferon (IFN)-aalpha;/bbeta; responses, has been implicated as an Ebola virus-encoded factor that contributes to suppression of dendritic cell (DC) function. We used wild-type and previously characterized VP35 mutants to clarify VP35-DC interactions. Our data demonstrate that VP35 is a general inhibitor of RIG-I-like receptor (RLR) signaling that blocks not only RIG-I but also MDA5-mediated induction of IFN-aalpha;/bbeta; responses. Further, in DCs VP35 also impairs RLR-mediated induction of proinflammatory cytokine production, upregulation of costimulatory markers and activation of T cells. These inhibitory activities require VP35 dsRNA binding activity, an activity previously correlated to VP35 RIG-I inhibitory function. In contrast, while VP35 can inhibit IFN-aalpha;/bbeta; production induced by TLR3 or TLR4 agonists, this occurs in a dsRNA-independent fashion, and VP35 does not inhibit TLR-mediated expression of proinflammatory cytokines. These data suggest strategies to overcome VP35 inhibition of DC function.

  • The ER stress sensor IRE1{alpha} protects cells from apoptosis induced by coronavirus infectious bronchitis virus [PublishAheadOfPrint]

  • The unfolded protein response (UPR) is a signal transduction cascade triggered by perturbation of the homeostasis of the endoplasmic reticulum (ER). UPR resolves ER stress by activating a cascade of cellular response including the induction of molecular chaperones, translational attenuation, ER-associated degradation and other mechanisms. Under prolonged and irremediable ER stress, however, UPR can also trigger apoptosis. Here we report that in cells infected with the avian coronavirus infectious bronchitis virus (IBV), ER stress was induced and the IRE1aalpha;-XBP1 pathway of UPR was activated. Knockdown and over-expression experiments demonstrated that IRE1aalpha; protects the infected cells from IBV-induced apoptosis, which required both its kinase and RNase activity. Our data also suggest that splicing of XBP1 mRNA by IRE1aalpha; appears to convert XBP1 from a pro-apoptotic XBP1u protein to a pro-survival XBP1s protein. Moreover, IRE1aalpha; antagonized IBV-induced apoptosis by modulating the phosphorylation status of the pro-apoptotic c-Jun N-terminal kinase (JNK) and the pro-survival RAC-alpha serine/threonine-protein kinase (Akt). Taken together, the ER stress sensor IRE1aalpha; is activated in IBV-infected cells and serves as a survival factor during coronavirus infection.

    Importance Animal coronaviruses are important veterinary viruses, which could cross the species barrier, becoming severe human pathogens. Molecular characterization of the interactions between coronaviruses and host cells is pivotal to the understanding of pathogenicity and species specificity of coronavirus infection. It has been well established that the endoplasmic reticulum (ER) is closely associated with coronavirus replication. Here we report that inositol-requiring protein-1 alpha (IRE1aalpha;), a key sensor of ER stress, is activated in cells infected with avian coronavirus infectious bronchitis virus (IBV). Moreover, IRE1aalpha; is shown to protect the infected cells from apoptosis by modulating the unfolded protein response (UPR) and two kinases related to cell survival. This study demonstrates that UPR activation constitutes a major aspect of coronavirus-host interactions. Manipulations of the coronavirus-induced UPR may provide novel therapeutic targets to the control of coronavirus infection and pathogenesis.

  • Gammaherpesvirus Latency Differentially Impacts The Generation Of Primary Versus Secondary Memory CD8+T Cells During Subsequent Infection [PublishAheadOfPrint]

  • Unlike laboratory animals, humans are infected with multiple pathogens, including the highly prevalent herpesviruses. The purpose of these studies was to determine the effect of gammaherpesvirus latency on T cell number and differentiation during subsequent heterologous viral infections. Mice were first infected with murine gammaherpesvirus 68 (MHV68), a model of Epstein-Barr virus (EBV) infection, and then after latency was established, they were challenged with the Armstrong strain of lymphocytic choriomeningitis virus (LCMV). The initial replication of LCMV was lower in latently infected mice and the maturation of dendritic cells was abated. Although the number of LCMV-specific effector CD8+ T cells was not altered, they were skewed to a memory phenotype. In contrast, LCMV-specific effector CD4+ T cells were increased in latently infected mice compared to those infected solely with LCMV. When the memory phase was reached, latently infected mice had a LCMV-specific memory T cell pool that was increased relative to that found in singly infected mice. Importantly, LCMV-specific memory CD8+ T cells had decreased CD27 and increased KLRG1 expression. Upon secondary challenge, LCMV-specific secondary effector CD8+ T cells expanded and cleared the infection. But, the LCMV-specific secondary memory CD8+ T cell pool was decreased in latently infected animals, abrogating the boosting effect normally observed following rechallenge. Taken together these results demonstrate ongoing gammaherpesvirus latency affects the number and phenotype of primary versus secondary memory CD8+ T cells during acute infection.

    Importance CD8+ T cells are critical for the clearance of intracellular pathogens including viruses, certain bacteria and tumors. However, current models for memory CD8+ T cell differentiation are derived from pathogen-free laboratory mice challenged with a single pathogen or vaccine vector. Unlike laboratory animals, all humans are infected with multiple acute and chronic pathogens, including the highly prevalent herpesviruses Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex viruses (HSV), and varicella zoster virus (VZV). The purpose of these studies was to determine the effect of gammaherpesvirus latency on T cell number and differentiation during subsequent heterologous viral infections. We observed that ongoing gammaherpesvirus latency affects the number and phenotype of primary versus secondary memory CD8+ T cells during acute infection. These results suggest that unlike pathogen free laboratory mice, infection or immunization of latently infected humans may result in the generation of T cells with limited potential for long-term protection.

  • Cellular Requirements for BIV Vif-Mediated Inactivation of Bovine APOBEC3 Proteins [PublishAheadOfPrint]

  • Human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) viral infectivity factor (Vif) form a CRL5 E3 ubiquitin ligase complex to suppress virus restriction by host APOBEC3 (A3) proteins. The primate lentiviral Vif complex is composed of the unique cofactor CBF-bbeta; and canonical ligase components Cullin5 (CUL5), ElonginB/C (ELOB/C), and RBX2. However, the mechanism by which the Vif protein of the related lentivirus bovine immunodeficiency virus (BIV) overcomes its host A3 proteins is less clear. In this study, we show that BIV Vif interacts with Cullin2 (CUL2), ELOB/C, and RBX1, but not with CBF-bbeta; or CUL5, to form a CRL2 E3 ubiquitin ligase and degrade the restrictive bovine A3 proteins (A3Z2Z3 and A3Z3). RNAi-mediated knockdown of ELOB or CUL2 inhibits BIV Vif-mediated degradation of these A3 proteins, whereas knockdown of CUL5 or CBF-bbeta; does not. BIV Vif with mutations in the BC box (Vif SLQ-AAA) or putative VHL box (Vif YI-AA), which cannot interact with ELOB/C or CUL2, respectively, lose the ability to counteract bovine A3 proteins. Moreover, CUL2 and UBE2M dominant-negative mutants competitively inhibit the BIV Vif-mediated degradation mechanism. Thus, although the general strategy for inhibiting A3 proteins is conserved between HIV-1/SIV and BIV, the precise mechanisms can differ substantially, with only the HIV-1/SIV Vif proteins requiring CBF-bbeta; as a cofactor, HIV-1/SIV Vif using CUL5/RBX2, and BIV Vif using CUL2/RBX1.

    IMPORTANCE Primate lentivirus HIV-1 and SIV Vif proteins form a ubiquitin ligase complex to target host anti-viral APOBEC3 proteins for degradation. However, the mechanism by which the non-primate lentivirus BIV Vif inhibits bovine APOBEC3 proteins is unclear. Here we determine the mechanism for BIV Vif-mediated degradation of bovine APOBEC3 proteins, which differs from the mechanism of HIV-1/SIV Vif by being CBF-bbeta; independent and requiring different ubiquitin ligase scaffolding proteins (CUL2/RBX1 instead of CUL5/RBX2). BIV Vif is the only known retroviral protein that can interact with CUL2. This information broadens our understanding of the distinct mechanisms by which the Vif proteins of different lentiviruses facilitate viral infection. This novel mechanism for assembly of the BIV Vif-APOBEC3 ubiquitin ligase complex advances our understanding of viral hijacking of host E3 ubiquitin ligases and illustrates the evolutionary flexibility of lentiviruses.

  • A Comprehensive Analysis of HSV-1 Entry Mediated by Zebrafish 3-O-Sulfotransferase Isoforms: Implications for the Development of a Zebrafish Model of HSV-1 Infection [PublishAheadOfPrint]

  • Binding of herpes simplex virus type-1 (HSV-1) envelope glycoprotein D (gD) to the receptor 3-O-sulfated heparan sulfate (3-OS HS) mediates viral entry. 3-O-sulfation of HS is catalyzed by 3-O-sulfotransferase (3-OST) enzyme. Multiple isoforms of 3-OST are differentially expressed in tissues in Zebrafish (ZF) embryos. Here, we performed a comprehensive analysis of the role of ZF 3-OST isoforms-1, -5, -6 and -7 in HSV-1 entry. We found that a group of 3-OST gene family (3-OST-2, -3, -4, and -6 isoforms) with conserved catalytic and substrate-binding residues of the enzyme mediates HSV-1 entry and spread, while the other group (3-OST-1, -5, and -7) lack these properties. These results demonstrate that HSV-1 entry can be recapitulated by certain ZF 3-OST enzymes, a significant step towards the establishment of a ZF model of HSV-1 infection and tissue-specific tropism.

  • Immunogenicity studies of proteins forming the T4 phage head surface [PublishAheadOfPrint]

  • Advances in phage therapy and novel applications of phages in biotechnology encourage interest in phage impact on human and animal immunity. Here we present comparative studies of immunogenic properties of T4 phage head surface proteins gp23*, gp24*, Hoc and Soc, both as elements of the phage capsid and as isolated agents. Studies comprise evaluation of specific antibodies in the human population, analysis of the proteins' impact on the primary and secondary response in mice, as well as the effect of specific antibodies on phage antibacterial activity in vitro and in vivo in mice.

    In humans, natural antibodies specific to T4-like phages were abundant (81% of investigated sera). Among those, significantly elevated levels of IgG antibodies only against major head protein (gp23*) were found, which probably reflected cross-reactions of T4 with antibodies induced by other T4-like phages. Both IgM and IgG antibodies were induced mostly by gp23* and Hoc, while weak (gp24*) and very weak (Soc) reactivity of other head proteins was noticed. Thus, T4 head proteins that markedly contribute to immunological memory to the phage are: highly antigenic outer capsid protein (Hoc) and major capsid protein (gp23*). Specific anti-gp23* and anti-Hoc antibodies substantially decreased T4 phage activity in vitro and to some extent in vivo. Cooperating with antibodies, the immune complement system also contributed to annihilating phages.

    Importance Current descriptions of phage immunogenicity and its biological consequences are still vague and incomplete thus the central problem of this work is timely and may have strong practical implications. Here, is presented the very first observation of bacteriophage proteins contribution to immunological memory of the phage. Understanding of interactions between phages and mammalian immunology may help in biotechnological adaptations of phages for therapeutic requirements as well as for better appreciation of phage ecology and their role in the biosphere.

  • Lysine 63-linked TANK-binding kinase 1 ubiquitination by mindbomb E3 ubiquitin protein ligase 2 is mediated by mitochondrial antiviral signaling protein [PublishAheadOfPrint]

  • Interferon beta (IFNbbeta;) is involved in a wide range of cellular functions, and its secretion must be tightly controlled to inhibit viral spreading while minimizing cellular damage. Intracellular viral replication triggers cellular signaling cascades leading to the activation of the transcription factors NFB and interferon regulatory factors 3 and 7 (IRF3/7), which synergistically bind to the IFNbbeta; gene promoter to induce its expression. The mitochondrial antiviral signaling protein (MAVS) is a governing adaptor protein that mediates signaling communications between virus sensing proteins and transcription factors. The activity of MAVS in the regulation of IFNbbeta; secretion is affected by many cellular factors. However, the mechanism of MAVS-mediated IRF3/7 activation is not completely understood. Here, we identified a highly conserved DLAIS motif at amino acid positions 438-442 of MAVS that is indispensable for IRF3/7 activation. Specifically, the L439S and A440R mutations suppress IRF3/7 activation. Pull-down experiments using wild-type and mutant MAVS showed that mindbomb E3 ubiquitin protein ligase 2 (MIB2) binds to the DLAIS motif. Furthermore, the DLAIS motif was found to be critical for MIB2 binding, the ligation of K63-linked ubiquitin to TANK-binding kinase 1, and phosphorylation-mediated IRF3/7 activation. Our results suggest that MIB2 plays a putative role in MAVS-mediated interferon signaling.

    Highlights Mitochondrial antiviral signaling protein (MAVS) mediates signaling from virus sensing proteins to transcription factors for the induction of interferon beta. However, the mechanism underlying MAVS-mediated interferon regulatory factors 3 and 7 (IRF3/7) activation is not completely understood. We found a highly conserved DLAIS motif in MAVS that is indispensable for IRF3/7 activation through TANK-binding kinase 1 (TBK1) and identified it as the binding site for mindbomb E3 ubiquitin protein ligase 2 (MIB2). The mutations that targeted the DLAIS motif abolished MIB2 binding, attenuated the K63-linked ubiquitination of TBK1, and decreased the phosphorylation-mediated activation of IRF3/7.

  • Chlorovirus PBCV-1 Encodes an Active Copper-Zinc Superoxide Dismutase [PublishAheadOfPrint]

  • Superoxide dismutases (SODs) are metalloproteins that protect organisms from toxic reactive oxygen species by catalyzing the conversion of superoxide anion to hydrogen peroxide and molecular oxygen. Sequence analysis of the Paramecium bursaria chlorella virus-1 (PBCV-1) genome identified a protein coding sequence that resembled a Cu-Zn SOD with all of the conserved amino acid residues for binding copper and zinc; the protein was named chlorella virus SOD (cvSOD). Recombinant cvSOD inhibited nitroblue tetrazolium reduction of superoxide anion generated in a xanthine-xanthine oxidase system in solution and also a riboflavin photochemical reduction system in a polyacrylamide gel assay. The inhibition was blocked by the Cu-Zn SOD inhibitor cyanide but not by azide, which inhibits Fe and Mn SODs. A kcat/Km value for cvSOD was determined by stop-flow spectrophotometry as 1.28 x 108 M-1s-1, suggesting that cvSOD catalyzed O2- dismutation was not a diffusion controlled encounter. The cvsod gene was expressed as a late gene and cvSOD activity was detected in purified virions. Superoxide accumulated rapidly during virus infection and circumstantial evidence indicates that cvSOD aids its decomposition to benefit virus replication. Cu-Zn SOD homologs have been described in 3 other families of large DNA viruses, poxviruses, baculoviruses and mimiviruses, which group as a clade. Interestingly, cvSOD does not group in the same clade as the other virus SODs but instead groups in an expanded clade that includes Cu-Zn SODs from many cellular organisms.

    IMPORTANCE Virus infection often leads to an increase in toxic reactive oxygen species in the host, which can be detrimental to virus replication. Viruses have developed various ways to overcome this barrier. As reported in this manuscript, the chloroviruses often encode and package a functional Cu-Zn superoxide dismutase in the virion that presumably lowers the concentration of reactive oxygen induced early during virus infection.

  • Human papillomavirus type 1 (HPV1) E1^E4 protein is a potent inhibitor of the serine-arginine (SR) protein kinase SRPK1 and inhibits phosphorylation of host SR proteins and of the viral transcription and replication regulator E2 [PublishAheadOfPrint]

  • The serine-arginine-specific protein kinase SRPK1 is a common binding partner of the E1^E4 protein.of diverse human papillomavirus types. Here, we show for the first time, that the interaction between HPV1 E1^E4 and SRPK1 leads to potent inhibition of SRPK1 phosphorylation of host SR proteins that have critical roles in mRNA metabolism, including pre-mRNA processing, mRNA export and translation. Furthermore, we show that SRPK1 phosphorylates serine residues of SR/RS dipeptides in the hinge region of the HPV1 E2 protein in in vitro kinase assays and HPV1 E1^E4 inhibits this phosphorylation. Following mutation of the putative phosphoacceptor serine residues, the localization of the E2 protein was altered in primary human keratinocytes; with a significant increase in the cell population showing intense E2 staining of the nucleolus. A similar effect was observed following co-expression of E2 and E1^E4 that is competent for inhibition of SRPK1 activity, suggesting that the nuclear localization of E2 is sensitive to E1^E4-mediated SRPK1 inhibition. Collectively, these data suggest that E1^E4 mediated inhibition of SRPK1 could affect the functions of host SR proteins and those of the virus transcription/replication regulator E2. We speculate that the novel E4 function identified here is involved in the regulation of E2 and SR protein function in posttranscriptional processing of viral transcripts.

    IMPORTANCE The HPV life cycle is tightly linked to the epithelial terminal differentiation programme, with the virion-producing phase restricted to differentiating cells. While the most abundant HPV protein expressed in this phase is the E4 protein, we do not fully understand the role of this protein. Few E4 interaction partners have been identified, but we had previously shown that E4 proteins from diverse papillomaviruses interact with the serine-arginine-specific protein kinase SRPK1, a kinase important in the replication cycles of a diverse range of DNA and RNA viruses. Here, we show that HPV1 E4 is a potent inhibitor of this host cell kinase. We show that E4 inhibits SRPK1 phosphorylation, not only of cellular SR proteins involved in regulating alternative splicing of RNA, but also the viral transcription/replication regulator E2. Our findings reveal a potential E4 function in regulation of viral late gene expression through inhibition of a host cell kinase.

  • Recombinant AAV utilizes cell-specific infectious entry mechanisms [PublishAheadOfPrint]

  • Understanding the entry and trafficking mechanism(s) of rAAV into host cells can lead to evolution in capsid and vector design and delivery methods, resulting in enhanced transduction and therapeutic gene expression. Variability of findings regarding the early entry pathway of rAAV supports the possibility that rAAV, like other viruses, can utilize more than one infectious entry pathway. We tested whether inhibition of macropinocytosis impacted rAAV transduction of HeLa cells as compared to hepatocellular carcinoma cell lines. We found that macropinocytosis inhibitor cytochalasin-D blocked rAAV transduction of HeLa cells (ggt;2-fold), but enhanced (10-fold) transduction in HepG2 and Huh7 lines. Similar results were obtained with another macropinocytosis inhibitor 5-(N-Ethyl-N-isopropyl) amiloride (EIPA). The augmented transduction was neither due to viral binding nor promoter activity, affected multiple rAAV serotypes (rAAV2, rAAV2-R585E, and rAAV8), and influenced both single stranded and self-complementary virions to comparable extents. Follow-up studies using CDC42 inhibitor ML141 and p21-activated kinase 1 (PAK1) siRNA knockdown also resulted in enhanced HepG2 transduction. Microscopy revealed that macropinocytosis inhibition correlated with expedited nuclear entry of the rAAV virions into HepG2 cells. Enhancement of hepatocellular rAAV transduction extended to the mouse liver in vivo (4-fold enhancement), but inversely blocked heart tissue transduction (13-fold). This evidence of host cell-specific rAAV entry pathways confers a potent means for controlling and enhancing vector delivery, and could help unify the divergent accounts of rAAV cellular entry mechanisms.

    IMPORTANCE There is a recognized need for improved rAAV vector targeting strategies that result in delivery of fewer total particles, averting untoward toxicity and/or an immune response against the vector. A critical step in rAAV transduction is entry and early trafficking through the host cellular machinery mmdash; the mechanisms of which are under continued study. However, should the early entry and trafficking mechanisms of rAAV differ across virus serotype or be dependent on host cell environment, this could expand our ability to target particular cells and tissue for selective transduction. Thus the observation that inhibiting macropinocytosis leads to cell-specific enhancement or inhibition of rAAV transduction that extends to the organismic level exposes a new means of modulating vector targeting.

  • Naturally arising strains of polyomaviruses with severely attenuated microRNA expression [PublishAheadOfPrint]

  • Several different polyomaviruses (PyVs) encode microRNAs (miRNAs) that regulate viral as well as host gene expression. However, the functions of polyomaviral miRNAs, particularly during in vivo infection, remain poorly understood. Here we identify rare naturally arising PyVs that are severely attenuated or null for miRNA expression. We identify hypomorphic or null strains for miRNA expression from rhesus macaque Simian Virus 40 (SV40) and human JC virus. These strains were isolated from immunocompromised hosts and derive from insertions or deletions in the viral DNA that preserve the amino acid reading frame of opposing strand large T antigen gene. Characterization of the SV40 miRNA hypomorph, K661, shows that it is inhibited at the early miRNA biogenesis step of Drosha-mediated processing. Despite having a non-rearranged enhancer, which a previous study has shown renders some PyVs more susceptible to the autoregulatory activities of the miRNA, restoring miRNA expression to K661 has little effect on virus growth in either immortalized or primary monkey kidney cells. Thus, in addition to any effect of accompanying genomic elements, these results suggest that cellular context also determines susceptibility to PyV miRNA-mediated effects. Combined, these results demonstrate that polyomaviruses lacking miRNAs can arise infrequently and that the functional importance of polyomaviral miRNAs is context-dependent, consistent with an activity connected to the immune status of the host.

    IMPORTANCE Diverse virus families encode miRNAs, yet much remains unknown about viral miRNA function and contribution to the infectious cycle. Polyomaviruses (PyVs) are small DNA viruses, long important as etiological agents of rare diseases and valuable models of DNA virus infection. Here, in immunosuppressed hosts, we uncover rare naturally-arising variants of different PyVs that have lost the ability to express miRNAs. This represents some of the only known natural viruses to have lost miRNA expression. By probing the biogenesis pathways of these variants, we uncover that miRNA expression is lost via small insertions or deletions that render the transcripts resistant to early steps of miRNA biogenesis, while preserving the reading frame of the opposing T antigen transcripts. Overall, our study informs how miRNA genes evolve/devolve in viruses, and suggests that miRNA function is exquisitely dependent not only on viral genomic context but also the cellular and host environment.

  • Dissection of the antibody response against herpes simplex virus glycoproteins in naturally infected humans [PublishAheadOfPrint]

  • Relatively little is known about the extent of the polyclonal antibody (PAb) repertoire elicited by HSV glycoproteins during natural infection and how these antibodies affect virus neutralization. Here, we examined IgGs from ten HSV-seropositive individuals originally classified as high or low virus shedders. All PAbs neutralized virus to varying extents. We determined which HSV entry glycoproteins these PAbs were directed against: glycoproteins gB, gD and gC were recognized by all sera but fewer reacted against gH/gL. We previously characterized multiple mouse monoclonal antibodies (MAbs) and mapped those with high neutralizing activity to the crystal structures of gD, gB and gH/gL. We used a biosensor competition assay to determine whether there were corresponding human antibodies to those epitopes. All ten samples had neutralizing IgGs to gD epitopes but there were variations in which epitopes were seen in individual samples. Surprisingly, only three samples contained neutralizing IgGs to gB epitopes. To further dissect the nature of these IgGs, we developed a method to select out gD- and gB-specific IgGs from four representative sera via affinity chromatography, allowing us to determine the contribution of antibodies against each glycoprotein to the overall neutralization capacity of the serum. In two cases, gD and gB accounted for all of the neutralizing activity against HSV-2, with a modest amount of HSV-1 neutralization directed against gC. In the other two samples, the dominant response was to gD.

    Importance Antibodies targeting functional epitopes on HSV entry glycoproteins mediate HSV neutralization. Virus neutralizing epitopes have been defined and characterized using murine monoclonal antibodies. However, it is largely unknown whether these same epitopes are targeted by the humoral response to HSV infection in humans. We have shown that during natural infection, virus-neutralizing antibodies are principally directed against gD and gB and to a lesser extent, gC. While several key HSV neutralizing epitopes within gD and gB are commonly targeted by human serum IgG, others fail to induce consistent responses. These data are particularly relevant to the design of future HSV vaccines.

  • Analysis of the highly diverse gene borders in Ebola virus reveals a distinct mechanism of transcriptional regulation [PublishAheadOfPrint]

  • Ebola virus (EBOV) belongs to the group of nonsegmented negative sense RNA viruses. The seven EBOV genes are separated by variable gene borders, including short (4-5 nucleotides) intergenic regions (IRs), a single long (144 nucleotides) IR and gene overlaps, where the neighboring gene end and start signals share five conserved nucleotides. The unique structure of the gene overlaps and the presence of a single long IR are conserved among all filoviruses. Here, we sought to determine the impact of the EBOV gene borders during viral transcription. We show that readthrough mRNA synthesis occurs in EBOV-infected cells irrespective of the structure of the gene border, indicating that the gene overlaps do not promote recognition of the gene end signal. However, two consecutive gene end signals at the VP24 gene might improve termination at the VP24/L gene border, ensuring efficient L gene expression. We further demonstrate that the long IR is not essential for, but regulates transcription reinitiation in a length-dependent, but sequence-independent manner. Mutational analysis of bicistronic minigenomes and recombinant EBOVs showed no direct correlation between IR length and reinitiation rates, but demonstrated that specific IR lengths, not found naturally in filoviruses, profoundly inhibit downstream gene expression. Intriguingly, although truncation of the 144 nucleotide long IR to 5 nucleotides did not substantially affect EBOV transcription, it led to a significant reduction of viral growth.

    Importance Our current understanding of EBOV transcription regulation is limited due to the requirement of high containment conditions to study this highly pathogenic virus. EBOV is thought to share many mechanistic features with well-analyzed prototype nonsegmented negative sense RNA viruses. A single polymerase entry site at the 3' end of the genome determines that transcription of the genes is mainly controlled by gene order and cis-acting signals found at the gene borders. Here, we examined the regulatory role of the structurally unique EBOV gene borders during viral transcription. Our data suggest that transcriptional regulation in EBOV is highly complex and differs from prototype viruses, and further the understanding of this most fundamental process in the filovirus replication cycle. Moreover, our results with recombinant EBOVs suggest a novel role of the long IR found in all filovirus genomes during the viral replication cycle.

  • Influenza A Virus Polymerase Is a Site for Adaptive Changes During Experimental Evolution in Bat Cells [PublishAheadOfPrint]

  • The recent identification of highly divergent influenza A viruses in bats revealed a new, geographically dispersed viral reservoir. To investigate the molecular mechanisms of host-restricted viral tropism and the potential for transmission of viruses between humans and bats, we exposed a panel of cell lines from bats of diverse species to a prototypical human-origin influenza A virus. All of the tested bat cell lines were susceptible to influenza A virus infection. Experimental evolution of human and avian-like viruses in bat cells resulted in efficient replication and created highly cytopathic variants. Deep sequencing of adapted human influenza A virus revealed a mutation in the PA polymerase subunit not previously described, M285K. Recombinant virus encoding PA M285K completely phenocopied the adapted virus. Adaptation of an avian-like virus resulted in the canonical PB2 E627K mutation that is required for efficient replication in other mammals. None of the adaptive mutations occurred in the viral hemagglutinin, a gene that frequently acquires changes to recognize host-specific variations in sialic acid receptors. We showed that human influenza A virus uses canonical sialic acid receptors to infect bat cells, even though bat influenza A viruses do not appear to use these receptors for virus entry. Our results demonstrate that bats are unique hosts that select for both a novel and a well-known adaptive mutation in the viral polymerase to support replication.

    IMPORTANCE Bats constitute well-known reservoirs for viruses that may be transferred into human populations, sometimes with fatal consequences. Influenza A viruses have recently been identified in bats, dramatically expanding the known host range of this virus. Here we investigate the replication of human influenza A virus in bat cell lines and the barriers the virus faces in this new host. Human influenza A and B viruses infected cells from geographically and evolutionarily diverse New and Old World bats. Viruses mutated during infections in bat cells resulting in increased replication and cytopathic effects. These mutations were mapped to the viral polymerase and shown to be solely responsible for adaptation to bat cells. Our data suggest that replication of human influenza A viruses in a non-native host drives evolution of new variants and may be an important source of genetic diversity.

  • Novel Denisovan and Neanderthal Retroviruses [PublishAheadOfPrint]

  • Following the recent availability of high-coverage genomes for Denisovan and Neanderthal man, we conducted a screen for endogenised retroviruses, identifying six novel, previously unreported HERV-K(HML2) elements. These elements are absent from the human genome (hg38), and appear to be unique to archaic hominids. These findings provide further evidence supporting the recent activity of the HERV-K(HML2) group, which has been implicated in human disease. They will also provide insights into the evolution of archaic hominids.

  • Trafficking of Bluetongue virus visualized by recovery of tetracysteine-tagged virion particles [PublishAheadOfPrint]

  • Bluetongue virus (BTV), a member of the Orbivirus genus in the Reoviridae family, is a double-capsid insect-borne virus enclosing a genome of 10 double-stranded RNA segments. As with other members of the family, BTV virions are non-enveloped particles containing two architecturally complex capsids. The two proteins of the outer capsid, VP2 and VP5, are involved in BTV entry and the delivery of the transcriptionally active core in the cell cytoplasm. Although the importance of endocytic pathway in BTV entry has been reported, a detailed analysis of entry and the role of each protein on virus trafficking have not been possible due to unavailability of a tagged virus. Here for the first time we report on the successful manipulation of a segmented genome of a non-enveloped capsid virus by the introduction of tags that were subsequently fluorescently visualized in infected cells. The genetically engineered fluorescent BTV particles were observed to enter live cells immediately after virus adsorption. Further, we showed separation of VP2 from VP5 during virus entry and confirmed that while VP2 is shed from virions in early endosomes, virus particles still consisting of VP5 were trafficked sequentially from early to late endosomes. Since BTV infects both mammalian and insect cells, the generation of tagged viruses will allow visualization of further downstream trafficking of BTV in different host cells. In addition, the tagging technology also has potential for transferable application on other non-enveloped complex viruses.

    Importance Live virus trafficking in host cells has been highly informative in understanding interactions between virus and host cells. Although insertion of fluorescent markers in viral genome have made it possible to study trafficking of enveloped viruses, the physical constraints of architecturally complex capsid viruses have led to practical limitations. In this study, we have successfully genetically engineered the segmented RNA genome of Bluetongue virus (BTV), a complex non-enveloped virus belonging to the Reoviridae family. The resulting fluorescent virus particles could be visualized in virus entry studies for both live and fixed cells. This is the first time a structurally complex capsid virus has been successfully genetically manipulated to generate virus particles that could be visualized in infected cells.

  • Intrabronchial infection of rhesus macaques with simian varicella virus results in a robust immune response in the lungs. [PublishAheadOfPrint]

  • Varicella zoster virus (VZV) is the etiological agent of varicella (chickenpox) and herpes zoster (shingles). Primary VZV infection is believed to occur via the inhalation of virus either in respiratory droplets, from shedding varicella lesions or by direct contact with infectious vesicular fluid. However, the ensuing immune response in the lungs remains incompletely understood. We have shown that intrabronchial inoculation of rhesus macaques with simian varicella virus (SVV), a homolog of VZV, recapitulates the hallmarks of acute and latent VZV infection in humans. In this study, we performed an in-depth analysis of the host immune response to acute SVV infection in the lungs and peripheral blood. We report that acute SVV infection results in a robust innate immune response in the lungs, characterized by the production of inflammatory cytokines, chemokines, and growth factors as well as an increased frequency of plasmacytoid DCs that corresponded with IFNaalpha; production and a rapid decrease in viral loads in the lungs. This is followed by T and B cell proliferation, antibody production, T cell differentiation and cytokine production, which correlate with the complete cessation of viral replication. Although terminally differentiated CD8 T cells became the predominant T cell population in bronchoalveolar lavage cells, a higher percentage of CD4 T cells were SVV-specific, which suggests a critical role for these cells in the resolution of primary SVV infection in the lungs. Given the homology between SVV and VZV, our data provide insight into the immune response to VZV within the lung.

    Importance Although primary VZV infection occurs primarily via the respiratory route, our understanding of the host response in the lungs and its contribution to the cessation of viral replication and establishment of latency remains poorly understood. The difficulty in accessing lung tissue and washes from individuals infected with VZV has hampered efforts to address this knowledge gap. SVV infection of rhesus macaques is an important model of VZV infection of humans; therefore we utilized this animal model to gain a comprehensive view of the kinetics of the immune response to SVV in the lung and its relationship to the resolution of acute infection in respiratory tissues. These data not only advance our understanding of host immunity to VZV, a critical step in developing new vaccines, but also provide additional insight into immunity to respiratory pathogens.

  • IMPACT OF CLADE, GEOGRAPHY AND AGE OF THE EPIDEMIC ON HIV-1 NEUTRALIZATION BY ANTIBODIES [PublishAheadOfPrint]

  • Neutralizing antibodies (nAbs) are a high priority for vaccines that aim to prevent the acquisition of HIV-1 infection. Vaccine effectiveness will depend on the extent to which induced antibodies neutralize the global diversity of circulating HIV-1 variants. Using large panels of genetically and geographically diverse HIV-1 Env-pseudotyped viruses and chronic infection plasma samples, we unambiguously show that cross-clade nAb responses are commonly induced in response to infection by any virus clade. Nonetheless, neutralization was significantly greater when the plasma clade matched the clade of the virus being tested. This within-clade advantage was diminished in older, more diverse epidemics in southern Africa, the US and Europe compared to more recent epidemics in Asia. It was most pronounced for circulating recombinant form (CRF) 07_BC, which is common in China and is the least divergent lineage studied; this was followed by the slightly more diverse Asian CRF01_AE. We found no evidence that transmitted/founder viruses are generally more susceptible to neutralization and are therefore easier targets for vaccination than chronic viruses. Features of the gp120 V1V2 loop, in particular length, net charge and number of N-linked glycans, were associated with Env susceptibility and plasma neutralization potency in a manner consistent with neutralization-escape being a force that drives viral diversification and plasma neutralization breadth. The overall susceptibility of Envs and potencies of plasmas were highly predictive of the neutralization outcome of any single virus/plasma combination. These findings highlight important considerations for the design and testing of candidate HIV-1 vaccines that aim to elicit effective nAbs.

    IMPORTANCE An effective HIV-1 vaccine will need to overcome the extraordinary variability of the virus, which is most pronounced in the envelope glycoproteins (Env) that are the sole targets for neutralizing antibodies (nAbs). Distinct genetic lineages, or clades, of HIV-1 occur in different locales that may require special consideration when designing and testing vaccines candidates. We show that nAb responses to HIV-1 infection are generally active across clades but are most potent within clades. Because effective vaccine-induced nAbs are likely to share these properties, optimal coverage of a particular clade or combination of clades may require clade-matched immunogens. Optimal within-clade coverage might be easier to achieve in regions such as China and Thailand, where the epidemic is more recent and the virus less diverse than in southern Africa, the US and Europe. Finally, features of the first and second hypervariable regions of gp120 (V1V2) may be critical for optimal vaccine design.

  • Residue 82 of the Chikungunya Virus E2 Attachment Protein Modulates Viral Dissemination and Arthritis in Mice [PublishAheadOfPrint]

  • Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that has reemerged to cause profound epidemics of fever, rash, and arthralgia throughout sub-Saharan Africa, Southeast Asia, and the Caribbean. Like other arthritogenic alphaviruses, mechanisms of CHIKV pathogenesis are not well defined. Using the attenuated CHIKV strain 181/25 and virulent strain AF15561, we identified a residue in the E2 viral attachment protein that is a critical determinant of viral replication in cultured cells and pathogenesis in vivo. Viruses containing an arginine at E2 residue 82 displayed enhanced infectivity in mammalian cells but reduced infectivity in mosquito cells and diminished virulence in a mouse model of CHIKV disease. Mice inoculated with virus containing an arginine at this position exhibited reduced swelling at the site of inoculation with a concomitant decrease in the severity of necrosis in joint-associated tissues. Viruses containing a glycine at E2 residue 82 produced higher titers in the spleen and serum at early times postinfection. Using wildtype and glycosaminoglycan (GAG)-deficient Chinese hamster ovary (CHO) cell lines and soluble GAGs, we found that an arginine at residue 82 conferred greater dependence on GAGs for infection of mammalian cells. These data suggest that CHIKV E2 interactions with GAGs diminish dissemination to lymphoid tissue, establishment of viremia, and activation of inflammatory responses early in infection. Collectively, these results suggest a function for GAG utilization in regulating CHIKV tropism and host responses that contribute to arthritis.

    IMPORTANCE CHIKV is a reemerging alphavirus of global significance with high potential to spread into new, immunologically naiiuml;ve populations. The severity of CHIKV disease, particularly its propensity for chronic musculoskeletal manifestations, emphasizes the need for identification of genetic determinants that dictate CHIKV virulence in the host. To better understand mechanisms of CHIKV pathogenesis, we probed the function of an amino acid polymorphism in the E2 viral attachment protein using a mouse model of CHIKV musculoskeletal disease. In addition to influencing glycosaminoglycan utilization, we identified roles for this polymorphism in differential infection of mammalian and mosquito cells and targeting of CHIKV to specific tissues within infected mice. These studies demonstrate a correlation between CHIKV tissue tropism and virus-induced pathology modulated by a single polymorphism in E2, which in turn illuminates potential targets for vaccine and antiviral drug development.

  • Sapovirus Translation Requires an Interaction Between VPg and the Cap Binding Protein eIF4E [PublishAheadOfPrint]

  • Sapoviruses of the Caliciviridae family of small RNA viruses, are emerging pathogens that cause gastroenteritis in humans and animals. Molecular studies on human sapovirus have been hampered due to the lack of a cell culture system. In contrast, porcine sapovirus (PSaV) can be grown in cell culture making it a suitable model for understanding the infectious cycle of sapoviruses and related-enteric caliciviruses. Caliciviruses are known to use a novel mechanism of protein synthesis that relies on the interaction of cellular translation initiation factors with the virus encoded VPg protein that is covalently linked to the 5' end of the viral genome. Using PSaV as a representative member of the Sapovirus genus, we characterized the role of the viral VPg protein in sapovirus translation. As observed for other caliciviruses, the PSaV genome was found to be covalently linked to VPg and this linkage was required for the translation and the infectivity of viral RNA. The PSaV VPg protein was associated with the eIF4F complex in infected cells and bound directly to the eIF4E protein. As have been previously demonstrated for feline calicivirus, a member of the Vesivirus genus, PSaV translation required eIF4E and the interaction between eIF4E and eIF4G. Overall our study provides new insights into the novel mechanism of sapovirus translation suggesting that sapovirus VPg can hijack the cellular translation initiation mechanism by recruiting the eIF4F complex through a direct eIF4E interaction.

    IMPORTANCE Sapoviruses, of the Caliciviridae family, are one of the causative agents of viral gastroenteritis in humans. However, human sapovirus remains non cultivable in the cell culture hampering the ability to characterize the virus infectious cycle. Here, we show that the VPg protein from porcine sapovirus, the only cultivatable sapovirus, is essential for viral translation and functions via a direct interaction with the cellular translation initiation factor eIF4E. This work provides new insights into the novel protein primed mechanism of calicivirus VPg-dependent translation initiation.

  • Establishment and characterization of a lethal mouse model for the Angola strain of Marburg virus [PublishAheadOfPrint]

  • Infections with Marburg (MARV) and Ebola virus (EBOV) cause severe hemorrhagic fever in humans and non-human primates (NHPs) with fatality rates up to 90%. A number of experimental vaccine and treatment platforms have previously been shown to be protective against EBOV infection. However, the rate of development for prophylactics and therapeutics against MARV has been slower in comparison, possibly because a small animal model is not widely available. Here we report the development of a mouse model for studying the pathogenesis of MARV Angola (MARV/Ang), the most virulent strain of MARV. Infection with the wild-type virus does not cause disease in mice, but the adapted virus (MARV/Ang-MA) recovered from liver homogenates after 24 serial passages in severe combined immunodeficient (SCID) mice caused severe disease when administered intranasally (IN) or intraperitoneally (IP). The LD50 was determined to be 0.015 TCID50 MARV/Ang-MA in SCID mice, and IP infection at a dose of 1000 x LD50 resulted in death between 6-8 days post-infection (dpi) in SCID mice. Similar results were obtained with immunocompetent BALB/c and C57BL/6 mice challenged IP with 2000 x LD50 of MARV/Ang-MA. Virological and pathological analyses in MARV/Ang-MA infected BALB/c mice revealed that the associated pathology was reminiscent of observations made in NHPs with MARV/Ang. MARV/Ang-MA infected mice showed most of the clinical hallmarks observed with Marburg hemorrhagic fever, including lymphopenia, thrombocytopenia, marked liver damage and uncontrolled viremia. Virus titers reached 108 TCID50/ml in the blood, and between 106-10 TCID50/g tissue in the intestines, kidney, lungs, brain, spleen, and liver. This model provides an important tool to screen candidate vaccines and therapeutics against MARV infections.

    Importance The Angola strain of Marburg virus (MARV/Ang) was responsible for the largest outbreak ever documented for Marburg viruses. At a 90% fatality rate, it is similar to Ebola virus, which makes it one of the most lethal viruses known to humans. There are currently no approved interventions for Marburg virus, in part because a small animal model that is vulnerable to MARV/Ang infection is not available to screen and test potential vaccines and therapeutics in a quick and economical manner. To address this need, we have adapted MARV/Ang so that it causes illness in mice resulting in death. The signs of disease in these mice are reminiscent of wild-type MARV/Ang infections in humans and nonhuman primates. We believe this will be of help in accelerating the development of life-saving measures against Marburg virus infections.

  • Epstein-Barr Virus Glycoproteins gB and gHgL Can Mediate Fusion and Entry in Trans; Heat Can Act as a Partial Surrogate for gHgL and Trigger a Conformational Change in gB [PublishAheadOfPrint]

  • Epstein-Barr virus (EBV) fusion with an epithelial cell requires virus glycoproteins gHgL and gB and is triggered by an interaction between gHgL and integrins aalpha;vbbeta;5, aalpha;vbbeta;6 or aalpha;vbbeta;8. Fusion with a B cell requires gHgL, gp42 and gB and is triggered by an interaction between gp42 and HLA class II. We report here that like alpha and betaherpesviruses, EBV, a gammaherpesvirus, can mediate cell fusion if gB and gHgL are expressed in trans. Entry of a gH-null virus into an epithelial cell is possible if the epithelial cell expresses gHgL and entry of the same virus, which phenotypically lacks gHgL and gp42, into a B cell expressing gHgL is possible in the presence of a soluble integrin. Heat is capable of inducing fusion of cells expressing only gB and the proteolytic digestion pattern of gB in virions changes in the same way following exposure of virus to heat or to soluble integrins. It is suggested that the Gibbs free energy released as a result of the high affinity interaction of gHgL with an integrin may contribute to the activation energy required to cause refolding of gB from a prefusion to a postfusion conformation.

    IMPORTANCE The core fusion machinery of herpesviruses consists of glycoproteins gB and gHgL. We demonstrate that as in alpha and betaherpesvirus, gB and gHgL of the gammaherpesvirus EBV can mediate fusion and entry when expressed in trans in opposing membranes implicating interactions between the ectodomains of the proteins in activation of fusion. We further show that heat and exposure to a soluble integrin, both of which activate fusion, result in the same changes in the proteolytic digestion pattern of gB possibly representing the refolding of gB from its prefusion to its post fusion conformation.

  • The HSV-2 Virion-associated Ribonuclease, vhs, Interferes with Stress Granule Formation [PublishAheadOfPrint]

  • In a previous study, it was observed that cells infected with herpes simplex virus type 2 (HSV-2) failed to accumulate stress granules (SGs) in response to oxidative stress induced by arsenite treatment. As a follow up to this observation, we demonstrate here that disruption of arsenite-induced SG formation by HSV-2 is mediated by a virion component. Through studies on SG formation in cells infected with HSV-2 strains carrying defective forms of UL41, the gene that encodes vhs, we identify vhs as a virion component required for this disruption. Cells infected with HSV-2 strains producing defective forms of vhs form SGs spontaneously late in infection. In addition to core SG components, these spontaneous SGs contain the viral immediate early protein ICP27 as well as the viral serine/threonine kinase Us3. As part of these studies, we re-examined the frameshift mutation known to reside within the UL41 gene of HSV-2 strain HG52. We demonstrate that this mutation is unstable and can rapidly revert to restore wild type UL41 following low multiplicity passaging. Identification of the involvement of virion-associated vhs in the disruption of SG formation will enable mechanistic studies on how HSV-2 is able to counteract antiviral stress responses early in infection. In addition, the ability of Us3 to localize to stress granules may indicate novel roles for this viral kinase in regulation of translation.

    IMPORTANCE Eukaryotic cells respond to stress by rapidly shutting down protein synthesis and storing mRNAs in cytoplasmic stress granules (SGs). Stoppages in protein synthesis are problematic for all viruses as they rely on host cell machinery to synthesize viral proteins. Thus, many viruses target SGs for disruption or modification. Infection by herpes simplex virus type 2 (HSV-2) was previously observed to disrupt SG formation induced by oxidative stress. In this follow up study, we identify virion host shutoff protein (vhs) as a viral protein involved in this disruption. Identification of a specific viral protein involved in disrupting SG formation is a key step towards understanding how HSV-2 interacts with these antiviral structures. Additionally, this understanding may provide insights into the biology of SGs that may find application in studies on human motor neuron degenerative diseases, like amyotrophic lateral sclerosis (ALS), which may arise as a result of disregulation of SG formation.

  • Organization of Capsid-associated Tegument Components in Kaposi's Sarcoma-associated Herpesvirus [PublishAheadOfPrint]

  • Capsid-associated tegument proteins have been identified in alpha- and beta-herpesviruses to play an essential role in viral DNA-packaging. Whether and how such tegument proteins exist in gamma-herpesviruses have been a mystery. Here we report a 6AAring; resolution cryoEM structure of Kaposi's sarcoma-associated herpesvirus (KSHV) virion, a member of the oncogenic gamma-herpesvirus subfamily. The KSHV virion structure reveals for the first time how capsid-associated tegument proteins are organized in a gamma-herpesvirus mmdash; with five tegument densities cap each penton vertex mmdash; a pattern highly similar to that in alpha-herpesvirus but completely different from that in beta-herpesvirus. Each KSHV tegument density can be divided into three prominent regions: a penton-binding globular region, a helix-bundle stalk region and a bbeta;-sheet-rich triplex-binding region. Fitting of crystal structure of the truncated HSV-1 UL25 (the KSHV ORF19 homolog) and secondary structure analysis of the full-length ORF19 establish that ORF19 constitutes the globular region with an N-terminal, 60 amino acid long helix extending into the stalk region. Matching secondary structural features resolved in the cryoEM density with secondary structures predicted by sequence analysis identifies the triplex-binding region to be ORF32, a homolog of alpha-herpesvirus UL17. Despite high-level of tegument structural similarities between KSHV and alpha-herpesvirus, an ORF19 monomer in KSHV, in contrast to a UL25 dimer in alpha-herpesviruses, binds each penton subunit, an observation that correlates with conformational difference in their pentons. This newly discovered organization of triplex-ORF32-ORF19 also resolves a long-standing mystery surrounding the virion location and conformation of the alpha-herpesvirus UL25.

    Importance Several capsid-associated tegument proteins have been identified in the alpha- and beta-herpesvirus subfamilies of the herpesviridae. These tegument proteins play essential roles in viral propagation and are potential drug targets for curbing herpesvirus infections. However, no such tegument proteins have been identified for gamma-herpesviruses, the third herpesvirus subfamily that contains members causing several human cancers. Here, by high-resolution cryo electron microscopy (cryoEM), we show the three-dimensional structure of the capsid-associated tegument proteins in the prototypical member of gamma-herpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV). The cryoEM structure reveals that the organization of KSHV capsid-associated tegument proteins is highly similar to that in alpha-herpesvirus, but completely different from that in beta-herpesvirus. Structural analyses further localize ORF19 and ORF32 proteins (the alpha-herpesvirus UL25 and UL17 homologs in KSHV respectively) in the KSHV capsid-associated tegument cryoEM structure. These findings also resolve a long-standing mystery regarding the location and conformation of alpha-herpesvirus UL25 inside the virion.

  • Autographa californica multiple nucleopolyhedrovirus orf132 encodes a nucleocapsid-associated protein required for budded virus and multiply enveloped occlusion-derived virus production [PublishAheadOfPrint]

  • Autographa californica multiple nucleopolyhedrovirus orf132, named ac132, has homologs in all genome-sequenced group I nucleopolyhedroviruses. Its role in viral replication cycle is unknown. In this study, ac132 was shown to express a protein of around 28 kD, which was determined to be associated with the nucleocapsids of both occlusion-derived virus and budded virus. Confocal microscopy showed that AC132 protein appeared in central region of the nucleus as early as 12 hours post infection with the virus. It formed a ring zone at the periphery of the nucleus by 24 hours post infection. To investigate its role in virus replication, ac132 was deleted from the viral genome by using a bacmid system. In the Sf9 cell culture transfected by the ac132-knochout bacmid, infection was restricted to single cells, and the titer of infectious budded virus reduced to undetectable level. However, viral DNA replication and the expression of late genes vp39 and odv-e25 and a reporter gene under control of the very late gene p10 promoter were unaffected. Electron microscopy showed that nucleocapsids, virions, and occlusion bodies were synthesized in the cells transfected by an ac132-knockout bacmid, but the formation of the virogenic stroma and occlusion bodies was delayed, the numbers of enveloped nucleocapsid was reduced and the occlusion bodies contained mainly singly enveloped nucleocapsids. AC132 was found to interact with envelope protein ODV-E18 and the viral DNA-binding protein P6.9. The data from this study suggest that ac132 possibly plays an important role in the assembly and envelopment of nucleocapsids.

    Importance To our knowledge, this is the first report on a functional analysis of ac132. The data presented here demonstrate that ac132 is required for production of the budded virus and multiply enveloped occlusion-derived virus of Autographa californica multiple nucleopolyhedrovirus. The paper revealed unique phenotypic changes induced by ac132 deletion on the virus and multiple new findings on ac132.

  • Catalytic Function and Substrate Specificity of the PLpro Domain of nsp3 from the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) [PublishAheadOfPrint]

  • The papain-like protease (PLpro) domain from the deadly Middle East Respiratory Syndrome coronavirus (MERS-CoV) was over-expressed and purified. MERS-CoV PLpro constructs with or without the putative ubiquitin-like (UBL) domain at the N-terminus were found to possess protease, deubiquitinating, deISGylating, and interferon antagonism activities in transfected HEK293T cells. The quaternary structure and substrate preferences of MERS-CoV PLpro were determined and compared to those of SARS-CoV PLpro, revealing prominent differences between these closely related enzymes. Steady-state kinetic analyses of purified MERS-CoV and SARS-CoV PLpros uncover significant differences in their rates of hydrolysis of 5-aminomethyl coumarin (AMC) from C-terminally labeled peptide, ubiquitin and ISG15 substrates, as well as in their rates of isopeptide bond cleavage of K48- and K63-linked polyubiquitin chains. MERS-CoV PLpro was found to have an 8-fold and 3,500-fold higher catalytic efficiency for hydrolysis of the ISG15-AMC over the Ub-AMC and Z-RLRGG-AMC substrates respectively. A similar trend is observed for SARS-CoV PLpro although it is much more efficient than MERS-CoV PLpro towards ISG15-AMC and peptide-AMC substrates. MERS-CoV PLpro was found to process K48- and K63-linked polyubiquitin chains with similar rates and debranching patterns producing monoubiquitin species. However, SARS-CoV PLpro much prefers K48-linked polyubiquitin chains to K63-linked chains, and it rapidly produces di-ubiquitin molecules from K48-linked chains. Finally, potent inhibitors of SARS-CoV PLpro were found to have no effect on MERS-CoV PLpro. A homology model of MERS-CoV PLpro structure was generated and compared to the X-ray structure of SARS-CoV PLpro to provide plausible explanations for differences in substrate and inhibitor recognition.

    IMPORTANCE Unlocking the secrets of how coronavirus (CoV) papain-like proteases (PLpros) perform their multifunctional roles during viral replication entails a complete mechanistic understanding of their substrate recognition and enzymatic activities. We show that the PLpro domains from the MERS and SARS coronaviruses can recognize and process the same substrates but with different catalytic efficiencies. The differences in substrate recognition between these closely related PLpros suggest that neither enzyme can be used as a generalized model to explain the kinetic behavior of all CoV PLpros. As a consequence, decoding the mechanisms of PLpro-mediated antagonism of the host innate immune response and the development of anit-CoV PLpro enzyme inhibitors will be a challenging undertaking. The results from this study provide valuable information for understanding how MERS-CoV PLpro-mediated antagonism of the host innate immune response is orchestrated and insight into the design of inhibitors against MERS-CoV PLpro./Background

  • Role of the hypervariable region 1 (HVR1) for the interplay of hepatitis C virus with entry factors and lipoproteins [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) particles associate with lipoproteins and infect cells using at least four cell entry factors. These include the scavenger receptor class B type I (SR-BI), CD81, claudin 1 (CLDN1) and occludin (OCLN). Little is known about specific functions of individual host factors during HCV cell entry and viral domains that mediate interaction with these factors. The HVR1 within the viral envelope protein 2 (E2) is involved in usage of SR-BI and conceals the viral CD81 binding site. Moreover, deletion of this domain alters the density of virions. We compared lipoprotein interaction, surface attachment, receptor usage and cell entry between wild type HCV and a viral mutant lacking this domain. Deletion of HVR1 did not affect CD81, CLDN1 and OCLN usage. However, unlike wild type HCV, HVR1-deleted viruses were not neutralized by antibodies and small molecules targeting SR-BI. Nevertheless, modulation of SR-BI cell surface expression altered infection efficiency of both viruses to similar levels. Analysis of affinity purified virions revealed comparable levels of ApoE incorporation into viruses with or without HVR1. However, ApoE incorporated into these viruses was differentially recognized by ApoE-specific antibodies. Thus, SR-BI has at least two functions during cell entry. One of them can be neutralized by SR-BI-targeting molecules and it is critical only for wild type HCV. The other one is important for both viruses, but apparently is not inactivated by those SR-BI-binding antibodies and small molecules evaluated here. In addition, HVR1 modulates the conformation and/or epitope exposure of virus particle assocated ApoE.

    Importance HCV cell entry is SR-BI-dependent irrespective of the presence or absence of HVR1. Moreover, this domain modulates the properties of ApoE on the surface of virus particles. These findings have implications for the development of SR-BI targeting antivirals. Furthermore, they highlight separable functions of SR-BI during HCV cell entry and reveal a novel role of HVR1 for the properties of virus associated lipoproteins.

  • Multiple groups of endogenous epsilon-like endogenous retroviruses conserved across primates. [PublishAheadOfPrint]

  • Several types of cancer in fish are caused by retroviruses, including those responsible for major outbreaks of disease, such as walleye dermal sarcoma virus and salmon swim bladder sarcoma virus. These viruses form a phylogenetic group often described as the "epsilonretrovirus" genus. Epsilon-like retroviruses have become endogenous retroviruses (ERVs) on several occasions, integrating into germline cells to become part of the host genome, and sections of fish and amphibian genomes are derived from epsilon-like retroviruses. However, epsilon-like ERVs have been identified in very few mammals.

    We have developed a pipeline to screen full genomes for ERVs and using this pipeline, we have located over 800 endogenous epsilon-like ERV fragments in primate genomes. Genomes from 32 species of mammals and birds were screened and epsilon-like ERV fragments were found in all primate and tree shrew genomes but no others. These viruses appear to have entered the genome of a common ancestor of old and new world monkeys between 42 million and 65 million years ago

    Based on these results, there is an ancient evolutionary relationship between epsilon-like retroviruses and primates. Clearly, these viruses had the potential to infect the ancestors of primates and were at some point a common pathogen in these hosts. Therefore, this result raises questions about the potential of epsilonretroviruses to infect humans and other primates and about the evolutionary history of these retroviruses.

    Importance Epsilonretroviruses are a group of retroviruses which cause several important diseases in fish. Retroviruses have the ability to become a permanent part of the DNA of their host by entering the germline as endogenous retroviruses (ERVs), where they lose their infectivity over time but can be recognised as retroviruses for millions of years. Very few mammals are known to have epsilon-like ERVs, however, we have identified over 800 fragments of endogenous epsilon-like ERVs in the genomes of all major groups of primates, including humans. These viruses seem to have circulated and infected primate ancestors 42 to 65 million years ago. We are now interested in how these viruses have evolved and whether they have the potential to infect modern humans or other primates.

  • Immunodeficient Mouse Models with Different Disease Profiles by in vivo Infection with the Same Clinical Isolate of Enterovirus 71 [PublishAheadOfPrint]

  • Like poliovirus, severe infection with enterovirus 71 (EV71) can cause neuropathology. Unlike poliovirus, EV71 is often associated with hand, foot, and mouth disease (HFMD). Here, we established three mouse models for experimental infection with the same clinical isolate of EV71. The NOD/SCID mouse model is unique in developing skin rash, an HFMD-like symptom. While the NOD/SCID model developed limb paralysis and death at near 100% efficiency, the interferon-gamma receptor knockout (ifngr KO) and the stat-1 knockout mice exhibited paralysis and death rates near 78% and 30%, respectively. Productive infection with EV71 depends on viral dose, host age, and inoculation routes. Infectious EV71, and VP1-specific RNA and protein in muscle, brain, and spinal cord, were compared side-by-side between NOD/SCID and stat-1 models before, during, and after disease onset. Spleen fibrosis and muscle degeneration are common in NOD/SCID and stat-1 models. Main differences between these two models include their disease manifestations and cytokine/chemokine profiles. Pathology of the NOD/SCID model includes 1) inflammation and expression of viral VP1 antigen in muscle; 2) increased neutrophils and decreased eosinophils and lymphocytes; 3) hair loss and skin rash. Characteristic pathology of the stat-1 model includes 1) a strong tropism of EV71 for central nervous system; 2) detection of VP1 protein in the Purkinje layer of cerebellar cortex, pons, brainstem, and spinal cord; 3) amplification of microglial cells; 4) dystrophy of intestinal villi. Our comparative studies on these new models by oral or intraperitoneal (i.p.) infection underscored the contribution of host immunity, including interferon-gamma receptor, to EV71 pathogenesis.

    IMPORTANCE In the past decade, enterovirus 71 (EV71) has emerged as a major threat to the public health in the Asia-Pacific regions. Disease manifestations include subclinical infection, common cold-like syndromes, hand-foot-and-mouth disease (HFMD), uncomplicated brainstem encephalitis, severe dysregulated autonomic nerve system, fatal pulmonary edema, and cardiopulmonary collapse. To date, no effective vaccine or treatment is available. A user-friendly and widely accessible animal model for researching EV71 infection and pathogenesis is urgently needed by the global community, both academia and industry.

  • Kinetic and phenotypic analysis of CD8+ T cell responses after priming with alphavirus replicons and homologous or heterologous booster immunizations [PublishAheadOfPrint]

  • Alphavirus replicons are potent inducers of CD8+ T cell responses and thus constitute an attractive vaccine vector platform for developing novel vaccines. However, the kinetics and memory phenotype of CD8+ T cell responses induced by alphavirus replicons are not well characterized. Furthermore, little is known how priming with alphavirus replicons affects booster immune responses induced by other vaccine modalities. We demonstrate that a single immunization with an alphavirus replicon, administered as viral particles or naked DNA, induced an antigen-specific CD8+ T cell response that had a sharp peak, followed by a rapid contraction. Administering a homologous boost before contraction had occurred did not further increase the response. In contrast, boosting after contraction when CD8+ T cells had obtained a memory phenotype (based on CD127/CD62L expression), resulted in maintenance of CD8+ T cells with a high recall capacity (based on CD27/CD43 expression). Increasing the dose of replicon particles promoted T effector memory (Tem) and inhibited T central memory (Tcm) development. Moreover, infection with a replicating alphavirus induced a similar distribution of CD8+ T cells as the replicon vector. Lastly, the distribution of T cell subpopulations induced by a DNA-launched alphavirus replicon could be altered by heterologous boosts. For instance, boosting with a poxvirus vector (MVA) favored expansion of the Tem compartment. In summary, we have characterized the antigen-specific CD8+ T cell response induced by alphavirus replicon vectors and demonstrated how it can be altered by homologous and heterologous boost immunizations.

    Importance Alphavirus replicons are promising vaccine candidates against a number of diseases and are by themselves developed as vaccines against for example chikungunya virus infection. Replicons are also considered to be used for priming followed by booster immunization using different vaccine modalities. In order to rationally design prime-boost immunization schedules with these vectors, characterization of the magnitude and phenotype of CD8+ T cell responses induced by alphavirus replicons is needed. Here, we demonstrate how factors such as timing and dose affect the phenotype of the memory T cell populations induced by immunization with alphavirus replicons. These findings are important for designing future clinical trials with alphaviruses, as they can be used to tailor vaccination regimens in order to induce a CD8+ T cell response that is optimal for control and/or clearance of a specific pathogen.

  • The temperature sensitive and attenuation phenotypes conferred by mutations in the influenza PB2, PB1, and NP genes are influenced by the species of origin of the PB2 gene in reassortant viruses derived from influenza A/California/07/2009 and A/WSN/33. [PublishAheadOfPrint]

  • Live attenuated influenza vaccines in the US are derived from a human virus that is temperature sensitive (ts), characterized by restricted (gge; 100-fold) replication at 39ddeg;C. The ts genetic signature (ts sig) has been mapped to 5 loci in 3 genes: PB1 (391E, 581G, and 661T), PB2 (265S) and NP (34G). However, when transferred into avian and swine influenza viruses, only partial ts and attenuation phenotypes occur. To investigate the reason for this, we introduced the ts sig into the human-origin virus A/WSN/33 (WSN), the avian-origin virus A/Vietnam/1203/04 (VN04), and the swine-origin triple reassortant 2009 pandemic H1N1 virus A/California/07/2009 (CA07), which contains gene segments from human-, avian- and swine-viruses. The VN04 ts sig and CA07 ts sig viruses replicated efficiently in MDCK cells at 39ddeg;C, but the replication of WSN ts sig was restricted gge; 100-fold compared to 33ddeg;C. Reassortant CA07 ts sig viruses were generated with individual polymerase gene segments from WSN, and vice versa. Only ts sig viruses with a PB2 gene segment derived from WSN were restricted in replication gge; 100-fold at 39ddeg;C. In ferrets, the CA07 ts sig virus replicated in the upper and lower respiratory tracts, but the replication of a reassortant CA07 ts sig virus with a WSN PB2 gene was severely restricted in the lungs. Taken together, these data suggest that the origin of the PB2 gene segment influences the ts phenotype in vitro and attenuation in vivo. This could have implications for the design of novel live vaccines against animal-origin influenza viruses.

    Importance Live attenuated influenza vaccines (LAIV) on temperature sensitive (ts) backbones derived from animal-origin influenza viruses are being sought for use in the poultry and swine industries and to protect people against animal-origin influenza. However, inserting the ts genetic signature from a licensed LAIV backbone fails to fully attenuate these viruses. Our data indicate this is associated with the presence of a PB2 gene segment derived from an avian influenza virus. We show that a reassortant 2009 pandemic H1N1 virus with the ts signature from a licensed LAIV donor virus is ts in vitro and attenuated in vivo when the PB2 gene is derived from a human-origin virus but not from an avian virus. Our study provides information that could benefit the rational design of alternative LAIV backbones against animal-origin influenza viruses.

  • Inhibition of breast cancer cell proliferation through disturbance of the Calcineurin/NFAT pathway by Human Herpesvirus-6B U54 tegument protein [PublishAheadOfPrint]

  • Nuclear factor of activated T cell (NFAT) proteins are key regulators involved in multiple physiological mechanisms such as immune response or cell growth. Selective calcineurin/NFAT inhibitors already demonstrated their capacity to decrease NFAT-dependent cancer cell progression, particularly in breast cancer. In this study, we report a role for the human herpesvirus 6B (HHV-6B) U54 tegument protein in inhibiting MCF-7 breast cancer cell line proliferation by inhibiting NFAT activation.

  • Inhibition of Interleukin-2 Gene Expression by Human Herpesvirus 6B U54 Tegument Protein [PublishAheadOfPrint]

  • Human Herpesvirus 6B (HHV-6B) is a ubiquitous pathogen causing life-long infections in approximately 95% of humans worldwide. To persist within its host, HHV-6B developed several immune evasion mechanisms such as latency during which minimal proteins are expressed and also by disturbing innate and adaptive immune responses. The primary cellular targets of HHV-6B are CD4+ T cells. Previous studies by Flamand et al. reported on the capacity of HHV-6A, as well as UV-irradiated HHV-6A at inhibiting interleukin-2 (IL-2) synthesis in CD4+ lymphocytes suggesting that viral structural components could be responsible for this effect. In the present study, we have identified the HHV-6B U54 tegument protein (U54) to be capable of inhibiting IL-2 expression. U54 binds the calcineurin (CaN) phosphatase enzyme causing improper dephosphorylation and nuclear translocation of nuclear factor of activated T cells (NFAT) proteins, resulting in sub-optimal IL-2 gene transcription. The U54 GISIT motif (aa 293-297), analogous to the NFAT PxIxIT motif, contributed to the inhibition of NFAT activation.

    IMPORTANCE Human herpesvirus-6A (HHV-6A) and HHV-6B are associated with an increasing number of pathologies. These viruses have developed strategies to avoid the immune response allowing them to persist into host. Several studies have illustrated mechanisms by which HHV-6A and HHV-6B are able to disrupt host defenses. Previous work informed us that HHV-6A is able to suppress interleukin-2 (IL-2) synthesis, a key immune growth factor essential for adequate T lymphocyte proliferation and expansion. We provide evidence that HHV-6B also inhibits IL-2 gene expression and identified the mechanisms by which it does so. Our work led us to the identification of U54, a virion-associated tegument protein, as being responsible for suppression of IL-2. Consequently, we have identified HHV-6B U54 protein as playing a role in immune evasion. These results further contribute to our understanding of HHV-6 interactions with its human host and the efforts deployed to ensure its long-term persistence.

  • The VP4 peptide of Hepatitis A Virus (HAV) Ruptures Membranes Through Formation of Discrete Pores [PublishAheadOfPrint]

  • Membrane active peptides, components of capsid structural proteins, assist viruses in overcoming the host membrane barrier in the initial stages of infection. Several such peptides have been identified and their roles in membrane fusion or disruption have been characterized through biophysical studies. In several members of the picornaviridae family, the role of the VP4 structural peptide in cellular membrane penetration is well established. However, there is not much information on the membrane penetrating capsid components of Hepatitis A Virus (HAV), an unusual member of this family. The VP4 peptide of HAV differs from its analogues in other picornaviruses by being significantly shorter in length, and also by lacking a signal for myristoylation, thought to be a critical requisite for VP4-mediated membrane penetration. Here we report, for the first time, that the atypical VP4 in HAV contains significant membrane-penetrating activity. Using a combination of biophysical assays and molecular dynamics simulation studies, we show that VP4 integrates into membrane vesicles through its N-terminal region, to finally form discrete pores of 5-9 nm diameter, which induces leakage in the vesicles without altering their overall size or shape. We further demonstrate that the membrane activity of VP4 is specific towards vesicles mimicking the lipid content of late endosomes, at acidic pH. Taken together, our data indicates that VP4 might be essential for the penetration of host endosomal membranes and release of genome during HAV entry.

    Importance Hepatitis A Virus (HAV) causes acute hepatitis in humans through the faecal-oral route, and is particularly prevalent in underdeveloped regions with poor hygienic conditions. Although a vaccine for HAV exists, its high cost makes it unsuitable for universal application in developing countries. Studies on host-virus interaction for HAV have been hampered due to lack of starting material, since the virus is extremely slow growing in culture. Among the unknown aspects of the HAV life cycle is its manner of host membrane penetration, which is one of the most important initial steps in viral infection. Here, we present data to suggest that a small peptide VP4, a component of the HAV structural polyprotein, might be essential in helping the viral genome cross cell membranes during entry. It is hoped that this work might help in elucidating the manner of initial host cell interaction by HAV.

  • Emergence of broadly neutralizing antibodies and viral co-evolution in two subjects during the early stages of infection with the human immunodeficiency virus type 1 [PublishAheadOfPrint]

  • Delineating the key early events that lead to the development of broadly neutralizing anti-HIV-1 antibodies during natural infection may help guide the development of immunogens and vaccine regimens to prevent HIV-1 infection. In this study, we followed two HIV-1 positive subjects, VC20013 and VC10014, over the course of infection from before they developed broadly neutralizing antibody (bNAb) activity until several years after breadth was detected in the plasma. Both subjects developed bNAb activity after approximately one year post infection, which ultimately mapped to the membrane proximal external region (MPER) in VC20013 and an epitope that overlaps the CD4 receptor binding site in VC10014. In subject VC20013, we were able to identify anti-MPER activity in the earliest plasma sample that exhibited no bNAb activity, indicating that this epitope specificity was acquired very early on, but that it was initially not able to mediate neutralization. Escape mutations within the bNAb epitopes did not arise in the circulating envelopes until bNAb activity was detectable in the plasma, indicating that this early response was not sufficient to drive viral escape. As bNAb activity began to emerge in both subjects, we observed a simultaneous increase in autologous anti-Envelope antibody binding affinity, indicating that antibody maturation was occurring as breadth was developing. Our findings illustrate one potential mechanism by which bNAbs develop during natural infection in which an epitope target is acquired very early on during the course of infection, but requires time and maturation to develop into broadly neutralizing activity.

    Importance One major goal of HIV-1 vaccine research is the development of a vaccine that can elicit broadly neutralizing antibodies (bNAbs). Although no such vaccine exists, bNAbs develop in approximately twenty percent of HIV-1-infected subjects, providing prototype of the bNAbs that must be re-elicited by vaccine. Thus, there is significant interest in understanding the mechanisms by which bNAbs develop during the course of infection. We studied the timing, the epitope specificity, and the evolution of the bNAb responses in two HIV-1 positive patients who developed bNAb activity within the first several years after infection. In one subject, antibodies to a broadly neutralizing epitope developed very early but were non-neutralizing. After several months neutralizing activity developed and the virus mutated to escape their activity. Our study highlights one mechanism for the development of bNAbs where early epitope acquisition followed by sufficient time for antibody maturation drives the epitope-specific antibody response toward broadly neutralizing activity.

  • Stearoyl-CoA Desaturase 1 Is Associated with Hepatitis C Virus Replication Complex and Regulates Viral Replication [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) life cycle is tightly regulated by lipid metabolism of host cells. In order to identify host factors involved in HCV propagation, we have recently screened the small interfering RNA (siRNA) library targeting host genes that control lipid metabolism and lipid droplet formation using cell culture grown HCV (HCVcc)-infected cells. We selected and characterized the gene encoding stearoyl-CoA desaturase 1 (SCD1). siRNA-mediated knockdown or pharmacological inhibition of SCD1 abrogated HCV replication in both subgenomic replicon and Jc1-infected cells, while exogenous supplementation of either oleate or palmitoleate, products of SCD1 activity, resurrected HCV replication in SCD1 knockdown cells. SCD1 was coimmunoprecipitated with HCV nonstructural proteins, and colocalized with both dsRNA and HCV nonstructural proteins, indicating that SCD1 is associated with HCV replication complex. Moreover, SCD1 was fractionated and enriched with HCV nonstructural proteins at detergent-resistant membrane. Electron microscopy data showed that SCD1 is required for NS4B-mediated intracellular membrane rearrangement. These data further support that SCD1 is associated with HCV replication complex and its products may contribute to the proper formation and maintenance of membranous web structures in HCV replication complex. Collectively, these data suggest that manipulation of SCD1 activity may represent a novel host-targeted antiviral strategy for the treatment of HCV infection.

    IMPORTANCE Stearoyl-CoA desaturase 1, a liver-specific enzyme, regulates HCV replication through its enzyme activity. HCV nonstructural proteins are associated with SCD1 at detergent-resistant membranes and SCD1 is enriched on the lipid raft by HCV infection. Therein SCD1 supports NS4B-mediated membrane rearrangement to provide a suitable microenvironment for HCV replication. We demonstrated that either genetic or chemical knockdown of SCD1 abrogated HCV replication in both replicon cells and HCV-infected cells. These findings provide novel mechanistic insights into the roles of SCD1 in HCV replication.

  • Apolipoprotein E likely contributes to a maturation step of infectious hepatitis C virus particles and interacts with viral envelope glycoproteins [PublishAheadOfPrint]

  • Assembly of infectious hepatitis C virus (HCV) particles is tightly linked to components of the very-low-density lipoprotein (VLDL) pathway. We and others have shown that apolipoprotein E (ApoE) plays a major role in production of infectious HCV particles. However, the mechanism by which ApoE contributes to virion assembly/release and how it gets associated with the HCV particle is poorly understood. We found that knock-down of ApoE reduces titers of infectious intra- and extracellular HCV, but not of the related Dengue virus. ApoE depletion also reduced amounts of extracellular HCV core protein without affecting intracellular core amounts. Moreover, we found that ApoE depletion neither affected formation of nucleocapsids nor their envelopment suggesting that ApoE acts at a late step of assembly such as particle maturation and infectivity. Importantly, we demonstrate that ApoE interacts with the HCV envelope glycoproteins, most notably E2. This interaction was independent from other viral proteins and required the transmembrane domain of E2 that was also required for recruitment of HCV envelope glycoproteins to detergent-resistant membrane fractions. These results suggest that ApoE plays an important role in HCV particle maturation, presumably by direct interaction with viral envelope glycoproteins.

    Importance The hepatitis C virus (HCV) replication cycle is tightly linked to host cell lipid pathways and components. This is best illustrated by the dependency of HCV assembly on lipid droplets and the very-low-density lipoprotein (VLDL) component apolipoprotein E (ApoE). Although the role of ApoE for production of infectious HCV particles is well established, it is still poorly understood how ApoE contributes to virion formation and how it gets associated with HCV particles. Here, we provide experimental evidence that ApoE likely is required for an intracellular maturation step of HCV particles. Moreover, we demonstrate that ApoE associates with the viral envelope glycoproteins. This interaction appears to be dispensable for envelopment of virus particles, but likely contributes to a quality control of secreted infectious virions. These results shed new light onto the exploitation of host cell lipid pathways by HCV and the link of viral particle assembly to the VLDL component ApoE.

  • Visualization of retroviral envelope spikes in complex with the V3 loop antibody 447-52D on intact viruses by cryo-electron tomography [PublishAheadOfPrint]

  • The gp120 portion of the envelope spike on human immunodeficiency virus -1 (HIV-1) plays a critical role in viral entry into host cells and is a key target for the humoral immune response, yet many structural details remain elusive. We have used cryoelectron tomography to visualize the binding of the broadly neutralizing monoclonal antibody (MAb) 447-52D to intact envelope spikes on virions of HIV-1 MN strain. Antibody 447-52D has previously been shown to bind to the tip of the V3 loop. Our results show antibody arms radiating from the sides of the gp120 protomers at a range of angles and place the antibody-bound V3 loop in an orientation that differs from that predicted by most current models but consistent with the idea that antibody binding dislodges the V3 loop from its location in the Env spike and making it flexible and disordered. These data reveal information on the position of the V3 loop, its relative flexibility, and suggest that 447-52D neutralizes HIV-1 MN by capturing the V3 loop, blocking its interaction with the co-receptor and altering the structure of the envelope spike.

    IMPORTANCE Antibody neutralization is one of the primary ways that the body fights infection with HIV. Because HIV is a highly mutable virus, the body must constantly produce new antibodies to counter new strains of HIV that the body itself is producing. Consequently, antibodies capable of neutralizing multiple HIV strains are comparatively few. An improved understanding of the mechanism of antibody neutralization might advance the development of immunogens. Most neutralizing antibodies target the Env glycoprotein spikes found on the virus surface. The broadly neutralizing antibody 447-52D targets the highly conserved bbeta;-turn of variable loop 3 (V3) of gp120. The importance of V3 lies in its contribution to the coreceptor binding site on the target cell. Here we show that 447-52D binding to V3 converts the Env conformation from closed to open and makes the V3 loop highly flexible, implying disruption of coreceptor binding and attachment to the target cell.

  • The N-terminus of the influenza B virus nucleoprotein is essential for virus viability, nuclear localization and optimal transcription and replication of the viral genome. [PublishAheadOfPrint]

  • The nucleoprotein (NP) of influenza viruses is a multifunctional protein with essential roles throughout viral replication. Despite influenza A and B viruses belonging to separate genera of the Orthomyxoviridae family, their NP proteins share a relatively high level of sequence conservation. However NP of influenza B viruses (BNP) contains an evolutionarily conserved N-terminal 50 amino acid extension that is absent from NP of influenza A viruses. There is conflicting evidence as to the functions of the BNP N-terminal extension, however this has never been assessed in the context of viral infection. We have used reverse genetics to assess the significance of this region on the functions of BNP and virus viability. Truncation of more than three amino acids prevented virus recovery suggesting that the N-terminal extension is essential for virus viability. Mutational analysis indicated that multiple regions of the protein are involved in nuclear localization of BNP with the entire N-terminal extension required for this to function efficiently. Viruses containing mutations in the first ten residues of BNP demonstrated little differences in nuclear localization, however the viruses exhibited significant reductions in viral mRNA transcription and genome replication resulting in significantly attenuated phenotypes. Mutations introduced to ablate a previously reported nuclear localization signal also resulted in a significant decrease in mRNA production during early stages of viral replication. Overall our results demonstrate that the N-terminal extension of BNP is essential to virus viability not only for directing nuclear localization of BNP, but also for regulating viral mRNA transcription and genome replication.

    Importance The multifunctional nucleoprotein (NP) of influenza viruses has roles throughout the viral replication cycle and is therefore essential for virus viability. Despite high levels of homology between the NP proteins of influenza A and B viruses the NP of influenza B virus (BNP) contains an evolutionarily conserved 50 amino acid N-terminal extension that is absent from the NP of influenza A viruses. In this study we show that this N-terminal extension is essential for virus viability and we confirm and expand upon recent findings that this region of BNP is required for nuclear localization of the protein. Furthermore we demonstrate for the first time that the N-terminus of BNP is involved in regulating viral mRNA transcription and replication of the viral genome. As the NP of influenza A virus lacks this N-terminal extension it suggests that these viruses have evolved separate mechanisms to regulate these processes.

  • High frequencies of polyfunctional CD8+ NK cells in chronic HIV-1 infection are associated with slower disease progression [PublishAheadOfPrint]

  • Summary: Natural killer (NK) cells are effector and regulatory innate immune cells and play a critical role in the first line of defense against various viral infections. Although previous reports indicated vital contributions of NK cells to HIV-1 immune control, non-genetic NK cell parameters directly associated with slower disease progression have not been defined yet. In a longitudinal, retrospective study of 117 untreated HIV-infected subjects we show that higher frequencies as well as absolute numbers of CD8+CD3- lymphocytes are linked to delayed HIV-1 disease progression. We show that the majority of these cells are well-described blood NK cells. In a subsequent cross-sectional study we demonstrate a significant loss of CD8+ NK cells in untreated HIV-infected individuals, which correlated with HIV viral loads and inversely correlated with CD4+ T cell counts. CD8+ NK cells had modestly higher frequencies of CD57-expressing cells compared to CD8- cells but no differences in the expression of a number of activating and inhibiting NK cell receptors. However, CD8+ NK cells exhibited a more functional profile as detected by cytokine production and degranulation.

    Importance: We demonstrate that the frequency of highly functional CD8+ NK cells is inversely associated with HIV-related disease markers and linked with delayed disease progression. These results thus indicate that CD8+ NK cells represent a novel NK cell-derived, innate immune correlate with an improved clinical outcome in HIV infection.

  • Epitope Mapping of the Hemagglutinin Molecule of A/(H1N1)pdm09 Virus by Using Monoclonal Antibody Escape Mutants [PublishAheadOfPrint]

  • We determined the antigenic structure of pandemic influenza A(H1N1)pdm09 virus hemagglutinin (HA) using 599 escape mutants that were selected using 16 anti-HA monoclonal antibodies (MAbs) against A/Narita/1/2009. The sequencing of mutant HA genes revealed 43 amino acid substitutions at 24 positions in three antigenic sites, Sa, Sb and Ca2, which were previously mapped onto A/Puerto Rico/8/34 (A/PR/8/34) HA (A.J. Caton, G.G. Brownlee, J.W. Yewdell, and W. Gerhard, Cell 31:417-427, 1982), and an undesignated site, i.e., amino acid residues 141, 142, 143, 171, 172, 174, 177 and 180 in the Sa site; residues 170, 173, 202, 206, 210, 211 and 212 in the Sb site; residues 151, 154, 156, 157, 158, 159, 200 and 238 in the Ca2 site; and residue 147 in the undesignated site (numbering begins at the first methionine). Sixteen MAbs were classified into four groups based on their cross-reactivity with the panel of escape mutants in the hemagglutination inhibition test. Among them, six MAbs targeting the Sa and Sb sites recognized both residues at positions 172 and 173. MAb n2 lost reactivity when mutations were introduced at positions 147, 159 (site Ca2), 170 (site Sb) and 172 (site Sa). We designated the site consisting of these residues as site Pa. From 2009 to 2013, no antigenic drift was detected for the A(H1N1)pdm09 viruses. However, if a novel variant carrying a mutation at a position involved in the epitopes of several MAbs, such as 172, appeared, such a virus would have the advantage of becoming a drift strain.

    IMPORTANCE The first influenza pandemic of the 21st century occurred in 2009 with the emergence of a novel virus originating from swine influenza, A(H1N1)pdm09. Although HA of A(H1N1)pdm09 has a common origin (1918 H1N1) with seasonal H1N1, the antigenic divergence of HA between the seasonal H1N1 and A(H1N1)pdm09 viruses gave rise to the influenza pandemic in 2009. To take precautions against the antigenic drift of the A(H1N1)pdm09 virus in the near future, it is important to identify its precise antigenic structure. To obtain various mutants that are not neutralized by MAbs, it is important to neutralize several plaque-cloned parent viruses rather than only a single parent virus. We characterized 599 escape mutants that were obtained by neutralizing four parent viruses of A(H1N1)pdm09 in the presence of 16 MAbs. Consequently, we were able to determine the details of the antigenic structure of HA, including a novel epitope.

  • Equine viperin restricts equine infectious anemia virus replication by inhibiting the production and/or release of viral Gag, Env and receptor by distorting the endoplasmic reticulum [PublishAheadOfPrint]

  • Viperin is an endoplasmic reticulum (ER)-associated multifunctional protein that regulates virus replication and possesses broad antiviral activity. In many cases, viperin interferes with the trafficking and budding of viral structural proteins by distorting the membrane transportation system. The lentivirus equine infectious anemia virus (EIAV) has been studied extensively. In this study, we examined the restrictive effect of equine viperin (eViperin) on EIAV replication and investigated the possible molecular basis of this restriction to obtain insights into the effect of this cellular factor on retroviruses. We demonstrated that EIAV infection of primary equine monocyte-derived macrophages (eMDMs) up-regulated the expression of eViperin. The overexpression of eViperin significantly inhibited the replication of EIAV in eMDMs, and knockdown of eViperin transcription enhanced the replication of EIAV in eMDMs by approximate 45.8%. Further experiments indicated that eViperin restricts EIAV at multiple steps of viral replication. The overexpression of eViperin inhibited EIAV Gag release. Both the aalpha;-helix domain and radical SAM domain were required for this activity. However, the essential motifs in SAM were different from those reported for the inhibition of HIV-1 Gag by human viperin. Furthermore, eViperin disrupted the synthesis of both EIAV Env and receptor, which consequently inhibited viral production and entry, respectively, and this disruption was dependent on the eViperin aalpha;-helix domain. Using immunofluorescence assays and electron microscopy, we demonstrated that the aalpha;-helix domain is responsible for the distortion of the endoplasmic reticulum (ER). Finally, EIAV did not exhibit counteracting eViperin at the protein level.

    IMPORTANCE In previously studies, viperin was indicated restricting virus replications primarily by the inhibition of virus budding. Here, we show that viperin may have multiple antiviral mechanisms including the reduction of EIAV Gag budding and Env expression, and these activities are dependent on different viperin domains. Especially, we demonstrate that overexpression of viperin inhibits EIAV entry by decreasing the virus receptor. Therefore, viperin restriction of viruses is largely determined by the dependence of virus on cellular membrane transportation system.

  • Type I interferon protects mice from fatal neurotropic infection with Langat virus by systemic and local anti-viral response [PublishAheadOfPrint]

  • Vector-borne flaviviruses such as tick-borne encephalitis virus (TBEV), West Nile virus and dengue virus cause millions of infections in humans. TBEV causes a broad range of pathological symptoms ranging from meningitis to severe encephalitis or even hemorrhagic fever with high mortality. Despite the availability of an effective vaccine, incidence of TBEV infections is increasing. Not much is known about the role of the innate immune system in the control of TBEV infections. Here, we show that the type I interferon (IFN) system is essential for protection against TBEV and Langat virus (LGTV) in mice. In the absence of a functional IFN system, mice rapidly develop neurological symptoms and succumb to LGTV and TBEV infections. Type I IFN system deficiency results in severe neuro-inflammation in LGTV-infected mice characterized by breakdown of the blood-brain barrier and infiltration of macrophages into the central nervous system (CNS). Using mice with tissue-specific IFN receptor deletions, we show that a coordinated activation of the type I IFN system in peripheral tissues as well as in the CNS is indispensable for viral control and protection against virus induced inflammation and fatal encephalitis.

    Importance The type I interferon (IFN) system is important to control viral infections, however, the interactions between tick-borne encephalitis virus (TBEV) and the type I IFN system is poorly characterized. TBEV causes severe infections in humans that are characterized by fever and debilitating encephalitis, which can progress to chronic illness or death. No treatment options are available. An improved understanding of antiviral innate immune responses is pivotal for the development of effective therapeutics. We show that type I IFN, an effector molecule of the innate immune system is responsible for the extended survival of TBEV and Langat virus (LGTV), an attenuated member of the TBE serogroup. IFN production and signaling appeared to be essential in two different phases during infection: first in the periphery, by reducing systemic LGTV replication and spreading into the central nervous system (CNS). Secondly, the local IFN response in the CNS prevents virus-induced inflammation and the development of encephalitis.

  • The matrix gene segment destabilizes the acid and thermal stability of the hemagglutinin of pandemic live attenuated influenza virus vaccines [PublishAheadOfPrint]

  • The threat of future influenza pandemics and their potential for rapid spread, morbidity, and mortality has led to the development of pandemic vaccines. We generated 7 reassortant pandemic live attenuated influenza vaccines (pLAIV) with the hemagglutinin (HA) and neuraminidase (NA) genes derived from animal influenza viruses on the backbone of the 6 internal protein gene segments of the temperature sensitive, cold-adapted (ca) A/Ann Arbor/60 (H2N2) virus (AA/60 ca) of the licensed seasonal LAIV. The pLAIVs were moderately to highly restricted in replication in seronegative adults; we sought to determine the biological basis for this restriction. Avian influenza viruses generally replicate at higher temperatures than human influenza viruses and although they shared the same backbone, the pLAIVs had a lower shut off temperature than seasonal LAIVs, suggesting the HA and NA influence the degree of temperature sensitivity. The pH of HA activation of highly pathogenic avian influenza viruses was greater than human and low pathogenicity avian influenza viruses, as reported by others. However, pLAIVs had a consistently higher pH of HA activation and reduced HA thermostability compared with the corresponding wild-type parental viruses. From studies with single gene reassortant viruses bearing one gene segment from the AA/60 ca virus in recombinant H5N1 or pH1N1 viruses, we found that the lower HA thermal stability and increased pH of HA activation were associated with the AA/60 M gene. Together, the impaired HA acid and thermal stability and temperature sensitivity likely contributed to the restricted replication of the pLAIVs we observed in seronegative adults.

    Significance: There is increasing evidence that HA stability of influenza viruses depends on the virus strain and host species and that HA stability can influence replication, virulence, and transmission of influenza A viruses in different species. We investigated the HA stability of pandemic live attenuated influenza vaccines (pLAIVs) and observed that the pLAIVs consistently had a less stable HA than the corresponding wild-type influenza viruses. The reduced HA stability and temperature sensitivity of the pLAIVs may account for their restricted replication in clinical trials.

  • Methyltransferase-defective avian metapneumovirus vaccines provide complete protection against challenge with homologous Colorado strain and heterologous Minnesota strain [PublishAheadOfPrint]

  • Avian metapneumovirus (aMPV), also known as avian pneumovirus or turkey rhinotracheitis virus, is the causative agent of turkey rhinotracheitis, and is associated with swollen head syndrome in chickens. Since its discovery in the 1970s, aMPV has been recognized as an economically important pathogen in the poultry industry worldwide. The conserved region VI (CR-VI) of the large (L) polymerase proteins of paramyxoviruses catalyzes methyltransferase (MTase) activities that typically methylate viral mRNAs at guanine N-7 (G-N-7) and ribose 2'-O positions. In this study, we generated a panel of recombinant aMPV (raMPV) Colorado strains carrying mutations in the S-adenosyl methionine (SAM) binding site in the CR-VI of L protein. These recombinant viruses were specifically defective in ribose 2'-O, but not G-N-7 methylation, and were genetically stable and highly attenuated in cell culture and viral replication in the upper and lower respiratory tracts of specific-pathogen-free (SPF) young turkeys. Importantly, turkeys vaccinated with these MTase-defective raMPVs triggered a high level of neutralizing antibody and were completely protected from challenge with homologous aMPV Colorado strain and heterologous aMPV Minnesota strain. Collectively, our results indicate that (i) aMPV lacking 2'-O methylation is highly attenuated in vitro and in vivo, and (ii) inhibition of mRNA cap MTase can serve as a novel target to rationally design live attenuated vaccines for aMPV, and perhaps other paramyxoviruses.

    Importance Paramyxoviruses include many economically and agriculturally important viruses such as avian metapneumovirus (aMPV), and Newcastle disease virus (NDV); human pathogens such as human respiratory syncytial virus, human metapneumovirus, human parainfluenza virus type 3, and measles virus; and highly lethal emerging pathogens such as Nipah virus, and Hendra virus. For many of them, there is no effective vaccine or antiviral drug. These viruses share common strategy for viral gene expression and replication. During transcription, paramyxoviruses produce capped, methylated, and polyadenylated mRNAs. Using aMPV as a model, we found that viral ribose 2'-O methyltransferase (MTase) is a novel approach to rationally attenuate the virus for vaccine purpose. Recombinant aMPV (raMPV) lacked 2'-O MTase were not only highly attenuated in turkeys, but also provided complete protection against the challenge of homologous and heterologous aMPV strains. This novel approach can be applicable to other avian and human paramyxoviruses for rationally designing live attenuated vaccines.

  • Modulation of hepatitis C virus genome replication by glycosphingolipids and FAPP2 protein [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) assembles its replication complex on cytosolic membrane vesicles often clustered in a membranous web (MW). During infection, HCV NS5A protein activates PI4KIIIaalpha; enzyme, causing massive production and redistribution of phosphatidylinositol 4-phosphate (PI4P) lipid to the replication complex. However, the role of PI4P in HCV lifecycle is not well understood. We postulated that PI4P recruits host effectors to modulate HCV genome replication or virus particles production. To test this hypothesis, we generated cell lines for doxycycline-inducible expression of shRNAs targeting PI4P effector, four-phosphate adaptor protein 2 or FAPP2. FAPP2 depletion attenuated HCV infectivity and impeded HCV RNA synthesis. Indeed, FAPP2 has two functional lipid-binding domains specific for PI4P and glycosphingolipids. While expression of the PI4P-binding mutant protein was expected to inhibit HCV replication, a marked drop in replication efficiency was unexpectedly observed with the glycosphingolipid-binding mutant protein. These data suggest that both domains are crucial for the role of FAPP2 in HCV genome replication. We also found that HCV significantly increases the level of some glycosphingolipids, whereas adding these lipids to FAPP2 depleted cells partially rescued replication, further arguing for the importance of glycosphingolipids in HCV RNA synthesis. Interestingly, FAPP2 is redistributed to the replication complex (RC) characterized by HCV NS5A, NS4B or dsRNA foci. Additionally, FAPP2 depletion disrupts the RC and alters the co-localization of HCV replicase proteins. Altogether, our study implies that HCV co-opts FAPP2 for virus genome replication via PI4P-binding and glycosphingolipids transport to the HCV RC.

    IMPORTANCE Like most viruses with a positive sense RNA genome, HCV replicates its RNA on remodeled host membranes composed of lipids hijacked from various internal membrane compartments. During infection, HCV induces massive production and retargeting of the PI4P lipid to its replication complex. However, the role of PI4P in HCV replication is not well understood. In this study, we have shown that FAPP2, a PI4P effector and glycosphingolipid-binding protein, is recruited to the HCV replication complex, required for HCV genome replication and replication complex formation. More importantly, this study demonstrates for the first time, the crucial role of glycosphingolipids in HCV lifecycle and suggests a link between PI4P and glycosphingolipids in HCV genome replication.

  • Cross-clade Ultrasensitive PCR-based Assays to Measure HIV Persistence in Large Cohort Studies [PublishAheadOfPrint]

  • A small pool of infected cells persists in HIV-infected individuals receiving antiretroviral therapy (ART). Here, we developed ultrasensitive assays to precisely measure the frequency of cells harbouring total HIV DNA, integrated HIV DNA and 2-LTR circles. These assays are performed on cell lysates, which circumvents the labour intensive step of DNA extraction and rely on the co-quantification of each HIV molecular form together with CD3 gene sequences to precisely measure cell input. Using primary isolates from HIV subtypes A, B, C, D, and CRF01_AE, we demonstrate that these assays can efficiently quantify low target copy numbers from diverse HIV subtypes. We further used these assays to measure total HIV DNA, integrated HIV DNA and 2-LTR circles in CD4+ T cells from HIV-infected subjects infected with subtype B. All samples obtained from ART-naiiuml;ve subjects were positive for the three HIV molecular forms (n=15). In ART-suppressed individuals, total HIV DNA, integrated HIV DNA and 2-LTR circles were detected in 100%, 94% and 77% of the samples. Higher levels of total HIV DNA and 2-LTR circles were detected in untreated subjects when compared to individuals on ART (p=0.0003 and p=0.0004, respectively), while the frequency of CD4+ T cells harbouring integrated HIV DNA did not differ between the two groups. These results demonstrate that these novel assays have the ability to quantify very low levels of HIV DNA from multiple HIV subtypes without the need of nucleic acid extraction, making them well-suited for monitoring viral persistence in large populations of HIV-infected individuals.

    Importance Since the discovery of viral reservoirs in HIV-infected subjects receiving suppressive ART, measuring the degree of viral persistence has been one of the greatest challenges in the field of HIV research. Here, we report the development and validation of ultrasensitive assays to measure HIV persistence in HIV-infected individuals from multiple geographical regions. These assays are relatively inexpensive, do not require DNA extraction and can be completed in a single day. Therefore, they are perfectly adapted to monitor HIV persistence in large cohorts of HIV infected individuals, and given their sensitivity, can be used to monitor the efficacy of therapeutic strategies aimed at interfering with HIV persistence after prolonged ART.

  • Trans-protease activity and structural insights into the active form of the alphavirus capsid protease [PublishAheadOfPrint]

  • The alphavirus capsid protein (CP) is a serine protease that possesses cis-proteolytic activity essential for its release from the nascent structural polyprotein. The released CP further participates in viral genome encapsidation, nucleocapsid core formation followed by its attachment to glycoproteins and virus budding. Thus, protease activity of the alphavirus capsid is a potential anti-alphaviral target to arrest capsid release, maturation and structural polyprotein processing. However, the discovery of capsid protease inhibitors has been hampered due to the lack of a suitable screening assay and crystal structure in its active form. Here we report the development of a trans-proteolytic activity assay for Aura virus capsid protease (AVCP) based on fluorescence resonance energy transfer (FRET) for screening protease inhibitors. Kinetic parameters using fluorogenic peptide substrates were estimated, and a KM value was found to be 2.63 pplusmn; 0.62 mmu;M, and a Kcat/KM value was 4.97 x 104 Mnndash;1 minnndash;1. Also, the crystal structure of the trans-active form of AVCP has been determined to 1.81 AAring; resolution. Structural comparisons of the active form with the crystal structures of available substrate-bound mutant and inactive blocked forms of the capsid protease identifies conformational changes in the active site, the oxyanion hole and the substrate specificity pocket residues, which could be critical for rational drug design.

    IMPORTANCE The alphavirus capsid protease is an attractive antiviral therapeutic target. In this study, we have described the formerly unappreciated trans-proteolytic activity of the enzyme and for the first time have developed a FRET based protease assay for screening capsid protease inhibitors. Our structural studies unveil the structural features of the trans-active protease which has been previously proposed to exist in the natively unfolded form (1). The different enzymatic forms have been structurally compared to reveal conformational variations in the active and substrate binding site. The flexible active site residue Ser218, the disordered C-terminal residues after His261, and the presence of a water molecule in the oxyanion hole of AVCP2 reveal the effect of the C-terminal Trp267 deletion on enzyme structure. New structural data reported in this study along with the fluorogenic assay will be useful in substrate specificity characterization, high-throughput protease inhibitor screening and structure-based development of antiviral drugs.

  • Gene expression profiling to predict and assess the consequences of therapy-induced virus eradication in chronic HCV [PublishAheadOfPrint]

  • Systems biology has proven to be a powerful tool to identify reliable predictors for treatment response in chronic HCV. In the present study, we studied chronic HCV patients who responded to IFN-based therapy as evidenced by absence of HCV RNA at the end of treatment, and focused on two issues that have not received much attention. Firstly, we evaluated whether specific genes or gene expression patterns in blood were able to distinguish responder patients with a viral relapse from responder patients who remained virus-negative after cessation of treatment. We found that chronic HCV patients who were sustained responders and relapsers to IFN-based therapy showed comparable baseline clinical parameters and immune composition in blood. However, at baseline, the gene expression profiles of a set of 18 genes predicted treatment outcome with an accuracy of 94%. Secondly, we examined whether patients with successful therapy-induced clearance of HCV still exhibited gene expression patterns characteristic for HCV, or whether normalization of their transcriptome was observed. We observed that the relatively high expression of IFN-stimulated genes (ISG) in chronic HCV patients prior to therapy was reduced after successful IFN-based antiviral therapy (at 24 weeks follow-up). These ISG included CXCL10, OAS1, IFI6, DDX60, TRIM5 and STAT1. In addition, 1428 differentially expressed non-ISG genes were identified in paired pre- and post-treatment samples from sustained responders, which included genes involved in TGF-bbeta; signaling, apoptosis, autophagy, and nucleic acid and protein metabolism. Interestingly, 1424 genes were identified with altered expression in responder patients after viral eradication in comparison to normal expression levels in healthy individuals. Additionally, aberrant expression of a subset of these genes, including IL-32, IL-16, CCND3 and RASSF1, was also observed at baseline.

    Our findings indicate that successful antiviral therapy of chronic HCV patients does not lead to normalization of their blood transcriptional signature. The altered transcriptional activity may reflect HCV-induced liver damage in previously infected individuals.

    Importance Tools to predict the efficacy of antiviral therapy of chronic HCV patients are important to select the optimal therapeutic strategy. Using a systems biology approach, we identify a set of 18 genes expressed in blood that predicts the recurrence of HCV RNA after cessation of therapy consisting of peginterferon and ribavirin. This set of genes may be applicable as a useful biomarker in clinical decision-making since the number of genes included in the predictor is small and the correct prediction rate is high (94%). In addition, we observe that the blood transcriptional profile in chronic HCV patients who were successfully treated is not normalized to the status observed in healthy individuals. Even 6 months after therapy-induced elimination of HCV RNA, gene expression profiles in blood are still altered in these chronic HCV patients, strongly suggesting long-term modulation of immune parameters in previously infected patients.

  • Isolation of dengue virus-specific memory B cells with live virus antigen from human subjects following natural infection reveals the presence of diverse novel functional groups of antibody clones [PublishAheadOfPrint]

  • Natural dengue virus (DENV) infection in humans induces antibodies (Abs) that neutralize the serotype of infection in a potent and type-specific manner, however most Abs generated in response to infection are serotype cross-reactive and poorly neutralizing. Such cross-reactive Abs may enhance disease during subsequent infection with virus of a different DENV serotype. Previous screening assays for DENV-specific human B cells and antibodies using viral and recombinant antigens mainly led to the isolation of dominant non-neutralizing B cell clones. To improve upon our ability to recover and study rare but durable and potently neutralizing DENV-specific Abs, we isolated human DENV-specific B cells using a primary screen of binding to live virus, followed by a secondary screen with a high-throughput flow cytometry-based neutralization assay to identify DENV-specific B cell lines prior to generation of hybridomas. Using this strategy, we identified several new classes of serotype-specific and serotype cross-neutralizing anti-DENV mAbs, including ultra-potent inhibitory antibodies with neutralizing activity concentrations less than 10 ng/mL. We isolated serotype-specific neutralizing Abs that target diverse regions of the E protein including epitopes present only on the intact fully-assembled viral particle. We also isolated a number of serotype cross-neutralizing mAbs, most of which recognized a region in the E protein domain I/II containing the fusion loop. These data provide insights into targets of the protective Ab-mediated immune response to natural DENV infection, which will prove valuable in the design and testing of new experimental DENV vaccines.

    Importance. Dengue virus infection is one of the most common mosquito-borne diseases and occurs in most countries of the world. Infection of humans with dengue virus induces a small number of antibodies that inhibit the infecting strain, but also induces a large number of antibodies that can bind but do not inhibit dengue virus strains of other serotypes. We used a focused screening strategy to discover a large number of rare potently inhibiting antibodies, and we mapped the regions on the virus that were recognized by such antibodies. The studies revealed that humans have the potential to generate very potent antibodies directed to diverse regions of the dengue virus surface protein. The studies provide important new information about protection from dengue infection that will be useful in the design and testing of new experimental dengue vaccines for humans.

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