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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]

  • Trunkloads of Viruses [Commentary]

  • 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 (EEHV1), which kills ~20% of Asian elephants (Elephas maximus) born in zoos and causes disease in the wild. EEHV1 is one of at least six distinct EEHV in a phylogenetic lineage that appears to represent an ancient but newly recognized subfamily (the Deltaherpesvirinae) in the family Herpesviridae.

  • 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 [Genetic Diversity and Evolution]

  • A family of novel endotheliotropic herpesviruses (EEHVs) 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 samples from six lethal cases and two survivors. Overall, the data obtained encompass 57 genes, including orthologues of 32 core genes common to all herpesviruses, 14 genes found in some other herpesviruses, plus 10 novel genes, including a single large putative transcriptional regulatory protein (ORF-L). On the basis of 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 ribonucleotide kinase B subunit (RRB), thymidine kinase (TK), and UL9-like origin binding protein (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 subgroups designated EEHV1A and EEHV1B with ancient partially chimeric features. Whereas large segments are nearly identical, three nonadjacent loci totaling 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. These viruses have caused a rapidly lethal hemorrhagic disease in 20% of all captive Asian elephant calves born in zoos in the United States 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 totaling 15 to 30% of the genomes from blood or necropsy tissue from eight different cases, we have determined that they fall into multiple types and chimeric subtypes of a novel Proboscivirus genus, and we propose that they should also be classified as the first examples of a new mammalian herpesvirus subfamily named the Deltaherpesvirinae.

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

  • The genomes of three types of novel endotheliotropic herpesviruses (elephant endotheliotropic herpesvirus 1A [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 samples or necropsy tissue samples from 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 analyzed differ from those of all other EEHVs by 37% and have a G+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 noncontiguous segments totaling 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 virus types likely initially diverged close to 100 million years ago when the ancestors of modern elephants split from all other placental mammals and then evolved into two major branches with high- or low-G+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 accompanying paper (L. K. Richman, J.-C. Zong, E. M. Latimer, J. Lock, R. C. Fleischer, S. Y. Heaggans, and G. S. Hayward, J. Virol. 88:13523nndash;13546, 2014, http://dx.doi.org/10.1128/JVI.01673-14). Here we have used PCR DNA sequence analysis from multiple segments of DNA amplified directly from blood or necropsy tissue samples 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.

  • The Conserved Disulfide Bond within Domain II of Epstein-Barr Virus gH Has Divergent Roles in Membrane Fusion with Epithelial Cells and B Cells [Virus-Cell Interactions]

  • 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 (DBs). The first is unique for EBV and closely related gammaherpesviruses. The second is conserved across the beta- and gammaherpesviruses 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, also was 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 posttransplant 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 the current studies, we found that mutants in the DB C278/C335 and the 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 a determinant for epithelial cell tropism during herpesvirus-induced membrane fusion and highlights a specific gH motif required for epithelial cell fusion.

  • Characterization of a Broadly Neutralizing Monoclonal Antibody That Targets the Fusion Domain of Group 2 Influenza A Virus Hemagglutinin [Vaccines and Antiviral Agents]

  • 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. In 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 few monoclonal antibodies (MAbs). 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 postexposure 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. Toward 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 (MAbs) 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.

  • Sendai Virus Pathogenesis in Mice Is Prevented by Ifit2 and Exacerbated by Interferon [Pathogenesis and Immunity]

  • 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 it 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 (Ifit2nndash;/nndash;) suffered severe weight loss and succumbed to intranasal infection with SeV strain 52 at a dose that killed only a few wild-type mice. Viral RNA was detectable only in lungs, and SeV titers were higher in Ifit2nndash;/nndash; mice than in 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 Ifit2nndash;/nndash; mice. Surprisingly, type I IFN receptor knockout (IFNARnndash;/nndash;) mice were less susceptible to SeV than Ifit2nndash;/nndash; 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/IFNARnndash;/nndash; double knockout mice. These mice were less susceptible to SeV than Ifit2nndash;/nndash; mice, although viral titers in their lungs were even higher. Our results indicate that high SeV replication in the lungs of infected Ifit2nndash;/nndash; 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 beneficial during virus infections. Here, we report two key findings. First, 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 Ifit2nndash;/nndash; lungs. Second, mortality of Ifit2nndash;/nndash; mice was reduced when mice also lacked the type I IFN receptor, while SeV loads in lungs still were 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.

  • Structural Plasticity of the Coiled-Coil Domain of Rotavirus NSP4 [Structure and Assembly]

  • Rotavirus (RV) nonstructural protein 4 (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 strain SA11 NSP4 forms a tetramer that binds Ca2+ at its core, the CCD of 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, it does not bind Ca2+ at low pH 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 nonstructural 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 [Virus-Cell Interactions]

  • Following entry into the target cell, human immunodeficiency virus type 1 (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 remains 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 postinfection, demonstrating that microtubule-facilitated uncoating is distinct from the previously reported role of viral reverse transcription in the uncoating process. We also find that pharmacological or small interfering RNA (siRNA)-mediated inhibition of cytoplasmic dynein or the kinesin 1 heavy chain KIF5B delays 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 the capsid motor interaction may reveal novel mechanisms of inhibition of 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 the cellular factors that facilitate uncoating, remains poorly understood. The main observation of this study is that normal uncoating requires intact microtubules and is facilitated by dynein and kinesin motors. Targeting these factors may either directly inhibit infection or delay it enough to trigger mediators of intrinsic immunity that recognize cytoplasmic capsid or DNA and subsequently induce an antiviral state in these cells.

  • The HERV-K Human Endogenous Retrovirus Envelope Protein Antagonizes Tetherin Antiviral Activity [Virus-Cell Interactions]

  • 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 the 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; therefore, it 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's 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 characterized 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, interindividual variations among HERV-K(HML2) Env genes may result in differences in immune responses to infection.

  • M27 Expressed by Cytomegalovirus Counteracts Effective Type I Interferon Induction of Myeloid Cells but Not of Plasmacytoid Dendritic Cells [Pathogenesis and Immunity]

  • 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 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 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 independently of TLR and RLH signaling, whereas in plasmacytoid dendritic cells, IFN-I induction was strictly TLR dependent. Macrophages and myeloid dendritic cells treated with either UV-inactivated MCMV or live MCMV that lacked the STAT2 antagonist M27 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 M27 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 evasins 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 induce IFN-I responses in fibroblasts and epithelial cells, 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 are all 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 9 (TLR9) dependently, whereas in myeloid cells, IFN-I induction is entirely TLR and RLH independent. MCMV-encoded M27 does not impair the IFN-I induction of plasmacytoid dendritic cells, while in myeloid dendritic cells, it reduces IFN-I responses. In macrophages, M27 plus other, not yet identified evasins profoundly inhibit the induction of IFN-I responses. Collectively, these results illustrate that MCMV has evolved diverse mechanisms to differentially modulate IFN-I responses in single immune cell subsets.

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

  • Poxviruses are composed of large double-stranded DNA (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 inversely 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 single nucleotide polymorphisms (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 suggests 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 evolutionary history of these virus pathogens may help predict their future evolutionary potential.

  • Establishment and Characterization of an Air-Liquid Canine Corneal Organ Culture Model To Study Acute Herpes Keratitis [Pathogenesis and Immunity]

  • Despite the clinical importance of herpes simplex virus (HSV)-induced ocular disease, the underlying pathophysiology of the disease remains poorly understood, in part due to the lack of adequate virusnndash;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, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays, and transmission electron microscopy (TEM). Next, canine corneas were inoculated with CHV-1 for 48 h, and at that time point postinfection, viral plaques could be visualized in the corneal epithelium and viral DNA copies were detected in both the infected corneas and 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 virusnndash;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. The advantages of this physiologically relevant model include the fact that (i) it provides a system in which ocular herpes can be studied in a virusnndash;natural-host setting and (ii) it reduces the number of experimental animals needed. In addition, this long-term explant culture model may also facilitate research in other fields where noninfectious and infectious ocular diseases of dogs and humans are being studied.

  • Crystal Structure of Herpes Simplex Virus 2 gD Bound to Nectin-1 Reveals a Conserved Mode of Receptor Recognition [Structure and Assembly]

  • Herpes simplex virus 1 (HSV-1) and 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 cocrystallography. Despite a high sequence identity between HSV-1 and HSV-2 gDs, the atomic intermolecule details for the HSV-2-gD/nectin-1 interaction remain elusive. Here, we report the crystal structures of both the unbound and the nectin-1-bound HSV-2 gDs. 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;-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 for HSV-1 and HSV-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 HSV-2 gDs, provided solid structural and functional evidence that HSV-1 and HSV-2 recognize 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 report the crystal structures of HSV-2 gD in both the free and the nectin-1-bound forms. The atomic intermolecule details for HSV-2-gD/nectin-1 interaction are 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 provide comprehensive structural and functional data demonstrating a conserved receptor-binding mode between HSV-1 and HSV-2 for nectin-1. Our results also indicate that the tropism difference between the two viruses likely arises from aspects other than the gD/nectin-1 binding features.

  • DDX3 DEAD-Box RNA Helicase Is a Host Factor That Restricts Hepatitis B Virus Replication at the Transcriptional Level [Genome Replication and Regulation of Viral Gene Expression]

  • 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 hepatitis B virus (HBV) polymerase (Pol) binding protein, and by using a transient transfection, we found that DDX3 inhibits HBV replication at the posttranscriptional level, perhaps following encapsidation. To determine the exact mechanism of the inhibition, we here employed a 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 the case with 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.

  • Vaginal Memory T Cells Induced by Intranasal Vaccination Are Critical for Protective T Cell Recruitment and Prevention of Genital HSV-2 Disease [Pathogenesis and Immunity]

  • Protective immunity against genital pathogens causing chronic infections, such as herpes simplex virus 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 kinase-deficient (TKnndash;) mutant of HSV-2 effectively induces HSV-2-specific gamma interferon (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 TKnndash; 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 antigen (Ag)-specific local effector T cells without introducing topical infection or inflammation.

    IMPORTANCE Intranasal (i.n.) vaccines against sexually transmitted diseases that are caused by viruses such as herpes simplex virus 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 i.n. immunization with HSV-2 will contribute to designing such a vaccine. Our study demonstrated that i.n. 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.

  • Analysis of the Evolution and Structure of a Complex Intrahost Viral Population in Chronic Hepatitis C Virus Mapped by Ultradeep Pyrosequencing [Genetic Diversity and Evolution]

  • Hepatitis C virus (HCV) causes chronic infection in up to 50% to 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 intrahost evolution are poorly understood. In this study, we used ultradeep pyrosequencing (UDPS) to map the 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 indicate that polyvirus infection of hepatocytes has occurred but that the fitness quotients of recombinant daughter virions are too low for the daughter virions 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 ultradeep pyrosequencing data sets derived from virus amplicons frequently relies on software tools that are not optimized 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 made use of a temporally matched reference sequence data set to inform error correction algorithms. Using this methodology, we were able to (i) detect multiple instances of hepatitis C virus intrasubtype recombination at the E1/E2 junction (a phenomenon rarely reported in the literature) and (ii) interrogate the longitudinal quasispecies complexity of the virome. Parallel to the UDPS, isolation of IgG-bound virions was found to coincide with the collapse of specific viral subpopulations.

  • Induced Maturation of Human Immunodeficiency Virus [Structure and Assembly]

  • HIV-1 assembles at the plasma membrane of virus-producing cells as an immature, noninfectious 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 virionmmdash;termed maturationmmdash;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 due mainly 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) washout from purified immature virions, thereby temporally uncoupling virus assembly and maturation. Drug washout resulted in the induction of proteolysis with cleavage efficiencies correlating with the off-rate of the respective PR-PI complex. Proteolysis of Gag was nearly complete and yielded the correct products with an optimal half-life (t1/2) of ~5 h, but viral infectivity was not recovered. Failure to gain infectivity following PI washout may be explained by the observed formation of aberrant viral capsids and/or by pronounced defects in processing of the reverse transcriptase (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 Gag and Gag-Pol HIV-1 polyproteins 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 washout strategy to synchronize activation of protease in wild-type virus. We demonstrated that nearly complete Gag processing and resolution of the immature virus architecture are 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.

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

  • Estimates for the risk of transmitting variant Creutzfeldt-Jakob disease (vCJD) via blood transfusion have relied largely 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 (i.c.) with brain tissues of patients with sporadic and familial CJD. We analyzed the data statistically by 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 (i.v.) and i.c. 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 nonleukoreduced red blood cells in the United Kingdom than 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 nonleukoreduced red blood cells and coagulation factor VIII manufactured from blood of United Kingdom 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.

  • Amino Acid Changes in the Influenza A Virus PA Protein That Attenuate Avian H5N1 Viruses in Mammals [Pathogenesis and Immunity]

  • 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 patients with 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's virulence and pathogenicity in mice. The PA protein thus plays a role in the pathogenicity of highly pathogenic H5N1 influenza viruses.

  • Characterization of an Alphamesonivirus 3C-Like Protease Defines a Special Group of Nidovirus Main Proteases [Genome Replication and Regulation of Viral Gene Expression]

  • 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 Coronaviridae and Roniviridae. The expression of these enzymes in virus-infected cells requires the 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', each are 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 to 16 kb) and large (26 to 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; thus, they are 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 mesoniviruses 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.

  • Molecular Mimicry between Dengue Virus and Coagulation Factors Induces Antibodies To Inhibit Thrombin Activity and Enhance Fibrinolysis [Pathogenesis and Immunity]

  • 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 antithrombin antibodies (ATAs) with both antithrombotic 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 preadsorption 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 subtropical 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.

  • Effects of Human Anti-Spike Protein Receptor Binding Domain Antibodies on Severe Acute Respiratory Syndrome Coronavirus Neutralization Escape and Fitness [Vaccines and Antiviral Agents]

  • The receptor binding domain (RBD) of the spike (S) glycoprotein of severe acute respiratory syndrome coronavirus (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 that overlaps the interface between the RBD and its receptor, angiotensin-converting enzyme 2 (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 strains, including neutralization escape viruses derived from other nAbs; however, no single nAb or 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 nAbs 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 has 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 immunostrategy for combating coronavirus 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 escape pathways of broad nAbs that target the receptor binding domain (RBD) of the virus. We found that neither single nAbs nor two nAbs in combination blocked escape. Our results suggest that targeting conserved regions with less plasticity and more structural constraint rather than the SARS-CoV RBD-like region(s) should have broader utility for antibody-based immunotherapy.

  • Transcriptome Responses of the Host Trichoplusia ni to Infection by the Baculovirus Autographa californica Multiple Nucleopolyhedrovirus [Cellular Response to Infection]

  • Productive infection of Trichoplusia ni cells by the baculovirus Autographa californica multiple nucleopolyhedrovirus (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 sequencing (RNA-seq) data set from infected and uninfected T. ni cells to generate and annotate a de novo transcriptome assembly of approximately 70,322 T. ni unigenes (assembled transcripts), representing the 48-h infection cycle. Using differential gene expression analysis, we found that the majority of host transcripts were downregulated after 6 h postinfection (p.i.) and throughout the remainder of the infection. In contrast, 5.7% (4,028) of the T. ni unigenes were upregulated during the early period (0 to 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 pathway members that were upregulated 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 they will stimulate new experimental approaches to understand, modify, and utilize baculoviruses for a variety of applications.

  • Chlorovirus Skp1-Binding Ankyrin Repeat Protein Interplay and Mimicry of Cellular Ubiquitin Ligase Machinery [Virus-Cell Interactions]

  • 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 an ancestral lineage with a broader group of related viruses, nucleo-cytoplasmic large DNA viruses (NCLDV). Chloroviruses encode an 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 (pox protein repeats of ankyrin at C-terminal) 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 Paramecium bursaria chlorella virus-1 (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 similarly to 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 types. Collectively, our results indicate that chloroviruses have evolved complementing 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 the 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 toward viral protein mimicry, substrate recognition, and key interactive domains controlling selective protein degradation. These findings may also further understanding of the evolution of other large DNA viruses, like poxviruses, that are reported to share the same monophyly lineage as chloroviruses.

  • The Exonuclease Domain of Lassa Virus Nucleoprotein Is Involved in Antigen-Presenting-Cell-Mediated NK Cell Responses [Pathogenesis and Immunity]

  • Lassa virus is an Old World Arenavirus which causes 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), low type I interferon (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 major histocompatibility complex (MHC) class I-related chains A and B and that they may produce interleukin-12 (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 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 the 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.

  • Overlapping and Distinct Molecular Determinants Dictating the Antiviral Activities of TRIM56 against Flaviviruses and Coronavirus [Virus-Cell Interactions]

  • 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 vesicular stomatitis virus (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 demonstrated that TRIM56's antiflavivirus 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 depends solely 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 viruses; 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.

  • The CD8+ Memory Stem T Cell (TSCM) Subset Is Associated with Improved Prognosis in Chronic HIV-1 Infection [Pathogenesis and Immunity]

  • Memory stem T cells (TSCM) constitute a long-lived, self-renewing lymphocyte population essential for the maintenance of 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 various degrees of viral control on and off highly active antiretroviral therapy (HAART). We observed that the frequency of CD8+ TSCM was decreased in all individuals with chronic, untreated HIV-1 infection and that HAART had 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 gut-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.

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

  • Tick-borne encephalitis (TBE) virus is an important human-pathogenic flavivirus endemic in large parts of Europe and 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. Postinfection and postvaccination 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, in contrast to what was found in mouse immunization studies with TBE and other flaviviruses, 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 and 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 are 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 postinfection and postvaccination 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.

  • p130Cas Scaffolds the Signalosome To Direct Adaptor-Effector Cross Talk during Kaposi's Sarcoma-Associated Herpesvirus Trafficking in Human Microvascular Dermal Endothelial Cells [Virus-Cell Interactions]

  • Kaposi's sarcoma-associated herpesvirus (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 focal adhesion kinase (FAK), Src, phosphoinositol 3-kinase (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 min postinfection. 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 small interfering RNA (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 suggest that simultaneous targeting of KSHV entry receptors with p130Cas would be an attractive potential avenue for therapeutic intervention in 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, extracellular matrix molecules, bacterial pathogens, and apoptotic cells. The present study identifies human microvascular dermal endothelial (HMVEC-d) cellular scaffold protein p130Cas (Crk-associated substrate) as a platform to promote Kaposi's sarcoma-associated herpesvirus (KSHV) trafficking. Early during KSHV de novo infection, p130Cas associates with lipid rafts and scaffolds EphrinA2 (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 cross talk of the KSHV entry-associated upstream signal complex from the immediate downstream trafficking-associated signalosome, consequently routing KSHV toward lysosomal degradation and eventually blocking KSHV infection and associated malignancies.

  • Severity of Clinical Disease and Pathology in Ferrets Experimentally Infected with Influenza Viruses Is Influenced by Inoculum Volume [Pathogenesis and Immunity]

  • Ferrets are a valuable model for influenza virus pathogenesis, virus transmission, and antiviral therapy studies. However, the contributions of the volume of inoculum administered and the ferret's respiratory tract anatomy to disease outcome have not been explored. We noted variations in clinical disease outcomes and the volume of inoculum administered and investigated these differences by administering two influenza viruses (A/California/07/2009 [H1N1 pandemic] and A/Minnesota/11/2010 [H3N2 variant]) to ferrets intranasally at a dose of 106 50% tissue culture infective doses in a range of inoculum volumes (0.2, 0.5, or 1.0 ml) and followed viral replication, clinical disease, and pathology over 6 days. Clinical illness and respiratory tract pathology were the most severe and most consistent when the viruses were administered in a volume of 1.0 ml. Using a modified micro-computed tomography 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 the 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 virus 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, it is highly desirable that the data from different laboratories be accurate and reproducible. We have found that, even when the same virus was administered at similar doses, different investigators reported a range of clinical disease outcomes, from asymptomatic infection to severe weight loss, ocular and nasal discharge, sneezing, and lethargy. We found that a wide range of inoculum volumes was 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.

  • Expanded Regulatory T Cells in Chronically Friend Retrovirus-Infected Mice Suppress Immunity to a Murine Cytomegalovirus Superinfection [Pathogenesis and Immunity]

  • 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 Friend virus (FV)-infected mice decreased murine cytomegalovirus (mCMV)-specific CD8+ T cell responses during acute mCMV superinfection. This suppression of mCMV-specific T cell immunity was found only in organs with FV-induced Treg expansion. Surprisingly, acute mCMV infection itself did not expand or activate Tregs.

  • 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 [Genetic Diversity and Evolution]

  • 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 the 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 the absence of the mutations in poultry/environmental samples, suggested that the mutations occurred during human infection and did not transmit further.

  • 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 [Vaccines and Antiviral Agents]

  • Extending our previous analyses to the most recently described monoclonal broadly neutralizing antibodies (bNAbs), we confirmed a drift of HIV-1 clade B variants over 2 decades toward higher resistance to bNAbs targeting almost all the identified gp120-neutralizing epitopes. In contrast, the sensitivity to bNAbs targeting the gp41 membrane-proximal external region remained stable, suggesting a selective pressure on gp120 preferentially. Despite this evolution, selected combinations of bNAbs remain capable of neutralizing efficiently most of the circulating variants.

  • Molecular Requirement for Sterols in Herpes Simplex Virus Entry and Infectivity [Virus-Cell Interactions]

  • Herpes simplex virus 1 (HSV-1) required cholesterol or desmosterol for virion-induced membrane fusion. HSV successfully entered DHCR24nndash;/nndash; cells, which lack a desmosterol-to-cholesterol conversion enzyme, indicating that 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.

  • Exonuclease Domain of the Lassa Virus Nucleoprotein Is Critical To Avoid RIG-I Signaling and To Inhibit the Innate Immune Response [Pathogenesis and Immunity]

  • 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 retinoic acid-inducible gene I (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.

  • Correction for Wilton et al., Effect of Formaldehyde Inactivation on Poliovirus [Author Correction]

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    JVI Accepts: Articles Published Ahead of Print

  • IMPACT AND REGULATION OF LAMBDA INTERFERON RESPONSE IN HUMAN METAPNEUMOVIRUS INFECTION [PublishAheadOfPrint]

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

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

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

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

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

  • Efficient infectious cell culture systems of the hepatitis C virus prototype strains HCV-1 and H77 [PublishAheadOfPrint]

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

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

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

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

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

  • Simian Hemorrhagic Fever Virus Cell Entry is Dependent on CD163 and Uses a Clathrin-mediated Endocytosis-like Pathway [PublishAheadOfPrint]

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

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

  • T cell Epitope Clustering in the Highly Immunogenic BZLF1 Antigen of Epstein-Barr virus [PublishAheadOfPrint]

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

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

  • The Megavirus chilensis Cu,Zn-Superoxide Dismutase: the first viral structure of a typical CCS-independent hyperstable dimeric enzyme [PublishAheadOfPrint]

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

    Importance Mimiviridae are giant viruses encoding more than 1000 proteins. The virion particles are loaded with proteins used by the virus to resist the vacuole oxidative stress. Megavirus chilensis virion contain a predicted copper, zinc superoxide dismutase (Cu,Zn-SOD). The corresponding gene is present in some Megavirus chilensis relatives but is absent from Mimivirus. This first crystallographic structure of a viral Cu,Zn-SOD highlights the common features and differences of the viral enzyme compared to cellular SODs. It corresponds to a very stable dimer of the apo form of the enzyme. We demonstrate that upon growth medium supplementation of Cu and Zn the recombinant protein is fully active, suggesting that the viral SOD activation is independent on a copper chaperone for SOD generally used by eukaryotic SODs.

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

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

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

  • High-Throughput RNA sequencing based virome analysis of 50 lymphoma cell lines from the Cancer Cell Line Encyclopedia project [PublishAheadOfPrint]

  • Using high-throughput RNA sequencing (RNA-seq) data from 50 common lymphoma cell culture models from the Cancer Cell Line Encyclopedia project, we performed an unbiased global interrogation for the presence of a panel of 740 viruses and strains known to infect human and other mammalian cells. This led to the findings of previously identified infections by Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV), and human T-lymphotropic virus 1 (HTLV-1). In addition, we also found a previously unreported infection of one cell line (DEL) with a murine leukemia virus. High expression of MuLV transcripts was observed in DEL cells and we identified 4 transcriptionally active integration sites, one being in the TNFRSF6B gene. We also found low levels of MuLV reads in a number of other cell lines and provided evidence suggesting cross-contamination during sequencing. Analysis of HTLV-1 integrations in two cell lines, HuT 102 and MJ, identified 14 and 66 transcriptionally active integration sites with potentially activating integrations in immune regulatory genes including IL15, IL6ST, STAT5B, HIVEP1, and IL9R. While KSHV and EBV do not typically integrate into the genome, we investigated a previously identified integration of EBV into the BACH2 locus in Raji cells. This analysis identified a BACH2 disruption mechanism involving splice donor sequestration. Through viral gene expression analysis, we detected expression of stable intronic RNAs from the EBV BamHI W repeats that may be part of long transcripts spanning the repeat region. We also observed transcripts at the EBV vIL10 locus exclusively in the Hodgkin's lymphoma cell line, Hs 611.T, the expression of which were uncoupled from other lytic genes. Assessment of the KSHV viral transcriptome in BCP-1 cells showed expression of the viral immune regulators, K2/vIL6, K4/vIL8-like vCCL1, and K5/E2-ubiquitin ligase 1 that was significantly higher than expression of the latency-associated nuclear antigen, LANA. Together, this investigation sheds light into the virus composition across these lymphoma model systems and provides insights into common viral mechanistic principles.

    Importance Viruses cause cancer in humans. In lymphomas the Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV) and human T-lymphotropic virus 1 (HTLV-1) are major contributors to oncogenesis. We assessed virus-host interactions using a high throughput sequencing method that facilitates the discovery of new virus-host associations and the investigation into how the viruses alter their host environment. We found a previously unknown murine leukemia virus infection in one cell line. We identified cellular genes including cytokine regulators that are disrupted by virus integration and we determined mechanisms through which virus integration causes deregulation of cellular gene expression. Investigation into the KSHV transcriptome in the BCP-1 cell line revealed high-level expression of immune signaling genes. EBV transcriptome analysis showed expression of vIL10 transcripts in a Hodgkin's lymphoma that was uncoupled from lytic genes. These findings illustrate unique mechanisms of viral gene regulation and to the importance of virus-mediated host immune signaling in lymphomas.

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

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

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

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

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

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

  • Uneven genetic robustness of HIV-1 integrase [PublishAheadOfPrint]

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

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

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

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

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

  • Modulation of Type I interferon-associated viral sensing during acute simian immunodefiency virus (SIV) infection in African green monkeys [PublishAheadOfPrint]

  • Natural hosts of simian immunodeficiency virus (SIV), such as African green monkeys (AGMs), do not progress to AIDS. This associates with an absence of a chronic type I interferon signature. It is unclear how the IFN-I response is downmodulated in AGMs. We longitudinally assessed the capacity of AGM blood cells to produce IFN-I in response to SIV and Herpes Simplex virus (HSV). Phenotype and function of plasmacytoid dendritic cells (pDCs) and other mononuclear blood cells were assessed by flow cytometry and expression of viral sensors measured by RT-PCR. pDC displayed low BDCA-2, CD40 and HLA-DR expression during acute SIVagm infection. BDCA-2 was required for sensing SIV, but not HSV, by pDCs. In acute infection, AGM PBMCs produced less IFN-I upon SIV stimulation. In chronic phase the production was normal, confirming that the lack of chronic inflammation is not due to a sensing defect of pDCs. In contrast to stimulation by SIV, more IFN-I was produced upon HSV stimulation of PBMC isolated during acute infection, while the frequency of AGM pDCs producing IFN-I upon in vitro stimulation with HSV was diminished. Indeed, other cells started producing IFN-I. This increased viral sensing by non-pDCs was associated with an upregulation of TLR3 and IFI16 caused by IFN-I in acute SIVagm infection. Our results suggest that, as in pathogenic SIVmac infection, SIVagm infection mobilizes bone marrow precursor pDC. Moreover, we show that SIV infection modifies the capacity of viral sensing in cells other than pDCs which could drive IFN-I production in specific settings.

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

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

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

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

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

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

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

  • APOBEC3A Functions as a Restriction Factor of Human Papillomavirus [PublishAheadOfPrint]

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

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

  • Shift in Monocyte Apoptosis with Increasing Viral Load and Change in Apoptosis-related ISG/Bcl2-family Gene Expression in Chronic HIV-1 Infected Subjects [PublishAheadOfPrint]

  • Although monocytes and macrophages are targets of HIV-1-mediated immunopathology, the impact of high viremia on activation-induced monocyte apoptosis relative to monocyte and macrophage activation changes remains undetermined. Here, we determined constitutive and oxidative stress-induced monocyte apoptosis in uninfected and HIV(+) individuals across a spectrum of viral load (n=35, range: 2,243 to 1,355,998 HIV-1 RNA copies/mL) and CD4 count (range: 26 to 801 cells/mm3). Both constitutive and oxidative stress-induced apoptosis were positively associated with viral load and negatively associated with CD4 with an elevation in apoptosis occurring in patients with greater than 40,000 (4.6 log) copies/mL. As expected, expression of Rb1 and interferon-stimulated genes (ISGs), plasma sCD163 concentration, and the proportion of CD14+/CD16+ intermediate monocytes were elevated in viremic patients compared to uninfected controls. Although most ISG expression, sCD14, sCD163, and CD14+/CD16+ frequencies were not directly associated with a change in apoptosis, sCD14 and ISG expression showed an association with increasing viral load. Multivariable analysis of clinical values and monocyte gene expression identified changes in IFI27, IFITM2, Rb1, and Bcl2 expression as determinants of constitutive apoptosis (p=3.77x10-5, adjusted-R2=0.5983), while changes in viral load, IFITM2, Rb1, and Bax expression were determinants of oxidative stress-induced apoptosis (p=5.59x10-5, adjusted-R2=0.5996). Our data demonstrate differential activation states in monocytes between levels of viremia in association with differences in apoptosis that may contribute to greater monocyte turnover with high viremia.

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

  • Polyreactivity and Autoreactivity among HIV-1 Antibodies [PublishAheadOfPrint]

  • It is generally acknowledged that human broadly neutralizing Abs (bNAbs) capable of neutralizing multiple HIV-1 clades are often poly- or autoreactive. Whereas poly-/autoreactivity has been proposed to be crucial for neutralization breadth, no systematic, quantitative study of self-reactivity among non-neutralizing HIV-1 Abs (nNAbs) has been performed to determine whether poly- or autoreactivity in bNAbs is a consequence of chronic antigen (Ag) exposure and/or inflammation or a fundamental property of neutralization. Here, we use protein microarrays to assess binding to ggt;9,000 human proteins and find that as a class, bNAbs are significantly more poly- and autoreactive than nNAbs. The poly- and autoreactive property is therefore not due to the infection milieu, but rather associated with neutralization. Our observations are consistent with a role of heteroligation for HIV-1 neutralization and/or structural mimicry of host Ags by conserved HIV-1 neutralization sites. Although bNAb are more mutated than nNAbs as a group, V(D)J mutation per se does not correlate with poly- and autoreactivity. Infrequent poly- or autoreactivity among nNAbs implies that their dominance in humoral responses is due to absence of negative control by immune regulation. Interestingly, four of nine bNAbs specific for the HIV-1 CD4 binding site (CD4bs) (VRC01, VRC02, CH106, and CH103) bind human ubiquitin ligase E3A (UBE3A) and UBE3A protein competitively inhibits gp120 binding to the VRC01 bNAb. Among these four bNAbs, avidity for UBE3A was correlated with neutralization breadth. Identification of UBE3A as a self-antigen recognized by CD4bs bNAbs offers a mechanism for the rarity of this bNAb class.

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

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

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

  • A newly-isolated reovirus has the simplest genomic and structural organization of any reovirus [PublishAheadOfPrint]

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

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

  • Mutation V111I in HIV-2 reverse transcriptase increases the fitness of the nucleoside analogue resistant K65R and Q151M viruses [PublishAheadOfPrint]

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

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

  • Amplified and Persistent Immune Responses Generated by Single Cycle Replicating Adenovirus Vaccines [PublishAheadOfPrint]

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

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

  • CD4+ T cell help is dispensable for protective CD8+ T cell memory against mousepox following vaccinia virus immunization [PublishAheadOfPrint]

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

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

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

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

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

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

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

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

  • Interaction of Infectious Spleen and Kidney Necrosis Virus ORF119L with PINCH Leads to Dominant-Negative Inhibition of ILK and Cardiovascular Defects in Zebrafish [PublishAheadOfPrint]

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

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

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

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

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

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

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

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

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

  • Role of ATM in the Formation of Replication Compartment During Lytic Replication of EBV in Nasopharyngeal Epithelial Cells [PublishAheadOfPrint]

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

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

  • Identification of capsid mutations that alter the rate of HIV-1 uncoating in infected cells [PublishAheadOfPrint]

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

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

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

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

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

  • The HIV-1 gp120 CD4-bound conformation is preferentially targeted by ADCC-mediating antibodies in sera from HIV-1-infected individuals. [PublishAheadOfPrint]

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

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

  • The sheep tetherin paralog, oBST2B, blocks envelope glycoprotein incorporation into nascent retroviral virions [PublishAheadOfPrint]

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

    Importance Bone marrow stromal cell antigen 2 (BST2) is a powerful cellular restriction factor against a wide range of enveloped viruses. Sheep possess two paralogs of the BST2 gene called oBST2A and oBST2B. Jaagsiekte sheep retrovirus (JSRV), the causative agent of a transmissible lung cancer of sheep, is known to be restricted by oBST2A. In this study we show that unlike oBST2A, oBST2B impairs the normal cellular trafficking of JSRV envelope glycoproteins by sequestering them within the Golgi apparatus. We also show that oBST2B decreases the incorporation of envelope glycoprotein into JSRV viral particles which in turn reduces virion infectivity. In conclusion, oBST2B exerts a novel antiviral activity that is distinct from BST2 proteins of other species.

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

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

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

  • Separate cellular localizations of HTLV-1 Env and GLUT1 are required for HTLV-1 Env-mediated fusion and infection [PublishAheadOfPrint]

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

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

  • Persistent HCV infection impairs ribavirin antiviral activity through clathrin-mediated trafficking of equilibrative nucleoside transporter 1 [PublishAheadOfPrint]

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

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

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

  • Human respiratory syncytial virus (RSV) lower respiratory tract infection can result in inflammation and mucus plugging of airways. RSV strain A2-line19F induces relatively high viral load and mucus in mice. The line19 fusion (F) protein harbors five unique residues compared to the non-mucus-inducing strains A2 and Long, at positions 79, 191, 357, 371, and 557. We hypothesized that differential fusion activity is a determinant of pathogenesis. In a cell-cell fusion assay, line19 F was more fusogenic than Long F. We changed the residues unique to line 19 F to the corresponding residues in Long F and identified residues 79 and 191 together as responsible for high fusion activity. Surprisingly, mutation of residues 357 or 357 with 371 resulted in gain of fusion activity. Thus, we generated RSV F mutants with a range of defined fusion activity and engineered these into recombinant viruses. We found a clear, positive correlation between fusion activity and early viral load in mice, however, we did not detect a correlation between viral loads and levels of airway mucin expression. The F mutant with the highest fusion activity, A2-line19F-K357T/Y371N, induced high viral loads, severe lung histopathology, and weight loss, but did not induce high levels of airway mucin expression. We defined residues 79/191 as critical for line19 F fusion activity and 357/371 as playing a role in A2-line19F mucus induction. Defining the molecular basis of the role of RSV F in pathogenesis may aid vaccine and therapeutic strategies aimed at this protein.

    Importance Human respiratory syncytial virus (RSV) is the most important lower respiratory tract pathogen of infants for which there is no vaccine. Elucidating mechanisms of RSV pathogenesis is important for rational vaccine and drug design. We defined specific amino acids in the fusion (F) protein of RSV strain line19 critical for fusion activity, and elucidated a correlation between fusion activity and viral load in mice. Further, we identified two distinct amino acids in F as contributing to the mucogenic phenotype of the A2-line19F virus. Taken together, these results illustrate a role for RSV F in virulence.

  • Human Cytomegalovirus UL97 Phosphorylates the Viral Nuclear Egress Complex [PublishAheadOfPrint]

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

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

  • Activation of ACLY by mTOR Signal Induces Disturbed Lipid Metabolism in HBV Pre-S2 Mutant Tumorigenesis [PublishAheadOfPrint]

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

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

  • Autophagy restricts HIV-1 infection by selectively degrading Tat in CD4+ T lymphocytes [PublishAheadOfPrint]

  • Autophagy is an ubiquitous mechanism involved in the lysosomal-mediated degradation of cellular components when engulfed in vacuoles called autophagosomes. Autophagy is also recognized as an important regulator of the innate and adaptive immune responses against numerous pathogens, which have, therefore, developed strategies to block or use the autophagy machinery to their own benefit.

    Upon Human Immunodeficiency Virus-1 (HIV-1) infection, viral envelope glycoproteins (Env) induce autophagy-dependent apoptosis of uninfected bystander CD4+ T lymphocytes, a mechanism likely contributing to the loss of CD4+ T cells. In contrast, in productively infected CD4+ T cells, HIV-1 is able to block Env-induced autophagy in order to avoid its antiviral effect. To date, nothing is known about how autophagy restricts HIV-1 infection in CD4+ T lymphocytes. Here we report that autophagy selectively degrades the HIV-1 transactivator Tat, a protein essential for viral transcription and virion production. We demonstrated that this selective autophagy-mediated degradation of Tat relies on its ubiquitin-independent interaction with the p62/SQSTM1 adaptor. Taken together, our results provide evidence that the anti-HIV effect of autophagy is specifically due to the degradation of the viral transactivator Tat, but this process is rapidly counteracted by the virus to favor its replication and spread.

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

  • Expression of neuronal CXCL10 induced by rabies virus infection initiates infiltration of inflammatory cells, production of chemokines/cytokines and enhancement of Blood-brain Barrier permeability [PublishAheadOfPrint]

  • It has been shown that enhancement of Blood-brain Barrier (BBB) permeability is modulated by expression of chemokines/cytokines and reduction of tight junction (TJ) proteins in the brains of mice infected with rabies virus (RABV). Since CXCL10 was found to be the most highly expressed chemokine, its temporal and spatial expression was determined in the present study. The expression of the chemokine CXCL10 was initially detected in neurons as early as 3 day post infection (dpi) before it was detected in microglia (6 dpi) and astrocytes (9 dpi) in the brain of RABV-infected mice. Neutralization of CXCL10 by treatment with anti-CXCL10 antibodies reduced IFN- production and Th17 cell infiltration as well as restored TJ protein expression and BBB permeability. Together, these data suggest that it is the neuronal CXCL-10 that initiates the cascade, leading to the activation of microglia/astrocytes, infiltration of inflammatory cells, expression of chemokines/cytokines, reduction of TJ protein expression, and enhancement of the BBB permeability.

  • Bindarit, an inhibitor of monocyte chemotactic proteins (MCPs) synthesis, protects against bone loss induced by Chikungunya virus infection [PublishAheadOfPrint]

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

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

  • Clonal evolution of CD8+ T cell responses against latent viruses: relationship between phenotype, localization and function. [PublishAheadOfPrint]

  • Human cytomegalovirus (hCMV) infection is characterized by a vast expansion of resting effector-type virus-specific T cells in the circulation. In mice, IL-7Raalpha;-expressing cells contain the precursors for long-lived antigen-experienced CD8+ T cells, but it is unclear if similar mechanisms operate to maintain these pools in humans. Here we studied whether IL-7Raalpha;-expressing cells obtained from peripheral blood (PB) or lymph nodes (LNs) sustain the circulating effector-type hCMV-specific pool.

    Using flowcytometry and functional assays we found that the IL-7Raalpha;+ hCMV-specific T cell population comprises cells that have a memory-phenotype and lack effector-features. We used next-generation sequencing of the T-cell receptor to compare the clonal repertoires of IL-7Raalpha;+ and IL-7Raalpha;- subsets. We observed limited overlap of clones between these subsets during acute infection and after one year. When comparing the hCMV-specific repertoire between PB and paired LN we found many identical clones, but also clones that were exclusively found in either compartment. New clones that were found in PB during antigenic recall were only seldomly identical to the unique LN clones.

    Thus, although PB IL-7Raalpha;-expressing and LN hCMV-specific CD8+ T cells show typical traits of memory-type cells, these populations do not seem to contain the precursors for the novel hCMV-specific CD8+ T-cell pool during latency or upon antigen recall. IL-7Raalpha;+ PB and LN hCMV-specific memory cells form separate virus-specific compartments, and precursors for these novel PB hCMV-specific CD8+ effector-type T cells are possibly located in other secondary lymphoid tissues or are being recruited from the naiiuml;ve CD8+ T cell pool.

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

  • Virulence Determinants in the PB2 Gene 1 of a Mouse-Adapted H9N2 Virus [PublishAheadOfPrint]

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

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

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

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

  • Entry mechanisms of Herpes Simplex Virus Type 1 into murine epidermis: Involvement of nectin-1 and HVEM as cellular receptors [PublishAheadOfPrint]

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

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

  • Selective acquisition of host-derived ICAM-1 by HIV-1 is a matrix-dependent process [PublishAheadOfPrint]

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

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

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

  • The herpes simplex virus 1 immediate-early protein ICP0 performs many functions during infection, including transactivation of viral gene expression, suppression of innate immune responses, and modification as well as eviction of histones from viral chromatin. Although these functions of ICP0 have been characterized, the detailed mechanisms underlying ICP0's complex role during infection warrant further investigation. We thus undertook an unbiased proteomic approach to identifying viral and cellular proteins that interact with ICP0 in the infected cell. Cellular candidates resulting from our analysis included the ubiquitin-specific protease USP7, the transcriptional repressor TRIM27, DNA repair proteins NBS1 and MRE11, regulators of apoptosis including BIRC6, and the proteasome. We also identified two HSV-1 early proteins involved in nucleotide metabolism, UL39 and UL50, as novel candidate interactors of ICP0. Because TRIM27 was the most statistically significant cellular candidate, we investigated the relationship between TRIM27 and ICP0. We observed rapid, ICP0-dependent loss of TRIM27 during HSV-1 infection. TRIM27 protein levels were restored by disrupting the RING domain of ICP0 or by inhibiting the proteasome, arguing that TRIM27 is a novel degradation target of ICP0. A mutant ICP0 lacking E3 ligase activity interacted with endogenous TRIM27 during infection as demonstrated by reciprocal co-immunoprecipitation and supported by immunofluorescence data. Surprisingly, ICP0-null mutant virus yields decreased upon TRIM27 depletion, arguing that TRIM27 has a positive effect on infection despite being targeted for degradation. These results illustrate a complex interaction between TRIM27 and viral infection with potential positive or negative effects of TRIM27 on HSV under different infection conditions.

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

  • Kaposi's Sarcoma-Associated Herpesvirus-Encoded RTA Impairs Innate Immunity via Ubiquitin-mediated Degradation of MyD88 [PublishAheadOfPrint]

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

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

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

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

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

  • Grouper heat-shock cognate protein 70 (GHSC70) is involved in the cellular entry of nervous necrosis virus (NNV) [PublishAheadOfPrint]

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

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

  • Swine IFITM proteins potently inhibit influenza A virus replication [PublishAheadOfPrint]

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

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

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

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

  • Profiling and characterization of potentially multidrug-resistant influenza neuraminidase 1 (N1) strains against neuraminidase inhibitors [PublishAheadOfPrint]

  • Neuraminidase inhibitors (NAIs) have been widely used to control influenza infection, but their increased use could promote global emergence of resistant variants. Although various mutations associated with NAI resistance have been identified, the amino acid substitutions that confer multidrug resistance with undiminished viral fitness remain poorly understood. We therefore screened known mutation(s) that could confer multidrug resistance to currently approved NAIs oseltamivir, zanamivir, and peramivir by assessing recombinant viruses with mutant NA genes (catalytic residues, R152K and R292K; framework residues, E119A/D/G, D198N, H274Y, and N294S) in the backbones of the 2009 pandemic H1N1 (pH1N1) and highly pathogenic avian influenza (HPAI) H5N1 viruses. Of the 14 single- and double-mutant viruses recovered in the backbone of pH1N1, 4 variants (E119D, E119A/D/G-H274Y) exhibited reduced inhibition to all the NAIs and 2 variants (E119D and E119D-H274Y) retained the overall properties of gene stability, replicative efficiency, pathogenicity, and transmissibility in vitro and in vivo. Of the 9 recombinant H5N1 viruses, 4 variants (E119D, E119A/D/G-H274Y) also showed reduced inhibition to all the NAIs, though their overall viral fitness was impaired in vitro and/or in vivo. Thus, single mutations or certain combination of the established mutations could confer potential multidrug resistance to pH1N1 or HPAI H5N1 viruses. Our findings emphasize the urgency of developing alternative drugs against influenza virus infection.

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

  • Murine cytomegalovirus is not restricted to the house mouse Mus musculus domesticus: prevalence and genetic diversity in the European house mouse hybrid zone. [PublishAheadOfPrint]

  • The murine cytomegalovirus (MCMV) is a betaherpesvirus of the house mouse, Mus musculus domesticus (Mmd). It is a common infection in wild mice and a highly studied pathogen of the laboratory mouse. Betaherpesviruses are specific to their hosts and it is unknown if other Mus taxa carry the MCMV or if it is restricted to Mmd. We sampled mice over a 145km transect of Bavaria-Bohemia crossing an hybrid zone between Mmd and M. m. musculus (Mmm), to investigate the occurrence of MCMV in two Mus subspecies and test the limit of specificity of the virus for its host. We hypothesized that if the two subspecies carry MCMV and if the virus is highly specific to its host, divergent MCMV lineages would have co-diverged with their hosts and would have a geographical distribution constrained by the host genetic background. 520 mice were tested by ELISA and/or nested PCR targeting the M94 gene. Seropositive and PCR positive individuals were found in both Mus subspecies. Seroprevalence was high, 79.4%, but viral DNA was only detected in 41.7% of mice. Sequencing revealed 20 haplotypes clustering in 3 clades that match the host genetic structure in the hybrid zone showing one and two MCMV lineages in Mmd and Mmm, respectively. The estimated MCMV time to most recent common ancestor (1.1 Mya) matches that of their hosts. In conclusion, MCMV has co-evolved with these hosts suggesting its diversity in nature may be underappreciated as other members of the subgenus Mus likely carry different MCMVs.

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

  • Slaying the Trojan horse: Natural killer cells exhibit robust anti-HIV-1 antibody-dependent activation and cytolysis against allogeneic T-cells [PublishAheadOfPrint]

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

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

  • Contact-induced mitochondria polarization supports HIV-1 virological synapse formation [PublishAheadOfPrint]

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

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

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

  • Susceptibility to alphavirus infection is age-dependent and host maturation is associated with decreased virus replication and less severe encephalitis. To identify factors associated with maturation-dependent restriction of virus replication, we studied AP-7 rat olfactory bulb neuronal cells that can be differentiated in vitro. Differentiation was associated with a 150- to 1000-fold decrease in replication of Sindbis and Venezuelan equine encephalitis alphaviruses, as well as La Crosse bunyavirus. Differentiation delayed synthesis of SINV RNA and protein, but did not alter the susceptibility of neurons to infection or virion maturation. Additionally, differentiation slowed virus-induced translation arrest and death of infected cells. Differentiation of uninfected AP-7 neurons was associated with changes in expression of antiviral genes. Expression of key transcription factors was increased, including interferon regulatory factor-3 and -7 (IRF-3 and IRF-7) and STAT-1, suggesting that neuronal maturation may enhance the capacity for antiviral signaling upon infection. IRF-7 produced by undifferentiated AP-7 neurons was exclusively the short dominant-negative isoform while that produced by differentiated neurons was the full-length aalpha; isoform. A similar switch in IRF-7 isoforms also occurred in the brains of maturing C57BL/6J mice. Silencing of IRF expression did not improve virus multiplication in differentiated neurons. Therefore, neuronal differentiation is associated with up regulation of transcription factors that activate antiviral signaling, but this alone does not account for maturation-dependent restriction of virus replication.

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

  • Identification of a neutralizing epitope within antigenic domain 5 of glycoprotein B of human cytomegalovirus [PublishAheadOfPrint]

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

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

  • Assessment of the internal genes of influenza A (H7N9) virus contributing to the high pathogenicity in mice [PublishAheadOfPrint]

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

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

  • Topical HSV-2 Vaccination with HPV Vectors Expressing gB/gD Ectodomains Induces Genital Tissue Resident Memory CD8+ T cells and Reduces Genital Disease and Viral Shedding After HSV-2 Challenge [PublishAheadOfPrint]

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

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

  • Super-infection with woodchuck hepatitis virus (WHV) strain WHVNY of the livers chronically infected with the strain WHV7. [PublishAheadOfPrint]

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

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

  • Malawi polyomavirus is a prevalent human virus that interacts with known tumor suppressors [PublishAheadOfPrint]

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

  • Merkel cell polyomavirus small T antigen mediates microtubule destabilisation to promote cell motility and migration [PublishAheadOfPrint]

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

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

  • An interleukin-1 beta encoding retrovirus exhibits enhanced replication in vivo. [PublishAheadOfPrint]

  • Interleukin-1 beta (IL-1bbeta;) is an inflammatory cytokine that is secreted in response to inflammasome activation by innate microbe-sensing pathways. Although some retroviruses can trigger IL-1bbeta; secretion through the DNA-sensing molecule IFI16, the effect of IL-1bbeta; on the course of infection is unknown. To test whether IL-1bbeta; secretion affects retroviral replication in vivo, I constructed a novel murine leukemia virus strain (FMLV-IL1bbeta;) that encodes the mature form of IL-1bbeta;. This virus replicated with kinetics similar to wild-type virus in tissue culture, but caused a dramatically more aggressive infection of both C57BL/6 and BALB/c mice. By 7dpi, mice infected with FMLV-IL1bbeta; exhibited splenomegaly and viral loads three hundred-fold higher than mice infected with wild-type FMLV. Furthermore, the enlarged spleens of FMLV-IL1bbeta;-infected mice correlated with a large expansion of Gr-1+CD11b+ myeloid-derived suppressor cells, as well as elevated levels of immune activation. Although FMLV-IL1bbeta; infection was controlled by C57BL/6 mice by 14dpi, FMLV-IL1bbeta; was able to establish a significant persistent infection and immune activation in BALB/c mice. These results demonstrate that IL-1bbeta; secretion is a powerful positive regulator of retroviral infection, and that FMLV-IL1bbeta; represents a new model of pro-inflammatory retroviral infection.

    Importance Interleukin-1 beta (IL-1bbeta;) is an inflammatory cytokine released in response to activation of innate pathogen-sensing pathways during microbial infection. To examine the potential impact of IL-1bbeta; on retroviral replication in vivo, I constructed a novel mouse retrovirus strain (FMLV-IL1bbeta;) that encodes IL-1bbeta; and promotes abundant IL-1bbeta; secretion from infected cells. This virus replicates with normal kinetics in cultured cells, but displays a dramatically enhanced ability to replicate in mice, and caused persistent infection and immune activation in the BALB/c strain of mice. These results establish IL-1bbeta; as a positive regulator of retroviral replication and pathogenesis, and suggest that targeting this pathway may have therapeutic benefits for pro-inflammatory retroviruses. This virus can also be used to further study the impact of pro-inflammatory pathways on retroviral infection.

  • RHABDOVIRAL-BASED VACCINE PLATFORMS AGAINST HENIPAVIRUSES [PublishAheadOfPrint]

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

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

  • G2/M cell cycle arrest correlates with primate lentiviral Vpr interaction with the SLX4 complex [PublishAheadOfPrint]

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

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

  • Venezuelan equine encephalitis virus variants lacking transcription inhibitory functions demonstrate highly attenuated phenotype [PublishAheadOfPrint]

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

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

  • Tetramer-enrichment reveals presence of phenotypically diverse hepatitis C virus specific CD8+ T cells in chronic infection [PublishAheadOfPrint]

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

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

  • Quantitative proteomics identifies host factors modulated during acute hepatitis E infection in swine model [PublishAheadOfPrint]

  • Hepatitis E virus (HEV) causes acute enterically-transmitted hepatitis. In industrialized countries, it is a zoonotic disease, swine being the major reservoir of human HEV contaminations. The occurrence and severity of the disease are variable, ranging from asymptomatic to self-limiting acute hepatitis, chronic infection or fulminant hepatitis. In the absence of a robust cell culture system or small animal models, the HEV life cycle and pathological process remain unclear. To characterize HEV pathogenesis and virulence mechanisms, a quantitative proteomic analysis was carried out to identify cellular factors and pathways modulated during acute infection of swine. Three groups of pigs were inoculated with 3 different strains of swine HEV to evaluate the possible role of viral determinants in pathogenesis. Liver samples were analyzed by a differential proteomic approach, 2D-DIGE and 61 modulated proteins were identified by mass spectroscopy. The results obtained show that the 3 HEV strains replicate similarly in swine and that they modulate several cellular pathways suggesting that HEV impairs several cellular processes, which can account for various disease expressions. Several proteins such as Heterogeneous nuclear ribonucleoprotein K, Apolipoprotein E and Prohibitin, known to be involved in other viral life cycles, were up-regulated in HEV-infected livers. Some differences were observed between the 3 strains, suggesting that HEV's genetic variability may induce variation in pathogenesis. This comparative analysis of liver proteome modulated during HEV infection with 3 different strains of genotype 3 provides an important basis for further investigations on factors involved in HEV replication and the mechanism of HEV pathogenesis.

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

  • A 2.5 kb deletion contaning a cluster of nine microRNAs in the LAT locus of the pseudorabies virus affects the host response of porcine trigeminal ganglia during established latency. [PublishAheadOfPrint]

  • The alphaherpesvirus Pseudorabies virus (PrV) establishes latency primarily in neurons of trigeminal ganglia when only transcription of the latency-associated transcript (LAT) locus is detected. Eleven microRNAs (miRNAs) cluster within LAT, suggesting a role in establishment and/or maintenance of latency.

    We generated a mutant PrV (M) deleted of nine miRNA genes which displayed almost identical properties with the parental PrV (WT) during propagation in vitro. Fifteen pigs were experimentally infected with either WT, M or mock infected.

    Similar levels of virus excretion and host antibody response were observed in all infected animals. At 62 days post infection trigeminal ganglia were excised and profiled by deep sequencing and RT-qPCR.

    Latency was established in all infected animals without evidence of viral reactivation demonstrating that miRNAs are not mandatory for this process. Lower levels of Large Latency Transcript (LLT) were found in ganglia infected by M compared to WT PrV. All PrV miRNAs were expressed, with highest expression found for prv-miR-LLT1, prv-miR-LLT2 (in WT-ganglia) and prv-miR-LLT10 (in both WT and M-ganglia). No evidence of differentially expressed porcine miRNAs was found. Fifty-four porcine genes were differentially expressed between WT, M and control ganglia. Both viruses triggered a strong host immune response, but in M- ganglia gene upregulation was prevalent. Pathway analyses indicated that several biofunctions, including those related to cell-mediated immune response and migration of dendritic cells, were impaired in M- ganglia. These findings are consistent with a function of the LAT locus in the modulation of host response for maintaining a latent state.

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

  • Basic motifs target PSGL-1, CD43, and CD44 to plasma membrane sites where HIV-1 assembles [PublishAheadOfPrint]

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

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

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

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

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

  • The human CD8+ T cell responses induced by a live attenuated tetravalent dengue vaccine are directed against highly conserved epitopes [PublishAheadOfPrint]

  • The incidence of infection with any of the four dengue virus serotypes (DENV 1-4) has increased dramatically in the last few decades, and the lack of a treatment or vaccine has contributed to significant morbidity and mortality worldwide. A recent comprehensive analysis of the human T cell response against wild-type DENV suggested an HLA-linked protective role for CD8+ T cells. We have collected one-unit blood donations from study participants receiving the monovalent or tetravalent live attenuated DENV vaccine (DLAV), developed by the U.S. National Institutes of Health. PBMCs from these donors were screened in IFN ELISPOT assays with pools of predicted, HLA matched, class I binding peptides covering the entire DENV proteome. Here, we characterize for the first time CD8+ T cell responses after live attenuated dengue vaccination and show that CD8+ T cell responses in vaccinees were readily detectable and comparable to natural dengue infection. Interestingly, while broad responses to structural and non-structural (NS) proteins were observed after monovalent vaccination, T cell responses following tetravalent vaccination were, dramatically, focused towards the highly conserved NS proteins. Epitopes were highly conserved in a vast variety of field isolates and able to elicit multifunctional T cell responses. Detailed knowledge of the T cell response will contribute to the identification of robust correlates of protection in natural immunity and following vaccination against DENV.

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

  • A novel mode of ISG15 mediated protection against influenza A virus and Sendai virus in mice [PublishAheadOfPrint]

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

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

  • Perturbation in the conserved methyltransferase-polymerase interface of flavivirus NS5 differentially affects polymerase initiation and elongation [PublishAheadOfPrint]

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

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

  • Proinflammatory Effects of Interferon Gamma in Mouse Adenovirus Type 1 Myocarditis [PublishAheadOfPrint]

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

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

  • Insight into the Mechanism of Inhibition of Recombinant Adeno-Associated Virus by the Mre11/Rad50/Nbs1 Complex [PublishAheadOfPrint]

  • Adeno-Associated virus (AAV) is a dependent virus of the family parvoviridae. Gene expression and replication of AAV and derived recombinant vectors (rAAV) are severely limited (ggt;10-fold) by the cellular DNA damage sensing complex made up of Mre11, Rad50, and Nbs1 (MRN). AAV does not encode the means to circumvent this block to productive infection, but relies on co-infecting helper-virus to do so. Using adenovirus helper proteins E1B55k andE4orf6, which enhance transduction of AAV via degradation of MRN, we investigate the mechanism through which this DNA damage complex inhibits gene expression from rAAV. We test substrate specificity of inhibition and the contribution of different functions of the MRN complex. Our results demonstrate that both single- and double-stranded rAAV vectors are inhibited by MRN, which is in contrast to the predominant model that inhibition is the result of a block to second-strand synthesis. Exploring the contribution of known functions of MRN, we found inhibition of rAAV does not require downstream DNA damage response factors, including signaling kinases ATM and ATR. The nuclease domain of Mre11 appears to play only a minor role in inhibition, while the DNA-binding domain makes a greater contribution. Additionally, mutation of the inverted terminal repeat of the rAAV genome, which has been proposed to be the signal for interaction with MRN, is tolerated by the mechanism of inhibition. These results articulate a model of inhibition of gene expression in which physical interaction is more important than enzymatic activity and several key downstream damage repair factors are dispensable.

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

  • Activation of p90 Ribosomal S6 Kinases (RSKs) by ORF45 of Kaposi Sarcoma-Associated Herpesvirus is Critical for Optimal Production of Infectious Viruses [PublishAheadOfPrint]

  • We have previously shown that ORF45, an immediate-early and tegument protein of Kaposi's sarcoma associated herpesvirus (KSHV), causes sustained activation of p90 ribosomal S6 kinases (RSKs) and ERK. We have now identified the critical region of ORF45 that is involved in RSK interaction and activation. Alanine scanning mutagenesis of this region revealed that a single F66A point mutation abolished binding of ORF45 to RSK or ERK, and consequently its ability to activate the kinases. We introduced the F66A mutation into BAC16 (a bacterial artificial chromosome clone containing the entire infectious KSHV genome), producing BAC16-45F66A. In parallel, we also repaired the mutation and obtained a revertant BAC16-45A66F. Reconstitution of these mutants in iSLK cells demonstrated that the ORF45-F66A mutant failed to cause sustained ERK and RSK activation during lytic reactivation, resulting in dramatic differences in the phosphoproteomic profile between the wild-type virus-infected cells and the mutant virus-infected cells. ORF45 mutation or deletion was also accompanied by noticeably decreased viral gene expression during lytic reactivation. The ORF45-F66A mutant consequently produced significantly fewer infectious progeny virions than the wild type or the revertant. These results suggest a critical role for ORF45-mediated RSK activation in KSHV lytic replication.

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

  • Variation of pH sensitivity, acid stability, and fusogenicity of three influenza H3 subtypes [PublishAheadOfPrint]

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

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

  • The putative HSV-1 chaperone protein UL32 modulates disulfide bond formation during infection. [PublishAheadOfPrint]

  • During DNA encapsidation, HSV-1 procapsids are converted to DNA-containing capsids by a process involving activation of the viral protease, expulsion of the scaffold proteins and the uptake of viral DNA. Encapsidation requires six minor capsid proteins (UL6, UL15, UL17, UL25, UL28 and UL33) and one viral protein, UL32, not found associated with capsids. Although functions have been assigned to each of the minor capsid proteins, the role of UL32 in encapsidation has remained a mystery. Using an HSV-1 variant containing a functional HA-tagged UL32, we demonstrated that UL32 was synthesized with true late kinetics and that it exhibited a previously unrecognized localization pattern. At 6 nndash; 9 h post infection (hpi), UL32 accumulated in viral replication compartments in the nucleus of the host cell while at 23 hpi it was additionally found in the cytoplasm. A newly generated UL32 null mutant was used to confirm that although B-capsids containing wild-type levels of capsid proteins were synthesized, these procapsids were unable to initiate the encapsidation process. Furthermore, we showed that UL32 is redox sensitive and identified two highly conserved oxidoreductase-like C-X-X-C motifs that are essential for protein function. In addition, the disulfide bond profiles of the viral proteins UL6, UL25, VP19C and the viral protease, VP24, were altered in the absence of UL32, suggesting that UL32 may act to modulate disulfide bond formation during procapsid assembly and maturation.

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

  • Complex TCR repertoire dynamics underlie the CD8+ T-cell response to HIV-1 [PublishAheadOfPrint]

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

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

  • The Autographa californica multiple nucleopolyhedrovirus ORF11 is essential for BV production and ODV envelopment [PublishAheadOfPrint]

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

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

  • NS5A and human RPA increase the processivity of HCV NS5B polymerase activity in vitro [PublishAheadOfPrint]

  • The precise role(s) and topological organization of different factors in the hepatitis C virus (HCV) RNA replication complex are not well understood. In order to elucidate the role of viral and host proteins in HCV replication, we have developed a novel in vitro replication system that utilizes a rolling circle RNA template. Under close-to-physiological salt conditions, HCV NS5B21, the RNA dependent RNA polymerase, has poor affinity for the RNA template. Human replication protein A (RPA) and HCV NS5A, recruit NS5B21 to the template. Subsequently, NS3 is recruited to the replication complex by NS5B21 resulting in RNA synthesis stimulation by helicase. Both RPA and NS5A(S25-C447), but not NS5A(S25-K215), enabled the NS5B21bbull;NS3 helicase complex to be stably associated with the template and synthesize RNA product in a highly processive manner in vitro. This new in vitro HCV replication system is a useful tool that may facilitate the study of other replication factors and aid in the discovery of novel inhibitors of HCV replication.

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

  • Structure-Function Relationships Underlying the Replication Fidelity of Viral RNA-dependent RNA Polymerases [PublishAheadOfPrint]

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

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

  • Expression of IFN{gamma} by a recombinant rabies virus strongly attenuates the pathogenicity of the virus via induction of type I interferon [PublishAheadOfPrint]

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

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

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

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

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

  • Blocking ESCRT-mediated envelopment inhibits microtubule-dependent trafficking of alphaherpesviruses in vitro. [PublishAheadOfPrint]

  • Herpes simplex virus and, as reported here, Pseudorabies virus utilize the ESCRT-apparatus to drive cytoplasmic envelopment of their capsids. Here we demonstrate that blocking ESCRT-mediated envelopment using the dominant negative inhibitor Vps4A-EQ reduced the ability of HSV and PRV particles to subsequently traffic along microtubules in vitro. HSV and PRV capsid-associated particles with bound GFP-Vps4A-EQ were readily detected by fluorescence microscopy in cytoplasmic extracts of infected cells. These Vps4A-EQ-associated capsid-containing particles bound to microtubules in vitro but were unable to traffic along them. Using a PRV strain expressing a fluorescent capsid and a fluorescently tagged form of the envelope protein gD, we found that similar numbers of gD-positive and gD-negative capsid-associated particles accumulated in cytoplasmic extracts under our conditions. Both classes of PRV particle bound to microtubules in vitro with comparable efficiency, and similar results were obtained for HSV using anti-gD immunostaining. The gD-positive and gD-negative PRV capsids were both capable of trafficking along microtubules in vitro, however motile gD-positive particles were less numerous and their trafficking was more sensitive to the inhibitory effects of Vps4A-EQ. We discuss our data in the context of microtubule-mediated trafficking of naked and enveloped alphaherpesvirus capsids.

    Importance The alphaherpesviruses include several important human pathogens. These viruses utilize microtubule-mediated transport to travel through the cell cytoplasm, however the molecular mechanisms of trafficking are not well understood. In this study we have used a cell-free system to examine the requirements for microtubule-trafficking, and have attempted to distinguish between the movement of "naked" and membrane-associated cytoplasmic alphaherpesvirus capsids.

  • Complex expression of the UL136 gene of human cytomegalovirus results in multiple protein isoforms with unique roles in replication [PublishAheadOfPrint]

  • Human cytomegalovirus is a complex DNA virus with a 230-kilobase genome encoding 170 to 750 proteins. The upper limit of this coding capacity suggests the evolution of complex mechanisms to substantially increase the coding potential from the 230-kb genome. Our work examines the complexity of one gene, UL136, encoded within the ULb' region of the genome that is lost during serial passage of HCMV in cultured fibroblasts. UL136 is expressed as five protein isoforms. We mapped these isoforms and demonstrate that they originate from both a complex transcriptional profile and possibly the usage of multiple translation initiation sites. Intriguingly, the pUL136 isoforms exhibit distinct subcellular distributions with varying association with the Golgi apparatus. The subcellular localization of membrane bound isoforms of UL136 differ when they are expressed exogenous compared to in the context of viral infection, suggesting that the trafficking of these isoforms is mediated by infection specific factors. While UL136, as most ULb' genes, was dispensable for replication in fibroblasts, the soluble 23-/19-kDa isoforms suppressed virus replication. In CD34+ hematopoietic progenitor cells (HPCs) infected in vitro, disruption of the 23-/19-kDa isoforms resulted in increased replication and a loss of latency phenotype, similar to the UL138 latency determinant encoded within the same genetic locus. Our work suggests complex interplay between the UL136 isoforms, which balances viral replication in multiple cell types and likely contributes to the cell-type dependent phenotypes of the UL133/8 locus and the outcome of HCMV infection.

    SIGNIFICANCE HCMV is a significant cause of morbidity in immunocompromised individuals, including transplant patients. Life-long persistence of the virus results in a high worldwide seroprevalence and may contribute to age related pathologies, such as atherosclerosis. Mechanisms of viral persistence are poorly understood; however, understanding the molecular basis of persistence is imperative for the development of new treatments. In this current work, we characterize a complex HCMV gene, UL136, which is expressed as five protein isoforms. These isoforms arise predominately from complex transcriptional mechanisms, which contribute to an increased coding capacity of the virus. Further, the UL136 isoforms oppose the activity of one another to balance HCMV replication in multiple cell types. We identify soluble isoforms of UL136 that function to suppress virus replication in fibroblasts and in CD34+ HPCs for latency.

  • Unabated Adenovirus replication following activation of the cGAS/STING dependent antiviral response in human cells [PublishAheadOfPrint]

  • The cGAS/STING DNA sensing complex has recently been established as a predominant pathogen recognition receptor (PRR) for DNA directed type I interferon innate immune activation. Using replication defective Adenovirus vectors and replication competent wildtype adenovirus, we have modeled the influence of the cGAS/STING cascade in permissive human cell lines (A549, HeLa, ARPE19, THP1). Wildtype adenovirus induces efficient early activation of the cGAS/STING cascade in a cell specific manner. In all responsive cell lines, cGAS/STING shRNA knockdown results in a loss of TBK1 and IRF3 activation, a lack of interferon bbeta; transcript induction, loss of interferon dependent STAT1 activation, and diminished induction of interferon-stimulated genes (ISGs). Adenoviruses that infect through the CAR (Ad2, Ad5), CD46 (Ad35), and Desmoglein-2 (Ad7) viral receptors all induce the cGAS/STING/TBK1/IRF3 cascade. The magnitude of the IRF3/IFN/ISG antiviral response, was strongly influenced by serotype with Ad35ggt;Ad7ggt;Ad2. For each serotype no enhancement of viral DNA replication or virus production occurred in cGAS or STING shRNA targeted cell line pools. We found no replication advantage in permissive cell lines that do not trigger the cGAS/STING cascade following infection. The cGAS/STING/TBK1/IRF3 cascade was not a direct target of viral anti-host strategies and we found no evidence that Ad stimulation of the cGAS/STING DNA response had an impact on viral replication efficiency.

    Significance statement: This study shows for the first time that the cGAS DNA sensor directs a dominant IRF3/IFN/ISG antiviral response to Adenovirus in human cell lines. Activation of cGAS occurs with viruses that infect through different high affinity receptors (CAR, CD46, and Desmoglein-2) and the magnitude of the cGAS/STING DNA response cascade is influenced by serotype specific functions. Furthermore, activation of the cGAS cascade occurred in a cell specific manner. Activation of the cGAS/STING response did not impact viral replication, and viral immune evasion strategies did not target the cGAS/STING/TBK1/IRF3 cascade. These studies provide novel insight into the early innate recognition response to adenovirus.

  • Putative Domain-Domain Interactions in the Vesicular Stomatitis Virus L Polymerase Protein Appendage Region [PublishAheadOfPrint]

  • The multidomain polymerase protein (L) of nonsegmented negative strand (NNS) RNA viruses catalyzes transcription and replication of the virus genome. The N-terminal half of the protein forms a ring-like polymerase structure while the C-terminal half encoding viral mRNA transcript modifications consists of a flexible appendage with three distinct globular domains. To gain insight into putative transient interactions between L domains during viral RNA synthesis, we exchanged each of the four distinct regions encompassing the appendage region of vesicular stomatitis virus (VSV) Indiana serotype L protein with their counterparts from VSV New Jersey, and analyzed effects on virus polymerase activity in a minigenome system. The methyltransferase domain exchange yielded a fully active polymerase protein, which functioned as well as wild type L in the context of a recombinant virus. Exchange of the downstream C-terminal non-conserved region abolished activity, but co-exchanging it with the methyltransferase domain generated a polymerase favoring replicase over transcriptase activity, providing strong evidence of interaction between these two regions. Exchange of the capping enzyme domain, or the adjacent non-conserved region thought to function as an "unstructured" linker, also abrogated polymerase activity, even when co-exchanged with other appendage domains. Further probing of the putative linker segment using in-frame EGFP insertions likewise abrogated activity. We discuss the implications of these findings with regard to L protein appendage domain structure and putative domain-domain interactions required for polymerase function.

    IMPORTANCE NNS viruses include many well-known human pathogens (e.g., rabies, measles, and Ebola viruses), as well as emerging viral threats (e.g., Nipah and Hendra viruses). These viruses all encode a large L polymerase protein similarly organized into multiple domains that work in concert to enable virus genome transcription and replication. But how the unique L protein carries out the multiplicity of individual steps in these two distinct processes is poorly understood. Using two different approaches, i.e., exchanging individual domains in the C-terminal appendage region of the protein between two closely related VSV serotypes, and inserting unrelated protein domains, we shed light on requirements for domain-domain interactions and domain contiguity in polymerase function. These findings further our understanding of the conformational dynamics of NNS L polymerase proteins, which play an essential role in pathogenic properties of these viruses and represent attractive targets for the development of antiviral measures.

  • Recombinant MVA generating excess early double-stranded RNA transiently activates PKR and triggers enhanced innate immune responses [PublishAheadOfPrint]

  • Double-stranded (ds) RNA is an important molecular pattern associated with viral infection and is detected by various extra- and intracellular recognition molecules. Poxviruses have evolved to avoid producing dsRNA early in infection but generate significant amounts of dsRNA late in infection due to convergent transcription of late genes. Protein kinase R (PKR) is activated by double-stranded (ds) RNA and triggers major cellular defenses against viral infection including protein synthesis shutdown, apoptosis and type I interferon (IFN-I) production. The poxviral E3 protein binds and sequesters viral dsRNA and is a major antagonist of the PKR pathway. We found that the highly replication-restricted modified vaccinia virus Ankara (MVA) engineered to produce excess amounts of dsRNA early in infection showed enhanced induction of IFN-bbeta; in murine and human cells in the presence of an intact E3L gene. IFN-bbeta; induction required a minimum overlap length of 300 bp between early complementary transcripts and was strongly PKR-dependent. Excess early dsRNA produced by MVAs activated PKR early but transiently in murine cells and induced enhanced systemic levels of IFN-aalpha;, IFN-, and other cytokines and chemokines in mice in a largely PKR-dependent manner. Replication-competent chorioallantois vaccinia virus Ankara (CVA) generating excess early dsRNA also enhanced IFN-I production and was apathogenic in mice even at very high doses, but showed no in vitro host range defect. Thus, genetically adjuvanting MVA and CVA to generate excess early dsRNA is an effective method to enhance innate immune stimulation by orthopoxvirus vectors and to attenuate replicating vaccinia virus in vivo.

    Importance Efficient cellular sensing of pathogen-specific components including double-stranded RNA (dsRNA) is an important prerequisite of an effective antiviral immune response. The prototype poxvirus vaccinia virus (VACV) and its derivative modified vaccinia virus Ankara (MVA) produce dsRNA as a byproduct of viral transcription. We found that inhibition of cellular dsRNA recognition established by the virus-encoded proteins E3 and K3 can be overcome by directing viral overexpression of dsRNA early in infection without compromising replication of MVA in permissive cells. Early dsRNA induced transient activation of the cellular dsRNA sensor protein kinase R (PKR), resulting in enhanced production of interferons and cytokines in cells and mice. Enhancing the capacity of MVA to activate the innate immune system is an important approach to further improve the immunogenicity of this promising vaccine vector.

  • The VP40 protein of Marburg virus exhibits impaired budding and increased sensitivity to human tetherin following mouse-adaptation [PublishAheadOfPrint]

  • The Marburg virus VP40 protein is a viral matrix protein that spontaneously buds from cells. It also functions as an interferon (IFN) signaling antagonist by targeting janus kinase 1 (JAK1). A previous study demonstrated that the VP40 protein of the Ravn strain of Marburg virus (Ravn virus or RAVV) failed to block IFN signaling in mouse cells, whereas the mouse-adapted RAVV (maRAVV) VP40 acquired the ability to inhibit IFN responses in mouse cells. The increased IFN-antagonist function of maRAVV VP40 mapped to residues 57 and 165, which were mutated during the mouse adaptation process. In the present study, we demonstrate that maRAVV VP40 lost the capacity to efficiently bud from human cell lines, despite the fact that both parental and maRAVV VP40s bud efficiently from mouse cell lines. The impaired budding in human cells corresponds with the appearance of protrusions on the surface of maRAVV VP40-expressing Huh7 cells and with an increased sensitivity of maRAVV VP40 to restriction by human tetherin but not mouse tetherin. However, transfer of the human tetherin cytoplasmic tail to mouse tetherin restored restriction of maRAVV VP40. Residues 57 and 165 were demonstrated to contribute to the failure of maRAVV VP40 to bud from human cells, and residue 57 was demonstrated to alter VP40 oligomerization, as assessed by co-precipitation assay, and to determine sensitivity to human tetherin. This suggests that RAVV VP40 acquired, during adaptation to mice, changes in its oligomerization potential that enhanced IFN-antagonist function. However, this new capacity impaired RAVV VP40 budding from human cells.

    IMPORTANCE Filoviruses, which include Marburg viruses and Ebola viruses, are zoonotic pathogens which cause severe disease in humans and non-human primates but do not cause similar disease in wild-type laboratory strains of mice unless first adapted to these animals. Although mouse adaptation has been used as a method to develop small animal models of pathogenesis, the molecular determinants associated with filovirus mouse-adaptation are poorly understood. Our study demonstrates how genetic changes which accrued during mouse-adaptation of the Ravn strain of Marburg virus have impacted the budding function of the viral VP40 matrix protein. Strikingly, we find impairment of mouse-adapted VP40 budding function in human but not mouse cell lines, and we correlate the impairment with an increased sensitivity of VP40 to restriction by human but not mouse tetherin and with changes in VP40 oligomerization. These data suggest that there are functional costs associated with filovirus adaptation to new hosts and implicate tetherin as a filovirus host restriction factor.

  • TNF-{alpha} and IL-6 facilitate corneal lymphangiogenesis in response to HSV-1 infection [PublishAheadOfPrint]

  • HSV-1 is a common human pathogen of clinical significance due to its association with vision impairment and encephalitis. In a mouse model of ocular neovascularization, we have previously identified HSV-1 elicits the genesis of lymphatic vessels into the cornea proper through epithelial cell expression of VEGFA dependent upon expression of VEGFR2 during acute infection. We hypothesized other factors may be involved in lymphangiogenesis with pro-inflammatory cytokines as the leading candidates. In the absence of infection or inflammation, intrastromal administration of TNF-aalpha; coupled with VEGFA elicited lymphatic vessel genesis significantly above either factor alone as well as vehicle control. Consistent with this observation, anti-TNF-aalpha; Ab blocked HSV-1-mediated corneal lymphangiogenesis within the first five days post infection. However, TNF-aalpha; deficient (TNF-aalpha;nndash;/nndash;) mice displayed a similar level of corneal vessel growth as wild type (WT) controls. To investigate the likely redundant nature of cytokines, PCR array analysis of HSV-1-infected TNF-aalpha;nndash;/nndash; mice revealed several factors elevated above that found in HSV-1-infected WT mice including IL-1bbeta;, platelet-derived growth factor, angiopoietin 2, insulin-like growth factor 2, and IL-6. Subconjunctival administration of neutralizing Ab to IL-6 blocked lymphangiogenesis in TNF-aalpha;nndash;/nndash; mice. Whereas the cornea levels of IL-6 were significantly reduced, there was no appreciable change in the level of IL-1bbeta; or other pro-angiogenic factors analyzed. Collectively, the results suggest in addition to VEGFA, TNF-aalpha; and IL-6 promotes and likely synergize with VEGFA in corneal lymphangiogenesis during acute HSV-1 infection.

    Importance We have identified at least two pro-inflammatory cytokines expressed locally that are involved in the genesis of lymphatic vessels in the normally avascular cornea in response to HSV-1 infection. This finding provides the basis to target IL-6 and TNF-aalpha; as additional pro-angiogenic factors in the cornea during the development of herpetic stromal keratitis as a means to alleviate further neovascularization and tissue pathology associated with the host immune response to the pathogen.

  • The Human Cytomegalovirus UL26 protein antagonizes NF{kappa}B activation [PublishAheadOfPrint]

  • Viral infection frequently triggers activation of host innate immune pathways that attempt to limit viral spread. The NFB pathway is a critical component that governs this response. We have found that the Human Cytomegalovirus (HCMV) UL26 protein antagonizes NFB activation. Upon infection, an HCMV strain lacking the UL26 gene (UL26) induced the nuclear translocation of the NFB RelB subunit, and activated expression and secretion of IL-6, an NFB target gene. The UL26 mutant was also more sensitive to challenge with TNFaalpha;, a canonical NFB inducer. Further, expression of UL26 in the absence of other viral proteins blocked NFB activation induced by either TNFaalpha; treatment or infection with Sendai virus (SeV). Our results indicate that UL26 expression is sufficient to block TNFaalpha;-induced NFB nuclear translocation and IB degradation. Lastly, UL26 blocks TNFaalpha;-induced IKappaB-kinase (IKK) phosphorylation, a key step in NFB activation. Combined, our results indicate that UL26 is part of a viral program to antagonize innate immunity through modulation of NFB signaling.

    Importance The NFB signaling pathway regulates innate immunity, an integral host process that limits viral pathogenesis. Viruses have evolved mechanisms to modulate NFB signaling to ensure their replication. Human Cytomegalovirus (HCMV) is a major cause of birth defects and disease in immunosuppressed populations. HCMV is known to actively target the NFB pathway, which is important for HCMV infection. Our results indicate that the HCMV UL26 gene is a key modulator of NFB pathway activity. We find the UL26 gene is both necessary and sufficient to block NFB activation upon challenge with anti-viral cytokines. Further, UL26 attenuates the phosphorylation and activation of a key NFB activating kinase complex, IKK. Our study provides new insight into how HCMV targets the NFB pathway. Given its importance to viral infection, the mechanisms through which viruses target the NFB pathway highlight areas of vulnerability that could be therapeutically targeted to attenuate viral replication.

  • RNA Populations in Immunocompromised Patients as Reservoirs for Novel Norovirus Variants [PublishAheadOfPrint]

  • Noroviruses are the leading cause of acute gastroenteritis outbreaks worldwide. The majority of norovirus outbreaks are caused by genogroup II.4 (GII.4) noroviruses. Novel GII.4 noroviruses emerge every 2-4 years and replace older variants as the dominant norovirus. The process of the emergence of novel variants is believed to be caused by a combination of recombination, genetic drift, and selection driven by population immunity, but how or where these novel variants emerge is not known. We detected two previously unknown novel GII.4 variants, termed GII.4 UNK1 and GII.4 UNK2, and a diverse norovirus population in fecal specimens from immunocompromised individuals with diarrhea after they had undergone bone-marrow transplantation. We hypothesized that immunocompromised individuals can serve as reservoirs for novel norovirus variants. To test our hypothesis, metagenomic analysis of viral RNA populations was combined with a full genome bioinformatic analysis of publicly available GII.4 noroviruses sequences from 1974 nndash; 2014 to identify converging sites. Localization analysis indicated that variable sites were more likely to be within two amino acids (Pllt; 0.05) of positively selected sites. Further analysis indicated polymorphic site distribution was random and its proximity to positively selected sites was dependent on the size of the norovirus genome and the number of positively selected sites. The results indicate that random mutations can have a positive impact on driving norovirus evolution and that immunocompromised individuals have the ability to serve as a potential reservoirs for novel GII.4 strains.

    Importance Norovirus is the most common cause of viral gastroenteritis in the US. Every two to three years novel norovirus variants emerge and rapidly disseminate throughout the world. The continual emergence of novel noroviruses is believed to be caused by a combination of genetic drift, population immunity, and recombination, but exactly how this emergence occurs remains unknown. In this study we identified two novel GII.4 variants in immunocompromised bone marrow transplant patients. Using metagenomic and bioinformatics analysis, we show that most genetic polymorphisms in the novel variants occur near, 0-2 amino acids, of positively selected sites, but the distribution of mutations was random; clustering of polymorphisms with positively selected sites was a result of genome size, number of mutations and positively selected sites. This study shows that immunocompromised patients can harbor infectious novel norovirus variants and although mutations in viruses are random they can have a positive effect in viral evolution.

  • Determinants of efficient degradation of APOBEC3 restriction factors by HIV-1 Vif [PublishAheadOfPrint]

  • The APOBEC3 deoxycytidine deaminases can restrict the replication of HIV-1 in cell culture to differing degrees. The effects of APOBEC3 enzymes are largely suppressed by HIV-1 Vif that interacts with host proteins to form a Cullin5-Ring E3 ubiquitin ligase that induces 48K-linked polyubiquitination (poly-Ub) and proteasomal degradation of APOBEC3 enzymes. Vif variants have differing abilities to induce degradation of APOBEC3 enzymes and the underlying biochemical mechanisms for these differences is not fully understood. We hypothesized that by characterizing the interaction of multiple APOBEC3 enzymes and Vif variants we could identify common features that resulted in Vif-mediated degradation and further define the determinants required for efficient Vif-mediated degradation of APOBEC3 enzymes. We used Vifs from HIV-1 NL4-3 (IIIB) and HXB2 to characterize their induced degradation of and interaction with APOBEC3G, APOBEC3G D128K, APOBEC3H, and APOBEC3B in 293T cells. We quantified the APOBEC3G-Vif and APOBEC3H-Vif interaction strengths in vitro using rotational anisotropy. Our biochemical and cellular analyses of the interactions support a model in which the degradation efficiency of VifIIIB and VifHXB2 correlated with both the binding strength of the APOBEC3-Vif interaction and the APOBEC3-Vif interface, which differs for APOBEC3G and APOBEC3H. Notably, Vif bound to APOBEC3H and APOBEC3B in the natural absence of Vif-induced degradation and the interaction resulted in 63K-linked poly-Ub of APOBEC3H and APOBEC3B, demonstrating additional functionality of the APOBEC3-Vif interaction apart from induction of proteasomal degradation.

    Importance APOBEC3 enzymes can potently restrict the replication of HIV-1 in the absence of HIV-1 Vif. Vif suppresses APOBEC3 action by inducing their degradation through a direct interaction with APOBEC3 enzymes and other host proteins. Vif variants from different HIV-1 strains have different effects on APOBEC3 enzymes. We used differing Vif degradation capacities of two Vif variants and various APOBEC3 enzymes with differential sensitivities to Vif to delineate determinants of the APOBEC3-Vif interaction that are required for inducing efficient degradation. Using a combined biochemical and cellular approach we identified that the strength of the APOBEC3-Vif binding interaction and the APOBEC3-Vif interface are determinants for degradation efficiency. Our results highlight the importance of using Vif variants with different degradation potential when delineating mechanisms of Vif-induced APOBEC3 degradation and identify features important for consideration in the development of HIV-1 therapies that disrupt the APOBEC3-Vif interaction.

  • Role of the short telomeric repeat region in Marek's disease virus (MDV) replication, genomic integration and lymphomagenesis [PublishAheadOfPrint]

  • Marek's disease virus (MDV) is a cell-associated alphaherpesvirus that causes generalized polyneuritis and T-cell lymphomas in chickens. MDV is able to integrate its genome into host telomeres, but the mechanism of integration is poorly understood. The MDV genome harbors two arrays of telomeric repeats (TMR) at the ends of its linear genome: multiple telomeric repeats (mTMR), with a variable number up to 100 repeats, and short telomeric repeats (sTMR), with a fixed number of 6 repeats. The mTMR have recently been shown to play an important role in MDV integration and tumor formation; however, the functions of the sTMR have remained unknown. In this study, we demonstrate that deletion of the sTMR in the MDV genome abrogates virus replication, while extensive mutation of the sTMR did not, indicating that presence but not the sTMR sequence itself is important. Furthermore, we generated a panel of truncation mutants to determine the minimal length of the sTMR and observed a direct correlation between sTMR length and MDV replication. To address the role of sTMR in MDV replication, integration and tumorigenesis, sTMR sequences were substituted by a scrambled repeated sequence (vsTMR_mut). vsTMR_mut replicated comparable to parental and revertant virus in vitro. In vivo, however, a significant reduction in disease and tumor incidence was observed in chickens infected with vsTMR_mut that also correlated with a reduced number of viral integration sites in tumor cells. Taken together, our data demonstrate that the sTMR play a central role in MDV genome replication, pathogenesis and MDV-induced tumor formation.

    Importance Marek's disease virus (MDV) is highly oncogenic alphaherpesvirus that infects chickens and causes high economic losses in poultry industry. MDV integrates its genetic material into host telomeres, a process that is crucial for efficient tumor formation. The MDV genome harbors two arrays of telomeric repeats (TMR) identical to host telomeres at the ends of its linear genome, termed mTMR and sTMR. mTMR have been recently shown to be involved in MDV integration, while the functions of sTMR remain unknown. Here we demonstrate that presence and length of sTMR sequence are crucial for MDV replication, but not the exact nucleotide sequence. Furthermore, the sTMR contribute to the high integration frequency of MDV and is important for MDV pathogenesis and tumor formation. As number of herpesviruses harbor arrays of telomeric repeats (TMR), MDV serves as a model to determine the role of the herpesvirus TMR in replication, integration and pathogenesis.

  • Linking pig-tailed macaque major histocompatibility complex class I haplotypes and cytotoxic T lymphocyte escape mutations in SIV infection [PublishAheadOfPrint]

  • The influence of MHC-I alleles on HIV diversity has been well characterised in humans at the population level. MHC-I alleles likely affect viral diversity in the SIV-infected pig-tailed macaque (M. nemestrina) model, but this is poorly characterised. We studied the evolution of SIV in pig-tailed macaques with a range of MHC-I haplotypes. SIVmac251 genomes were amplified from the plasma of 44 pig-tailed macaques infected with SIVmac251 at 4-10 months after infection and characterized by Illumina deep sequencing. MHC-I typing was performed on cellular RNA using Roche/454 pyrosequencing. MHC-I haplotypes and viral sequence polymorphisms were linked using in-house bioinformatics pipelines, both at individual mutations and groups of mutations spanning 10 amino acid segments, since CTL escape can occur at different amino acids within the same epitope in different animals. The approach successfully identified 6 known CTL escape mutations within 3 Mane-A1*084-restricted epitopes. The approach also identified over 70 new SIV polymorphisms linked to a variety of MHC-I haplotypes. Using functional CD8 T cell assays we confirmed that one of these associations, a Mane-B028 haplotype-linked mutation in Nef, corresponded to a CTL epitope. We also identified mutations associated with the Mane-B017 haplotype that were previously described as CTL epitopes restricted by Mamu-B*017:01 in rhesus macaques. This detailed study of pig-tailed macaque MHC-I genetics and SIV polymorphisms will enable a refined level of analysis for future vaccine design and treatment strategies for HIV.

    Importance Cytotoxic T lymphocytes select for virus escape mutants of HIV and SIV and this limits the effectiveness of vaccines and immunotherapies against these viruses. Patterns of immune escape variants are similar in HIV-1 infected human subjects that share the same MHC-I genes, but this has not been studied for SIV infection of macaques. By studying SIV sequence diversity in 44 MHC-typed SIV-infected pigtail macaques, we defined over 70 sites within SIV where mutations were common in macaques sharing particular MHC-I genes. Further, pigtail macaques sharing near-identical MHC-I genes with rhesus macaques responded to the same CTL epitope and forced immune escape. This allows many reagents developed to study rhesus macaque reagents to also be used to study pigtail macaques. Overall, our study defines sites of immune escape in SIV in pigtailed macaques and this enables a more refined level of analysis of future vaccine design and treatment strategies for HIV.

  • Genome analysis of the first Marseilleviridae representative from Australia indicates that most of its genes contribute to the virus fitness. [PublishAheadOfPrint]

  • The Marseilleviridae family consists of Acanthamoeba-infecting large DNA viruses with icosahedral particles ~0.2 micrometer in diameter and genome sizes in the 346-380 kb range. Since the isolation of Marseillevirus from a cooling tower in Paris (France) in 2009, the Marseilleviridae family rapidly expanded with representatives from Europe and Africa. Five members have been fully sequenced that are distributed among 3 emerging Marseilleviridae lineages. One comprises Marseillevirus and Cannes8virus, another one includes Insectomime and Tunisvirus, the third one corresponding to the more distant Lausannevirus. We now report the genomic characterization of Melbournevirus, the first Marseilleviridae representative isolated in Australia, from a fresh water pond in Melbourne. Despite the large distance separating this sampling point from France, Melbournevirus is remarkably similar to Cannes8virus and Marseillevirus with most orthologous genes exhibiting more than 98% identical nucleotide sequences. We took advantage of this optimal evolutionary distance to evaluate the selection pressure as the ratio of non-synonymous over synonymous mutations for various categories of genes. This ratio was found to be smaller than one for all of them, including those solely shared by the closest Melbournevirus and Cannes8virus isolates and absent from Lausannevirus. This suggests that most of the 403 protein coding genes composing the large Melbournevirus genome are under negative/purifying selection and must thus significantly contribute to the virus fitness. This conclusion contrasts with the more common view that many of the genes of the usually more diverse large DNA viruses might be (almost) dispensable.

    Importance A pervasive view is that viruses are fast evolving parasites and carry the smallest possible genomic information required to highjack the host cell machinery and perform their replication. This notion, probably inherited from the study of RNA viruses, is being gradually undermined by the discovery of DNA viruses with increasingly large gene content. These viruses also encode a variety of DNA repair functions presumably slowing down their evolution by preserving their genomes from random alterations. On the other hand, these viruses also encode a majority of proteins without cellular homologs, including many only shared between closest members of the same family. One may thus question the actual contribution of these anonymous and/or quasi-orphan genes to the virus fitness. Genomic comparisons of Marseilleviridae, including a new Marseillevirus isolated in Australia, demonstrate that most of their genes, irrespective of their functions and conservation across families, are evolving under negative selection.

  • Different roles of the three loops forming the adhesive interface of nectin-4 in measles virus binding and cell entry, nectin-4 homodimerization, and heterodimerization with nectin-1 [PublishAheadOfPrint]

  • Many viruses utilize cell adhesion molecules of the immunoglobulin superfamily as receptors. In particular, viruses of different classes exploit nectins. The large DNA viruses, herpes simplex and pseudorabies, use ubiquitous nectins 1 and 2. The negative strand RNA virus measles (MeV) uses tissue-specific nectin-4, and the positive strand RNA virus polio uses nectin-like 5 (necl-5), also known as poliovirus receptor. These viruses contact the BC, C'C'' and FG loops on the upper tip of their receptor's most membrane-distal domain. This location corresponds to the newly defined canonical adhesive interface of nectins, but how viruses utilize this interface has remained unclear. Here we show that the same key residues in the BC and FG loops of nectin-4 govern binding to the MeV attachment protein hemagglutinin (H) and cell entry, nectin-4 homodimerization, and heterodimerization with nectin-1. On the other hand, residues in the C'C'' loop necessary for homo- and heterotypic interactions are dispensable for MeV-induced fusion and cell entry. Remarkably, the C'C'' loop governs dissociation of the nectin-4 and H ectodomains. We provide formal proof that H can interfere with the formation of stable nectin-1/nectin-4 heterodimers. Finally, while developing an alternative model to study MeV spread, we observed that polarized primary pig airway epithelial sheets cannot be infected. We show that a single amino acid variant in the BC loop of pig nectin-4 fully accounts for restricted MeV entry. Thus, the three loops forming the adhesive interface of nectin-4 have different roles in supporting MeV H association and dissociation, and MeV-induced fusion.

    Importance Different viruses utilize nectins as receptors. Nectins are immunoglobulin superfamily glycoproteins that mediate cell-cell adhesion in vertebrate tissues. They interact through an adhesive interface located at the top of their membrane-distal domain. How viruses utilize the three loops forming this interface has remained unclear. We demonstrate that while nectin-nectin interactions require residues in all three loops, the association of nectin-4 with the measles virus hemagglutinin only requires the BC and FG loops. However, we discovered that residues in the C'C'' loop modulate the dissociation of nectin-4 from the viral hemagglutinin. Analogous mechanisms may support cell entry of other viruses that utilize nectins or other cell adhesion molecules of the immunoglobulin superfamily as receptors.

  • Differential contribution of adeno-associated virus type 2 Rep protein expression and nucleic acid elements to the inhibition of adenoviral replication in cis and in trans [PublishAheadOfPrint]

  • The helper-dependent adeno-associated virus (AAV-2) exhibits complex interactions with its helper adenovirus. Whereas AAV-2 is dependent on adenoviral functions for productive replication, it conversely inhibits adenoviral replication, both when its genome is present in trans after coinfection with both viruses or when it is present in cis as in the production of recombinant adenovirus (rAd)/AAV-2 hybrid vectors. The notion that AAV-mediated inhibition of adenoviral replication is predominantly due to the expression of the AAV-2 Rep proteins has recently been challenged by successful Rep78 expression in a rAd5 vector through recoding of the Rep ORF. We closely analyzed the relative contribution of AAV-2 nucleic acid elements and Rep protein expression to the inhibition of adenoviral replication in both of the above scenarios. When present in cis, a sequence element in the 3'-part of the rep gene comprising only the AAV-2 p40 promoter and the AAV-2 intron sequence, which we termed RIS-Ad, completely blocks adenoviral replication. p5/p19 promoter-driven Rep protein expression, on the other hand, only weakly inhibits rAd/AAV-2 vector propagation and by inactivation of the RIS-Ad it is feasible to generate first-generation rAd vectors expressing functional Rep proteins. The RIS-Ad plays no role in the inhibition of adenoviral replication in trans in a model closely mimicking AAV-2/Ad co-infection. Here expression of the Rep proteins is required, but additionally the presence of an amplifiable ITR containing template. Thus very different AAV-2 elements and mechanisms are involved in inhibition of adenoviral replication during rAd/AAV-2 vector propagation and after Ad/AAV coinfection.

    Importance This is the first study to systematically compare the contribution of AAV-2 protein expression and AAV-2 nucleic acid elements to the inhibition of adenoviral replication in rAd/AAV-2 hybrid vector generation and in AAV-2/adenovirus coinfection. It shows that the two inhibitory processes are very different with regard to the AAV-2 functions and the mechanisms involved. Whereas inhibition of rAd/AAV-2 hybrid vector propagation mostly involves a 3'-nucleic acid element in the rep gene, inhibition of an adenoviral genome in trans requires the Rep proteins and the AAV-ITRs. These findings have important implications both for the basic understanding of the AAV replication cycle and for generation of rAd/AAV-2 hybrid vectors expressing the nonstructural and structural proteins of AAV-2.

  • Evolution of co-circulating varicella zoster virus genotypes during a chickenpox outbreak in Guinea Bissau [PublishAheadOfPrint]

  • Varicella zoster virus (VZV), a double-stranded DNA alphaherpesvirus, is associated with seasonal outbreaks of varicella in non-immunised populations. Little is known about whether these outbreaks are associated with a single or multiple viral genotypes and if new mutations rapidly accumulate during transmission. Here, we take advantage of a well-characterised population cohort in Guinea-Bissau and produce a unique set of 23 full length genome sequences, collected over seven months from eight households. Comparative sequence analysis reveals that four distinct genotypes co-circulated amongst the population, three of which were present during the first week of the outbreak, although no patients were co-infected, which indicates that exposure to infectious virus from multiple sources is common during VZV outbreaks. Transmission of VZV was associated with length polymorphisms in the R1 repeat region and the origin of DNA replication. In two cases, these were associated with the formation of distinct lineages and point to the possible co-evolution of these loci, despite the lack of any known functional link in VZV or related herpesviruses. We show that these and all other sequenced clade 5 viruses possess a distinct R1 repeat motif that increases the acidity of an ORF 11p protein domain and postulate that this has either arisen or been lost following divergence of the major clades. Thus, sequencing of whole VZV genomes collected during an outbreak has provided novel insights into VZV biology, transmission patterns and (recent) natural history.

    Importance VZV is a highly infectious virus and the causative agent of chickenpox and shingles, the latter being particularly associated with the risk of painful complications. Seasonal outbreaks of chickenpox are very common amongst young children, yet little is known about the dynamics of the virus during person-to-person to transmission or whether multiple distinct viruses seed and/or co-circulate during an outbreak. In this study we have sequenced chickenpox viruses from an outbreak in Guinea Bissau that are supported by detailed epidemiological data. Our data show that multiple different virus strains seeded and were maintained throughout the six month outbreak period, and that viruses transmitted between individuals accumulated new mutations in specific genomic regions. Of particular interest is the potential co-evolution of two distinct parts of the genomes and our calculations of the rate of viral mutation, both of which increase our understanding of how VZV evolves over short periods of time in human populations.

  • HIV-1 Nef and Vpu are Functionally Redundant Broad-Spectrum Modulators of Cell Surface Receptors Including Tetraspanins [PublishAheadOfPrint]

  • HIV-1 Nef and Vpu are thought to optimize virus replication in the infected host, at least in part via their ability to interfere with vesicular host cell trafficking. Despite the use of distinct molecular mechanisms, Nef and Vpu share the specificity for some molecules such as CD4 and MHC-I, while disruption of intracellular transport of the host cell restriction factor CD317/tetherin represents a specialized activity of Vpu not exerted by HIV-1 Nef. To establish a profile of host cell receptors whose intracellular transport is affected by Nef, Vpu or both, we comprehensively analyzed the effect of these accessory viral proteins on cell surface receptors levels on A3.01 T lymphocytes. 36 out of 105 detectable receptors were significantly downregulated by HIV-1 Nef, revealing a previously unappreciated scope with which HIV-1 Nef remodels the cell surface of infected cells. Remarkably, the effects of HIV-1 Vpu on host cell receptor exposure largely matched those of HIV-1 Nef in breadth and specificity (32 of 105, all also targeted by Nef), even though the magnitude was generally less pronounced. Of particular note, cell surface exposure of all members of the tetraspanin (TSPAN) protein family analyzed was reduced by both Nef and Vpu, and the viral proteins triggered the enrichment of TSPANs in a perinuclear area of the cell. While Vpu displayed significant colocalization and physical association with TSPANs, interactions of Nef with TSPANs were less robust. TSPANs thus emerge as a major target of deregulation in host cell vesicular transport by HIV-1 Nef and Vpu. The conservation of this activity in two independent accessory proteins suggests its importance for spread of HIV-1 in the infected host.

    Importance In this manuscript we define that HIV-1 Nef and Vpu display a surprising functional overlap and affect the cell surface exposure of a previously unexpected breadth of cellular receptors. Our analyses furthermore identify the tetraspanin protein family as a previously unrecognized target of Nef and Vpu activity. These findings have implications for the interpretation of effects detected for these accessory gene products on individual host cell receptors and illustrate the co-evolution of Nef and Vpu function.

  • Role of Mason-Pfizer monkey virus CA-NC spacer peptide-like domain in assembly of immature particles [PublishAheadOfPrint]

  • The hexameric lattice of an immature retroviral particle consists of Gag polyprotein, which is the precursor of all viral structural proteins. Lentiviral and alpharetroviral Gag contains a peptide sequence called the spacer peptide (SP), which is localized between the capsid (CA) and nucleocapsid (NC) domains. SP plays a critical role in intermolecular interactions during the assembly of immature particles of several retroviruses. Published models of supramolecular structures of immature particles suggest that in lentiviruses and alpharetroviruses, SP adopts a rod-like six-helix bundle organization. In contrast, Mason-Pfizer monkey virus (M-PMV), a betaretrovirus that assembles in the cytoplasm, does not contain a distinct SP sequence, and the CA-NC connecting region is not organized into a clear rod-like structure. Nevertheless, the CA-NC junction comprises a sequence critical for assembly of immature M-PMV particles. In the present work, we characterize this region, called the SP-like domain, in detail. We provide biochemical data confirming the critical role of M-PMV SP-like domain in immature particle assembly, release, processing and infectivity. Circular dichroism spectroscopy revealed that, in contrast to the SP regions of other retroviruses, a short SP-like domain-derived peptide (SPLP) does not form a purely helical structure in aqueous or helix-promoting solution. Using the 8 AAring; cryo-electron microscopy density maps of immature M-PMV particles, we prepared computational models of the SP-like domain and indicate the structural features required for M-PMV immature particle assembly.

    IMPORTANCE Retroviruses such as HIV-1 are of great medical importance. Using Mason-Pfizer monkey virus (M-PMV) as a model retrovirus, we provide biochemical and structural data confirming the general relevance of a short segment of the structural polyprotein Gag for retrovirus assembly and infectivity. Although this segment is critical for assembly of immature particles of lentiviruses, alpharetroviruses and betaretroviruses, the organization of this domain is strikingly different. A previously published electron microscopic structure of an immature M-PMV particle allowed us to model this important region into the electron density map. The data presented here help explain the different packing of the Gag segment of various retroviruses, such as HIV, RSV and M-PMV. Such knowledge contributes to understanding the importance of this region and its structural flexibility among retroviral species. The region might play a key role in Gag-Gag interactions, leading to different morphological pathways of immature particle assembly.

  • Anterograde Glycoprotein Dependent Transport of Newly Generated Rabies Virus in Dorsal Root Ganglion Neurons [PublishAheadOfPrint]

  • Rabies virus (RABV) spread is widely accepted to occur only by retrograde axonal transport. However, examples of anterograde RABV spread in peripheral neurons such as dorsal root ganglion (DRG) neurons indicated a possible bidirectional transport by an uncharacterized mechanism. Here, we analyzed the axonal transport of fluorescence labeled RABV in DRG neurons by live-cell microscopy. Both, entry-related retrograde transport of RABV after infection at axon endings and post-replicative transport of newly formed virus were visualized in compartmentalized DRG neuron cultures. Whereas entry-related transport at 1.5 mmu;m/sec occurred only retrogradually, after two days of infection multiple particles were observed in axons moving in both the anterograde and retrograde directions. The dynamics of post-replicative retrograde transport (1.6 mmu;m/sec) were similar to entry-related retrograde transport. In contrast, anterograde particle transport at 3.4 mmu;m/sec was faster, indicating active particle transport. Interestingly, RABV missing the glycoproteins did not move anterogradually within the axon. Thus, anterograde RABV particle transport depended on the RABV glycoprotein. Moreover, co-localization of GFP-labeled ribonucleoproteins (RNP) and glycoprotein in distal axonal regions as well as co-transport of labeled RNPs with membrane anchored mCherry reporter confirmed that either complete enveloped virus particles, or vesicle associated RNPs were transported. Our data show that anterograde RABV movement in peripheral DRG neurons occurs by active motor protein dependent transport. We propose two models for post-replicative long distance transport in peripheral neurons: either transport of complete virus particles or co-transport of RNPs and G-containing vesicles through axons to release virus at distal sites of infected DRG neurons.

    Importance Rabies virus retrograde axonal transport by dynein motors supports virus spread over long distances and lethal infection of the central nervous system. Though active rabies virus has been widely accepted to be unidirectional, evidence for anterograde spread in peripheral neurons supports the hypothesis that in some neurons RABV also enters the anterograde pathway by so far unknown mechanisms.

    By live microscopy we visualized fast anterograde axonal transport of rabies virus. The velocities exceeded those of retrograde movements, suggesting that active, most likely kinesin-dependent transport machineries are involved. Dependency of anterograde transport on the expression of virus glycoprotein G and co-transport with vesicles further suggest, that complete enveloped virus particles or co-transport of virus ribonucleoprotein and G-containing vesicles occurred. These data provide first insight in the mechanism of anterograde rabies virus transport and substantially contributes to the understanding of RABV replication and spread of newly formed virus in peripheral neurons.

  • HIV-1 Envelope Glycan Moieties Modulate HIV-1 Transmission [PublishAheadOfPrint]

  • HIV-1 envelope protein (Env) is heavily glycosylated with approximately 50% of the Env molecular mass contributed by N-glycans. HIV-1 Env N-glycans shield the protein backbone and have been shown to play key roles in determining Env structure, surface exposure, and, consequently, antigenicity, infectivity, antibody neutralization, and carbohydrate and receptor binding. Studies of HIV-1 glycosylation have focused mainly on the position of glycosylation, rather than the types of glycans. Also, the role of Env glycan moieties on HIV-1 transmission has not been systematically defined. Using viruses with modified Env glycan content and heterogeneity, we examined the effects of Env glycan moieties on the major events of HIV-1 transmission. Compared to viruses with less oligomannose and more complex Env glycans, viruses with more oligomannose and less complex glycans more efficiently (a) transcytose across an epithelial cell monolayer, (b) attach to monocyte-derived macrophages (MDMs), (c) bind monocyte-derived dendritic cells (MoDCs), and (d) trans-infect primary lymphocytes via MoDCs. However, viruses with more oligomannose and less complex glycans displayed impaired infectivity in TZMbl cells, MDMs, primary lymphocytes and fresh human intestinal tissue. Thus, N-linked Env glycans display discordant effects on the major events of HIV-1 transmission, with mature oligosaccharide structures on Env playing crucial role in HIV-1 infection. Env glycosylation should be taken into consideration in the development of vaccine strategies to interdict HIV-1 transmission.

    IMPORTANCE HIV-1 Env N-glycans shield the protein backbone and play key roles in determining Env structure and surface exposure, thereby impacting Env vantigenicity, infectivity, antibody neutralization, and carbohydrate and receptor binding. Studies of HIV-1 glycosylation have focused mainly on the position of glycosylation, rather than the types of glycans. In this report, we investigated systematically the role of Env glycan moieties on HIV-1 transmission. We show that N-linked Env glycans display discordant effects on the major events of HIV-1 transmission. These data indicate that Env glycan moieties impact HIV-1 transmission and that modulation of Env glycan moieties offers a potential strategy for the development of therapeutic or prophylactic vaccines against HIV-1.

  • Vaccine-Induced CD107a+ CD4+ T Cells Are Resistant to Depletion Following AIDS Virus Infection [PublishAheadOfPrint]

  • CD4+ T-cell responses are crucial for effective antibody and CD8+ T-cell induction following virus infection. However, virus-specific CD4+ T cells can be preferential targets for human immunodeficiency virus (HIV) infection. HIV-specific CD4+ T-cell induction by vaccination may thus result in enhancement of virus replication following infection. In the present study, we show that vaccine-elicited CD4+ T cells expressing CD107a are relatively resistant to depletion in a macaque AIDS model. Comparison of virus-specific CD107a, macrophage inflammatory protein-1bbeta;, interferon-, tumor necrosis factor-aalpha;, and interleukin-2 responses in CD4+ T cells of vaccinated macaques pre- and 1-week post-challenge showed a significant reduction in the CD107anndash; but not the CD107a+ subset after virus exposure. Those vaccinees that failed to control viremia showed a more marked reduction and exhibited significantly higher viral loads at week 1 than unvaccinated animals. Our results indicate that vaccine-induced CD107anndash; CD4+ T cells are depleted following virus infection, suggesting a rationale for avoiding virus-specific CD107anndash; CD4+ T-cell induction in HIV vaccine design.

    Importance Induction of effective antibody and/or CD8+ T-cell responses is a principal vaccine strategy against human immunodeficiency virus (HIV) infection. CD4+ T-cell responses are crucial for effective antibody and CD8+ T-cell induction. However, virus-specific CD4+ T cells can be preferential targets for HIV infection. Here, we show that vaccine-induced virus-specific CD107anndash; CD4+ T cells are largely depleted following infection in a macaque AIDS model. While CD4+ T-cell responses are important in viral control, our results indicate that virus-specific CD107anndash; CD4+ T-cell induction by vaccination may not lead to efficient CD4+ T-cell responses following infection but rather be detrimental and accelerate viral replication in the acute phase. This suggests that HIV vaccine design should avoid virus-specific CD107anndash; CD4+ T-cell induction. Conversely, this study found that vaccine-induced CD107a+ CD4+ T cells are relatively resistant to depletion following virus challenge, implying that induction of these cells may be an alternative approach toward HIV control.

  • Exposure of epitope residues on the outer face of the chikungunya virus envelope trimer determines antibody neutralizing efficacy [PublishAheadOfPrint]

  • Chikungunya virus (CHIKV) is a re-emerging alphavirus that causes a debilitating arthritic disease, infects millions of people, and has no specific treatment. Like many alphaviruses, the structural targets on CHIKV that elicit a protective humoral immune response in humans are poorly defined. Here we used phage display against virus-like particles (VLPs) to isolate seven human monoclonal antibodies (MAbs) against the CHIKV envelope glycoproteins E2 and E1. One MAb, IM-CKV063, was highly neutralizing (IC50 7.4 ng/ml), demonstrated high-affinity binding (320 pM), and was capable of therapeutic and prophylactic protection in multiple animal models, up to 24 h post-exposure. Epitope mapping using a comprehensive shotgun mutagenesis library of 910 E2/E1 alanine mutations demonstrated that IM-CKV063 binds to an inter-subunit conformational epitope on domain A, a functionally important region of E2. MAbs against the highly conserved fusion loop have not previously been reported, but were also isolated in our studies. Fusion loop MAbs were broadly cross-reactive against diverse alphaviruses, but were non-neutralizing. Fusion loop MAb reactivity was affected by temperature and reactivity conditions, suggesting that the fusion loop is hidden in infectious virions. Visualizing the binding sites of 15 different MAbs on the structure of E2/E1 reveals that all epitopes are located at the membrane distal region of the E2/E1 spike. Interestingly, epitopes on the exposed top-most and outer surfaces of the E2/E1 trimer structure are neutralizing whereas epitopes facing the interior of the trimer are not, providing a rationale for vaccine design and therapeutic MAb development using the intact CHIKV E2/E1 trimer.

    IMPORTANCE CHIKV is the most important alphavirus affecting humans, resulting in a chronic arthritic condition that can persist for months or years. In recent years, millions of people have been infected globally and spread to the Americas is now beginning, with over 100,000 cases in the Caribbean within six months of arrival. Our study reports seven human MAbs against CHIKV envelope, including a highly protective MAb and rarely isolated fusion loop MAbs. Epitope mapping of these MAbs demonstrates how some E2/E1 epitopes are exposed or hidden from the human immune system and suggests a structural mechanism by which these MAbs protect (or fail to protect) against CHIKV infection. Our results suggest that the membrane distal end of CHIKV E2/E1 is the primary target for the humoral immune response to CHIKV and antibodies targeting the exposed top-most and outer surfaces of the E2/E1 trimer determine the neutralizing efficacy of this response.

  • A recombinant HCV envelope glycoprotein vaccine elicits antibodies targeting multiple epitopes on the envelope glycoproteins associated with broad cross-neutralization [PublishAheadOfPrint]

  • Although effective Hepatitis C Virus (HCV) antivirals are on the horizon, a global prophylactic vaccine for HCV remains elusive. The diversity of the virus is a major concern for vaccine development; there are 7 major genotypes of HCV found globally. Therefore, a successful vaccine will need to protect against HCV infection of all genotypes. Despite the diversity, many monoclonal antibodies (mAbs) with broadly cross-neutralizing activity have been described suggesting the presence of conserved epitopes that can be targeted to prevent infection. Similarly, a vaccine comprising recombinant envelope glycoproteins (rE1E2) derived from the genotype 1a HCV-1 strain has been shown to be capable of eliciting cross-neutralizing antibodies in guinea pigs, chimpanzees, and healthy human volunteers. In order to investigate the basis for this cross-neutralization, epitope mapping of anti-E1E2 antibodies present within antisera from goats and humans immunized with HCV-1 rE1E2 was conducted through peptide mapping and competition studies with a panel of cross-neutralizing mAbs targeting various epitopes within E1E2. The immunized goat antisera was shown to compete with the binding of all mAbs tested (AP33, HC33.4, HC84.26, 1:7, AR3B, AR4A, AR5A, IGH526, A4). Antisera showed the best competition against HC84.26/AR3B and the weakest competition against AR4A. Furthermore, antisera from five immunized human vaccinees were shown to compete with five pre-selected mAbs (AP33, AR3B, AR4A, AR5A, IGH526). These data show that immunization with HCV-1 rE1E2 elicits antibodies targeting multiple cross-neutralizing epitopes. Our results further support the use of such a vaccine antigen to induce cross-genotype neutralization.

    Importance An effective prophylactic vaccine for HCV is needed for optimal control of the disease burden. The high diversity of HCV has posed a challenge for developing vaccines that elicit neutralizing antibodies for protection against infection. Despite this, we have previously shown that a vaccine comprising recombinant envelope glycoproteins derived from a single genotype 1a strain was capable of eliciting a cross-neutralizing antibody response in human volunteers. Here, we have used competition binding assays and peptide binding assays to show that antibodies present in the vaccinated antisera bind epitopes overlapping with those of a variety of well-characterized cross-neutralizing monoclonal antibodies. This provides a mechanism for the cross-neutralizing human antisera: antibodies present in the antisera bind to conserved regions associated with cross-neutralization. Importantly, this work provides further support for a vaccine comprising recombinant envelope glycoproteins mmdash; perhaps in a formulation with a vaccine component eliciting strong anti-HCV CD4+ and CD8+ T cell responses.

  • Cooperativity Among Rev-Associated Nuclear Export Signals Regulates HIV-1 Gene Expression and is a Determinant of Virus Species Tropism [PublishAheadOfPrint]

  • Murine cells exhibit a profound block to HIV-1 virion production that was recently mapped to a species-specific structural attribute of the murine version of the CRM1 (mCRM1) nuclear export receptor, and rescued by expression of human CRM1 (hCRM1). In human cells, the HIV-1 Rev protein recruits hCRM1 to intron-containing viral mRNAs encoding the Rev response element (RRE), thereby facilitating viral late gene expression. Here we exploited murine 3T3 fibroblasts as a gain-of-function system to study hCRM1's species-specific role in regulating Rev's effector functions. We show that Rev is rapidly exported from the nucleus by mCRM1 despite only weak contributions to HIV-1's post-transcriptional stages. Indeed, Rev preferentially accumulates in the cytoplasm of murine 3T3 cells with or without hCRM1 expression, in contrast to human HeLa cells where Rev exhibits striking en masse transitions between the nuclear and cytoplasmic compartments. Efforts to bias Rev's trafficking either into or out of the nucleus revealed that Rev encoding a second CRM1 binding domain (Rev-2xNES), or Rev-dependent viral gag-pol mRNAs bearing tandem RREs (GP-2xRRE), rescue virus particle production in murine cells even in the absence of hCRM1. Combined, these results suggest a model wherein Rev-associated nuclear export signals cooperate to regulate the number or quality of CRM1's interactions with viral Rev/RRE ribonucleoprotein complexes in the nucleus. This mechanism regulates CRM1-dependent viral gene expression and is a determinant of HIV-1's capacity to produce virions in non-human cell types.

    Importance Cells derived from mice and other non-human species exhibit profound blocks to HIV-1 replication. Here we elucidate a block to HIV-1 gene expression attributable to the murine version of the CRM1 (mCRM1) nuclear export receptor. In human cells, hCRM1 regulates the nuclear export of viral intron-containing mRNAs through the activity of the viral Rev adapter protein that forms a multimeric complex on these mRNAs prior to recruiting hCRM1. We demonstrate that Rev-dependent gene expression is poor in murine cells despite the finding that, surprisingly, the bulk of Rev interacts efficiently with mCRM1 and is rapidly exported from the nucleus. Instead, we map the mCRM1 defect to the apparent inability of this factor to engage Rev multimers in the context of large viral Rev/RNA ribonucleoprotein complexes. These findings shed new light on HIV-1 gene regulation and could inform the development of novel antiviral strategies that target viral gene expression.

  • Viral Reprogramming of the Daxx-Histone H3.3 Chaperone During EBV Early Infection [PublishAheadOfPrint]

  • Host chromatin assembly can function as a barrier to viral infection. Epstein-Barr virus (EBV) establishes latent infection as chromatin-assembled episomes in which all but a few viral genes are transcriptionally silent. The factors that control chromatin assembly and guide transcription regulation during the establishment of latency are not well understood. Here, we demonstrate that the EBV tegument protein BNRF1 binds the histone H3.3 chaperone Daxx to modulate histone mobility and chromatin assembly on the EBV genome during the early stages of primary infection. We demonstrate that BNRF1 substitutes for the repressive co-chaperone ATRX to form a ternary complex of BNRF1-Daxx-H3.3-H4, using co-immunoprecipitation and size exclusion chromatography with highly purified components. Fluorescence recovery after photobleaching (FRAP) assays were used to demonstrate that BNRF1 promotes global mobilization of cellular histone H3.3. Mutation of putative nucleotide binding motifs on BNRF1 attenuates the displacement of ATRX from Daxx. We also show by immunofluorescence (IF) combined with fluorescence in situ hybridization (FISH) that BNRF1 is important for the dissociation of ATRX and Daxx from nuclear bodies during de novo infection of primary B-lymphocytes. Virion-delivered BNRF1 suppresses Daxx-ATRX-mediated H3.3 loading on viral chromatin as measured by chromatin immunoprecipitation (ChIP) assays and enhances viral gene expression during early infection. We propose that EBV tegument protein BNRF1 replaces ATRX to reprogram Daxx-mediated H3.3 loading, in turn generating chromatin suitable for latent gene expression.

    Importance Epstein-Barr Virus (EBV) is a human herpesvirus that efficiently establishes latent infection in primary B-lymphocytes. Cellular chromatin assembly plays an important role in regulating the establishment of EBV latency. We show that the EBV tegument protein BNRF1 functions to regulate chromatin assembly on the viral genome during early infection. BNRF1 alters the host cellular chromatin assembly to prevent anti-viral repressive chromatin, and establish chromatin structure permissive for viral gene expression and the establishment of latent infection.

  • The 2'-5' oligoadenylate synthetase 3 (OAS3) enzyme potently synthesizes the 2'-5' oligoadenylates required for RNase L activation [PublishAheadOfPrint]

  • The oligoadenylate synthetase (OAS) family of proteins are antiviral restriction factors that target a wide range of RNA and DNA viruses. They function as intracellular double stranded RNA (dsRNA) sensors that upon binding to dsRNA undergo a conformational change and are activated to synthesize 2'-5' linked oligoadenylates (2-5A). 2-5As of sufficient length act as second messengers to activate RNase L and thereby restrict viral replication. We expressed human OAS3 using the baculovirus system and purified it to homogeneity. We show that recombinant OAS3 is activated at a substantially lower concentration of dsRNA compared to OAS1, making it a potent in vivo sensor of dsRNA. Moreover, we find that OAS3 synthesizes considerably longer 2-5As than previously reported and that OAS3 can activate RNase L intracellularly. The combined high affinity for dsRNA and the capability to produce 2-5As of sufficient length to activate RNase L suggests that OAS3 is a potent activator of RNase L. In addition, we provide experimental evidence to support one active site of OAS3 located in the C-terminal OAS domain and generate a low resolution structure of OAS3 using SAXS.

    IMPORTANCE We are the first to purify the OAS3 enzyme to homogeneity, which allowed us to characterize the mechanism utilized by OAS3 and identify the active site. We provide compelling evidence that OAS3 can produce 2'-5' oligoadenylates of sufficient length to activate RNase L. This is contrary to what is described in the current literature but agrees with recent in vivo data showing that OAS3 harbors an antiviral activity requiring RNase L. Thus our work redefines our understanding of the biological role of OAS3. Furthermore, we used a combination of mutagenesis and small angle X-ray scattering to describe the active site and low resolution structure of OAS3.

  • A Combination of HA and PA Mutations Enhances Virulence in a Mouse-adapted H6N6 Influenza A Virus [PublishAheadOfPrint]

  • H6N6 viruses are commonly isolated from domestic ducks and avian-to-swine transmissions of H6N6 viruses have been detected in China. Whether subsequent adaptation of H6N6 viruses in mammals would increase their pathogenicity towards humans is not known. To address this, we generated a mouse-adapted swine influenza H6N6 virus (GDK6-MA) which exhibited greater virulence than the wild-type virus (GDK6). Amino acid substitutions in PB2 (E627K), PA (I38M) and HA (L111F, H156N and S263R) occurred in GDK6-MA. HA H156N resulted in enlarged plaque sizes on MDCK cells and enhanced early stage viral replication in mammalian cells. PA I38M raised polymerase activity in vitro but did not change virus replication in either mammalian cells or mice. These single substitutions had only limited effects on virulence, however, a combination of HA H156N, S263R and PA I38M in the GDK6 backbone led to a significantly more virulent variant. This suggests these substitutions can compensate for the lack of PB2-627K and modulate virulence, revealing a new determinant of pathogenicity for H6N6 viruses in mice, which might also pose a threat to human health.

    Importance Avian H6N6 influenza viruses are enzootic in domestic ducks and have been detected in swine in China. Infections of mammals by H6N6 viruses raise the possibility of viral adaptation and increasing pathogenicity in the new hosts. To examine the molecular mechanisms of adaptation, a mouse-adapted avian-origin swine influenza H6N6 virus (GDK6-MA), which had higher virulence than its parental virus, was generated. Specific mutations were found in PB2 (E627K), PA (I38M) and HA (L111F, H156N, S263R) and were assessed for their virulence in mice. The combination of HA H156N, S263R and PA I38M compensated for the lack of PB2-627K and showed increased pathogenicity in mice, revealing a novel mechanism that can affect the virulence of influenza viruses. H6N6 viruses should be monitored for more virulent forms in the field that could threaten human health.

  • Kaposi's Sarcoma Herpesvirus downregulates Transforming Growth Factor-{beta}2 to promote the enhanced stability of capillary-like tube formation [PublishAheadOfPrint]

  • Kaposi's Sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's Sarcoma (KS), the most common tumor of AIDS patients world-wide. A key characteristic of KS tumors is extremely high levels of vascular slits and extravasated red blood cells making neo-angiogenesis a key component of the tumor. The main KS tumor cell is the spindle cell, a cell of endothelial origin that maintains KSHV predominantly in the latent state. In cultured endothelial cells, latent KSHV infection induces angiogenic phenotypes including longer term stabilization of capillary-like tube formation in matrigel, a basement membrane matrix. The present studies show that KSHV infection of endothelial cells strongly downregulates TGF-bbeta;2. This downregulation allows the stabilization of capillary-like tube formation during latent infection, as the addition of exogenous TGF-bbeta;2 inhibits the KSHV-induced stability of these structures. While two KSHV microRNAs are sufficient to downregulate TGF-bbeta;2 in endothelial cells, they are not required during KSHV infection. However, activation of the gp130 cell surface receptor is both necessary and sufficient for downregulation of TGF-bbeta;2 in KSHV infected cells.

    Importance: Kaposi's Sarcoma is a highly vascularized, endothelial cell based tumor supporting large amounts of angiogenesis. There is evidence that KSHV, the etiologic agent of KS, induces aberrant angiogenesis. For example, KSHV induces stabilization of capillary-like tube formation in cultured endothelial cells. A clearer understanding of how KSHV regulates angiogenesis could provide potential therapeutic targets for KS. We found that KSHV downregulates TGF-bbeta;2, a cytokine related to TGF-bbeta;1, that is known to inhibit angiogenesis. The downregulation of this inhbitor promotes the stability of capillary-like tube formation insofar as adding back TGF-bbeta;2 to infected cells blocks KSHV induced long-term tubule stability. Therefore, KSHV downregulation of TGF-bbeta;2 may increase aberrent vascularization in KS tumors through increased capillary formation and thereby aid in KS tumor promotion

  • Expression of the Human Cytomegalovirus UL11 Glycoprotein in Viral Infection and Evaluation of its Effect on Virus-Specific CD8 T Cells [PublishAheadOfPrint]

  • The human cytomegalovirus (CMV) UL11 open reading frame (ORF) encodes a putative type I transmembrane glycoprotein, which displays remarkable amino acid sequence variability among different CMV isolates, suggesting that it represents an important virulence factor. In a previous study we have shown that UL11 can interact with the cellular receptor tyrosine phosphatase CD45, which has a central role for signal transduction in T cells, and treatment of T cells with high amounts of a soluble UL11 protein inhibited their proliferation. In order to analyze UL11 expression in CMV-infected cells, we constructed CMV recombinants that either encode tagged UL11 versions or carry a stop mutation in the UL11 ORF. Moreover, we examined whether UL11 affects the function of virus-specific cytotoxic T lymphocytes (CTL). We found that the UL11 ORF gives rise to several proteins due to both posttranslational modification and alternative translation initiation sites. Biotin labelling of surface proteins on infected cells indicated that only highly glycosylated UL11 forms are present at the plasma membrane, whereas low glycosylated UL11 forms were found in the endoplasmic reticulum. We did not find evidence of UL11 cleavage and secretion of a soluble UL11 version. Co-cultivation of CTLs recognizing different CMV epitopes with fibroblasts infected with a UL11 deletion mutant or the parental strain revealed that under the conditions applied UL11 did not influence the activation of CMV-specific CD8 T cells. For further studies we propose to investigate the interaction of UL11 with CD45 and the functional consequences in other immune cells expressing CD45.

    IMPORTANCE Human cytomegalovirus belongs to those viruses that extensively interfere with the host immune response. Yet, the precise function of many putative immunomodulatory CMV proteins remains elusive. Previously, we have shown that the CMV UL11 protein interacts with the leukocyte common antigen CD45, a cellular receptor tyrosine phosphatase with a central role for signal transduction in T cells. Here, we examined the proteins expressed by the UL11 gene in CMV infected cells and found that at least one form of UL11 is present at the cell surface, enabling it to interact with CD45 on immune cells. Surprisingly, CMV-expressed UL11 did not affect the activity of virus-specific CD8 T cells. This finding warrants to investigate the impact of UL11 on CD45 functions in other leukocyte subpopulations.

  • Nipah Virion Entry Kinetics, Composition, and Conformational Changes Determined by Enzymatic VLPs and New Flow Virometry Tools [PublishAheadOfPrint]

  • Viral-cell membrane fusion is essential for enveloped virus infections. However, mechanistic viral membrane fusion studies have predominantly focused on cell-cell fusion models, largely due to the low availability of technologies capable of characterizing actual viral-cell membrane fusion. Although cell-cell fusion assays are valuable, they do not fully recapitulate all the variables of viral-cell membrane fusion. Drastic differences between viral vs. cellular membrane lipid and protein compositions and curvatures exist. For BSL4 pathogens such as the deadly Nipah virus (NiV), viral-cell fusion mechanistic studies are notably cumbersome. To circumvent these limitations we used enzymatic Nipah virus-like-particles (NiVLPs) and developed new flow virometric tools. NiV's attachment (G) and fusion (F) envelope glycoproteins mediate viral binding to the ephrinB2/ephrinB3 cell receptors and viral-cell membrane fusion, respectively. The NiV matrix protein (M) can autonomously induce NiV assembly and budding. Using a bbeta;-Lactamase (bbeta;La) reporter/NiV-M chimeric protein, we produced NiVLPs expressing NiV-G and wild-type or mutant NiV-F on their surfaces. By pre-loading target cells with the bbeta;La fluorescent substrate CCF2-AM, we obtained viral entry kinetic curves that correlated with the NiV-F fusogenic phenotypes, validating NiVLPs as suitable viral entry kinetic tools, and suggesting overall relatively slower viral entry than cell-cell fusion kinetics. Additionally, the proportions of F and G on individual NiVLPs and the extent of receptor-induced conformational changes in NiV-G were measured via flow virometry, allowing the proper interpretation of the viral entry kinetic phenotypes. The significance of these findings in the viral entry field extends beyond NiV to other paramyxoviruses and enveloped viruses.

    IMPORTANCE Viral-cell membrane fusion is essential for enveloped virus infections. However, mechanistic viral membrane fusion studies have predominantly focused on cell-cell fusion models, largely due to the low availability of technologies capable of characterizing actual viral-cell membrane fusion. Although cell-cell fusion assays are valuable, they do not fully recapitulate all the variables of viral-cell membrane fusion. For example, drastic differences between viral vs. cellular membrane lipid and protein compositions and curvatures exist. For biosafety level 4 (BSL4) pathogens such as the deadly Nipah virus (NiV), viral-cell fusion mechanistic studies are especially cumbersome. To circumvent these limitations we used enzymatic Nipah virus-like-particles (NiVLPs) and developed new flow virometric tools. Our new tools allowed us the high-throughput measurement of viral entry kinetics, glycoprotein proportions on individual viral particles, and receptor-induced conformational changes in viral glycoproteins on viral surfaces. The significance of these findings extends beyond NiV to other paramyxoviruses and enveloped viruses.

  • 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.

  • 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.

  • 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 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.

  • 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.

  • 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.

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