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Jean-Yves Sgro
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Table of Contents for this page:

  • Current Issue
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  • Current Issue of Journal of Virology

    JVI Current Issue

  • Editorial Board [Masthead]

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

  • Engineering Enhanced Vaccine Cell Lines To Eradicate Vaccine-Preventable Diseases: the Polio End Game [Vaccines and Antiviral Agents]

  • Vaccine manufacturing costs prevent a significant portion of the world's population from accessing protection from vaccine-preventable diseases. To enhance vaccine production at reduced costs, a genome-wide RNA interference (RNAi) screen was performed to identify gene knockdown events that enhanced poliovirus replication. Primary screen hits were validated in a Vero vaccine manufacturing cell line using attenuated and wild-type poliovirus strains. Multiple single and dual gene silencing events increased poliovirus titers ggt;20-fold and ggt;50-fold, respectively. Host gene knockdown events did not affect virus antigenicity, and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9-mediated knockout of the top candidates dramatically improved viral vaccine strain production. Interestingly, silencing of several genes that enhanced poliovirus replication also enhanced replication of enterovirus 71, a clinically relevant virus to which vaccines are being targeted. The discovery that host gene modulation can markedly increase virus vaccine production dramatically alters mammalian cell-based vaccine manufacturing possibilities and should facilitate polio eradication using the inactivated poliovirus vaccine.

    IMPORTANCE Using a genome-wide RNAi screen, a collection of host virus resistance genes was identified that, upon silencing, increased poliovirus and enterovirus 71 production by from 10-fold to ggt;50-fold in a Vero vaccine manufacturing cell line. This report provides novel insights into enterovirus-host interactions and describes an approach to developing the next generation of vaccine manufacturing through engineered vaccine cell lines. The results show that specific gene silencing and knockout events can enhance viral titers of both attenuated (Sabin strain) and wild-type polioviruses, a finding that should greatly facilitate global implementation of inactivated polio vaccine as well as further reduce costs for live-attenuated oral polio vaccines. This work describes a platform-enabling technology applicable to most vaccine-preventable diseases.

  • The Interferon Type I/III Response to Respiratory Syncytial Virus Infection in Airway Epithelial Cells Can Be Attenuated or Amplified by Antiviral Treatment [Vaccines and Antiviral Agents]

  • Human respiratory syncytial virus (RSV) is a single-stranded RNA virus that causes acute, and occasionally fatal, lower respiratory illness in young infants, the elderly, and immunocompromised patients. Therapeutic interventions able to cut short viral replication and quickly return the airways to normal function are needed. An understanding of antiviral activities and their effects on host defense mechanisms is important for the design of safe and effective therapy. We targeted functionally and temporally distinct steps within the viral life cycle using small-molecule RSV inhibitors and studied their antiviral activities and their effects on innate interferon responses of airway epithelial cells in vitro. Antivirals acting upstream of RSV polymerase activity (i.e., compounds targeting the fusion protein or the nucleoprotein) reduced viral load immediately postinfection and partially attenuated interferon responses. In contrast, antivirals directed to the RSV polymerase demonstrated activity throughout the viral replication cycle and specifically modulated the RIG-I/mitochondrial antiviral signaling protein (MAVS)/TBK1/IRF3/interferon-stimulated gene (ISG) axis, causing either an upregulation or a downregulation of interferon responses, depending on the mechanism of polymerase inhibition. Notably, polymerase inhibition leading to the accumulation of abortive RNA products correlated with the amplification of interferon-stimulated genes to up to 10 times above normal infection levels. Understanding how antiviral activities and their modulation of innate immunity may affect recovery from RSV infection will help guide the development of safe and effective therapies.

    IMPORTANCE RSV circulates seasonally, causing acute lower respiratory disease. Therapeutic interventions with efficacy throughout the viral replication cycle, rapid viral clearance, and prevention of potentially harmful inflammatory responses are desirable. Compounds targeting the RSV polymerase inhibited virus replication late in the viral life cycle and, depending on the functional domain targeted, either attenuated or amplified RIG-I and downstream interferon pathways in infected cells. These data will help guide the development of safe and effective therapies by providing new molecular evidence that the mechanism of inhibition by an antiviral compound can directly impact innate antiviral immune responses in the airway epithelium.

  • NP1 Protein of the Bocaparvovirus Minute Virus of Canines Controls Access to the Viral Capsid Genes via Its Role in RNA Processing [Genome Replication and Regulation of Viral Gene Expression]

  • Minute virus of canines (MVC) is an autonomous parvovirus in the genus Bocaparvovirus. It has a single promoter that generates a single pre-mRNA processed via alternative splicing and alternative polyadenylation to produce at least 8 mRNA transcripts. MVC contains two polyadenylation sites, one at the right-hand end of the genome, (pA)d, and another complex site, (pA)p, within the capsid-coding region. During viral infection, the mRNAs must extend through (pA)p and undergo additional splicing of the immediately upstream 3D/3A intron to access the capsid gene. MVC NP1 is a 22-kDa nuclear phosphoprotein unique to the genus Bocaparvovirus of the Parvovirinae which we have shown governs suppression of (pA)p independently of viral genome replication. We show here that in addition to suppression of (pA)p, NP1 is also required for the excision of the MVC 3D/3A intron, independently of its effect on alternative polyadenylation. Mutations of the arginine/serine (SR) di-repeats within the intrinsically disordered amino terminus of NP1 are required for splicing of the capsid transcript but not suppression of polyadenylation at (pA)p. 3'-end processing of MVC mRNAs at (pA)p is critical for viral genome replication and the optimal expression of NP1 and NS1. Thus, a finely tuned balance between (pA)p suppression and usage is necessary for efficient virus replication. NP1 is the first parvovirus protein implicated in RNA processing. Its characterization reveals another way that parvoviruses govern access to their capsid protein genes, namely, at the RNA level, by regulating the essential splicing of an intron and the suppression of an internal polyadenylation site.

    IMPORTANCE The Parvovirinae are small nonenveloped icosahedral viruses that are important pathogens in many animal species, including humans. Although parvoviruses have only subtle early-to-late expression shifts, they all regulate access to their capsid genes. Minute virus of canines (MVC) is an autonomous parvovirus in the genus Bocaparvovirus. It has a single promoter generating a single pre-mRNA which is processed via alternative splicing and alternative polyadenylation to generate at least 8 mRNA transcripts. MVC contains two polyadenylation sites, one at the right-hand end of the genome, (pA)d, and another, (pA)p, within the capsid-coding region. It had not been clear how the potent internal polyadenylation motif is suppressed to allow processing, export, and accumulation of the spliced capsid protein-encoding mRNAs. We show here that MVC NP1, the first parvovirus protein to be implicated in RNA processing, governs access to the MVC capsid gene by facilitating splicing and suppressing internal polyadenylation of MVC pre-mRNAs.

  • Hepatitis B Virus X Protein Induces Hepatic Steatosis by Enhancing the Expression of Liver Fatty Acid Binding Protein [Cellular Response to Infection]

  • Hepatitis B virus (HBV) has been implicated as a potential trigger of hepatic steatosis although molecular mechanisms involved in the pathogenesis of HBV-associated hepatic steatosis still remain elusive. Our prior work has revealed that the expression level of liver fatty acid binding protein 1 (FABP1), a key regulator of hepatic lipid metabolism, was elevated in HBV-producing hepatoma cells. In this study, the effects of HBV X protein (HBx) mediated FABP1 regulation on hepatic steatosis and the underlying mechanism were determined. mRNA and protein levels of FABP1 were measured by quantitative RT-PCR (qPCR) and Western blotting. HBx-mediated FABP1 regulation was evaluated by luciferase assay, coimmunoprecipitation, and chromatin immunoprecipitation. Hepatic lipid accumulation was measured by using Oil-Red-O staining and the triglyceride level. It was found that expression of FABP1 was increased in HBV-producing hepatoma cells, the sera of HBV-infected patients, and the sera and liver tissues of HBV-transgenic mice. Ectopic overexpression of HBx resulted in upregulation of FABP1 in HBx-expressing hepatoma cells, whereas HBx abolishment reduced FABP1 expression. Mechanistically, HBx activated the FABP1 promoter in an HNF3bbeta;-, C/EBPaalpha;-, and PPARaalpha;-dependent manner, in which HBx increased the gene expression of HNF3bbeta; and physically interacted with C/EBPaalpha; and PPARaalpha;. On the other hand, knockdown of FABP1 remarkably blocked lipid accumulation both in long-chain free fatty acids treated HBx-expressing HepG2 cells and in a high-fat diet-fed HBx-transgenic mice. Therefore, FABP1 is a key driver gene in HBx-induced hepatic lipid accumulation via regulation of HNF3bbeta;, C/EBPaalpha;, and PPARaalpha;. FABP1 may represent a novel target for treatment of HBV-associated hepatic steatosis.

    IMPORTANCE Accumulating evidence from epidemiological and experimental studies has indicated that chronic HBV infection is associated with hepatic steatosis. However, the molecular mechanism underlying HBV-induced pathogenesis of hepatic steatosis still remains to be elucidated. In this study, we found that expression of liver fatty acid binding protein (FABP1) was dramatically increased in the sera of HBV-infected patients and in both sera and liver tissues of HBV-transgenic mice. Forced expression of HBx led to FABP1 upregulation, whereas knockdown of FABP1 remarkably diminished lipid accumulation in both in vitro and in vivo models. It is possible that HBx promotes hepatic lipid accumulation through upregulating FABP1 in the development of HBV-induced nonalcoholic fatty liver disease. Therefore, inhibition of FABP1 might have therapeutic value in steatosis-associated chronic HBV infection.

  • ORF33 and ORF38 of Kaposi's Sarcoma-Associated Herpesvirus Interact and Are Required for Optimal Production of Infectious Progeny Viruses [Structure and Assembly]

  • We recently showed that the interaction between Kaposi's sarcoma-associated herpesvirus (KSHV) tegument proteins ORF33 and ORF45 is crucial for progeny virion production, but the exact functions of KSHV ORF33 during lytic replication were unknown (J. Gillen, W. Li, Q. Liang, D. Avey, J. Wu, F. Wu, J. Myoung, and F. Zhu, J Virol 89:4918nndash;4931, 2015, Therefore, here we investigated the relationship between ORF33 and ORF38, whose counterparts in both alpha- and betaherpesviruses interact with each other. Using specific monoclonal antibodies, we found that both proteins are expressed during the late lytic cycle with similar kinetics and that both are present in mature virions as components of the tegument. Furthermore, we confirmed that ORF33 interacts with ORF38. Interestingly, we observed that ORF33 tightly associates with the capsid, whereas ORF38 associates with the envelope. We generated ORF33-null, ORF38-null, and double-null mutants and found that these mutants apparently have identical phenotypes: the mutations caused no apparent effect on viral gene expression but reduced the yield of progeny virion by about 10-fold. The progeny virions also lack certain virion component proteins, including ORF45. During viral lytic replication, the virions associate with cytoplasmic vesicles. We also observed that ORF38 associates with the membranes of vesicles and colocalizes with the Golgi membrane or early endosome membrane. Further analyses of ORF33/ORF38 mutants revealed the reduced production of virion-containing vesicles, suggesting that ORF33 and ORF38 are involved in the transport of newly assembled viral particles into cytoplasmic vesicles, a process important for viral maturation and egress.

    IMPORTANCE Herpesvirus assembly is an essential step in virus propagation that leads to the generation of progeny virions. It is a complicated process that depends on the delicate regulation of interactions among virion proteins. We previously revealed an essential role of ORF45-ORF33 binding for virus assembly. Here, we report that ORF33 and its binding partner, ORF38, are required for infectious virus production due to their important role in the tegumentation process. Moreover, we found that both ORF33 and ORF38 are involved in the transportation of virions through vesicles during maturation and egress. Our results provide new insights into the important roles of ORF33 and ORF38 during viral assembly, a process critical for virus propagation that is intimately linked to KSHV pathobiology.

  • Episodic Diversifying Selection Shaped the Genomes of Gibbon Ape Leukemia Virus and Related Gammaretroviruses [Genetic Diversity and Evolution]

  • Gibbon ape leukemia viruses (GALVs) are part of a larger group of pathogenic gammaretroviruses present across phylogenetically diverse host species of Australasian mammals. Despite the biomedical utility of GALVs as viral vectors and in cancer gene therapy, full genome sequences have not been determined for all of the five identified GALV strains, nor has a comprehensive evolutionary analysis been performed. We therefore generated complete genomic sequences for each GALV strain using hybridization capture and high-throughput sequencing. The four strains of GALV isolated from gibbons formed a monophyletic clade that was closely related to the woolly monkey virus (WMV), which is a GALV strain that likely originated in a gibbon host. The GALV-WMV clade in turn formed a sister group to the koala retroviruses (KoRVs). Genomic signatures of episodic diversifying selection were detected among the gammaretroviruses with concentration in the env gene across the GALV strains that were particularly oncogenic and KoRV strains that were potentially exogenous, likely reflecting their adaptation to the host immune system. In vitro studies involving vectors chimeric between GALV and KoRV-B established that variable regions A and B of the surface unit of the envelope determine which receptor is used by a viral strain to enter host cells.

    IMPORTANCE The gibbon ape leukemia viruses (GALVs) are among the most medically relevant retroviruses due to their use as viral vectors for gene transfer and in cancer gene therapy. Despite their importance, full genome sequences have not been determined for the majority of primate isolates, nor has comprehensive evolutionary analysis been performed, despite evidence that the viruses are facing complex selective pressures associated with cross-species transmission. Using hybridization capture and high-throughput sequencing, we report here the full genome sequences of all the GALV strains and demonstrate that diversifying selection is acting on them, particularly in the envelope gene in functionally important domains, suggesting that host immune pressure is shaping GALV evolution.

  • Dimerization of the SP1 Region of HIV-1 Gag Induces a Helical Conformation and Association into Helical Bundles: Implications for Particle Assembly [Structure and Assembly]

  • HIV-1 immature particle (virus-like particle [VLP]) assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We previously investigated the role of SP1, a "spacer" between CA and NC, in VLP assembly. We found that small changes in SP1 drastically disrupt assembly and that a peptide representing the sequence around the CA-SP1 junction is helical at high but not low concentrations. We suggested that by virtue of such a concentration-dependent change, this region could act as a molecular switch to activate HIV-1 Gag for VLP assembly. A leucine zipper domain can replace NC in Gag and still lead to the efficient assembly of VLPs. We find that SP1 mutants also disrupt assembly by these Gag-Zip proteins and have now studied a small fragment of this Gag-Zip protein, i.e., the CA-SP1 junction region fused to a leucine zipper. Dimerization of the zipper places SP1 at a high local concentration, even at low total concentrations. In this context, the CA-SP1 junction region spontaneously adopts a helical conformation, and the proteins associate into tetramers. Tetramerization requires residues from both CA and SP1. The data suggest that once this region becomes helical, its propensity to self-associate could contribute to Gag-Gag interactions and thus to particle assembly. There is complete congruence between CA/SP1 sequences that promote tetramerization when fused to zippers and those that permit the proper assembly of full-length Gag; thus, equivalent interactions apparently participate in VLP assembly and in SP1-Zip tetramerization.

    IMPORTANCE Assembly of HIV-1 Gag into virus-like particles (VLPs) appears to require an interaction with nucleic acid, but replacement of its principal nucleic acid-binding domain with a dimerizing leucine zipper domain leads to the assembly of RNA-free VLPs. It has not been clear how dimerization triggers assembly. Results here show that the SP1 region spontaneously switches to a helical state when fused to a leucine zipper and that these helical molecules further associate into tetramers, mediated by interactions between hydrophobic faces of the helices. Thus, the correct juxtaposition of the SP1 region makes it "association competent." Residues from both capsid and SP1 contribute to tetramerization, while mutations disrupting proper assembly in Gag also prevent tetramerization. Thus, this region is part of an associating interface within Gag, and its intermolecular interactions evidently help stabilize the immature Gag lattice. These interactions are disrupted by proteolysis of the CA-SP1 junction during virus maturation.

  • Influenza A Virus-Induced Expression of a GalNAc Transferase, GALNT3, via MicroRNAs Is Required for Enhanced Viral Replication [Virus-Cell Interactions]

  • Influenza A virus (IAV) affects the upper and lower respiratory tracts and rapidly induces the expression of mucins, which are common O-glycosylated proteins, on the epithelial surfaces of the respiratory tract. Although mucin production is associated with the inhibition of virus transmission as well as characteristic clinical symptoms, little is known regarding how mucins are produced on the surfaces of respiratory epithelial cells and how they affect IAV replication. In this study, we found that two microRNAs (miRNAs), miR-17-3p and miR-221, which target GalNAc transferase 3 (GALNT3) mRNA, are rapidly downregulated in human alveolar basal epithelial cells during the early stage of IAV infection. We demonstrated that the expression of GALNT3 mRNA is upregulated in an IAV replication-dependent fashion and leads to mucin production in bronchial epithelial cells. A lectin microarray analysis revealed that the stable expression of GALNT3 by human alveolar basal epithelial cells induces mucin-type O-glycosylation modifications similar to those present in IAV-infected cells, suggesting that GALNT3 promotes mucin-type O-linked glycosylation in IAV-infected cells. Notably, analyses using short interfering RNAs and miRNA mimics showed that GALNT3 knockdown significantly reduces IAV replication. Furthermore, IAV replication was markedly decreased in embryonic fibroblast cells obtained from galnt3-knockout mice. Interestingly, IAV-infected galnt3-knockout mice exhibited high mortality and severe pathological alterations in the lungs compared to those of wild-type mice. Our results demonstrate not only the molecular mechanism underlying rapid mucin production during IAV infection but also the contribution of O-linked glycosylation to the replication and propagation of IAV in lung cells.

    IMPORTANCE Viral infections that affect the upper or lower respiratory tracts, such as IAV, rapidly induce mucin production on the epithelial surfaces of respiratory cells. However, the details of how mucin-type O-linked glycosylation is initiated by IAV infection and how mucin production affects viral replication have not yet been elucidated. In this study, we show that levels of two miRNAs that target the UDP-GalNAc transferase GALNT3 are markedly decreased during the early stage of IAV infection, resulting in the upregulation of GALNT3 mRNA. We also demonstrate that the expression of GALNT3 initiates mucin production and affects IAV replication in infected cells. This is the first report demonstrating the mechanism underlying the miRNA-mediated initiation of mucin-type O-glycosylation in IAV-infected cells and its role in viral replication. Our results have broad implications for understanding IAV replication and suggest a strategy for the development of novel anti-influenza approaches.

  • Antibody Binding Modulates Conformational Exchange in Domain III of Dengue Virus E Protein [Structure and Assembly]

  • Domain III of dengue virus E protein (DIII) participates in the recognition of cell receptors and in structural rearrangements required for membrane fusion and ultimately viral infection; furthermore, it contains epitopes for neutralizing antibodies and has been considered a potential vaccination agent. In this work, we addressed various structural aspects of DIII and their relevance for both the dengue virus infection mechanism and antibody recognition. We provided a dynamic description of DIII at physiological and endosomal pHs and in complex with the neutralizing human antibody DV32.6. We observed conformational exchange in the isolated DIII, in regions important for the packing of E protein dimers on the virus surface. This conformational diversity is likely to facilitate the partial detachment of DIII from the other E protein domains, which is required to achieve fusion to the host cellular membranes and to expose the epitopes of many anti-DIII antibodies. A comparison of DIII of two dengue virus serotypes revealed many common features but also some possibly unexpected differences. Antibody binding to DIII of dengue virus serotype 4 attenuated the conformational exchange in the epitope region but, surprisingly, generated exchange in other parts of DIII through allosteric effects.

    IMPORTANCE Many studies have provided extensive structural information on the E protein and particularly on DIII, also in complex with antibodies. However, there is very scarce information regarding the molecular dynamics of DIII, and almost nothing is available on the dynamic effect of antibody binding, especially at the quantitative level. This work provides one of the very rare descriptions of the effect of antibody binding on antigen dynamics.

  • Influenza Virus Infection Induces Platelet-Endothelial Adhesion Which Contributes to Lung Injury [Cellular Response to Infection]

  • Lung injury after influenza infection is characterized by increased permeability of the lung microvasculature, culminating in acute respiratory failure. Platelets interact with activated endothelial cells and have been implicated in the pathogenesis of some forms of acute lung injury. Autopsy studies have revealed pulmonary microthrombi after influenza infection, and epidemiological studies suggest that influenza vaccination is protective against pulmonary thromboembolism; however, the effect of influenza infection on platelet-endothelial interactions is unclear. We demonstrate that endothelial infection with both laboratory and clinical strains of influenza virus increased the adhesion of human platelets to primary human lung microvascular endothelial cells. Platelets adhered to infected cells as well as to neighboring cells, suggesting a paracrine effect. Influenza infection caused the upregulation of von Willebrand factor and ICAM-1, but blocking these receptors did not prevent platelet-endothelial adhesion. Instead, platelet adhesion was inhibited by both RGDS peptide and a blocking antibody to platelet integrin aalpha;5bbeta;1, implicating endothelial fibronectin. Concordantly, lung histology from infected mice revealed viral dose-dependent colocalization of viral nucleoprotein and the endothelial marker PECAM-1, while platelet adhesion and fibronectin deposition also were observed in the lungs of influenza-infected mice. Inhibition of platelets using acetylsalicylic acid significantly improved survival, a finding confirmed using a second antiplatelet agent. Thus, influenza infection induces platelet-lung endothelial adhesion via fibronectin, contributing to mortality from acute lung injury. The inhibition of platelets may constitute a practical adjunctive strategy to the treatment of severe infections with influenza.

    IMPORTANCE There is growing appreciation of the involvement of the lung endothelium in the pathogenesis of severe infections with influenza virus. We have recently shown that the virus can infect human lung endothelial cells, but the functional consequences of this infection are unknown (S. M. Armstrong, C. Wang, J. Tigdi, X. Si, C. Dumpit, S. Charles, A. Gamage, T. J. Moraes, and W. L. Lee, PLoS One 7:e47323, 2012, Here, we show that this infection causes platelets to adhere to the lung endothelium. Importantly, blocking platelets using two distinct antiplatelet drugs improved survival in a mouse model of severe influenza infection. Thus, platelet inhibition may constitute a novel therapeutic strategy to improve the host response to severe infections with influenza.

  • Equine Infectious Anemia Virus Gag Assembly and Export Are Directed by Matrix Protein through trans-Golgi Networks and Cellular Vesicles [Structure and Assembly]

  • Gag intracellular assembly and export are very important processes for lentiviruses replication. Previous studies have demonstrated that equine infectious anemia virus (EIAV) matrix (MA) possesses distinct phosphoinositide affinity compared with HIV-1 MA and that phosphoinositide-mediated targeting to peripheral and internal membranes is a critical factor in EIAV assembly and release. In this study, we compared the cellular assembly sites of EIAV and HIV-1. We observed that the assembly of EIAV particles occurred on interior cellular membranes, while HIV-1 was targeted to the plasma membrane (PM) for assembly. Then, we determined that W7 and K9 in the EIAV MA N terminus were essential for Gag assembly and release but did not affect the cellular distribution of Gag. The replacement of EIAV MA with HIV-1 MA directed chimeric Gag to the PM but severely impaired Gag release. MA structural analysis indicated that the EIAV and HIV-1 MAs had similar spatial structures but that helix 1 of the EIAV MA was closer to loop 2. Further investigation indicated that EIAV Gag accumulated in the trans-Golgi network (TGN) but not the early and late endosomes. The 9 N-terminal amino acids of EIAV MA harbored the signal that directed Gag to the TGN membrane system. Additionally, we demonstrated that EIAV particles were transported to the extracellular space by the cellular vesicle system. This type of EIAV export was not associated with multivesicular bodies or microtubule depolymerization but could be inhibited by the actin-depolymerizing drug cytochalasin D, suggesting that dynamic actin depolymerization may be associated with EIAV production.

    IMPORTANCE In previous studies, EIAV Gag was reported to localize to both the cell interior and the plasma membrane. Here, we demonstrate that EIAV likely uses the TGN as the assembly site in contrast to HIV-1, which is targeted to the PM for assembly. These distinct assembly features are determined by the MA domain. We also identified two sites in the N terminus of EIAV MA that were important for Gag assembly and release. Furthermore, the observation of EIAV transport by cellular vesicles but not by multivesicular bodies sheds light on the mechanisms underlying EIAV cellular replication.

  • Crystal Structure of Marburg Virus VP40 Reveals a Broad, Basic Patch for Matrix Assembly and a Requirement of the N-Terminal Domain for Immunosuppression [Structure and Assembly]

  • Marburg virus (MARV), a member of the filovirus family, causes severe hemorrhagic fever with up to 90% lethality. MARV matrix protein VP40 is essential for assembly and release of newly copied viruses and also suppresses immune signaling in the infected cell. Here we report the crystal structure of MARV VP40. We found that MARV VP40 forms a dimer in solution, mediated by N-terminal domains, and that formation of this dimer is essential for budding of virus-like particles. We also found the N-terminal domain to be necessary and sufficient for immune antagonism. The C-terminal domains of MARV VP40 are dispensable for immunosuppression but are required for virus assembly. The C-terminal domains are only 16% identical to those of Ebola virus, differ in structure from those of Ebola virus, and form a distinct broad and flat cationic surface that likely interacts with the cell membrane during virus assembly.

    IMPORTANCE Marburg virus, a cousin of Ebola virus, causes severe hemorrhagic fever, with up to 90% lethality seen in recent outbreaks. Molecular structures and visual images of the proteins of Marburg virus are essential for the development of antiviral drugs. One key protein in the Marburg virus life cycle is VP40, which both assembles the virus and suppresses the immune system. Here we provide the molecular structure of Marburg virus VP40, illustrate differences from VP40 of Ebola virus, and reveal surfaces by which Marburg VP40 assembles progeny and suppresses immune function.

  • TRIM5{alpha}-Mediated Ubiquitin Chain Conjugation Is Required for Inhibition of HIV-1 Reverse Transcription and Capsid Destabilization [Virus-Cell Interactions]

  • Rhesus macaque TRIM5aalpha; (rhTRIM5aalpha;) is a retroviral restriction factor that inhibits HIV-1 infection. Previous studies have revealed that TRIM5aalpha; restriction occurs via a two-step process. The first step is restriction factor binding, which is sufficient to inhibit infection. The second step, which is sensitive to proteasome inhibition, prevents the accumulation of reverse transcription products in the target cell. However, because of the pleotropic effects of proteasome inhibitors, the molecular mechanisms underlying the individual steps in the restriction process have remained poorly understood. In this study, we have fused the small catalytic domain of herpes simplex virus UL36 deubiquitinase (DUb) to the N-terminal RING domain of rhTRIM5aalpha;, which results in a ubiquitination-resistant protein. Cell lines stably expressing this fusion protein inhibited HIV-1 infection to the same degree as a control fusion to a catalytically inactive DUb. However, reverse transcription products were substantially increased in the DUb-TRIM5aalpha; fusion relative to the catalytically inactive control or the wild-type (WT) TRIM5aalpha;. Similarly, expression of DUb-rhTRIM5aalpha; resulted in the accumulation of viral cores in target cells following infection, while the catalytically inactive control and WT rhTRIM5aalpha; induced the abortive disassembly of viral cores, indicating a role for ubiquitin conjugation in rhTRIM5aalpha;-mediated destabilization of HIV-1 cores. Finally, DUb-rhTRIM5aalpha; failed to activate NF-B signaling pathways compared to controls, demonstrating that this ubiquitination-dependent activity is separable from the ability to restrict retroviral infection.

    IMPORTANCE These studies provide direct evidence that ubiquitin conjugation to rhTRIM5aalpha;-containing complexes is required for the second step of HIV-1 restriction. They also provide a novel tool by which the biological activities of TRIM family proteins might be dissected to better understand their function and underlying mechanisms of action.

  • In Vitro Reactivation of Replication-Competent and Infectious HIV-1 by Histone Deacetylase Inhibitors [Virus-Cell Interactions]

  • The existence of long-lived HIV-1-infected resting memory CD4 T cells is thought to be the primary obstacle to HIV-1 eradication. In the search for novel therapeutic approaches that may reverse HIV-1 latency, inhibitors of histone deacetylases (HDACis) have been tested to reactivate HIV-1 replication with the objective of rendering HIV-1-infected cells susceptible to elimination either by HIV-specific CD8 T cells or through virus-mediated cytopathicity. In the present study, we evaluated the efficiency of HDACis to reactivate HIV-1 replication from resting memory CD4 T cells isolated from aviremic long-term-treated HIV-1-infected subjects. We demonstrate that following prolonged/repeated treatment of resting memory CD4 T cells with HDACis, HIV-1 replication may be induced from primary resting memory CD4 T cells isolated from aviremic long-term-treated HIV-1-infected subjects. More importantly, we demonstrate that HIV-1 reactivated in the cell cultures was not only replication competent but also infectious. Interestingly, givinostat, an HDACi that has not been investigated in clinical trials, was more efficient than vorinostat, panobinostat, and romidepsin in reversing HIV-1 latency in vitro. Taken together, these results support further evaluation of givinostat as a latency-reversing agent (LRA) in aviremic long-term-treated HIV-1-infected subjects.

    IMPORTANCE The major barrier to HIV cure is the existence of long-lived latently HIV-1-infected resting memory CD4 T cells. Latently HIV-1-infected CD4 T cells are transcriptionally silent and are therefore not targeted by conventional antiretroviral therapy (ART) or the immune system. In this context, one strategy to target latently infected cells is based on pharmacological molecules that may force the virus to replicate and would therefore render HIV-1-infected cells susceptible to elimination either by HIV-specific CD8 T cells or through virus-mediated cytopathicity. In this context, we developed an experimental strategy that would allow the evaluation of latency-reversing agent (LRA) efficiency in vitro using primary CD4 T cells. In the present study, we demonstrate that HDACis are potent inducers of replication-competent and infectious HIV-1 in resting memory CD4 T cells of long-term ART-treated patients and identify givinostat as the most efficient LRA tested.

  • Glycine at Position 622 in PB1 Contributes to the Virulence of H5N1 Avian Influenza Virus in Mice [Pathogenesis and Immunity]

  • We isolated two H5N1 viruses, A/duck/Hunan/S4020/2008 (DK/08) and A/chicken/Guangxi/S2039/2009 (CK/09), from live-bird markets during routine surveillance and found that these two viruses are genetically similar but differ in their replication and virulence in mice. The CK/09 virus is lethal for mice with a 50% mouse lethal dose (MLD50) of 1.6 log10 50% egg infectious doses (EID50), whereas the DK/08 virus is nonpathogenic for mice with an MLD50 value of 6.2 log10 EID50. We explored the genetic basis of the virulence difference of these two viruses by generating a series of reassortant viruses and mutants in the lethal virus CK/09 background and evaluating their virulence in mice. We found that the PB1 gene of the DK/08 virus dramatically attenuated the virulence of the CK/09 virus and that the amino acid at position 622 in PB1 made an important contribution. We further demonstrated that the mutation of glycine (G) to aspartic acid (D) at position 622 in PB1 partially impaired the binding of PB1 to viral RNA, thereby dramatically decreasing the polymerase activity and attenuating H5N1 virus virulence in mice. Our results identify a novel virulence-related marker of H5N1 influenza viruses and provide a new target for live attenuated vaccine development.

    IMPORTANCE H5N1 avian influenza viruses have caused the deaths of nearly 60% of the humans that they have infected since 1997 and clearly represent a threat to public health. A thorough understanding of the genetic basis of virulence determinants will provide important insights for antiviral drug and live attenuated vaccine development. Several virulence-related markers in the PB2, PA, M1, and NS1 proteins of H5N1 viruses have been identified. In this study, we isolated two H5N1 avian influenza viruses that are genetically similar but differ in their virulence in mice, and we identified a new virulence-related marker in the PB1 gene. We found that the mutation of glycine (G) to aspartic acid (D) at position 622 in PB1 partially impairs the binding of PB1 to viral RNA, thereby attenuating H5N1 virus virulence in mice. This newly identified virulence-related marker could be applied to the development of live attenuated vaccines against H5N1 influenza.

  • Signatures in Simian Immunodeficiency Virus SIVsmE660 Envelope gp120 Are Associated with Mucosal Transmission but Not Vaccination Breakthrough in Rhesus Macaques [Pathogenesis and Immunity]

  • Mucosal surfaces are vulnerable to human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection and thus are key sites for eliciting vaccine-mediated protection. Vaccine protocols carried out at the Yerkes Primate Research Center utilized SIVmac239-based immunization strategies with intrarectal and intravaginal SIVsmE660 challenge of rhesus macaques. We investigated whether there were genetic signatures associated with SIVsmE660 intrarectal and intravaginal transmissions in vaccinated and unvaccinated monkeys. When transmitted/founder (T/F) envelope (Env) sequences from 49 vaccinated and 15 unvaccinated macaques were compared to each other, we were unable to identify any vaccine breakthrough signatures. In contrast, when the vaccinated and control T/F Envs were combined and compared to the challenge stock, residues at gp120 positions 23, 45, 47, and 70 (Ile-Ala-Lys-Asn [I-A-K-N]) emerged as signatures of mucosal transmission. However, T/F Envs derived from intrarectal and intravaginal infections were not different. Our data suggest that the vaginal and rectal mucosal environments both imposed a strong selection bias for SIVsmE660 variants carrying I-A-K-N that was not further enhanced by immunization. These findings, combined with the strong conservation of A-K-N in most HIV-2/SIVsmm isolates and the analogous residues in HIV-1/SIVcpz isolates, suggest that these residues confer increased transmission fitness to SIVsmE660.

    IMPORTANCE Most HIV-1 infections occur across a mucosal barrier, and it is therefore important to understand why these sites are vulnerable and how to protect them with a vaccine. To gain insight into these questions, we studied rhesus macaques that were vaccinated with SIVmac239 and unvaccinated controls to determine whether the SIVsmE660 viral variants that infected these two groups were different. We did not find differences between viral variants in the absence versus presence of vaccination-induced immunity, but we did find that the SIVsmE660 viral variants that infected the monkeys, regardless of vaccination, were different from the dominant population found in the viral challenge inoculum. Our data suggest that the mucosal environments of the vagina and rectum both impose a strong selection for the SIVsmE660 variants in the challenge inoculum that are most like SIV and HIVs that circulate in nature.

  • Predicting Disease Severity and Viral Spread of H5N1 Influenza Virus in Ferrets in the Context of Natural Exposure Routes [Pathogenesis and Immunity]

  • Although avian H5N1 influenza virus has yet to develop the capacity for human-to-human spread, the severity of the rare cases of human infection has warranted intensive follow-up of potentially exposed individuals that may require antiviral prophylaxis. For countries where antiviral drugs are limited, the World Health Organization (WHO) has developed a risk categorization for different levels of exposure to environmental, poultry, or human sources of infection. While these take into account the infection source, they do not account for the likely mode of virus entry that the individual may have experienced from that source and how this could affect the disease outcome. Knowledge of the kinetics and spread of virus after natural routes of exposure may further inform the risk of infection, as well as the likely disease severity. Using the ferret model of H5N1 infection, we compared the commonly used but artificial inoculation method that saturates the total respiratory tract (TRT) with virus to upper respiratory tract (URT) and oral routes of delivery, those likely to be encountered by humans in nature. We show that there was no statistically significant difference in survival rate with the different routes of infection, but the disease characteristics were somewhat different. Following URT infection, viral spread to systemic organs was comparatively delayed and more focal than after TRT infection. By both routes, severe disease was associated with early viremia and central nervous system infection. After oral exposure to the virus, mild infections were common suggesting consumption of virus-contaminated liquids may be associated with seroconversion in the absence of severe disease.

    IMPORTANCE Risks for human H5N1 infection include direct contact with infected birds and frequenting contaminated environments. We used H5N1 ferret infection models to show that breathing in the virus was more likely to produce clinical infection than swallowing contaminated liquid. We also showed that virus could spread from the respiratory tract to the brain, which was associated with end-stage disease, and very early viremia provided a marker for this. With upper respiratory tract exposure, infection of the brain was common but hard to detect, suggesting that human neurological infections might be typically undetected at autopsy. However, viral spread to systemic sites was slower after exposure to virus by this route than when virus was additionally delivered to the lungs, providing a better therapeutic window. In addition to exposure history, early parameters of infection, such as viremia, could help prioritize antiviral treatments for patients most at risk of succumbing to infection.

  • Transcriptional Regulation in Ebola Virus: Effects of Gene Border Structure and Regulatory Elements on Gene Expression and Polymerase Scanning Behavior [Genome Replication and Regulation of Viral Gene Expression]

  • The highly pathogenic Ebola virus (EBOV) has a nonsegmented negative-strand (NNS) RNA genome containing seven genes. The viral genes either are separated by intergenic regions (IRs) of variable length or overlap. The structure of the EBOV gene overlaps is conserved throughout all filovirus genomes and is distinct from that of the overlaps found in other NNS RNA viruses. Here, we analyzed how diverse gene borders and noncoding regions surrounding the gene borders influence transcript levels and govern polymerase behavior during viral transcription. Transcription of overlapping genes in EBOV bicistronic minigenomes followed the stop-start mechanism, similar to that followed by IR-containing gene borders. When the gene overlaps were extended, the EBOV polymerase was able to scan the template in an upstream direction. This polymerase feature seems to be generally conserved among NNS RNA virus polymerases. Analysis of IR-containing gene borders showed that the IR sequence plays only a minor role in transcription regulation. Changes in IR length were generally well tolerated, but specific IR lengths led to a strong decrease in downstream gene expression. Correlation analysis revealed that these effects were largely independent of the surrounding gene borders. Each EBOV gene contains exceptionally long untranslated regions (UTRs) flanking the open reading frame. Our data suggest that the UTRs adjacent to the gene borders are the main regulators of transcript levels. A highly complex interplay between the different cis-acting elements to modulate transcription was revealed for specific combinations of IRs and UTRs, emphasizing the importance of the noncoding regions in EBOV gene expression control.

    IMPORTANCE Our data extend those from previous analyses investigating the implication of noncoding regions at the EBOV gene borders for gene expression control. We show that EBOV transcription is regulated in a highly complex yet not easily predictable manner by a set of interacting cis-active elements. These findings are important not only for the design of recombinant filoviruses but also for the design of other replicon systems widely used as surrogate systems to study the filovirus replication cycle under low biosafety levels. Insights into the complex regulation of EBOV transcription conveyed by noncoding sequences will also help to interpret the importance of mutations that have been detected within these regions, including in isolates of the current outbreak.

  • Crystal Structure of Feline Infectious Peritonitis Virus Main Protease in Complex with Synergetic Dual Inhibitors [Structure and Assembly]

  • Coronaviruses (CoVs) can cause highly prevalent diseases in humans and animals. Feline infectious peritonitis virus (FIPV) belongs to the genus Alphacoronavirus, resulting in a lethal systemic granulomatous disease called feline infectious peritonitis (FIP), which is one of the most important fatal infectious diseases of cats worldwide. No specific vaccines or drugs have been approved to treat FIP. CoV main proteases (Mpros) play a pivotal role in viral transcription and replication, making them an ideal target for drug development. Here, we report the crystal structure of FIPV Mpro in complex with dual inhibitors, a zinc ion and a Michael acceptor. The complex structure elaborates a unique mechanism of two distinct inhibitors synergizing to inactivate the protease, providing a structural basis to design novel antivirals and suggesting the potential to take advantage of zinc as an adjunct therapy against CoV-associated diseases.

    IMPORTANCE Coronaviruses (CoVs) have the largest genome size among all RNA viruses. CoV infection causes various diseases in humans and animals, including severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). No approved specific drugs or vaccinations are available to treat their infections. Here, we report a novel dual inhibition mechanism targeting CoV main protease (Mpro) from feline infectious peritonitis virus (FIPV), which leads to lethal systemic granulomatous disease in cats. Mpro, conserved across all CoV genomes, is essential for viral replication and transcription. We demonstrated that zinc ion and a Michael acceptor-based peptidomimetic inhibitor synergistically inactivate FIPV Mpro. We also solved the structure of FIPV Mpro complexed with two inhibitors, delineating the structural view of a dual inhibition mechanism. Our study provides new insight into the pharmaceutical strategy against CoV Mpro through using zinc as an adjuvant therapy to enhance the efficacy of an irreversible peptidomimetic inhibitor.

  • Pan-genotypic Hepatitis C Virus Inhibition by Natural Products Derived from the Wild Egyptian Artichoke [Vaccines and Antiviral Agents]

  • Hepatitis C virus (HCV) infection is the leading cause of chronic liver diseases. Water extracts of the leaves of the wild Egyptian artichoke (WEA) [Cynara cardunculus L. var. sylvestris (Lam.) Fiori] have been used for centuries in the Sinai Peninsula to treat hepatitis symptoms. Here we isolated and characterized six compounds from the water extracts of WEA and evaluated their HCV inhibition capacities in vitro. Importantly, two of these compounds, grosheimol and cynaropicrin, inhibited HCV with half-maximal effective concentrations (EC50s) in the low micromolar range. They inhibited HCV entry into target cells and were active against both cell-free infection as well as cell-cell transmission. Furthermore, the antiviral activity of both compounds was pan-genotypic as HCV genotypes 1a, 1b, 2b, 3a, 4a, 5a, 6a, and 7a were inhibited. Thus, grosheimol and cynaropicrin are promising candidates for the development of new pan-genotypic entry inhibitors of HCV infection.

    IMPORTANCE Because there is no preventive HCV vaccine available today, the discovery of novel anti-HCV cell entry inhibitors could help develop preventive measures against infection. The present study describes two compounds isolated from the wild Egyptian artichoke (WEA) with respect to their structural elucidation, absolute configuration, and quantitative determination. Importantly, both compounds inhibited HCV infection in vitro. The first compound was an unknown molecule, and it was designated "grosheimol," while the second compound is the known molecule cynaropicrin. Both compounds belong to the group of sesquiterpene lactones. The mode of action of these compounds occurred during the early steps of the HCV life cycle, including cell-free and cell-cell infection inhibition. These natural compounds present promising candidates for further development into anti-HCV therapeutics.

  • The Dual Nature of Nek9 in Adenovirus Replication [Virus-Cell Interactions]

  • To successfully replicate in an infected host cell, a virus must overcome sophisticated host defense mechanisms. Viruses, therefore, have evolved a multitude of devices designed to circumvent cellular defenses that would lead to abortive infection. Previous studies have identified Nek9, a cellular kinase, as a binding partner of adenovirus E1A, but the biology behind this association remains a mystery. Here we show that Nek9 is a transcriptional repressor that functions together with E1A to silence the expression of p53-inducible GADD45A gene in the infected cell. Depletion of Nek9 in infected cells reduces virus growth but unexpectedly enhances viral gene expression from the E2 transcription unit, whereas the opposite occurs when Nek9 is overexpressed. Nek9 localizes with viral replication centers, and its depletion reduces viral genome replication, while overexpression enhances viral genome numbers in infected cells. Additionally, Nek9 was found to colocalize with the viral E4 orf3 protein, a repressor of cellular stress response. Significantly, Nek9 was also shown to associate with viral and cellular promoters and appears to function as a transcriptional repressor, representing the first instance of Nek9 playing a role in gene regulation. Overall, these results highlight the complexity of virus-host interactions and identify a new role for the cellular protein Nek9 during infection, suggesting a role for Nek9 in regulating p53 target gene expression.

    IMPORTANCE In the arms race that exists between a pathogen and its host, each has continually evolved mechanisms to either promote or prevent infection. In order to successfully replicate and spread, a virus must overcome every mechanism that a cell can assemble to block infection. On the other hand, to counter viral spread, cells must have multiple mechanisms to stifle viral replication. In the present study, we add to our understanding of how the human adenovirus is able to circumvent cellular roadblocks to replication. We show that the virus uses a cellular protein, Nek9, in order to block activation of p53-regulated gene GADD45A, which is an important player in stress response and p53-mediated cell cycle arrest. Importantly, our study also identifies Nek9 as a transcriptional repressor.

  • Mutations of Conserved Residues in the Major Homology Region Arrest Assembling HIV-1 Gag as a Membrane-Targeted Intermediate Containing Genomic RNA and Cellular Proteins [Structure and Assembly]

  • The major homology region (MHR) is a highly conserved motif that is found within the Gag protein of all orthoretroviruses and some retrotransposons. While it is widely accepted that the MHR is critical for assembly of HIV-1 and other retroviruses, how the MHR functions and why it is so highly conserved are not understood. Moreover, consensus is lacking on when HIV-1 MHR residues function during assembly. Here, we first addressed previous conflicting reports by confirming that MHR deletion, like conserved MHR residue substitution, leads to a dramatic reduction in particle production in human and nonhuman primate cells expressing HIV-1 proviruses. Next, we used biochemical analyses and immunoelectron microscopy to demonstrate that conserved residues in the MHR are required after assembling Gag has associated with genomic RNA, recruited critical host factors involved in assembly, and targeted to the plasma membrane. The exact point of inhibition at the plasma membrane differed depending on the specific mutation, with one MHR mutant arrested as a membrane-associated intermediate that is stable upon high-salt treatment and other MHR mutants arrested as labile, membrane-associated intermediates. Finally, we observed the same assembly-defective phenotypes when the MHR deletion or conserved MHR residue substitutions were engineered into Gag from a subtype B, lab-adapted provirus or Gag from a subtype C primary isolate that was codon optimized. Together, our data support a model in which MHR residues act just after membrane targeting, with some MHR residues promoting stability and another promoting multimerization of the membrane-targeted assembling Gag oligomer.

    IMPORTANCE The retroviral Gag protein exhibits extensive amino acid sequence variation overall; however, one region of Gag, termed the major homology region, is conserved among all retroviruses and even some yeast retrotransposons, although the reason for this conservation remains poorly understood. Highly conserved residues in the major homology region are required for assembly of retroviruses; however, when these residues are required during assembly is not clear. Here, we used biochemical and electron microscopic analyses to demonstrate that these conserved residues function after assembling HIV-1 Gag has associated with genomic RNA, recruited critical host factors involved in assembly, and targeted to the plasma membrane but before Gag has completed the assembly process. By revealing precisely when conserved residues in the major homology region are required during assembly, these studies resolve existing controversies and set the stage for future experiments aimed at a more complete understanding of how the major homology region functions.

  • Molecular Basis of the Divergent Immunogenicity of Two Pediatric Tick-Borne Encephalitis Virus Vaccines [Vaccines and Antiviral Agents]

  • Studies evaluating the immunogenicity of two pediatric tick-borne encephalitis virus (TBEV) vaccines have reported contradictory results. These vaccines are based on two different strains of the European TBEV subtype: FSME-Immun Junior is based on the Neudörfl (Nd) strain, whereas Encepur Children is based on the Karlsruhe (K23) strain. The antibody (Ab) response induced by these two vaccines might be influenced by antigenic differences in the envelope (E) protein, which is the major target of neutralizing antibodies. We used an established hybrid virus assay platform to compare the levels of induction of neutralizing antibodies against the two vaccine virus strains in children aged 1 to 11 years who received two immunizations with FSME-Immun Junior or Encepur Children. The influence of amino acid differences between the E proteins of the Nd and K23 vaccine strains was investigated by mutational analyses and three-dimensional computer modeling. FSME-Immun Junior induced 100% seropositivity and similar neutralizing antibody titers against hybrid viruses containing the TBEV E protein of the two vaccine strains. Encepur Children induced 100% seropositivity only against the hybrid virus containing the E protein of the homologous K23 vaccine strain. Antibody responses induced by Encepur Children to the hybrid virus containing the E protein of the heterologous Nd strain were substantially and significantly (P llt; 0.001) lower than those to the K23 vaccine strain hybrid virus. Structure-based mutational analyses of the TBEV E protein indicated that this is due to a mutation in the DI-DII hinge region of the K23 vaccine strain E protein which may have occurred during production of the vaccine seed virus and which is not present in any wild-type TBE viruses.

    IMPORTANCE Our data suggest that there are major differences in the abilities of two European subtype pediatric TBEV vaccines to induce antibodies capable of neutralizing heterologous TBEV strains. This is a result of a mutation in the DI-DII hinge region of the E protein of the K23 vaccine virus strain used to manufacture Encepur Children which is not present in the Nd strain used to manufacture FSME-Immun Junior or in any other known naturally occurring TBEVs.

  • Visualization of Host-Polerovirus Interaction Topologies Using Protein Interaction Reporter Technology [Virus-Cell Interactions]

  • Demonstrating direct interactions between host and virus proteins during infection is a major goal and challenge for the field of virology. Most protein interactions are not binary or easily amenable to structural determination. Using infectious preparations of a polerovirus (Potato leafroll virus [PLRV]) and protein interaction reporter (PIR), a revolutionary technology that couples a mass spectrometric-cleavable chemical cross-linker with high-resolution mass spectrometry, we provide the first report of a host-pathogen protein interaction network that includes data-derived, topological features for every cross-linked site that was identified. We show that PLRV virions have hot spots of protein interaction and multifunctional surface topologies, revealing how these plant viruses maximize their use of binding interfaces. Modeling data, guided by cross-linking constraints, suggest asymmetric packing of the major capsid protein in the virion, which supports previous epitope mapping studies. Protein interaction topologies are conserved with other species in the Luteoviridae and with unrelated viruses in the Herpesviridae and Adenoviridae. Functional analysis of three PLRV-interacting host proteins in planta using a reverse-genetics approach revealed a complex, molecular tug-of-war between host and virus. Structural mimicry and diversifying selectionmmdash;hallmarks of host-pathogen interactionsmmdash;were identified within host and viral binding interfaces predicted by our models. These results illuminate the functional diversity of the PLRV-host protein interaction network and demonstrate the usefulness of PIR technology for precision mapping of functional host-pathogen protein interaction topologies.

    IMPORTANCE The exterior shape of a plant virus and its interacting host and insect vector proteins determine whether a virus will be transmitted by an insect or infect a specific host. Gaining this information is difficult and requires years of experimentation. We used protein interaction reporter (PIR) technology to illustrate how viruses exploit host proteins during plant infection. PIR technology enabled our team to precisely describe the sites of functional virus-virus, virus-host, and host-host protein interactions using a mass spectrometry analysis that takes just a few hours. Applications of PIR technology in host-pathogen interactions will enable researchers studying recalcitrant pathogens, such as animal pathogens where host proteins are incorporated directly into the infectious agents, to investigate how proteins interact during infection and transmission as well as develop new tools for interdiction and therapy.

  • Type I Interferon Response Limits Astrovirus Replication and Protects against Increased Barrier Permeability In Vitro and In Vivo [Pathogenesis and Immunity]

  • Little is known about intrinsic epithelial cell responses against astrovirus infection. Here we show that human astrovirus type 1 (HAstV-1) infection induces type I interferon (beta interferon [IFN-bbeta;]) production in differentiated Caco2 cells, which not only inhibits viral replication by blocking positive-strand viral RNA and capsid protein synthesis but also protects against HAstV-1-increased barrier permeability. Excitingly, we found similar results in vivo using a murine astrovirus (MuAstV) model, providing new evidence that virus-induced type I IFNs may protect against astrovirus replication and pathogenesis in vivo.

    IMPORTANCE Human astroviruses are a major cause of pediatric diarrhea, yet little is known about the immune response. Here we show that type I interferon limits astrovirus infection and preserves barrier permeability both in vitro and in vivo. Importantly, we characterized a new mouse model for studying astrovirus replication and pathogenesis.

  • Molecular Evolution and Intraclade Recombination of Enterovirus D68 during the 2014 Outbreak in the United States [Genetic Diversity and Evolution]

  • In August 2014, an outbreak of enterovirus D68 (EV-D68) occurred in North America, causing severe respiratory disease in children. Due to a lack of complete genome sequence data, there is only a limited understanding of the molecular evolution and epidemiology of EV-D68 during this outbreak, and it is uncertain whether the differing clinical manifestations of EV-D68 infection are associated with specific viral lineages. We developed a high-throughput complete genome sequencing pipeline for EV-D68 that produced a total of 59 complete genomes from respiratory samples with a 95% success rate, including 57 genomes from Kansas City, MO, collected during the 2014 outbreak. With these data in hand, we performed phylogenetic analyses of complete genome and VP1 capsid protein sequences. Notably, we observed considerable genetic diversity among EV-D68 isolates in Kansas City, manifest as phylogenetically distinct lineages, indicative of multiple introductions of this virus into the city. In addition, we identified an intersubclade recombination event within EV-D68, the first recombinant in this virus reported to date. Finally, we found no significant association between EV-D68 genetic variation, either lineages or individual mutations, and a variety of demographic and clinical variables, suggesting that host factors likely play a major role in determining disease severity. Overall, our study revealed the complex pattern of viral evolution within a single geographic locality during a single outbreak, which has implications for the design of effective intervention and prevention strategies.

    IMPORTANCE Until recently, EV-D68 was considered to be an uncommon human pathogen, associated with mild respiratory illness. However, in 2014 EV-D68 was responsible for more than 1,000 disease cases in North America, including severe respiratory illness in children and acute flaccid myelitis, raising concerns about its potential impact on public health. Despite the emergence of EV-D68, a lack of full-length genome sequences means that little is known about the molecular evolution of this virus within a single geographic locality during a single outbreak. Here, we doubled the number of publicly available complete genome sequences of EV-D68 by performing high-throughput next-generation sequencing, characterized the evolutionary history of this outbreak in detail, identified a recombination event, and investigated whether there was any correlation between the demographic and clinical characteristics of the patients and the viral variant that infected them. Overall, these results will help inform the design of intervention strategies for EV-D68.

  • The SD1 Subdomain of Venezuelan Equine Encephalitis Virus Capsid Protein Plays a Critical Role in Nucleocapsid and Particle Assembly [Structure and Assembly]

  • Venezuelan equine encephalitis virus (VEEV) is an important human and animal pathogen, for which no safe and efficient vaccines or therapeutic means have been developed. Viral particle assembly and budding processes represent potential targets for therapeutic intervention. However, our understanding of the mechanistic process of VEEV assembly, RNA encapsidation, and the roles of different capsid-specific domains in these events remain to be described. The results of this new study demonstrate that the very amino-terminal VEEV capsid-specific subdomain SD1 is a critical player in the particle assembly process. It functions in a virus-specific mode, and its deletion, mutation, or replacement by the same subdomain derived from other alphaviruses has strong negative effects on infectious virus release. VEEV variants with mutated SD1 accumulate adaptive mutations in both SD1 and SD2, which result in a more efficiently replicating phenotype. Moreover, efficient nucleocapsid and particle assembly proceeds only when the two subdomains, SD1 and SD2, are derived from the same alphavirus. These two subdomains together appear to form the central core of VEEV nucleocapsids, and their interaction is one of the driving forces of virion assembly and budding. The similar domain structures of alphavirus capsid proteins suggest that this new knowledge can be applied to other alphaviruses.

    IMPORTANCE Alphaviruses are a group of human and animal pathogens which cause periodic outbreaks of highly debilitating diseases. Despite significant progress made in understanding the overall structure of alphavirus and VEEV virions, and glycoprotein spikes in particular, the mechanistic process of nucleocapsid assembly, RNA encapsidation, and the roles of different capsid-specific domains in these processes remain to be described. Our new data demonstrate that the very amino-terminal subdomain of Venezuelan equine encephalitis virus capsid protein, SD1, plays a critical role in the nucleocapsid assembly. It functions synergistically with the following SD2 (helix I) and appears to form a core in the center of nucleocapsid. The core formation is one of the driving forces of alphavirus particle assembly.

  • Antibody-Dependent Cellular Cytotoxicity against Reactivated HIV-1-Infected Cells [Pathogenesis and Immunity]

  • Lifelong antiretroviral therapy (ART) for HIV-1 does not diminish the established latent reservoir. A possible cure approach is to reactivate the quiescent genome from latency and utilize immune responses to eliminate cells harboring reactivated HIV-1. It is not known whether antibodies within HIV-1-infected individuals can recognize and eliminate cells reactivated from latency through antibody-dependent cellular cytotoxicity (ADCC). We found that reactivation of HIV-1 expression in the latently infected ACH-2 cell line elicited antibody-mediated NK cell activation but did not result in antibody-mediated killing. The lack of CD4 expression on these HIV-1 envelope (Env)-expressing cells likely resulted in poor recognition of CD4-induced antibody epitopes on Env. To examine this further, cultured primary CD4+ T cells from HIV-1+ subjects were used as targets for ADCC. These ex vivo-expanded primary cells were modestly susceptible to ADCC mediated by autologous or heterologous HIV-1+ serum antibodies. Importantly, ADCC mediated against these primary cells could be enhanced following incubation with a CD4-mimetic compound (JP-III-48) that exposes CD4-induced antibody epitopes on Env. Our studies suggest that with sufficient reactivation and expression of appropriate Env epitopes, primary HIV-1-infected cells can be targets for ADCC mediated by autologous serum antibodies and innate effector cells. The results of this study suggest that further investigation into the potential of ADCC to eliminate reactivated latently infected cells is warranted.

    IMPORTANCE An HIV-1 cure remains elusive due to the persistence of long-lived latently infected cells. An HIV-1 cure strategy, termed "shock and kill," aims to reactivate HIV-1 expression in latently infected cells and subsequently eliminate the reactivated cells through immune-mediated killing. While recent research efforts have focused on reversing HIV-1 latency, it remains unclear whether preexisting immune responses within HIV-1+ individuals can efficiently eliminate the reactivated cells. HIV-1-specific antibodies can potentially eliminate cells reactivated from latency via Fc effector functions by recruiting innate immune cells. Our study highlights the potential role that antibody-dependent cellular cytotoxicity might play in antilatency cure approaches.

  • Abortively Infected Astrocytes Appear To Represent the Main Source of Interferon Beta in the Virus-Infected Brain [Pathogenesis and Immunity]

  • Interferon beta (IFN-bbeta;) is a key component of cellular innate immunity in mammals, and it constitutes the first line of defense during viral infection. Studies with cultured cells previously showed that almost all nucleated cells are able to produce IFN-bbeta; to various extents, but information about the in vivo sources of IFN-bbeta; remains incomplete. By applying immunohistochemistry and employing conditional-reporter mice that express firefly luciferase under the control of the IFN-bbeta; promoter in either all or only distinct cell types, we found that astrocytes are the main producers of IFN-bbeta; after infection of the brain with diverse neurotropic viruses, including rabies virus, Theiler's murine encephalomyelitis virus, and vesicular stomatitis virus. Analysis of a panel of knockout mouse strains revealed that sensing of viral components via both RIG-I-like helicases and Toll-like receptors contributes to IFN induction in the infected brain. A genetic approach to permanently mark rabies virus-infected cells in the brain showed that a substantial number of astrocytes became labeled and, therefore, must have been infected by the virus at least transiently. Thus, our results strongly indicate that abortive viral infection of astrocytes can trigger pattern recognition receptor signaling events which result in secretion of IFN-bbeta; that confers antiviral protection.

    IMPORTANCE Previous work indicated that astrocytes are the main producers of IFN after viral infection of the central nervous system (CNS), but it remained unclear how astrocytes might sense those viruses which preferentially replicate in neurons. We have now shown that virus sensing by both RIG-I-like helicases and Toll-like receptors is involved. Our results further demonstrate that astrocytes get infected in a nonproductive manner under these conditions, indicating that abortive infection of astrocytes plays a previously unappreciated role in the innate antiviral defenses of the CNS.

  • Bovine Herpesvirus 4 Modulates Its {beta}-1,6-N-Acetylglucosaminyltransferase Activity through Alternative Splicing [Virus-Cell Interactions]

  • Carbohydrates play major roles in host-virus interactions. It is therefore not surprising that, during coevolution with their hosts, viruses have developed sophisticated mechanisms to hijack for their profit different pathways of glycan synthesis. Thus, the Bo17 gene of Bovine herpesvirus 4 (BoHV-4) encodes a homologue of the cellular core 2 protein bbeta;-1,6-N-acetylglucosaminyltransferase-mucin type (C2GnT-M), which is a key player for the synthesis of complex O-glycans. Surprisingly, we show in this study that, as opposed to what is observed for the cellular enzyme, two different mRNAs are encoded by the Bo17 gene of all available BoHV-4 strains. While the first one corresponds to the entire coding sequence of the Bo17 gene, the second results from the splicing of a 138-bp intron encoding critical residues of the enzyme. Antibodies generated against the Bo17 C terminus showed that the two forms of Bo17 are expressed in BoHV-4 infected cells, but enzymatic assays revealed that the spliced form is not active. In order to reveal the function of these two forms, we then generated recombinant strains expressing only the long or the short form of Bo17. Although we did not highlight replication differences between these strains, glycomic analyses and lectin neutralization assays confirmed that the splicing of the Bo17 gene gives the potential to BoHV-4 to fine-tune the global level of core 2 branching activity in the infected cell. Altogether, these results suggest the existence of new mechanisms to regulate the activity of glycosyltransferases from the Golgi apparatus.

    IMPORTANCE Viruses are masters of adaptation that hijack cellular pathways to allow their growth. Glycans play a central role in many biological processes, and several studies have highlighted mechanisms by which viruses can affect glycosylation. Glycan synthesis is a nontemplate process regulated by the availability of key glycosyltransferases. Interestingly, bovine herpesvirus 4 encodes one such enzyme which is a key enzyme for the synthesis of complex O-glycans. In this study, we show that, in contrast to cellular homologues, this virus has evolved to alternatively express two proteins from this gene. While the first one is enzymatically active, the second results from the alternative splicing of the region encoding the catalytic site of the enzyme. We postulate that this regulatory mechanism could allow the virus to modulate the synthesis of some particular glycans for function at the location and/or the moment of infection.

  • Evolution of Newcastle Disease Virus Quasispecies Diversity and Enhanced Virulence after Passage through Chicken Air Sacs [Genetic Diversity and Evolution]

  • It has been reported that lentogenic Newcastle disease virus (NDV) isolates have the potential to become velogenic after their transmission and circulation in chickens, but the underlying mechanism is unclear. In this study, a highly velogenic NDV variant, JS10-A10, was generated from the duck-origin lentogenic isolate JS10 through 10 consecutive passages in chicken air sacs. The velogenic properties of this selected variant were determined using mean death time (MDT) assays, intracerebral pathogenicity index (ICPI), the intravenous pathogenicity index (IVPI), histopathology, and the analysis of host tissue tropism. In contrast, JS10 remained lentogenic after 20 serial passages in chicken eggs (JS10-E20). The JS10, JS10-A10, and JS10-E20 genomes were sequenced and found to be nearly identical, suggesting that both JS10-A10 and JS10-E20 were directly generated from JS10. To investigate the mechanism for virulence enhancement, the partial genome covering the F0 cleavage site of JS10 and its variants were analyzed using ultradeep pyrosequencing (UDPS) and the proportions of virulence-related genomes in the quasispecies were calculated. Velogenic NDV genomes accumulated as a function of JS10 passaging through chicken air sacs. Our data suggest that lentogenic NDV strains circulating among poultry might be a risk factor to future potential velogenic NDV outbreaks in chickens.

    IMPORTANCE An avirulent isolate, JS10, was passaged through chicken air sacs and embryos, and the pathogenicity of the variants was assessed. A virulent variant, JS10-A10, was generated from consecutive passage in air sacs. We developed a deep-sequencing approach to detect low-frequency viral variants across the NDV genome. We observed that virulence enhancement of JS10 was due to the selective accumulation of velogenic quasispecies and the concomitant disappearance of lentogenic quasispecies. Our results suggest that because it is difficult to avoid contact between natural waterfowl reservoirs and sensitive poultry operations, circulating lentogenic NDV strains may represent a potential reservoir for emergent velogenic NDV strains that could cause outbreaks in chickens.

  • Sensing of HIV-1 Infection in Tzm-bl Cells with Reconstituted Expression of STING [Cellular Response to Infection]

  • Production of proinflammatory cytokines indicative of potent recognition by the host innate immune system has long been recognized as a hallmark of the acute phase of HIV-1 infection. The first components of the machinery by which primary HIV target cells sense infection have recently been described; however, the mechanistic dissection of innate immune recognition and viral evasion would be facilitated by an easily accessible cell line model. Here we describe that reconstituted expression of the innate signaling adaptor STING enhanced the ability of the well-established HIV reporter cell line Tzm-bl to sense HIV infection and to convert this information into nuclear translocation of IRF3 as well as expression of cytokine mRNA. STING-dependent immune sensing of HIV-1 required virus entry and reverse transcription but not genome integration. Particularly efficient recognition was observed for an HIV-1 variant lacking expression of the accessory protein Vpr, suggesting a role of the viral protein in circumventing STING-mediated immune signaling. Vpr as well as STING significantly impacted the magnitude and breadth of the cytokine mRNA expression profile induced upon HIV-1 infection. However, cytoplasmic DNA sensing did not result in detectable cytokine secretion in this cell system, and innate immune recognition did not affect infection rates. Despite these deficits in eliciting antiviral effector functions, these results establish Tzm-bl STING and Tzm-bl STING IRF3.GFP cells as useful tools for studies aimed at dissecting mechanisms and regulation of early innate immune recognition of HIV infection.

    IMPORTANCE Cell-autonomous immune recognition of HIV infection was recently established as an important aspect by which the host immune system attempts to fend off HIV-1 infection. Mechanistic studies on host cell recognition and viral evasion are hampered by the resistance of many primary HIV target cells to detailed experimental manipulation. We describe here that expression of the signaling adaptor STING renders the well-established HIV reporter cell line Tzm-bl competent for innate recognition of HIV infection. Key characteristics reflected in this cell model include nuclear translocation of IRF3, expression of a broad range of cytokine mRNAs, and an antagonistic activity of the HIV-1 protein Vpr. These results establish Tzm-bl STING and Tzm-bl STING IRF3.GFP cells as a useful tool for studies of innate recognition of HIV infection.

  • A Novel DNA Motif Contributes to Selective Replication of a Geminivirus-Associated Betasatellite by a Helper Virus-Encoded Replication-Related Protein [Genome Replication and Regulation of Viral Gene Expression]

  • Rolling-circle replication of single-stranded genomes of plant geminiviruses is initiated by sequence-specific DNA binding of the viral replication-related protein (Rep) to its cognate genome at the replication origin. Monopartite begomovirus-associated betasatellites can be trans replicated by both cognate and some noncognate helper viruses, but the molecular basis of replication promiscuity of betasatellites remains uncharacterized. Earlier studies showed that when tomato yellow leaf curl China virus (TYLCCNV) or tobacco curly shoot virus (TbCSV) is coinoculated with both cognate and noncognate betasatellites, the cognate betasatellite dominates over the noncognate one at the late stages of infection. In this study, we constructed reciprocal chimeric betasatellites between tomato yellow leaf curl China betasatellite and tobacco curly shoot betasatellite and assayed their competitiveness against wild-type betasatellite when coinoculated with TYLCCNV or TbCSV onto plants. We mapped a region immediately upstream of the conserved rolling-circle cruciform structure of betasatellite origin that confers the cognate Rep-mediated replication advantage over the noncognate satellite. DNase I protection and in vitro binding assays further identified a novel sequence element termed Rep-binding motif (RBM), which specifically binds to the cognate Rep protein and to the noncognate Rep, albeit at lower affinity. Furthermore, we showed that RBM-Rep binding affinity is correlated with betasatellite replication efficiency in protoplasts. Our data suggest that although strict specificity of Rep-mediated replication does not exist, betasatellites have adapted to their cognate Reps for efficient replication during coevolution.

    IMPORTANCE Begomoviruses are numerous circular DNA viruses that cause devastating diseases of crops worldwide. Monopartite begomoviruses are frequently associated with betasatellites which are essential for induction of typical disease symptoms. Coexistence of two distinct betasatellites with one helper virus is rare in nature. Our previous research showed that begomoviruses can trans replicate cognate betasatellites to higher levels than noncognate ones. However, the molecular mechanisms of betasatellites selective replication remain largely unknown. We investigated the interaction between the begomovirus replication-associated protein and betasatellite DNA. We found that the replication-associated protein specifically binds to a motif in betasatellites, with higher affinity for the cognate motif than the noncognate motif. This preference for cognate motif binding determines the selective replication of betasatellites. We also demonstrated that this motif is essential for betasatellite replication. These findings shed new light on the promiscuous yet selective replication of betasatellites by helper geminiviruses.

  • Porcine Epidemic Diarrhea Virus 3C-Like Protease Regulates Its Interferon Antagonism by Cleaving NEMO [Cellular Response to Infection]

  • Porcine epidemic diarrhea virus (PEDV) is an enteropathogenic coronavirus causing lethal watery diarrhea in piglets. Since 2010, a PEDV variant has spread rapidly in China, and it emerged in the United States in 2013, posing significant economic and public health concerns. The ability to circumvent the interferon (IFN) antiviral response, as suggested for PEDV, promotes viral survival and regulates pathogenesis of PEDV infections, but the underlying mechanisms remain obscure. Here, we show that PEDV-encoded 3C-like protease, nsp5, is an IFN antagonist that proteolytically cleaves the nuclear transcription factor kappa B (NF-B) essential modulator (NEMO), an essential adaptor bridging interferon-regulatory factor and NF-B activation. NEMO is cleaved at glutamine 231 (Q231) by PEDV, and this cleavage impaired the ability of NEMO to activate downstream IFN production and to act as a signaling adaptor of the RIG-I/MDA5 pathway. Mutations specifically disrupting the cysteine protease activity of PEDV nsp5 abrogated NEMO cleavage and the inhibition of IFN induction. Structural analysis suggests that several key residues outside the catalytic sites of PEDV nsp5 probably impact NEMO cleavage by modulating potential interactions of nsp5 with their substrates. These data show that PEDV nsp5 disrupts type I IFN signaling by cleaving NEMO. Previously, we and others demonstrated that NEMO is also cleaved by 3C or 3C-like proteinases of picornavirus and artertivirus. Thus, NEMO probably represents a prime target for 3C or 3C-like proteinases of different viruses.

    IMPORTANCE The continued emergence and reemergence of porcine epidemic diarrhea virus (PEDV) underscore the importance of studying how this virus manipulates the immune responses of its hosts. During coevolution with its hosts, PEDV has acquired mechanisms to subvert host innate immune responses for its survival advantage. At least two proteins encoded by PEDV have been identified as interferon (IFN) antagonists, papain-like protease (PLP) and N protein. Here, we report that the PEDV nsp5 gene, which encodes the 3C-like protease of PEDV, is another IFN antagonist. Mechanistically, the cysteine protease activity of PEDV nsp5 mediates proteolysis of NEMO, the key adaptor for IFN synthesis, and NEMO is cleaved at glutamine 231 (Q231). The new molecular details and determinants impacting NEMO scission by PEDV nsp5 delineated in this study are fundamental to our understanding of critical virus-host interactions that determine PEDV pathogenesis.

  • The Basic Domain of Herpes Simplex Virus 1 pUS9 Recruits Kinesin-1 To Facilitate Egress from Neurons [Structure and Assembly]

  • The alphaherpesviral envelope protein pUS9 has been shown to play a role in the anterograde axonal transport of herpes simplex virus 1 (HSV-1), yet the molecular mechanism is unknown. To address this, we used an in vitro pulldown assay to define a series of five arginine residues within the conserved pUS9 basic domain that were essential for binding the molecular motor kinesin-1. The mutation of these pUS9 arginine residues to asparagine blocked the binding of both recombinant and native kinesin-1. We next generated HSV-1 with the same pUS9 arginine residues mutated to asparagine (HSV-1pUS9KBDM) and then restored them being to arginine (HSV-1pUS9KBDR). The two mutated viruses were analyzed initially in a zosteriform model of recurrent cutaneous infection. The primary skin lesion scores were identical in severity and kinetics, and there were no differences in viral load at dorsal root ganglionic (DRG) neurons at day 4 postinfection (p.i.) for both viruses. In contrast, HSV-1pUS9KBDM showed a partial reduction in secondary skin lesions at day 8 p.i. compared to the level for HSV-1pUS9KBDR. The use of rat DRG neuronal cultures in a microfluidic chamber system showed both a reduction in anterograde axonal transport and spread from axons to nonneuronal cells for HSV-1pUS9KBDM. Therefore, the basic domain of pUS9 contributes to anterograde axonal transport and spread of HSV-1 from neurons to the skin through recruitment of kinesin-1.

    IMPORTANCE Herpes simplex virus 1 and 2 cause genital herpes, blindness, encephalitis, and occasionally neonatal deaths. There is also increasing evidence that sexually transmitted genital herpes increases HIV acquisition, and the reactivation of HSV increases HIV replication and transmission. New antiviral strategies are required to control resistant viruses and to block HSV spread, thereby reducing HIV acquisition and transmission. These aims will be facilitated through understanding how HSV is transported down nerves and into skin. In this study, we have defined how a key viral protein plays a role in both axonal transport and spread of the virus from nerve cells to the skin.

  • Structure of an HIV-2 gp120 in Complex with CD4 [Structure and Assembly]

  • HIV-2 is a nonpandemic form of the virus causing AIDS, and the majority of HIV-2-infected patients exhibit long-term nonprogression. The HIV-1 and HIV-2 envelope glycoproteins, the sole targets of neutralizing antibodies, share 30 to 40% identity. As a first step in understanding the reduced pathogenicity of HIV-2, we solved a 3.0-AAring; structure of an HIV-2 gp120 bound to the host receptor CD4, which reveals structural similarity to HIV-1 gp120 despite divergence in amino acid sequence.

  • Acute Viral Escape Selectively Impairs Nef-Mediated Major Histocompatibility Complex Class I Downmodulation and Increases Susceptibility to Antiviral T Cells [Virus-Cell Interactions]

  • Nef-specific CD8+ T lymphocytes (CD8TL) are associated with control of simian immunodeficiency virus (SIV) despite extensive nef variation between and within animals. Deep viral sequencing of the immunodominant Mamu-B*017:01-restricted Nef165nndash;173IW9 epitope revealed highly restricted evolution. A common acute escape variant, T170I, unexpectedly and uniquely degraded Nef's major histocompatibility complex class I (MHC-I) downregulatory capacity, rendering the virus more vulnerable to CD8TL targeting other epitopes. These data aid in a mechanistic understanding of Nef functions and suggest means of immunity-mediated control of lentivirus replication.

  • A Highly Conserved Residue of the HIV-1 gp120 Inner Domain Is Important for Antibody-Dependent Cellular Cytotoxicity Responses Mediated by Anti-cluster A Antibodies [Pathogenesis and Immunity]

  • Previous studies have shown that sera from HIV-1-infected individuals contain antibodies able to mediate antibody-dependent cellular cytotoxicity (ADCC). These antibodies preferentially recognize envelope glycoprotein (Env) epitopes induced upon CD4 binding. Here, we show that a highly conserved tryptophan at position 69 of the gp120 inner domain is important for ADCC mediated by anti-cluster A antibodies and sera from HIV-1-infected individuals.

  • Suppression of NYVAC Infection in HeLa Cells Requires RNase L but Is Independent of Protein Kinase R Activity [Virus-Cell Interactions]

  • Protein kinase R (PKR) and RNase L are host cell components that function to contain viral spread after infections. In this study, we analyzed the role of both proteins in the abortive infection of human HeLa cells with the poxvirus strain NYVAC, for which an inhibition of viral A27L and B5R gene expression is described. Specifically, the translation of these viral genes is independent of PKR activation, but their expression is dependent on the RNase L activity.

  • Changes in the Length of the Neuraminidase Stalk Region Impact H7N9 Virulence in Mice [Pathogenesis and Immunity]

  • The neuraminidase stalk of the newly emerged H7N9 influenza virus possesses a 5-amino-acid deletion. This study focuses on characterizing the biological functions of H7N9 with varied neuraminidase stalk lengths. Results indicate that the 5-amino-acid deletion had no impact on virus infectivity or replication in vitro or in vivo compared to that of a virus with a full-length stalk, but enhanced virulence in mice was observed for H7N9 encoding a 19- to 20-amino-acid deletion, suggesting that N9 stalk length impacts virulence in mammals, as N1 stalk length does.

  • Kaposi's Sarcoma-Associated Herpesvirus Latency Locus Compensates for Interleukin-6 in Initial B Cell Activation [Pathogenesis and Immunity]

  • Interleukin 6 (IL-6) is considered a proliferation and survival factor for B cells. To assess the role of IL-6 in Kaposi sarcoma-associated herpesvirus (KSHV) latency, KSHV latency locus-transgenic mice (referred to as latency mice) lacking IL-6 were evaluated. IL-6nndash;/nndash; latency mice had the same phenotypes as the latency mice, i.e., increased frequency of marginal zone B cells, hyperplasia, and hyperglobulinemia, indicating that the KSHV latency locus, which includes all viral microRNAs (miRNAs), can compensate for lack of IL-6 in premalignant B cell activation.

  • Complexities of Estimating Evolutionary Rates in Viruses [Letters to the Editor]

  • Reply to "Complexities of Estimating Evolutionary Rates in Viruses" [Letters to the Editor]

  • Correction for Chen et al., Divergent MicroRNA Targetomes of Closely Related Circulating Strains of a Polyomavirus [Author Corrections]

  • Correction for Vigdorovich et al., Expression and Characterization of a Soluble, Active Form of the Jaagsiekte Sheep Retrovirus Receptor, Hyal2 [Author Corrections]

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    JVI Accepts: Articles Published Ahead of Print

  • Prime, shock, and kill: Priming CD4 T cells from HIV patients with a BCL-2 antagonist before HIV reactivation reduces HIV reservoir size [PublishAheadOfPrint]

  • Understanding how some HIV infected cells resist the cytotoxicity of HIV replication is crucial to enabling HIV cure efforts. HIV killing of CD4 T cells that replicate HIV can involve HIV protease-mediated cleavage of procaspase 8 to generate a fragment (Casp8p41) that directly binds and activates the mitochondrial pro-apoptotic protein BAK. Here we demonstrate that Casp8p41 also binds with nanomolar affinity to the anti-apoptotic protein Bcl-2, which sequesters Casp8p41 and prevents apoptosis. Further, we show that central memory CD4 T cells (TCM) from HIV infected individuals have heightened expression of BCL-2 relative to procaspase 8, possibly explaining the persistence of HIV-infected TCM despite generation of Casp8p41. Consistent with this hypothesis, the selective BCL-2 antagonist venetoclax induced minimal killing of uninfected CD4 T cells but markedly increased the death of CD4 T cells and diminished cell-associated HIV DNA when CD4 T cells from antiretroviral therapy (ART)-suppressed HIV patients were induced with aalpha;CD3/aalpha;CD28 to reactivate HIV ex vivo. Thus, priming CD4 T cells from ART suppressed HIV patients with a BCL-2 antagonist followed by HIV reactivation achieves reductions in cell associated HIV DNA, whereas HIV reactivation alone does not.

    Importance HIV infection is incurable due to a long lived reservoir of HIV+ memory CD4 T cells, and no clinically relevant interventions have been identified that reduce the number of these HIV DNA containing cells. Since post integration HIV replication can result in HIV protease generation of Casp8p41, which activates BAK causing infected CD4 T cell death, we sought to determine whether this occurs in memory CD4 T cells. Herein we demonstrate that memory CD4 T cells can generate Casp8p41, yet are intrinsically resistant to death induced by diverse stimuli, including Casp8p41. Furthermore, BCL2 expression is relatively increased in these cells and directly binds and inhibits Casp8p41's proapoptotic effects. Antagonizing BCL2 with venetoclax derepresses this antagonism resulting in death preferentially in HIV DNA containing cells, as only these cells generate Casp8p41. Thus BCL2 antagonism is a clinically relevant intervention with the potential to reduce HIV reservoir size in patients.

  • {alpha}v{beta}3-integrin boosts the innate response elicited in epithelial cells through plasma membrane and endosomal Toll-like receptors [PublishAheadOfPrint]

  • We report that aalpha;vbbeta;3-integrin strongly affects the innate response in epithelial cells. aalpha;vbbeta;3-integrin greatly increased the response elicited via plasma-membrane TLRs by HSV or bacterial ligands. The endosomal TLR3, not the cytosolic sensor IFI16, was also boosted by aalpha;vbbeta;3-integrin. The boosting was specifically exerted by aalpha;vbbeta;3-integrin but not by aalpha;vbbeta;6- or aalpha;vbbeta;8-integrins. Current and previous work indicates that the integrin-TLR cooperation occurs in epithelial and monocytic cells. The TLR response should be considered as integrin-TLR response.

  • Promoter targeted histone acetylation of chromatinized parvoviral genome is essential for infection progress [PublishAheadOfPrint]

  • The association of host histones with parvoviral DNA is poorly understood. We analyzed the chromatinization and histone acetylation of canine parvovirus DNA during infection by confocal imaging and in situ proximity ligation assay combined with chromatin immunoprecipitation and high-throughput sequencing. We found that at late infection parvovirus replication bodies were rich in histones bearing modifications characteristic of transcriptionally active chromatin, i.e. histone H3 lysine 27 acetylation (H3K27ac). The H3K27ac, in particular, was located in close proximity to the viral DNA-binding protein NS1. Importantly, our results show for the first time that in the chromatinized parvoviral genome, particularly the two viral promoters were rich in H3K27ac. Histone acetyltransferase (HAT) inhibitor efficiently interfered with expression of viral proteins and infection progress. Altogether, our data suggest that acetylation of histones on parvoviral DNA is essential for viral gene expression and completion of viral life cycle.

    IMPORTANCE Viral DNA introduced into cell nuclei is exposed to cellular responses to foreign DNA including chromatinization and epigenetic silencing, both of which determine the outcome of infection. How the incoming parvovirus resists cellular epigenetic down-regulation of its genes is not understood. Here, the critical role of epigenetic modifications in regulation of parvovirus infection was demonstrated. We showed for the first time that a successful parvovirus infection is characterized by deposition of nucleosomes with active histone acetylation on the viral promoter areas. The results provide new insights to regulation of parvoviral gene expression which is an important aspect in the development of parvovirus-based virotherapy.

  • MOV10 is a Restriction Factor of Influenza A Virus by Inhibiting the Nuclear Import of the Viral Nucleoprotein [PublishAheadOfPrint]

  • The viral ribonucleoprotein (vRNP) complex of influenza A viruses (IAVs) contains an RNA-dependent RNA polymerase complex (RdRp) and nucleoprotein (NP), and is the functional unit for viral RNA transcription and replication. The vRNP complex is an important determinant of virus pathogenicity and host adaptation, implying its function can be affected by host factors. In our study, we identified host protein Moloney leukemia virus 10 (MOV10) as an inhibitor for IAV replication, since depletion of MOV10 resulted in a significant increase in virus yield. MOV10 inhibited the polymerase activity in a minigenome system through RNA-mediated interaction with the NP subunit of vRNP complex. Importantly, we found that the interaction between MOV10 and NP prevented the binding of NP to importin-aalpha;, resulting in the retention of NP in the cytoplasm. Both the binding of MOV10 to NP and its inhibitory effect on polymerase activity were independent on its helicase activity. These results suggest that MOV10 acts as an anti-influenza virus factor through specifically inhibiting the nuclear transportation of NP and subsequently inhibiting the function of the vRNP complex.

    IMPORTANCE The interaction between the influenza vRNP complex and host factors is a major determinant of viral tropism and pathogenicity. Our study identified MOV10 as a novel host restriction factor for influenza viral life cycle since it inhibited the viral growth rate. Conversely, importin-aalpha; has been shown as a determinant for influenza tropism and a positive regulator for viral polymerase activity in mammalian cells, but not in avian cells. MOV10 disrupted the interaction between NP and importin-aalpha;, suggesting that MOV10 could also be an important host factor for influenza viral transmission and pathogenicity. Importantly, as a member of interferon (IFN)-inducible protein, MOV10 exerted a novel mechanism for IFNs to inhibit the replication of influenza viruses. Furthermore, our study potentially provides a new drug design strategy, the use of molecules that mimic the antiviral mechanism of MOV10.

  • A regulatory element near the 3 -end of the AAV rep gene inhibits adenovirus replication in cis by means of p40 promoter-associated short transcripts [PublishAheadOfPrint]

  • Adeno-associated virus (AAV) has long been known to inhibit helper adenovirus (Ad) replication in dependence of AAV Rep protein expression. More recently, replication of Ad5/AAV-2 hybrid vectors was shown to be inhibited in cis by a sequence near the 3rrsquo; end of AAV rep, termed RIS-Ad (Rep inhibition sequence for adenoviral replication). RIS-Ad functions independently of Rep protein expression. Here we demonstrate that inhibition of adenoviral replication by RIS-Ad requires an active AAV p40 promoter and the 5rrsquo; half of the intron. In addition, Ad inhibition is critically dependent on the integrity of the p40 transcription start site (TSS) leading to short p40-associated transcripts. These do not give rise to effector molecules capable of inhibiting adenoviral replication in trans, like small polypeptides or microRNAs. Our data point to RNA Pol II pausing directly downstream the p40 promoter leading to interference of the stalled Pol II transcription complex with the adenoviral replication machinery. Whereas inhibition by RIS-Ad is mediated exclusively in cis, it can be overcome by providing a replication-competent adenoviral genome in trans. Moreover, the inhibitory effect of RIS-Ad is not limited to AAV-2, but could also be shown for the corresponding regions of other AAV serotypes including AAV-5. These findings have important implications for the future generation of Ad5/AAV hybrid vectors.

    Importance Insertion of sequences from the 3rrsquo; -part of the rep gene of adeno-associated virus (AAV) into the genome of its helper adenovirus strongly reduces adenoviral genome replication. We could show that this inhibition is mediated exclusively in cis without the involvement of trans-acting regulatory RNAs or polypeptides, but nevertheless requires an active AAV-2 p40 promoter and p40-associated short transcripts. Our results suggest a novel inhibitory mechanism not described for AAV so far, which involves stalled RNA polymerase II complexes and their interference with adenoviral DNA replication. Such a mechanism would have important implications both for the generation of adenoviral vectors expressing the AAV rep and cap genes and for the regulation of AAV gene expression in the absence and presence of helpervirus.

  • Nuclear Translocation Sequence and Regions in AcMNPV ME53 Are Important for Optimal Baculovirus Production [PublishAheadOfPrint]

  • Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is in the family Baculoviridae, genus Alphabaculovirus. AcMNPV me53 is a highly conserved immediate early gene in all lepidopteran baculoviruses that have been sequenced, and is transcribed up to late times post-infection. Although me53 is not essential for viral DNA synthesis, infectious budded virus (BV) production is greatly attenuated when deleted. ME53 associates with the nucleocapsid on both budded virus and occlusion-derived virus, but not with the virus envelope. ME53 co-localizes in plasma membrane foci with the envelope glycoprotein GP64 in a GP64-dependent manner. ME53 localizes in the cytoplasm early post-infection, and despite lack of a reported nuclear localization signal (NLS), ME53 translocates to the nucleus at late times post-infection. To map determinants of ME53 that facilitate its nuclear translocation, recombinant AcMNPV bacmids containing a series of ME53 truncations, internal deletions and peptides fused with HA or GFP tags were constructed. Intracellular localization studies identified residues within amino acids 109 to 137 at the N-terminus of ME53 that acted as the nuclear translocation sequence (NTS) facilitating its nuclear transport at late times post-infection. The first 100 N-terminal amino acids and the last 50 C-terminal amino acids of ME53 are dispensable for high levels of budded virus production. The region within amino acids 101 to 398, which also contains the NTS, is critical for optimal levels of budded virus production.

    Importance Baculovirus me53 is a conserved immediate early gene found in all sequenced lepidopteran alpha- and betabaculoviruses. We first identified residues within amino acids 109 to 137 at the N-terminus that act as the ME53 nuclear translocation sequence (NTS) to facilitate its nuclear translocation, and defined an internal region within amino acids 101 to 398, which includes the NTS, as being necessary for optimal budded virus production. Altogether these results indicate a previously unidentified nuclear role that ME53 plays in virus replication.

  • Hepatitis B virus capsids have diverse structural responses to small molecule ligands bound to the HAP pocket [PublishAheadOfPrint]

  • Though the Hepatitis B virus (HBV) core protein is an important participant in many aspects of the viral life cycle, its best-characterized activity is self-assembly into 240-monomer capsids. Small-molecules that target core protein (Core protein Allosteric Modulators, CpAMs) represent a promising antiviral strategy. To better understand the structural basis of CpAM mechanism, we determined the crystal structure of the HBV capsid in complex with HAP18. HAP18 accelerates assembly, increases protein-protein association by more than 100-fold, and induces assembly of non-icosahedral macro-structures. In a preformed capsid, HAP18 is found at quasi-equivalent subunit-subunit interfaces. In detailed comparison to the two other extant CpAM structures, we find that the HAP18-capsid structure presents a paradox. Where the two other structures expanded the capsid diameter by up to 10AAring;, HAP18 caused only minor changes in quaternary structure and actually decreased capsid diameter by ~3AAring;. These results indicate that CpAMs do not have a single allosteric effect on capsid structure. We suggest that HBV capsids present an ensemble of states that can be trapped by CpAMs, indicating a more complex basis for antiviral drug design.

    IMPORTANCE Hepatitis B Virus core protein has multiple roles in the viral lifecycle - assembly, compartment for reverse transcription, intracellular trafficking, nuclear functions - making it an attractive antiviral target. Core protein allosteric modulators (CpAMs), are an experimental class of antiviral that bind core protein. The most accessible CpAM activity is that they accelerate core protein assembly and strengthen interactions between subunits. In this study, we observe that the CpAM-binding pocket has multiple conformations. We compare structures of capsids co45 crystallized with different CpAMs and find that they also affect quaternary structure in different ways. These results suggest that the capsid breathes and is trapped in different states by the drug and crystallization. Understanding that the capsid is a moving target will aid drug design and improve our understanding of HBV interaction with its environment.

  • Development of broadly neutralizing antibodies and their mapping by monomeric gp120 in HIV-1 infected humans and SHIVSF162P3N infected macaques [PublishAheadOfPrint]

  • To improve our understanding of the similarities and differences between neutralizing antibodies elicited by SHIV-infected rhesus macaques and HIV-1-infected humans, we examined the plasma of 13 viremic macaques infected with SHIVSF162P3N and 85 HIV-1-infected humans with known time of infection. We identified 5 macaques (38%) from 1-2 years post infection (YPI) with broadly neutralizing antibodies (bnAbs) against tier-2 HIV-1. In comparison, only 2 out of 42 (5%) human plasmas collected at a similar time frame of 1-3 YPI exhibited comparable neutralizing breadth and potency, with this number increasing to 7 out of 21 (30%) after 3 YPI. Plasma mapping with monomeric gp120 identified only 2 out of 9 humans and 2 out of 4 macaques that contain gp120-reactive neutralizing antibodies, indicating distinct specificities in these plasmas, with most of them recognizing the envelope trimer (including gp41) rather than gp120 monomer. Indeed, a total of 20 gp120-directed monoclonal antibodies (mAbs) isolated from a human subject AD358 and a Chinese rhesus macaque GB40 displayed none or limited neutralizing activity against tier-2 strains. These isolated mAbs, mapped to the CD4-binding site, the V3-loop, the inner domain, and the C5 region of gp120, revealed genetic similarity between the human and macaque immunoglobulin genes used to encode some V3-directed mAbs. These results also emphasize the use of envelope trimer probes for efficient isolation of HIV-1 bnAbs.

    Importance HIV-1 vaccine research can benefit from understanding the development of broadly neutralizing antibodies (bnAbs) in rhesus macaques commonly used to assess vaccine immunogenicity and efficacy. Here we examined 85 HIV-1-infected humans and 13 SHIVSF162P3N-infected macaques for bnAbs and found that similar to HIV-1-infected humans, bnAbs in SHIV-infected macaques are also rare, but their development may be faster in some of the studied macaques. Plasma mapping with monomeric gp120 indicated that most bnAbs bind to the envelope trimer rather than gp120 monomer. In support, none of the isolated gp120-reactive monoclonal antibodies (mAbs) displayed the neutralization breadth observed in the corresponding plasma. However, the mAb sequences revealed similarity between human and macaque genes used to encode some V3-directed mAbs. Our study sheds light on the timing and development of bnAbs in SHIV-infected macaques in comparison to HIV-1-infected humans and highlights the use of envelope trimer probes for efficient recovery of bnAbs.

  • Genetic Stability of BAC-Derived Human Cytomegalovirus During Culture In Vitro [PublishAheadOfPrint]

  • Clinical human cytomegalovirus (HCMV) strains invariably mutate when propagated in vitro. Mutations in gene RL13 are selected in all cell types, whereas in fibroblasts mutants in the UL128 locus (UL128L; genes UL128, UL130, and UL131A) are also selected. In addition, sporadic mutations are selected elsewhere in the genome in all cell types. We sought to investigate conditions under which HCMV can be propagated without incurring genetic defects. Bacterial artificial chromosomes (BACs) provide a stable, genetically-defined source of viral genome. Viruses were generated from BACs containing the genomes of strains TR, TB40, FIX, and Merlin, as well as from Merlin-BAC recombinants containing variant nucleotides in UL128L from TB40-BAC4 or FIX-BAC. Propagation of viruses derived from TR-BAC, TB40-BAC4, and FIX-BAC in either fibroblast or epithelial cells was associated with the generation of defects around the prokaryotic vector, which is retained in the US region of viruses. This was not observed for Merlin-BAC, from which the vector is excised in derived viruses, however propagation in epithelial cells was consistently associated with mutations in the UL/b' region, all impacting on gene UL141. Viruses derived from Merlin-BAC in fibroblasts mutated in UL128L, but this occurred less frequently with recombinants containing UL128L nucleotides from TB40-BAC4 or FIX-BAC. Viruses derived from a Merlin-BAC derivative in which RL13 and UL128L were either mutated or repressed were remarkably stable in fibroblasts. Thus, HCMV containing a wild-type gene complement can be generated in vitro by deriving virus from a self-excising BAC in fibroblasts, and repressing RL13 and UL128L.

    Importance Researchers should aim to study viruses that accurately represent the causative agents of disease. This is problematic for HCMV, because clinical strains mutate rapidly when propagated in vitro, becoming less cell-associated, altered in tropism, more susceptible to natural killer cells, and less pathogenic. Following isolation from clinical material, HCMV genomes can be stabilized by cloning into bacterial artificial chromosomes (BACs), then virus regenerated by DNA transfection. However, mutations can occur not only during isolation prior to BAC cloning, but also when virus is regenerated. We have identified conditions under which BAC-derived viruses containing an intact, wild-type genome can be propagated in vitro with minimal risk of mutants being selected, enabling studies of viruses expressing the gene complement of a clinical strain. However, even under these optimized conditions, sporadic mutations can occur, highlighting the advisability of sequencing the HCMV stocks used in experiments.

  • Expansion of inefficient HIV-specific CD8+ T cells during acute infection [PublishAheadOfPrint]

  • Attrition within the CD4+ T cell compartment, high viremia and a cytokine storm characterize the early days after HIV infection. When the first emerging HIV-specific CD8+ T cell responses gain control over viral replication, it is incomplete, and clearance of HIV infection is not achieved even in the rare cases of individuals who spontaneously control viral replication to near to immeasurable low levels. Thus, despite their partial ability to control viremia, HIV-specific CD8+ T cell responses are insufficient to clear HIV infection. Studying individuals in the first few days of acute HIV infection, we detected the emergence of a unique population of CD38+CD27-CD8+ T cells characterized by a low expression of the CD8 receptor (CD8dim). Interestingly, while a high frequency of HIV-specific CD8+ T cell responses are within the CD38+CD27-CD8dim T cell population, the minority populations of CD8bright T cells are significantly more effective in inhibiting HIV replication. Furthermore, the frequency of CD8dim T cells directly correlates with viral load and clinical predictors of more rapid disease progression. We found that a canonical burst of proliferative cytokines coincides with the emergence CD8dim T cells and the size of this population inversely correlates with the acute loss of CD4+ T cells. These data indicate, for the first time, that early CD4+ T cell loss coincides with the expansion of a functionally impaired HIV specific CD8dim T cell population less efficient in controlling HIV viremia.

    Importance: A distinct population of activated CD8+ T cells appears during acute HIV infection with diminished capacity to inhibit HIV replication and is predictive of viral set-point, offering the first immunologic evidence of CD8+ T cell dysfunction during acute infection.

  • Replication of Human Herpes Viruses is Associated with Greater HIV DNA Levels during Antiretroviral Therapy Started during Early HIV Infection [PublishAheadOfPrint]

  • Background: Asymptomatic replication of human herpesviruses (HHV) is frequent in HIV-infected men, and is associated with increased T-cell activation and HIV disease progression. We hypothesized that presence of cytomegalovirus (CMV) and Epstein Barr virus (EBV) replication (the most frequent HHV) might influence HIV DNA decay during antiretroviral therapy (ART).

    Methods: We investigated 607 peripheral blood mononuclear cell (PBMC) samples from 107 CMV-seropositive, HIV-infected men who have sex with men, who started ART within a median of 3 months from their estimated date of infection (EDI), and were followed for a median of 19 months thereafter. Levels of HIV, CMV and EBV DNA, and cellular HIV RNA were measured by ddPCR for each time-point. Using a general linear mixed-effect regression model, we evaluated associations between the frequency of detectable CMV DNA, EBV DNA levels, HIV DNA decay and cellular HIV RNA levels, while adjusting for peak HIV RNA, nadir CD4+ count, CD4:CD8 ratio, CMV IgG levels, time from EDI to ART initiation, time from ART initiation to virologic suppression, detectable CMV DNA pre-ART, and age.

    Results: The presence of intermittent CMV DNA in PBMC during ART was significantly associated with slower decay of HIV DNA levels (p=0.011) but not with increased cellular HIV RNA transcription or 2-LTR circles. Levels of EBV DNA were also associated with higher levels of HIV DNA (pllt;0.001) and unspliced cellular HIV RNA (p=0.010).

    Conclusions: These observations suggest that replication of HHV may help maintain a larger HIV DNA reservoir but the underlying mechanisms remain unclear.

    Importance Over three-fourths of HIV-infected men have at least one actively replicating human herpesvirus (HHV) in their mucosal secretions at any one time. Cytmegalovirus (CMV) and Epstein Barr virus (EBV) are the most common, and although it is often asymptomatic, such CMV and EBV replication is associated with higher levels of immune activation and HIV disease progression. We hypothesize that HHV-associated activation of HIV-infected CD4+ T-cells might lead to increased HIV DNA. This study found that detectable CMV in blood cells of HIV-infected men was associated with slower decay of HIV DNA even during antiretroviral therapy (ART) that was started during early HIV infection. Similarly, levels of EBV DNA were associated with higher levels of HIV DNA during ART. If this observation points to a causal pathway, interventions that control CMV and EBV replication may be able to reduce the HIV reservoir, which might be relevant to current HIV cure efforts.

  • Virus and host mechanics support membrane penetration and cell entry [PublishAheadOfPrint]

  • Viruses are quasi-inert macromolecular assemblies. Their meta-stable conformation changes during entry into cells, when chemical and mechanical host cues expose viral membrane-interacting proteins. This leads to membrane rupture or fusion, and genome uncoating. Importantly, virions tune their physical properties and enhance penetration and uncoating. For example, influenza virus softens at low pH to uncoat. Stiffness and pressure of adenovirus control uncoating and membrane penetration. Virus and host mechanics thus present new opportunities for anti-viral therapy.

  • The Herpes Simplex Virus LAT Gene is Associated with a Broader Repertoire of Virus-Specific Exhausted CD8+ T Cells Retained Within The Trigeminal Ganglia of Latently Infected HLA Transgenic Rabbits [PublishAheadOfPrint]

  • Persistent pathogens, such as herpes simplex virus type 1 (HSV-1), have evolved a variety of immune evasion strategies to avoid being detected and destroyed by the host's immune system. A dynamic cross talk appears to occur between the HSV-1 Latency-Associated Transcript (LAT), the only viral gene that is abundantly transcribed during latency, and the CD8+ T cells that reside in HSV-1 latently infected human and rabbit trigeminal ganglia (TG). The reactivation phenotype of TG that are latently infected with wild type HSV-1 or with LAT rescued mutant (i.e. LAT(+) TG) is significantly higher than TG latently infected with LAT null mutant (i.e. LAT(-) TG). Whether LAT promotes virus reactivation by selectively shaping a unique repertoire of HSV-specific CD8+ T cells in LAT(+) TG is unknown. In the present study, we assessed the frequency, function, and exhaustion status of TG-resident CD8+ T cells specific to 40 epitopes derived from HSV-1 gB, gD, VP11/12 and VP13/14 proteins, in Human Leukocyte Antigen (HLA-A*0201) transgenic rabbits infected ocularly with LAT(+) vs. LAT(-) virus. Compared to CD8+ T cells from LAT(-) TG, CD8+ T cells from LAT(+) TG: (i) recognized a broader selection of non-overlapping HSV-1 epitopes; (ii) expressed higher levels of PD-1, TIM-3 and CTLA-4 markers of exhaustion; and (iii) produced less TNF-aalpha;, IFN-, and GzmB. These results suggest a novel immune evasion mechanism by which the HSV-1 LAT may contribute to the shaping of a broader repertoire of exhausted HSV-specific CD8+ T cells in latently infected TG, thus, allowing for increased viral reactivation.

    IMPORTANCE A significantly larger repertoire of dysfunctional (exhausted) HSV-specific CD8+ T cells were found in the TG of HLA transgenic rabbits latently infected with wild type HSV-1 or with LAT rescued mutant (i.e. LAT(+) TG), compared to a more restricted repertoire of functional HSV-specific CD8+ T cells in the TG of HLA transgenic rabbits latently infected with LAT null mutant (i.e. LAT (-) TG). These findings suggest that the HSV-1 LAT locus interferes with the host cellular immune response by shaping a broader repertoire of exhausted HSV-specific CD8+ T cells within the latency/reactivation TG site.

  • The barrier to autointegration factor: interlocking antiviral defense with genome maintenance [PublishAheadOfPrint]

  • Intrinsic defenses targeting foreign DNA are one facet of the cellular armament tasked with protecting host genomic integrity. The DNA binding protein BAF (barrier to autointegration factor) contributes to multiple aspects of genome maintenance and intercepts retrovirus, poxvirus, and herpesvirus genomes during infection. In this GEM, we discuss the unique position BAF occupies at the virus-host interface and how both viral and cellular mechanisms may regulate its capacity to act as a pro- or antiviral effector targeting viral DNA.

  • gD-independent superinfection exclusion of alphaherpesviruses [PublishAheadOfPrint]

  • Many viruses have the capacity to prevent a cell from being infected by a second virus, often termed superinfection exclusion. Alphaherpesviruses, including the human pathogen Herpes Simplex virus type 1 (HSV-1) and the animal herpesviruses pseudorabies virus (PRV), encode a membrane bound glycoprotein, gD, that can interfere with subsequent virion entry. We sought to characterize the timing and mechanism of superinfection exclusion during HSV-1 and PRV infection. To this end, we utilized recombinant viruses expressing fluorescent protein (FP) markers of infection that allowed visualization of viral infections by microscopy and flow cytometry as well as differentiation of viral progeny. Our results demonstrated the majority of HSV-1 and PRV infected cells establish superinfection exclusion by two hours post infection. Modification of viral infections by virion inactivation, phosphonoacetic acid, cycloheximide and actinomycin D treatments, indicated new protein synthesis is needed to establish superinfection exclusion. Primary infection with gene deletion PRV recombinants identified that new gD expression is not required to establish superinfection exclusion of secondary viral inoculum. We also identified the timing of co-infection events during axon-to-cell spread, with most occurring within a 2-hour window, suggesting a role for cellular superinfection exclusion during neuroinvasive spread of infection. In summary, we have characterized a gD-independent mechanism of superinfection exclusion established by two members of the alphaherpesvirus family and identified a potential role of exclusion during pathogenic spread of infection.

    Importance Superinfection exclusion is a widely observed phenomenon initiated by a primary viral infection to prevent further viruses from infecting the same cell. The capacity for alphaherpesviruses to infect the same cell impacts rates of interviral recombination and disease. Interviral recombination allows genome diversification, facilitating development of resistance to antiviral therapeutics and evasion of vaccine-mediated immune responses. Our results demonstrate superinfection exclusion occurs early, through a gD-independent process, and is important in the directed spread of infection. Identifying when and where in an infected host viral genomes are more likely to co-infect the same cell and generate viral recombinants will enhance development of effective antiviral therapies and interventions.

  • miR-33a-5p modulates Japanese Encephalitis Virus Replication by targeting Eukaryotic Translation Elongation Factor 1A1 [PublishAheadOfPrint]

  • Japanese encephalitis virus (JEV) is a typical mosquito-borne flavivirus responsible for acute encephalitis and meningitis in humans. However, the molecular mechanism for JEV pathogenesis is still unclear. MicroRNAs (miRNAs) are small noncoding RNAs that act as gene regulators. They are directly or indirectly involved in many cellular functions owing to their ability to target mRNAs for degradation or translational repression. However, how cellular miRNAs are regulated and their functions during JEV infection are largely unknown. In the present study, we found that JEV infection downregulated the expression of endogenous cellular miR-33a-5p. Notably, artificially transfecting with miR-33a-5p mimics led to a significant decrease in viral replication, suggesting that miR-33a-5p acts as a negative regulator of JEV replication. A dual-luciferase reporter assay identified eukaryotic translation elongation factor 1A1 (EEF1A1) as one of the miR-33a-5p target genes. Our study further demonstrated that EEF1A1 can interact with the JEV proteins NS3 and NS5 in replicase complex. Through this interaction, EEF1A1 can stabilize the components of viral replicase complex, and thus facilitates viral replication during JEV infection. Taken together, these results suggest that miR-33a-5p is downregulated during JEV infection, which contributes to viral replication by increasing the intracellular level of EEF1A1, an interaction partner of JEV NS3 and NS5. This study provides a better understanding of the molecular mechanisms of JEV pathogenesis.

    IMPORTANCE MiRNAs are critical regulators of gene expression that utilize sequence complementarity to bind to and modulate the stability or translation efficiency of target mRNAs. Accumulating data suggest that miRNAs regulate a wide variety of molecular mechanisms in the host cells during viral infections. JEV, a neurotropic flavivirus, is one of the major causes of acute encephalitis in humans worldwide. The roles of cellular miRNAs during JEV infections are widely unexplored. The present study explores a novel role of miR-33a-5p as a negative regulator of JEV replication. We found EEF1A1 as a direct target of miR-33a-5p. We also demonstrated that EEF1A1 interacts with and stabilize the components of JEV replicase complex, which positively regulates JEV replication. These findings suggest a new insight into the molecular mechanism of JEV pathogenesis, and provide a possible therapeutic entry point for viral encephalitis.

  • Induction of Epstein-Barr Virus Oncoprotein Latent Membrane Protein 1 (LMP1) by Transcription Factors Activating Protein 2 (AP-2) and Early B Cell Factor (EBF) [PublishAheadOfPrint]

  • Latent membrane protein 1 (LMP1) is a major oncogene essential for primary B cell transformation by Epstein-Barr virus (EBV). Previous studies suggested that some transcription factors, such as PU.1, RBP-J, NKB, and STAT, are involved in this expression, but the underlying mechanism is unclear. Here, we identified binding sites for PAX5, AP-2, and EBF in the proximal LMP1 promoter (ED-L1p). We first confirmed the significance of PU.1 and POU domain transcription factor binding for activation of the promoter in latency III. We then focused on the transcription factors AP-2 and EBF. Interestingly, among the three AP-2-binding sites in the LMP1 promoter, two motifs were also bound by EBF. Overexpression, knockdown, and mutagenesis in the context of the viral genome indicated that AP-2 plays an important role in LMP1 expression in latency II in epithelials. In latency III B cells on the other hand, the B cell-specific transcription factor EBF binds to the ED-L1p and activates LMP1 transcription from the promoter.

    IMPORTANCE Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is crucial for B cell transformation and oncogenesis of other EBV-related malignancies, such as nasopharyngeal carcinoma and T/NK lymphoma. Its expression is largely dependent on the cell type or condition, and some transcription factors have been implicated in its regulation. However, these previous reports evaluated the significance of specific factors mostly by reporter assay. In this study, we prepared point-mutated EBV at the binding sites of such transcription factors and confirmed the importance of AP-2, EBF, PU.1 and POU domain factors. Our results will provide insight into the transcriptional regulation of the major oncogene LMP1.

  • Functional Comparison of HBZ and the Related APH-2 Protein Provide Insight into HTLV-1 Pathogenesis [PublishAheadOfPrint]

  • Human T-cell leukemia virus type 1 (HTLV-1) and type 2 (HTLV-2) are highly related retroviruses that transform T-cells in vitro, but have distinct pathological outcomes in vivo. HTLV-1 encodes a protein from the antisense strand of its proviral genome, the HTLV-1 basic leucine zipper factor (HBZ), which inhibits Tax-1-mediated viral transcription and promotes cell proliferation, high proviral load, and persistence in vivo. In Adult T-cell Leukemia (ATL) cell lines and patient T-cells, the HBZ gene product is often the only viral gene expressed. HTLV-2 also encodes a protein from the antisense strand of its proviral genome, termed APH-2. Like HBZ, APH-2 is able to inhibit Tax-2-mediated viral transcription and is detectable in most primary lymphocytes from HTLV-2-infected patients. However, unlike HBZ, loss of APH-2 in vivo results in increased viral replication and proviral loads, suggesting that HBZ and APH-2 modulate the virus and cellular pathways differently. Herein, we examined the effect of APH-2 on several known HBZ-modulated pathways: NF-B (p65) transactivation, TGF-bbeta; signaling, and IRF-1 transactivation. Like HBZ, APH-2 has the ability to inhibit p65 transactivation. Conversely, HBZ and APH-2 have divergent effects on TGF-bbeta; signaling and IRF-1 transactivation. qPCR and protein half-life experiments revealed a substantial disparity between HBZ and APH-2 transcript levels and protein stability, respectively. Taken together, our data further elucidate the functional differences between HBZ and APH-2 and how these differences can have profound effects on infected cell survival and ultimately, pathogenesis.

    IMPORTANCE Human T-cell leukemia virus type 1 (HTLV-1) and type 2 (HTLV-2) are highly related retroviruses that have distinct pathological outcomes in infected hosts. Functional comparisons between HTLV-1 and HTLV-2 viral proteins provide a better understanding about how HTLV-1 infection is associated with disease while HTLV-2 infection is not. The HTLV genome antisense strand genes, HBZ and APH-2, are often the only viral gene products expressed in HTLV-infected T-cells. Previously, our group found HTLV-1 HBZ and HTLV-2 APH-2 had distinct effects in vivo and hypothesized the differences in interactions of HBZ and APH-2 with important cell signaling pathways dictate whether cells undergo proliferation, apoptosis, or senescence. Ultimately, these functional differences may affect how HTLV-1 causes disease while HTLV-2 generally does not. In the current study, we compared the effects of HBZ and APH-2 on several HTLV-relevant cellular pathways, including TGF-bbeta; signaling, NF-B activation, and IRF-1 transactivation.

  • Spatiotemporal analysis of the genetic diversity of seal influenza A(H10N7) virus, Northwestern Europe [PublishAheadOfPrint]

  • Influenza A viruses are major pathogens for humans, domestic animals and wildlife that cross the species barrier occasionally. In spring 2014, increased mortality of harbor seals (Phoca vitulina) was reported in Sweden and Denmark, associated with infection with an influenza A(H10N7) virus. Within a few months, this virus spread to seals of the coastal waters of Germany and the Netherlands, causing the death of thousands of animals. Genetic analysis of the hemagglutinin (HA) and neuraminidase (NA) genes of this seal influenza A(H10N7) virus revealed that it was most closely related to various avian influenza A(H10N7) viruses. The collection of samples from infected seals during the course of the outbreak provided an unique opportunity to follow the adaptation of this avian virus to its new seal host. Sequence data was obtained from samples collected from 41 different seals from four different countries between April 2014 and January 2015 using Sanger sequencing and next-generation sequencing to describe the molecular epidemiology of the seal influenza A(H10N7) virus. The majority of sequence variation occurred in the HA gene, and some mutations corresponded with amino acid changes not found in H10 viruses isolated from Eurasian birds. Also, sequence variation in the HA gene was greater at the beginning than at the end of the epidemic, when a number of the mutations observed earlier had been fixed. These results imply that when an avian influenza virus jumps the species barrier from birds to seals, amino acid changes in the HA may occur rapidly and are important for virus adaptation to its new mammalian host.

    Importance Influenza A viruses are major pathogens for humans, domestic animals and wildlife. In addition to the continuous circulation of influenza A viruses among various host species, cross-species transmission of influenza A viruses occurs occasionally. Wild waterfowl and shorebirds are the main reservoir for most influenza A virus subtypes, and spillover of influenza A viruses from birds to humans or other mammalian species may result in major outbreaks. In the present study, various sequencing methods were used to elucidate the genetic changes that occurred after the introduction and subsequent spread of an avian influenza A(H10N7) among harbor seals of Northwestern Europe using various samples collected during the outbreak. Such detailed knowledge of genetic changes necessary for introduction and adaptation of avian influenza A viruses to mammalian hosts is important for a rapid risk assessment of such viruses soon after they cross the species barrier.

  • A hepatitis C virus envelope polymorphism confers resistance to neutralization by polyclonal sera and broadly neutralizing monoclonal antibodies [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) infection is a global health problem with millions of chronically-infected individuals at risk for cirrhosis and hepatocellular carcinoma. HCV vaccine development is vital in the effort toward disease control and eradication, an undertaking aided by an increased understanding of the mechanisms of resistance to broadly neutralizing antibodies (bNAbs). In this study, we identify HCV codons that vary deep in a phylogenetic tree of HCV sequences and show that a polymorphism at one of these positions renders Bole1a, a computationally-derived, ancestral genotype 1a HCV virus, resistant to neutralization by both polyclonal HCV-infected plasma and multiple broadly neutralizing monoclonal antibodies with unique binding epitopes. This bNAb resistance mutation reduces replicative fitness, which may explain persistence of both neutralization sensitive and resistant variants in circulating viral strains. This work identifies an important determinant of bNAb resistance in an ancestral, representative HCV genome, which may inform HCV vaccine development.

    Importance Worldwide, more than 170 million people are infected with hepatitis C virus (HCV), the leading cause of hepatocellular carcinoma and liver transplantation in the United States. Despite recent significant advances in HCV treatment, a vaccine is needed. Control of the HCV pandemic with drug treatment alone is likely to fail due to limited access to treatment, reinfections in high risk individuals, and potential for resistance to direct acting antivirals (DAAs). Broadly neutralizing antibodies (bNAbs) block infection by diverse HCV variants and therefore serve as a useful guide for vaccine development, but our understanding of resistance to bNAbs is incomplete. In this study, we identify a viral polymorphism conferring resistance to neutralization by both polyclonal plasma and broadly neutralizing monoclonal antibodies, which may inform HCV vaccine development.

  • Human cytomegalovirus pTRS1 and pIRS1 antagonize PKR to facilitate virus replication. [PublishAheadOfPrint]

  • Human cytomegalovirus (HCMV) counteracts host defenses that otherwise act to limit viral protein synthesis. One such defense is the antiviral kinase PKR, which inactivates the eIF2 translation initiation factor upon binding to viral double-stranded RNAs. Previously the viral TRS1 and IRS1 proteins were found to antagonize the antiviral kinase PKR outside the context of HCMV infection, and the expression of either pTRS1 or pIRS1 was shown to be necessary for HCMV replication. In this study we found that expression of either pTRS1 or pIRS1 is necessary to prevent PKR activation during HCMV infection, and that antagonism of PKR is critical for efficient viral replication. Consistent with a previous study, we observed decreased overall levels of protein synthesis, reduced viral protein expression and diminished virus replication in the absence of both pTRS1 and pIRS1. In addition both PKR and eIF2aalpha; were phosphorylated during infection when pTRS1 and pIRS1 were absent. We also found that expression of pTRS1 was both necessary and sufficient to prevent stress granule formation in response to eIF2aalpha; phosphorylation. Depletion of PKR prevented eIF2aalpha; phosphorylation, rescued HCMV replication and protein synthesis, and reversed the accumulation of stress granules in infected cells. Infection with an HCMV mutant lacking the pTRS1 PKR binding domain resulted in PKR activation, suggesting that pTRS1 inhibits PKR through a direct interaction. Together our results show that antagonism of PKR by HCMV pTRS1 and pIRS1 is critical for viral protein expression and efficient HCMV replication.

    IMPORTANCE To successfully replicate viruses must counteract host defenses that limit viral protein synthesis. We have identified inhibition of the antiviral kinase PKR by the viral proteins TRS1 and IRS1 and shown that this is a critical step in HCMV replication. Our results suggest that inhibiting pTRS1 and pIRS1 function or restoring PKR activity during infection may be a successful strategy to limit HCMV disease.

  • ESCRT-0 Component Hrs Promotes Macropinocytosis of Kaposi's Sarcoma-Associated Herpesvirus in Human Dermal Microvascular Endothelial Cells [PublishAheadOfPrint]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) enters human dermal microvascular endothelial cells (HMVEC-d), its natural in vivo target cells, by lipid raft dependent macropinocytosis. The internalized viral envelope fuses with the macropinocytic membrane and released capsid is transported to the nuclear vicinity resulting in the nuclear entry of viral DNA. The Endosomal Sorting Complexes Required for Transport (ESCRT) proteins which include ESCRT-0, -I, -II, and -III play a central role in endosomal trafficking and sorting of internalized and ubiquitinated receptors. Here, we examined the role of ESCRT-0 component Hrs (Hepatocyte growth factor regulated tyrosine kinase substrate) in KSHV entry into HMVEC-d cells by macropinocytosis. Knockdown of Hrs by shRNA transduction results in significant decrease in KSHV entry and viral gene expression. Immunofluorescence analysis (IFA) and plasma membrane isolation and proximity ligation assay (PLA) demonstrate the translocation of Hrs from the cytosol to the plasma membranes of infected cells and association with aalpha;-actinin-4. In addition, infection induces the plasma membrane translocation and activation of the serine/threonine kinase ROCK1, a downstream target of the RhoA GTPase. Hrs knockdown reduces these associations suggesting that the recruitment of ROCK1 is an Hrs mediated event. Interaction between Hrs and ROCK1 is essential for the ROCK1 induced phosphorylation of NHE1 (Na+/H+ exchanger 1) involved in the regulation of intracellular pH. Thus, our studies demonstrate the plasma membrane association of ESCRT protein Hrs during macropinocytosis and suggest that KSHV entry requires both Hrs and ROCK1 dependent mechanisms, and ROCK1 mediated phosphorylation of NHE1 and pH change is an essential event required for the macropinocytosis of KSHV.

    IMPORTANCE Macropinocytosis is the major entry pathway of KSHV in human dermal microvascular endothelial cells, the natural target cells of KSHV. Although the role of ESCRT protein Hrs has been extensively studied with respect to endosomal movement and sorting of ubiquitinated proteins into lysosomes, its function in macropinocytosis is not known. In the present study, we demonstrate for the first time that upon KSHV infection the endogenous Hrs localizes to the plasma membrane and the membrane associated Hrs facilitates assembly of signaling molecules, macropinocytosis and virus entry. Hrs recruits ROCK1 to the membrane which is required for the activation of NHE1 and an increase in submembraneous intracellular pH occurring during macropinocytosis. These studies demonstrate that the localization of Hrs from the cytosol to the plasma membrane is important for coupling membrane dynamics to the cytosolic signaling events during macropinocytosis of KSHV.

  • Generation and characterization of monoclonal antibodies against a cyclic variant of Hepatitis C Virus E2 epitope 412-422. [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) E2 envelope glycoprotein is crucial for virus entry into hepatocytes. A conserved region of E2 encompassing amino acids 412-423 and containing Trp420, a residue critical for virus entry, is recognized by several broadly neutralizing antibodies. Peptides embodying this epitope I sequence adopt a bbeta;-hairpin conformation when bound to neutralizing monoclonal antibodies (MAbs) AP33 and HCV-1. We therefore generated new mouse MAbs that were able to bind to a cyclic peptide containing E2 residues 412-422 (C-Epitope I) but not to the linear counterpart. These MAbs bound to purified E2 with affinities of about 50 nM, but they were unable to neutralize virus infection. Structural analysis of the complex between C-Epitope I and one of our MAbs (C2) show that the Trp420 side chain is largely buried in the combining site and that the Asn417 side chain, which is glycosylated in E2 and solvent-exposed in other complexes, is slightly buried upon C2 binding. Also, the orientation of the cyclic peptide in the antibody combining site is rotated by 180ddeg; compared to other complexes. All these structural features, however, do not explain the lack of neutralization activity. This is instead ascribed to the high selectivity of the new MAbs for the cyclic epitope and to their inability to interact with the epitope in more flexible and extended conformations, which recent data suggest play a role in the mechanisms of neutralization escape.

    IMPORTANCE Hepatitis C virus (HCV) remains a major health care burden affecting almost 3% of the global population. The conserved epitope I comprising residues 412-423 of the viral E2 glycoprotein, is a valid vaccine candidate because antibodies recognizing this region exhibit potent neutralizing activity. This epitope adopts a bbeta;-hairpin conformation when bound to neutralizing MAbs. We explored the potential of cyclic peptides mimicking this structure to elicit anti-HCV antibodies. MAbs that specifically recognize a cyclic variant of the epitope bind to soluble E2 with lower affinity than other blocking antibodies and don't neutralize virus. The structure of the complex between one such MAb and the cyclic epitope, together with new structural data showing the linear peptide bound to neutralizing MAbs in extended conformations, suggests that the epitope displays a conformational flexibility that contributes to neutralization escape. Such features can be of major importance for the design of epitope-based anti-HCV vaccines.

  • Primary B lymphocytes infected with KSHV can be expanded in vitro and are recognized by LANA-specific CD4+ T cells. [PublishAheadOfPrint]

  • Kaposi sarcoma-associated herpesvirus (KSHV) has a tropism for B lymphocytes in which it establishes latency and can also cause lymphoproliferative disorders of these cells manifesting as primary effusion lymphoma (PEL) and multi-centric Castleman disease (MCD). T cell immunity is vital for the control of KSHV infection and disease however few models of B lymphocyte infection exist to study immune recognition of such cells. Here we developed a model of B lymphocyte infection with KSHV where infected tonsillar B lymphocytes were expanded by providing mitogenic stimuli and these challenged with KSHV-specific CD4+ T cells. Infected cells expressed viral proteins found in PELs namely LANA and vIRF3, albeit at lower levels, with similar patterns of gene expression for the major latency, vIL-6 and vIRF3 transcripts. Despite low level expression of ORF50, transcripts for the immune evasion genes K3 and K5 were detected, with some downregulation of cell surface expressed CD86 and ICAM. The vast majority of infected lymphocytes expressed IgM and the Ig light chain, recapitulating these features seen in infected cells in MCD. We assessed the ability of the infected lymphocytes to be targeted by a panel of MHC class II matched CD4+ T cells and found that LANA-specific T cells restricted to different epitopes recognized these infected cells. Given that at least some KSHV latent antigens are thought to be poor targets for CD8+ T cells, we suggest that CD4+ T cells are potentially important effectors for the in vivo control of KSHV infected B lymphocytes.

    Importance KSHV establishes a latent reservoir within B lymphocytes but few models exist to study KSHV-infected B cells other than the transformed PEL cell lines which have likely accrued mutations during the transformation process. We developed a model of KSHV-infected primary B lymphocytes which recapitulate features seen in PEL and MCD by gene expression and cell phenotype analysis, allowing the study of T cell recognition of these cells. Challenge of KSHV-infected B cells with CD4+ T cells specific for LANA, a protein expressed in all KSHV-infected cells and malignancies in vivo, showed that these effectors could efficiently recognize such targets. Given the virus expresses immune evasion genes or use proteins with intrinsic properties such as LANA that minimize epitope recognition by CD8+ T cells, CD4+ T cell immunity to KSHV may be important for maintaining the virus host balance.

  • The L-, M- and S-segments of Rift Valley fever virus MP-12 vaccine independently contribute to a temperature-sensitive phenotype [PublishAheadOfPrint]

  • Rift Valley fever (RVF) is endemic to Africa, and the mosquito-borne disease is characterized by "abortion storms" in ruminants and hemorrhagic fever, encephalitis and blindness in humans. Rift Valley fever virus (RVFV: family Bunyaviridae, genus Phlebovirus) has a tripartite negative-stranded RNA genome (L-, M- and S-segments). A live-attenuated vaccine for RVF, the MP-12 vaccine, is conditionally licensed for veterinary use in the U.S. MP-12 is fully attenuated by the combination of the partially attenuated L-, M- and S-segments. Temperature-sensitivity (ts) limits viral replication at a restrictive temperature and may be involved with viral attenuation. In this study, we aimed to characterize the ts mutations for MP-12. The MP-12 vaccine showed a restricted replication at 38ddeg;C and replication shut-off (100-fold or more reduction in virus titer compared to that at 37ddeg;C) at 39ddeg;C in Vero and MRC-5 cells. Using rZH501 reassortants encoding either the MP-12 L-, M-, or S-segment, we found that all three segments encode a temperature-sensitive phenotype. However, the ts phenotype of S-segment was weaker than that of the M- or L-segment. We identified Gn-Y259H, Gc-R1182G, L-V172A, and L-M1244I as major ts mutations for MP-12. The ts mutations in the L-segment decreased viral RNA synthesis, while those in the M-segment delayed progeny production from infected cells. We also found that a lack of NSs and/or 78kD/NSm protein expression minimally affected the ts phenotype. Our study revealed that MP-12 is a unique vaccine encoding ts mutations in the L-, M- and S-segments.

    IMPORTANCE Rift Valley fever (RVF) is a mosquito-borne viral disease endemic to Africa, characterized by high-rates of abortion in ruminants and severe diseases in humans. Vaccination is important to prevent the spread of disease, and a live-attenuated MP-12 vaccine is currently the only vaccine with conditional license in the U.S. This study determined the temperature-sensitivity (ts) of MP-12 vaccine to understand virologic characteristics. Our study revealed that MP-12 vaccine encodes ts mutations independently in the L-, M- and S-segments, and MP-12 displays a restrictive replication at 38ddeg;C.

  • Suppression of type I interferon production by human T-cell leukemia virus type 1 oncoprotein Tax through inhibition of IRF3 phosphorylation [PublishAheadOfPrint]

  • Infection with human T-cell leukemia virus type 1 (HTLV-1) is associated with adult T-cell leukemia (ATL) and tropical spastic paraparesis. Type I interferons (IFNs) are key effectors of innate antiviral response and IFN-aalpha; combined with nucleoside reverse transcriptase inhibitor zidovudine is considered the standard first-line therapy for ATL. HTLV-1 oncoprotein Tax is known to suppress innate IFN production and response but the underlying mechanisms remain to be fully established. In this study we report on the suppression of type I IFN production by HTLV-1 Tax through interaction with and inhibition of TBK1 kinase that phosphorylates IRF3. Induced transcription of IFN-bbeta; was severely impaired in HTLV-1-transformed ATL cells and freshly infected T lymphocytes. The ability to suppress IRF3 activation was ascribed to Tax. Expression of Tax alone sufficiently repressed the induction of IFN production by RIG-I plus PACT, cGAMP synthase plus STING, TBK1, IKK, IRF3 and IRF7, but not by IRF3-5D, a dominant active phosphomimetic mutant. This suggests that Tax perturbs IFN production at the step of IRF3 phosphorylation. Tax mutants deficient for CREB or NF-B activation were fully competent in the suppression of IFN production. Co-immunoprecipitation experiments confirmed the association of Tax with TBK1, IKK, STING and IRF3. In vitro kinase assay indicated an inhibitory effect of Tax on TBK1-mediated phosphorylation of IRF3. Taken together, our findings suggested a new mechanism by which HTLV-1 oncoprotein Tax circumvents the production of type I IFNs in infected cells. Our findings have implications in therapeutic intervention of ATL.

    IMPORTANCE Human T-cell leukemia virus type 1 (HTLV-1) is the cause of adult T-cell leukemia (ATL), an aggressive and fatal blood cancer, as well as another chronic disabling disease of the spinal cord. Treatments are unsatisfactory and options are limited. A combination of antiviral cellular protein interferon-aalpha; and zidovudine, which is an inhibitor of a viral enzyme called reverse transcriptase, has been recommended as the standard first-line therapy for ATL. Exactly how HTLV-1 interacts with the cellular machinery for interferon production and action is not well understood. Our work sheds light on the mechanism of action for the inhibition of interferon production by an HTLV-1 oncogenic protein called Tax. Our findings might help to improve interferon-based anti-HTLV-1 and anti-ATL therapy.

  • Chimeric Filoviruses for Identification and Characterization of Monoclonal Antibodies [PublishAheadOfPrint]

  • Recent experiments suggest that some glycoprotein (GP) specific monoclonal antibodies (mAbs) can protect experimental animals against the filovirus Ebola virus (EBOV). There is a need for isolation of mAbs capable of neutralizing multiple filoviruses. Antibody neutralization assays for filoviruses frequently use surrogate systems such as the rhabdovirus vesicular stomatitis Indiana virus (VSV), lentiviruses or gammaretroviruses with their envelope proteins replaced with EBOV glycoprotein (GP) or pseudotyped with EBOV GP. It is optimal for both screening and in-depth characterization of newly identified neutralizing mAbs to generate recombinant filoviruses that express a reporter fluorescent protein in order to more easily monitor and quantify the infection. Our study showed that unlike neutralization-sensitive chimeric VSV, authentic filoviruses are highly resistant to neutralization by mAbs. We used reverse genetics techniques to replace EBOV GP with its counterpart from the heterologous filoviruses Bundibugyo virus (BDBV), Sudan virus (SUDV), and even Marburg virus (MARV) and Lloviu virus (LLOV), which belong to the heterologous genera in the filovirus family. This work resulted in generation of multiple chimeric filoviruses, demonstrating the ability of filoviruses to tolerate swapping of the envelope protein. The sensitivity of chimeric filoviruses to neutralizing mAbs was similar to that of authentic biologically derived filoviruses with the same GP. Moreover, disabling the expression of the secreted GP (sGP) resulted in an increased susceptibility of an engineered virus to the BDBV52 mAb isolated from a BDBV survivor, suggesting a role for sGP in evasion of antibody neutralization in the context of a human filovirus infection.

    IMPORTANCE The study demonstrated that chimeric rhabdoviruses in which G protein is replaced with filovirus GP, widely used as surrogate targets for characterization of filovirus neutralizing antibodies, do not accurately predict the ability of antibodies to neutralize authentic filoviruses, which appeared to be resistant to neutralization. However, a recombinant EBOV expressing a fluorescent protein tolerated swapping of GP with counterparts from heterologous filoviruses, allowing high-throughput screening of B cell lines to isolate mAbs of any filovirus specificity. Human mAb BDBV52, that was isolated from a survivor of BDBV infection, was capable of partially neutralizing a chimeric EBOV carrying BDBV GP in which expression of sGP was disabled. In contrast, the parental virus expressing sGP was resistant to the mAb. Thus, the ability of filoviruses to tolerate swapping of GP can be used for identification of neutralizing mAbs specific to any filovirus and for characterization of mAb specificity and mechanism of action.

  • Dissecting the molecular mechanisms of the tropism of varicella-zoster virus for human T cells [PublishAheadOfPrint]

  • Studies of VZV tropism for T cells support their role in viral transport to skin during primary infection. Instead of preferentially infecting skin homing T cells, multiparametric single cell mass cytometry demonstrates that VZV alters cell signaling and remodels surface proteins to enhance T cell skin trafficking. Viral proteins dispensable in skin, such as ORF66, are necessary in T cells. Interfering with VZV T cell tropism may offer novel strategies for drug and vaccine design.

  • Infectious entry pathway mediated by the human endogenous retrovirus K envelope protein [PublishAheadOfPrint]

  • Endogenous retroviruses (ERVs) comprise approximately 8% of the human genome, the majority of which exist as degraded remnants of ancient viruses. The youngest human ERVs (HERVs) belong to the HERV-K(HML-2) subgroup and were endogenized within the past one million years. The viral envelope protein (ENV) facilitates the earliest events of endogenization (cellular attachment and entry), and here we characterize the requirements for HERV-K ENV to mediate infectious cell entry. Cell-cell fusion assays indicate that a minimum of two events are required for fusion, proteolytic processing by furin-like proteases and exposure to acidic pH. We generated an infectious autonomously replicating recombinant vesicular stomatitis virus (VSV) in which the glycoprotein was replaced by HERV-K ENV. HERV-K ENV imparts an endocytic entry pathway that requires dynamin-mediated membrane scission and endosomal acidification but is distinct from clathrin dependent or macropinocytic uptake pathways. The lack of impediments to the replication of the VSV core in eukaryotic cells allowed us to broadly survey the HERV-K ENV-dictated tropism. Unlike extant betaretroviral envelopes, which impart a narrow species tropism, we found that HERV-K ENV mediates a broad tropism encompassing cells from multiple mammalian and non-mammalian species. We conclude that HERV-K ENV dictates an evolutionarily conserved entry pathway, and that the restriction of HERV-K to primate genomes reflects downstream stages of the viral replication cycle.

    Importance Approximately 8% of the human genome is of retroviral origin. While many of those viral genomes have become inactivated, some copies of the most recently endogenized human retrovirus, HERV-K, can encode individual functional proteins. Here we characterize the envelope protein (ENV) of this virus to define how it mediates infection of cells. We demonstrate that HERV-K ENV undergoes a proteolytic processing step and triggers membrane fusion in response to acidic pH nndash; a strategy common to many viral fusogens. Our data suggest that the infectious entry pathway mediated by this ENV requires endosomal acidification and the GTPase dynamin but does not require clathrin dependent uptake. In marked contrast to other betaretroviruses, HERV-K ENV imparts a broad species tropism in cultured cells. This work provides new insights into the entry pathway of an extinct human virus and provides a powerful tool to further probe the endocytic route by which HERV-K infects cells.

  • Codon deletions in the influenza A virus PA gene generate temperature-sensitive viruses [PublishAheadOfPrint]

  • The influenza virus RNA-dependent RNA polymerase which is composed of three subunits, PB1, PB2, and PA, catalyses genome replication and transcription within the cell nucleus. The PA linker (residues 197-256) can be altered by nucleotide substitutions to engineer temperature-sensitive (ts) and attenuated mutants that display a defect in the transport of the PA-PB1 complex to the nucleus at restrictive temperature. In this study, we investigated the ability of the PA linker to tolerate deletion mutations for further in vitro and in vivo characterization. Four single-codon deletion viable mutants were generated; all of them exhibited a ts-phenotype that was associated to a reduced efficiency of replication/transcription of a pseudoviral reporter RNA in a minireplicon assay. Using a fluorescent-tagged PB1, we observed that the deletion mutants did not efficiently recruit PB1 to reach the nucleus at restrictive temperature (39.5ddeg;C). Mice infections showed that the four mutants were attenuated and induced antibodies able to protect from a lethal homologous wild-type virus challenge. Serial in vitro passages of two deletion mutants at 39.5ddeg;C and 37ddeg;C did not allowed to restore a wt-phenotype among virus progeny. Thus, our results identify codons that can be deleted in the PA gene to engineer genetically stable ts-mutants that could be used to design novel attenuated vaccines.

    IMPORTANCE In order to generate genetically stable live influenza A vaccines, we constructed viruses with single-codon deletion in a discrete domain of the RNA-polymerase PA gene. The four rescued viruses exhibited a temperature-sensitive phenotype that we found associated to a defect of transport of the PA-PB1 dimer to the nucleus where viral replication occurs. These deletion ts-mutants were shown to be attenuated, able to produce antibodies in mice and protect from a lethal challenge. Assays to select revertants able to grow efficiently at restrictive temperature failed, showing that these deletion mutants are genetically more stable than conventional substitution mutants. These results are of interest for the design of influenza genetically stable live vaccines.

  • The Role of Mitochondrial Membrane Spherules in Flock House Virus Replication [PublishAheadOfPrint]

  • Viruses that generate double-stranded RNA (dsRNA) during replication must overcome host defense systems designed to detect this infection intermediate. All positive-sense RNA viruses studied to date modify host membranes to help facilitate the sequestration of dsRNA from host defenses and concentrate replication factors to enhance RNA production. Flock House virus (FHV) is an attractive model for the study of these processes since it is well characterized and infects Drosophila cells, which are known to have a highly effective RNA silencing system. During infection, FHV modifies the outer membrane of host mitochondria to form numerous membrane invaginations, called spherules, that are ~50 nm in diameter and known to be the site of viral RNA replication. While previous studies have outlined basic structural features of these invaginations, very little is known about the mechanism underlying their formation. Here we describe the optimization of an experimental system for the analysis of FHV host membrane modifications using crude mitochondrial preparations from infected Drosophila cells. These preparations can be programmed to synthesize both single- and double-stranded FHV RNA. The system was used to demonstrate that dsRNA is protected from nuclease digestion by virus-induced membrane invaginations and that spherules play an important role in stimulating RNA replication. Finally, we show that spherules generated during FHV infection appear to be dynamic as evidenced by their ability to form or disperse based on the presence or absence of RNA synthesis.

    IMPORTANCE It is well established that positive-sense RNA viruses induce significant membrane rearrangements in infected cells. However, the molecular mechanisms underlying these rearrangements, particularly membrane invagination and spherule formation, remain essentially unknown. How the formation of spherules enhances viral RNA synthesis is also not understood, although it is assumed to be partly a result of evading host defense pathways. To help interrogate some of these questions we optimized a cell-free replication system consisting of mitochondria isolated from Flock House virus-infected Drosophila cells for use in biochemical and structural studies. Our data suggest that spherules generated during Flock House virus replication are dynamic, protect double-stranded RNA and enhance RNA replication in general. Cryo-electron microscopy suggests that the samples are amenable to detailed structural analyses of spherules engaged in RNA synthesis. This system thus provides a foundation for understanding the molecular mechanisms underlying spherule formation, maintenance and function during positive-sense viral RNA replication.

  • Mutations in a highly conserved motif of nsp1{beta} protein attenuate the innate immune suppression function of porcine reproductive and respiratory syndrome virus (PRRSV) [PublishAheadOfPrint]

  • PRRSV nonstructural protein 1bbeta; (nsp1bbeta;) is a multifunctional viral protein, which involves in suppressing innate immune response and activating a unique -2/-1 programmed ribosomal frameshifting (PRF) signal for the expression of frameshifting products. In this study, site-directed mutagenesis analysis showed that R128A or R129A mutation introduced in a highly conserved motif (123GKYLQRRLQ131) reduced the ability of nsp1bbeta; to suppress IFN-bbeta; activation and also impaired nsp1bbeta;'s function as PRF transactivator. Three recombinant viruses, vR128A, vR129A and vRR129AA, carrying single or double mutations in the GKYLQRRLQ motif were characterized. In comparison to the wild type (WT) virus, vR128A and vR129A showed slightly reduced growth ability, while vRR129AA mutant had significantly reduced growth ability in infected cells. Consistent with the attenuated growth phenotype in vitro, the pigs infected with nsp1bbeta; mutants had lower level of viremia than that of WT virus-infected pigs. Comparing to WT virus in infected cells, all of the three mutated viruses stimulated higher level of IFN-aalpha; expression and exhibited reduced ability in suppressing mRNA expression of selected ISGs. In pigs infected with nsp1bbeta; mutants, IFN-aalpha; production was increased in the lungs during early time points of post-infection, which was correlated with an increased innate NK cell function. Furthermore, augmented innate response was consistent with increased production of IFN- in those mutated viruses-infected pigs. These data demonstrate that R128 and R129 residues are critical for nsp1bbeta; function, and modifying these key residues in the GKYLQRRLQ motif attenuates virus growth ability and improve the innate and adaptive immune responses in infected animals.

    IMPORTANCE PRRSV infection induces poor anti-viral innate IFN and cytokine responses, which results in weak adaptive immunity. One of the strategies in next generation vaccine construction is to manipulate viral proteins/genetic elements involved in antagonizing host immune response. The PRRSV nsp1bbeta; was identified to be a strong innate immune antagonist. In this study, two basic amino acids, R128 and R129, in a highly conserved GKYLQRRLQ motif were determined to be critical for nsp1bbeta; function. Mutations introduced into these two residues attenuated virus growth and improved the innate and adaptive immune responses in infected animals. Technologies developed in this study could be broadly applied to current commercial PRRSV MLV vaccines and other candidate vaccines.

  • Human cytomegalovirus encoded cmvIL-10 amplifies its immunomodulatory potential by upregulating human IL-10 in monocytes [PublishAheadOfPrint]

  • The human cytomegalovirus (HCMV) gene UL111A encodes cmvIL-10, a homolog of the potent immunomodulatory cytokine, human interleukin 10 (hIL-10). This viral homolog exhibits a range of immunomodulatory functions, including suppression of pro-inflammatory cytokine production and dendritic cell (DC) maturation, as well as inhibition of MHC class I and class II. Here, we present data showing that cmvIL-10 upregulates hIL-10 and identify CD14+ monocytes and monocyte derived macrophages and DCs as major sources of hIL-10 secretion in response to cmvIL-10. Monocyte activation was not a pre-requisite for cmvIL-10 mediated upregulation of hIL-10, which was dose dependent and controlled at the transcriptional level. Furthermore, cmvIL-10 upregulated expression of tumor progression locus 2 (TPL2), which is a regulator of the positive hIL-10 feedback loop, whereas expression of a negative regulator of the hIL-10 feedback loop, dual-specificity phosphatase 1 (DUSP1), remained unchanged. Engagement of the hIL-10 receptor (hIL-10R) by cmvIL-10 led to upregulation of heme oxygenase 1 (HO-1), an enzyme linked with suppression of inflammatory responses, and this upregulation was required for cmvIL-10 mediated upregulation of hIL-10. We also demonstrate an important role for both PI3K and STAT3 in the upregulation of HO-1 and hIL-10 by cmvIL-10. In addition to upregulating hIL-10, cmvIL-10 could exert a direct immunomodulatory function, as demonstrated by its capacity to upregulate expression of cell surface CD163 when hIL-10 was neutralized. This study identifies a mechanistic basis for cmvIL-10 function, including the capacity of this viral cytokine to potentially amplify its immunosuppressive impact by upregulating hIL-10 expression.

    IMPORTANCE Human cytomegalovirus (HCMV) is a large, double stranded DNA virus that causes significant human disease, particularly in the congenital setting and in solid organ and hematopoietic stem cell transplant patients. A prominent feature of HCMV is the wide range of viral gene products that it encodes which function to modulate host defenses. One of these is cmvIL-10, which is a homolog of the potent immunomodulatory cytokine human interleukin 10 (hIL-10). In this study we report that in addition to exerting a direct biological impact, cmvIL-10 upregulates the expression of hIL-10 by primary blood derived monocytes, and that it does so by modulating existing cellular pathways. This capacity of cmvIL-10 to upregulate hIL-10 represents a mechanism by which HCMV may amplify its immunomodulatory impact during infection.

  • Amino acid substitutions that affect receptor binding and stability of the hemagglutinin of influenza A/H7N9 virus [PublishAheadOfPrint]

  • Receptor-binding preference and stability of hemagglutinin have been implied as crucial determinants of airborne transmission of influenza viruses. Here, amino acid substitutions previously identified to affect these traits were tested in the context of an A/H7N9 virus. Some combinations of substitutions, most notably G219S and K58I, resulted in relatively high affinity for aalpha;2,6-linked sialic acid receptor and acid and temperature stability. Thus, the hemagglutinin of the A/H7N9 virus may adopt traits associated with airborne transmission.

  • During Infection Theiler's Virions are Cleaved by Caspases and Disassembled into Pentamers [PublishAheadOfPrint]

  • Infected macrophages in spinal cords of mice persistently infected with Theiler's murine encephalomyelitis virus (TMEV) undergo apoptosis, resulting in restricted virus yields as do infected macrophages in culture. Apoptosis of murine macrophages in culture occurs via the intrinsic pathway later in infection (ggt;10 h post-infection, pi) after maximal virus titers (150 to 200 pfu/cell) have been reached, with loss of most infectious virus (llt;5 pfu/cell) by 20 to 24 h pi. Here, we show that BeAn virus RNA replication, translation, polyprotein processing into final protein products and assembly of protomers and pentamers in infected M1-D macrophages did not differ from those processes in TMEV-infected BHK-21 cells, which undergo necroptosis. However, the initial difference from BHK-21 cell infection was seen at 10 to 12 h pi where virions from the 160S peak in sucrose gradients had incompletely processed VP0 (compared to that in infected BHK-21 cells). Thereafter, there was a gradual loss of the 160S virion peak in sucrose gradients with replacement by a 216S peak that was observed to contain pentamers among lipid debris in negatively stained grids by electron microscopy. After infection or incubation of purified virions with activated caspase-3 in vitro, 13- and 17-kDa capsid peptide fragments were observed and were predicted by algorithms of cleavage sites within proteins by cysteine-dependent aspartate-directed proteases. These findings suggest that caspase cleavage of sites in exposed capsid loops of assembled virions occurs contemporaneously with the onset and progression of apoptosis later in the infection.

    IMPORTANCE Theiler's murine encephalomyelitis virus (TMEV) infection in mice results in establishment of virus persistence in the central nervous system and chronic inflammatory demyelinating disease, providing an experimental animal model for multiple sclerosis. Virus persistence primarily takes place in macrophages recruited into the spinal cord that undergo apoptosis and in turn may facilitate viral spread via infected apoptotic blebs. Infection of murine macrophages in culture results in restricted virus yields late in infection. Here it is shown that the early steps of the virus life cycle in infected macrophages in vitro do not differ from these processes in TMEV-infected BHK-21 cells, which undergo necroptosis. However, the findings late in infection suggest that caspases cleave sites in exposed capsid loops and possibly internal sites of assembled virions occurring contemporaneously with onset and progression of apoptosis. Mechanistically, this would explain the dramatic loss in virus yields during TMEV-induced apoptosis and attenuate the virus enabling persistence.

  • DDX3 interacts with influenza A NS1 and NP proteins and exerts antiviral function through regulation of stress granule formation [PublishAheadOfPrint]

  • DDX3 belongs to the DEAD box RNA helicase family and is a multifunctional protein affecting the life cycle of a variety of viruses. However, its role in influenza virus infection is unknown. In this study, we explored the potential role of DDX3 in influenza virus life cycle and discovered that DDX3 is an antiviral protein. Since many host proteins affect virus life cycle by interacting with certain components of the viral machinery, we first verified whether DDX3 has any viral interaction partners. Immunoprecipitation studies revealed NS1 and NP as direct interaction partners of DDX3. Stress granules (SGs) are known to be antiviral and do form in influenza infected cells expressing defective NS1 protein. Additionally, a recent study showed that DDX3 is an important SG nucleating factor. We thus explored whether DDX3 plays a role in influenza virus infection through regulation of SGs. Our results showed that SGs were formed in infected cells upon infection with a mutant influenza virus lacking functional NS1 (del NS1) protein and DDX3 co-localized with NP in SGs. We further identified that DDX3 helicase domain did not interact with NS1 and NP; however, it was essential for DDX3 localization in virus induced SGs. Knockdown of DDX3 resulted in impaired SG formation and led to increased virus titers. Taken together, our results identified DDX3 as an antiviral protein with a role in virus induced SG formation.

    Importance DDX3 is a multifunctional RNA helicase and has been reported to be involved in regulating various virus life cycles. However, its function during influenza A virus infection remains unknown. In this study, we demonstrated that DDX3 is capable of interacting with influenza NS1 and NP proteins; DDX3 and NP co-localize in the del NS1 virus induced SGs. Furthermore, knockdown of DDX3 impaired SG formation and led to a decreased virus titer. Thus, we provided evidence that DDX3 is an antiviral protein during influenza virus infection and its antiviral activity is through regulation of SG formation. Our findings provide knowledge about the function of DDX3 in influenza virus life cycle and information for future work on manipulating the SG pathway and its components to fight influenza infection.

  • The MHC class II transactivator CIITA inhibits the persistent activation of NF-kB by Human T cell Lymphotropic Virus type-1 Tax-1 oncoprotein [PublishAheadOfPrint]

  • Human T cell Lymphotropic Virus type-1 (HTLV-1) Tax-1, a key protein of HTLV-1-induced T-cell transformation, deregulates diverse cell-signalling pathways. Among them, the NF-kB pathway is constitutively activated by Tax-1, which binds to NF-kB proteins and activates the IkB-kinase (IKK). Upon phosphorylation-dependent IkB degradation, NF-kB migrates into the nucleus mediating Tax-1-stimulated gene expression. We show that the transcriptional regulator of Major Histocompatibility Complex Class II genes CIITA, endogenously or ectopically expressed in different cells, inhibits the activation of the canonical NF-kB pathway by Tax-1 and map the region mediating this effect. CIITA affects the subcellular localization of Tax-1, which is mostly retained in the cytoplasm and this correlates with an impaired migration of RelA into the nucleus. Cytoplasmic and nuclear CIITA mutants reveal that CIITA exploits different strategies to suppress Tax-1-mediated NF-kB activation in both subcellular compartments. CIITA interacts with Tax-1 without preventing Tax-1 binding to both IKK and RelA. Nevertheless, CIITA affects Tax-1-induced IKK activity causing the retention of the inactive p50/RelA/IkB complex in the cytoplasm. Nuclear CIITA associates with Tax-1/RelA in nuclear bodies blocking Tax-1-dependent activation of NF-kB-responsive genes. Thus, CIITA inhibits cytoplasmic and nuclear steps of Tax-1-mediated NF-kB activation. These results, together with our previous finding that CIITA acts as a restriction factor inhibiting Tax-1-promoted HTLV-1 gene expression and replication, indicate that CIITA is a versatile molecule that might also counteract Tax-1 transforming activity. Unveiling the molecular basis of CIITA-mediated inhibition of Tax-1 functions may be important in defining new strategies to control HTLV-1 spreading and oncogenic potential.

    IMPORTANCE HTLV-1 is the causative agent of the human adult T cell leukaemia-lymphoma (ATLL). The viral transactivator Tax-1 plays a central role in the onset of ATLL mostly by deregulating the NF-kB pathway. We demonstrate that CIITA, a key regulator of adaptive immunity, suppresses Tax-1-dependent activation of NF-kB by acting at several levels: it retains most of Tax-1 and RelA in the cytoplasm and it inhibits their residual functional activity in the nucleus. Importantly, this inhibition occurs in cells that are target of HTLV-1 infection. These findings are of interest in the field of virology because they expand the current knowledge on the functional relationship between viral products and cellular interactors, and provide the basis for better understanding the molecular countermeasures adopted by the host cell to antagonize HTLV-1 spreading and transforming properties. Within this frame, our results may contribute to establish novel strategies against HTLV-1 infection and virus-dependent oncogenic transformation.

  • Extensive positive selection drives the evolution of non-structural proteins in lineage C Betacoronaviruses [PublishAheadOfPrint]

  • Middle East Respiratory Syndrome-related Coronavirus (MERS-CoV) spread to humans via the zoonotic transmission from camels. MERS-CoV belongs to the lineage C of Betacoronaviruses (betaCoVs), which also includes viruses isolated from bats and hedgehogs. A large portion of the betaCoV genome consists of two open reading frames (ORF1a and ORF1b) that are translated into polyproteins. These are cleaved by viral proteases to generate 16 non-structural proteins (nsp1-16) which compose the viral replication-transcription complex. We investigated the evolution of ORF1a and ORF1b in lineage C betaCoVs. Results indicated widespread positive selection, acting mostly on ORF1a. The proportion of positively selected sites in ORF1a was much higher than that previously reported for the surface-exposed spike protein. Selected sites were unevenly distributed, with nsp3 representing the preferential target. Several pairs of co-evolving sites were also detected, possibly indicating epistatic interactions; most of these were located in nsp3. Adaptive evolution at nsp3 is ongoing in MERS-CoV strains and two selected sites (G720 and R911) were detected in the protease domain. Whereas position 720 is variable in camel-derived viruses, suggesting that the selective event does not represent a specific adaptation to humans, the R911C substitution was only observed in human-derived MERS-CoV isolates, including the viral strain responsible for the recent South Korean outbreak. It will be extremely important to assess whether these changes affect host range or other viral phenotypes. More generally, data herein indicate that CoV nsp3 represents a major selection target and nsp3 sequencing should be envisaged in monitoring programs and field surveys.

    Importance Both SARS-CoV and MERS-CoV originated in bats and spread to humans via an intermediate host. This clearly highlights the potential for coronavirus host shifting and the relevance of understanding the molecular events underling the adaptation to new host species. We investigated the evolution of ORF1a and ORF1b in lineage C betaCoVs and in 87 sequenced MERS-CoV isolates. Results indicated widespread positive selection, stronger in ORF1a than in ORF1b. Several selected sites were found to be located in functionally relevant protein regions and some of them corresponded to functional mutations in other coronaviruses. The proportion of selected sites we identified in ORF1a is much higher than that for the surface-exposed spike protein. This observation suggests that adaptive evolution in ORF1a might contribute to host shifts or immune evasion. Data herein also indicate that genetic diversity at non-structural proteins should be taken into account when antiviral compounds are developed.

  • Glycoprotein-specific antibodies produced by DNA vaccination protect guinea pigs from lethal Argentine and Venezuelan hemorrhagic fever [PublishAheadOfPrint]

  • Several members of the Arenavirdae can cause acute febrile diseases in humans often resulting in lethality. Convalescent human plasma is an effective treatment in humans infected with arenaviruses, particularly species found in South America. Despite this, little work has focused on developing potent and defined immunotherapeutics against arenaviruses. Here, we produced arenavirus neutralizing antibodies by DNA vaccination of rabbits with plasmids encoding the full-length glycoprotein precursor of Juniiacute;n virus (JUNV), Machupo virus (MACV) and Guanarito virus (GTOV). Geometric mean neutralizing antibody titers, as measured by 50% plaque reduction neutralization test (PRNT50) exceeded 5000 against homologous viruses. Antisera against each targeted virus exhibited limited cross-species binding and, to a lesser extent, cross-neutralization. Anti-JUNV glycoprotein rabbit antiserum protected Hartley guinea pigs from lethal intraperitoneal infection with JUNV strain Romero when administered 2 days after challenge and provided some protection (~30%) when administered 4 days after challenge. Treatment starting on day 6 did not protect animals. We further formulated an IgG antibody cocktail by combining anti-JUNV, MACV and GTOV antibodies produced in DNA-vaccinated rabbits. This cocktail protected 100% of guinea pigs against JUNV and GTOV lethal disease. We then expanded on this cocktail approach by simultaneously vaccinating rabbits with a combination of plasmids encoding glycoprotein genes from JUNV, MACV, GTOV and Sabia virus (SABV). Sera collected from vaccinated rabbits with the combination vaccine neutralized all four targets. These findings support the concept of using a DNA vaccine approach to generate a potent pan-arenavirus immunotherapeutic.

    IMPORTANCE Arenaviruses are an important family of emerging viruses. In infected humans, convalescent plasma containing neutralizing antibodies can mitigate disease severity caused by arenaviruses, particularly species found in South America. Because of variation in potency of the human-derived product, limited availability and safety concerns this treatment option has been essentially abandoned. Accordingly, despite being an effective post-infection treatment option research on novel approaches to produce potent polyclonal antibody-based therapies have been deficient. Here, we show that DNA-based vaccine technology can be used to make potently neutralizing antibodies in rabbits that exclusively target the glycoproteins of several human pathogenic areanviruses found in South America, including JUNV, MACV, GTOV and SABV. These antibodies protected guinea pigs from lethal disease when given post-virus challenge. We also generated a purified antibody cocktail with antibodies targeting three arenavirus and demonstrated protective efficacy against all three targets. Our findings demonstrate that use of the DNA vaccine technology could be used to produce candidate anti-arenavirus neutralizing antibody-based products.

  • Venezuelan Equine Encephalitis Virus Induces Apoptosis through the Unfolded Protein Response Activation of EGR1 [PublishAheadOfPrint]

  • Venezuelan equine encephalitis virus (VEEV) is a previously weaponized arthropod-borne virus responsible for causing acute and fatal encephalitis in animal and human hosts. The increased circulation and spread in the Americas of VEEV, and other encephalitic arboviruses such as Eastern equine encephalitis virus and West Nile virus, underscores the need for research aimed at characterizing the pathogenesis of viral encephalomyelitis for the development of novel medical countermeasures. The host-pathogen dynamics of VEEV-TrD infected human astrocytoma U87MG cells were determined by carrying out RNA sequencing (RNA-Seq) of poly(A) + mRNAs. To identify critical alterations in the host transcriptome that take place following VEEV infection, samples were collected at 4, 8, and 16 hours post-infection and RNA-Seq data acquired using the Ion Torrent PGM platform. Differential expression of interferon responsive genes, stress response factors, and components of the unfolded protein response (UPR) were observed. The PERK arm of the UPR was activated as both ATF4 and CHOP (DDIT3), critical regulators of the pathway, were altered after infection. The transcription factor Early Growth Response 1 (EGR1) was induced in a PERK dependent manner. EGR1 -/- MEFs demonstrated lower susceptibility to VEEV induced cell death as compared to isogenic wild type MEFs, indicating that EGR1 modulates pro-apoptotic pathways following VEEV infection. The influence of EGR1 is of great importance as neuronal damage can lead to long-term sequelae in individuals who have survived VEEV infection.

    Importance Alphaviruses represent a group of clinically relevant viruses transmitted by mosquitoes to humans. In severe cases, viral spread targets neuronal tissue, resulting in significant and life threatening inflammation dependent on a combination of viral-host interactions. Currently there are no therapeutics for encephalitic alphaviruses due to an incomplete understanding of their molecular pathogenesis. Venezuelan equine encephalitis virus (VEEV) is an alphaviruses prevalent in the Americas capable of infecting horses and humans. Here we utilized next generation RNA sequencing to identify differential alterations in VEEV infected astrocytes. Our results indicated that transcripts associated with interferon and the unfolded protein response pathways were altered following infection, and demonstrated that Early Growth Response 1 (EGR1) contributed to VEEV induced cell death.

  • Hepatocyte factor JMJD5 regulates HBV replication through interaction with HBx [PublishAheadOfPrint]

  • Hepatitis B virus (HBV) is a causative agent for chronic liver diseases such as hepatitis, cirrhosis and hepatocellular carcinoma (HCC). HBx protein encoded by the HBV genome plays crucial roles not only in pathogenesis but also in replication of HBV. Although HBx has been shown to bind to a number of host proteins, the molecular mechanisms by which HBx regulates HBV replication are largely unknown. In this study, we identified jumonji C domain-containing 5 (JMJD5) as a novel binding partner of HBx interacting in the cytoplasm. DNA microarray analysis revealed that JMJD5-knockout (JMJD5KO) Huh7 cells exhibited a significant reduction in the expression of transcriptional factors involved in hepatocyte differentiation, such as HNF4A, CEBPA and FOXA3. We found that hydroxylase activity of JMJD5 participates in the regulation of these transcriptional factors. Moreover, JMJD5KO Huh7 cells exhibited a severe reduction in HBV replication, and complementation of HBx expression failed to rescue replication of a mutant HBV deficient in HBx, suggesting that JMJD5 participates in HBV replication through an interaction with HBx. We also found that substitution of Gly135 to Glu in JMJD5 abrogates binding with HBx and replication of HBV. Moreover, the hydroxylase activity of JMJD5 was crucial for HBV replication. Collectively, these results suggest that direct interaction of JMJD5 with HBx facilitates HBV replication through the hydroxylase activity of JMJD5.

    Importance HBx protein encoded by hepatitis B virus (HBV) plays important roles in pathogenesis and replication of HBV. We identified jumonji-C domain containing-5 (JMJD5) as a novel binding partner to HBx. JMJD5 was shown to regulate several transcriptional factors to maintain hepatocyte function. Although HBx had been shown to support HBV replication, deficiency of JMJD5 abolished contribution of HBx in HBV replication, suggesting that HBx mediated HBV replication is largely dependent on JMJD5. We showed that hydroxylase activity of JMJD5 in the C-terminus region is crucial for expression of HNF4A and replication of HBV. Furthermore, a mutant JMJD5 substituted Gly135 to Glu failed to interact with HBx and to rescue the replication of HBV in JMJD5-knockout cells. Taken together, our data suggest that interaction of JMJD5 with HBx facilitates HBV replication through the hydroxylase activity of JMJD5.

  • The role of viral RNA and co-opted cellular ESCRT-I and ESCRT-III factors in formation of tombusvirus spherules harboring the tombusvirus replicase [PublishAheadOfPrint]

  • Plus-stranded RNA viruses induce membrane deformations in infected cells in order to build viral replication complexes (VRCs). Tomato bushy stunt virus (TBSV) co-opts cellular ESCRT (endosomal sorting complexes required for transport) proteins to induce the formation of vesicle (spherule-)-like structures in the peroxisomal membrane with tight openings towards the cytosol. In this paper, we show using double-deletion vps23bro1 mutant of yeast (Saccharomyces cerevisiae) that the Vps23p ESCRT-I protein (Tsg101 in mammals) and Bro1p (ALIX) ESCRT-associated protein, both of which bind to the viral p33 replication protein, play partially complementary roles in TBSV replication in cells and in cell-free extracts. Dual-expression of dominant-negative versions of Arabidopsis homologs of Vps23p and Bro1p inhibited tombusvirus replication to greater extent than individual expression in Nicotiana benthamiana leaves. We also demonstrate the critical role of Snf7p (CHMP4), Vps20p and Vps24p ESCRT-III proteins in tombusvirus replication in yeast and in vitro. Electron microscopic imaging of vps23 yeast reveals the lack of tombusvirus-induced spherule-like structures, while crescent-like structures are formed in ESCRT-III deletion yeasts replicating TBSV RNA. In addition, we also show that the length of the viral RNA affects the sizes of spherules formed in N. benthamiana cells. The 4.8 kb genomic RNA is needed for the formation of spherules 66 nm in diameter, while spherules formed during the replication of the ~600 nt long defective-interfering RNA in the presence of p33 and p92 replication proteins are 42 nm. We propose that the viral RNA serves as a "measuring string" during VRC assembly and spherule formation.

    IMPORTANCE Plant positive strand RNA viruses, similar to animal positive strand RNA viruses, replicate in membrane-bound viral replicase complexes in the cytoplasm of infected cells. Identification of cellular and viral factors affecting the formation of the membrane-bound viral replication complex is a major frontier in current virology research. In this paper, the authors have dissected the functions of co-opted cellular ESCRT-I (endosomal sorting complexes required for transport-I) and ESCRT-III proteins and the viral RNA in tombusvirus replicase complex formation using in vitro, yeast and plant-based approaches. Electron microscopic imaging has revealed the lack of tombusvirus-induced spherule-like structures in ESCRT-I or ESCRT-III deletion yeasts replicating TBSV RNA, demonstrating the requirement of these co-opted cellular factors in tombusvirus replicase formation. The work could be of broad interest in virology and beyond.

  • C-terminal DxD-containing Sequences within Paramyxovirus Nucleocapsid Proteins Determine Matrix Protein Compatibility, and Can Direct Foreign Proteins into Budding Particles [PublishAheadOfPrint]

  • Paramyxovirus particles are formed by a budding process coordinated by viral matrix (M) proteins. M proteins coalesce at sites underlying infected cell membranes and induce other viral components, including viral glycoproteins and viral ribonucleoprotein complexes (vRNPs), to assemble at these locations from which particles will bud. M proteins interact with the nucleocapsid (NP or N) components of vRNPs, and these interactions enable production of infectious, genome-containing virions. For the paramyxoviruses parainfluenza virus 5 (PIV5) and mumps virus, M-NP interaction also contributes to efficient production of virus-like particles (VLPs) in transfected cells. A DLD sequence near the C-terminal end of PIV5 NP protein was previously found to be necessary for M-NP interaction and efficient VLP production. Here, we demonstrate that 15 residue-long, DLD-containing sequences derived from either the PIV5 or Nipah virus nucleocapsid protein C-terminal ends are sufficient to direct packaging of a foreign protein, Renilla luciferase, into budding VLPs. Mumps virus NP protein harbors DWD in place of the DLD sequence found in PIV5 NP protein, and consequently, PIV5 NP protein is incompatible with mumps virus M protein. A single amino acid change converting DLD to DWD within PIV5 NP protein induced compatibility between these proteins and allowed efficient production of mumps VLPs. Our data suggests a model in which paramyxoviruses share an overall common strategy for directing M-NP interactions, but with important variations contained within DLD-like sequences that play key roles in defining M/NP protein compatibilities.

    IMPORTANCE Paramyxoviruses are responsible for a wide range of diseases that affect both humans and animals. Paramyxovirus pathogens include measles virus, mumps virus, human respiratory syncytial virus, and the zoonotic paramyxoviruses Nipah virus and Hendra virus. Infectivity of paramyxovirus particles depends on matrix-nucleocapsid protein interactions which enable efficient packaging of encapsidated viral RNA genomes into budding virions. In this study, we have defined regions near the C-terminal ends of paramyxovirus nucleocapsid proteins that are important for matrix protein interaction and that are sufficient to direct a foreign protein into budding particles. These results advance our basic understanding of paramyxovirus genome packaging interactions, and also have implications for the potential use of virus-like particles as protein delivery tools.

  • Permissive sense and antisense transcription from the 5 and 3 long terminal repeats of Human T-cell Leukemia Virus type 1 [PublishAheadOfPrint]

  • Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus, and as such, its genome becomes chromosomally integrated following infection. The resulting provirus contains identical 5rrsquo; and 3rrsquo; peripheral long terminal repeats (LTRs) containing bidirectional promoters. Antisense transcription from the 3rrsquo; LTR regulates expression of a single gene, hbz, while sense transcription from the 5rrsquo; LTR controls expression of all other viral genes including tax. Both the HBZ and Tax proteins are implicated in the development of Adult T-cell Leukemia (ATL), a T-cell malignancy caused by HTLV-1 infection. However, these proteins appear to harbor opposing molecular functions, indicating that they may act independently and at different time points prior to leukemogenesis. Here we used bidirectional reporter constructs to test whether transcriptional interference serves as a mechanism that inhibits simultaneous expression of Tax and HBZ. We found that sense transcription did not interfere with antisense transcription from the 3rrsquo; LTR and vice versa, even with strong transcription emanating from the opposing direction. Therefore, bidirectional transcription across the provirus might not restrict hbz or tax expression. Single-cell analyses revealed that antisense transcription predominates in the absence of Tax, which trans-activates viral sense transcription. Interestingly, a population of Tax-expressing cells exhibited antisense but not activated sense transcription. Consistent with the ability of Tax to induce cell cycle arrest, this population was arrested in G0/G1 phase. These results imply that cell-cycle arrest inhibits Tax-mediated activation of sense transcription without affecting antisense transcription, which may be important for long-term viral latency.

    IMPORTANCE The chromosomally integrated form of the retrovirus, Human T-cell Leukemia Virus type 1 (HTLV-1), contains identical DNA sequences, known as a long terminal repeats (LTRs), at its 5rrsquo; and 3rrsquo; ends. The LTRs modulate transcription in both forward (sense) and reverse (antisense) directions. We found that sense transcription from the 5rrsquo; LTR does not interfere with antisense transcription from the 3rrsquo; LTR, allowing viral genes encoded on opposite DNA strands to be simultaneously transcribed. Two such genes are tax and hbz, and while they are thought to function at different times during the course of infection to promote leukemogenesis of infected T-cells, our results indicate that they can be simultaneously transcribed. We also found that the ability of Tax to induce cell cycle arrest inhibits its fundamental function of activating viral sense transcription, but does not affect antisense transcription. This regulatory mechanism may be important for long-term HTLV-1 infection.

  • Two populations of viral minichromosomes are present in a geminivirus-infected plant showing symptom remission (recovery). [PublishAheadOfPrint]

  • Geminiviruses are important plant pathogens characterized by circular, ssDNA genomes. However, in the nuclei of infected cells, viral dsDNA associates with host histones to form a minichromosome. In phloem-limited geminiviruses, the characterization of viral minichromosomes is hindered by the low concentration of recovered complexes due to the small number of infected cells. Nevertheless, geminiviruses are both inducers and targets of the host PTGS and TGS machinery. We have previously characterized a "recovery" phenomenon observed in pepper plants infected with Pepper golden mosaic virus (PepGMV) that is associated with a reduction of viral DNA and RNA levels, the presence of virus-related siRNAs, and an increase in the levels of viral DNA methylation. Initial micrococcal nuclease-based assays pinpointed the presence of different viral chromatin complexes in symptomatic and recovered tissues. Using the pepper-PepGMV system, we developed a methodology to obtain a viral minichromosome-enriched fraction that does not disturb the basic chromatin structural integrity, as evaluated by the detection of core histones. Using this procedure, we have further characterized two populations of viral minichromosomes in PepGMV-infected plants. After further purification using sucrose gradient sedimentation, we also observed that minichromosomes isolated from symptomatic tissue showed a relaxed conformation (based on their sedimentation rate), are associated to a chromatin activation marker (H3K4me3) and present a low level of DNA methylation. The minichromosome population obtained from recovered tissue, on the other hand, sedimented as a compact structure, is associated to a chromatin repressive marker (H3K9me2) and presents a high level of DNA methylation.

    Importance Viral minichromosomes have been reported in several animal and plant models. However, in the case of geminiviruses, there has been some recent discussion about the importance of this structure and the significance of the epigenetic modifications that can undergo during the infective cycle. Major problems in this type of studies are the low concentration of these complexes in an infected plant, as well as the asynchronicity of infected cells along the process, therefore, the complexes isolated in a given moment usually represent a mixture of cells at different infection stages. The recovery process observed in PepGMV-infected plants and the isolation procedure described here provides two distinct populations of minichromosomes that will allow a more precise characterization of the modifications of viral DNA and its host proteins associated along the infective cycle. This structure could be also an interesting model to study several processes involving plant chromatin.

  • The P1N-PISPO Trans-frame Gene of Sweet Potato Feathery Mottle Potyvirus is Produced During Virus Infection and Functions as RNA Silencing Suppressor [PublishAheadOfPrint]

  • The positive-sense RNA genome of Sweet potato feathery mottle virus (SPFMV, genus Potyvirus, family Potyviridae) contains a large open reading frame (ORF) of 3494 codons translatable as a polyprotein, and two embedded shorter ORFs in the -1 frame: PISPO, of 230 codons, and PIPO, of 66 codons, located respectively in the P1 and P3 regions. PISPO is specific of some sweet potato-infecting potyviruses, while PIPO is present in all potyvirids. In SPFMV these two extra ORFs are preceded by conserved G2A6 motifs. We have shown recently that a polymerase slippage mechanism at these sites could produce transcripts bringing these ORFs in frame with the upstream polyprotein, thus leading to P1N-PISPO and P3N-PIPO products (B. Rodamilans, A. Valli, A. Mingot, D. San Leon, D.B. Baulcombe, J.J. Lopez-Moya, and J.A. Garcia, J Virol 89:6965-6967, 2015, doi: 10.1128/JVI.00337-15). Here, we demonstrate by liquid chromatography coupled to mass spectrometry that both P1 and P1N-PISPO are produced during viral infection and coexist in SPFMV-infected Ipomoea batatas plants. Interestingly, transient expression of SPFMV gene products co-agroinfiltrated with a reporter gene in Nicotiana benthamiana revealed that P1N-PISPO acts as a RNA silencing suppressor, a role normally associated with HCPro in other potyviruses. Moreover, mutation of WG/GW motifs present in P1N-PISPO abolished its silencing suppression activity, suggesting that the function might require interaction with Argonaute components of the silencing machinery, as it was shown for other viral suppressors. Altogether, our results reveal a further layer of complexity of the RNA silencing suppression activity within the Potyviridae family.

    Importance Gene products of potyviruses include P1, HCPro, P3, 6K1, CI, 6K2, VPg/NIaPro, NIb and CP, all derived from the proteolytic processing of a large polyprotein, and an additional P3N-PIPO product, with the PIPO segment encoded in a different frame within the P3 cistron. In Sweet potato feathery mottle virus (SPFMV), another out of frame element (PISPO) was predicted within the P1 region. We have shown recently that a polymerase slippage mechanism can generate the transcript variants with extra nucleotides that could be translated into P1N-PISPO and P3N-PIPO. Now, we demonstrate by mass spectromety analysis that P1N-PISPO is indeed produced in SPFMV infected plants, in addition to P1. Interestingly, while in other potyviruses the suppressor of RNA silencing is HCPro, we show here that P1N-PISPO exhibited this activity in SPFMV, revealing how the complexity of the gene content could contribute to supply this essential function in members of the Potyviridae family.

  • An evolutionary view of the arms race between protein kinase R and large DNA viruses. [PublishAheadOfPrint]

  • To establish productive infections, viruses must counteract numerous cellular defenses that are poised to recognize viruses as non-self and to activate anti-viral pathways. The opposing goals of host and viral factors lead to evolutionary arms races that can be illuminated by evolutionary and computational methods and tested in experimental models. Here we illustrate how this perspective has been contributing to our understanding of the interactions of the protein kinase R pathway with large DNA viruses.

  • Influenza virus and the chromatin: Role of CHD1 chromatin remodeler on virus life cycle [PublishAheadOfPrint]

  • Influenza A virus requires ongoing cellular transcription to carry out the cap-snatching process. Chromatin remodelers modify chromatin structure to produce an active or inactive conformation, which enables or prevents recruitment of transcriptional complexes to specific genes; viral transcription thus depends on chromatin dynamics. Influenza virus polymerase associates with chromatin components of the infected cell, such as RNA polymerase II (RNAP II) or the CHD6 chromatin remodeler. Here we show that other CHD family member, CHD1 protein, also interacts with the influenza virus polymerase complex. CHD1 recognizes the H3K4me3 histone modification, a hallmark of active chromatin. Downregulation of CHD1 causes a reduction in viral polymerase activity, viral RNA transcription, and production of infectious particles. Despite influenza virus dependence on cellular transcription, RNAP II is degraded when viral transcription is complete, and recombinant viruses unable to degrade RNAP II show decreased pathogenicity in the murine model. We describe the CHD1-RNAP II association, as well as the parallel degradation of both proteins along infection with viruses showing full or reduced induction of degradation. The H3K4me3 histone mark also decreased during influenza virus infection, whereas a histone mark of inactive chromatin, H3K27me3, remained unchanged. Our results indicate that CHD1 is a positive regulator of influenza virus multiplication and suggest a role for chromatin remodeling in the control of the influenza virus life cycle.

    IMPORTANCE Although influenza virus does not integrate into the genome of the infected cell, it needs continuous cellular transcription to synthesize viral mRNA. This mechanism implies functional association with host genome expression and thus depends on chromatin dynamics. Influenza virus polymerase associates with transcription-related factors such as the RNA polymerase II and with chromatin remodelers such as CHD6. We identified association of viral polymerase with another chromatin remodeler, the CHD1 protein, which positively modulated viral polymerase activity, viral RNA transcription and virus multiplication. Once viral transcription is complete, RNAP II is degraded in infected cells, probably as a virus-induced mechanism to reduce the antiviral response. CHD1 associated with RNAP II and paralleled its degradation during infection with viruses that induce full or reduced degradation. These findings suggest that RNAP II and CHD1 degradation cooperate to reduce the antiviral response.

  • Ecotropic Murine Leukemia Virus Infection of Glial Progenitors Interferes with Oligodendrocyte Differentiation: Implications for Neurovirulence [PublishAheadOfPrint]

  • Certain murine leukemia viruses (MLVs) are capable of inducing fatal progressive spongiform motor neuron disease in mice that is largely mediated by viral Env glycoprotein expression within CNS glia. While the etiologic mechanisms and the glial subtypes involved remain unresolved, NG2 glia infection was recently observed to correlate spatially and temporally with altered neuronal physiology and spongiogenesis. Since one role of NG2 cells is to serve as oligodendrocyte (OL) progenitor cells (OPCs), we examined herein whether their infection by neurovirulent (FrCasE) or non-neurovirulent (Fr57E) ecotropic MLVs influenced their viability and/or differentiation. Herein, we demonstrate that OPCs, but not OLs, were major CNS targets of both FrCasE and Fr57E. We also show that MLV infection of neural progenitor cells (NPCs) in culture, did not affect survival, proliferation or OPC progenitor marker expression, but suppressed certain glial differentiation markers. Assessment of glial differentiation in vivo using transplanted transgenic NPCs showed that while MLVs did not affect cellular engraftment or survival, they did inhibit OL differentiation, irrespective of MLV neurovirulence. In addition, in chimeric brains where FrCasE-NPC transplants caused neurodegeneration, the transplanted NPCs proliferated. These results suggest that MLV infection is not directly cytotoxic to OPCs, but rather acts to interfere with OL differentiation. Since both FrCasE and Fr57E viruses restrict OL differentiation, but only FrCasE induces overt neurodegeneration, restriction of OL maturation alone cannot account for neuropathogenesis. Instead neurodegeneration may involve a two-hit scenario where interference with OPC differentiation combined with glial Env-induced neuronal hyperexcitability precipitate disease.

    Importance A variety of human and animal retroviruses are capable of causing central nervous system (CNS) neurodegeneration manifest as motor and cognitive deficits. These retroviruses infect a variety of CNS cell types, however, the specific role each cell type plays in neuropathogenesis remains to be established. The NG2 glia, whose CNS functions are only now emerging, are a newly appreciated viral target in murine leukemia virus (MLV)-induced neurodegeneration. Since one role of NG2 glia is that of oligodendrocyte progenitor cell (OPC), we investigated here whether their infection by the neurovirulent MLV, FrCasE, contributed to neurodegeneration by affecting OPC viability and/or development. Our results show that both neurovirulent and non-neurovirulent MLVs interfere with oligodendrocyte differentiation. Thus, NG2 glial infection could contribute to neurodegeneration by preventing myelin formation and/or repair, and by suspending OPCs in a state of persistent susceptibility to excitotoxic insult mediated by neurovirulent virus affects on other glial subtypes.

  • Acidianus tailed spindle virus: a new archaeal large tailed spindle virus discovered by culture-independent methods [PublishAheadOfPrint]

  • The field of viral metagenomics has expanded our understanding of viral diversity from all three domains of life (Archaea, Bacteria and Eukarya). Traditionally, viral metagenomic studies provide information about viral gene content, but rarely provide knowledge about virion morphology and or cellular host identity. Here we describe a new virus, Acidianus tailed spindle virus (ATSV), initially identified by bioinformatic analysis of viral metagenomic datasets from a high temperature (80ddeg; C) acidic (pH 2) hot spring located in Yellowstone National Park, USA followed by more detailed characterization using only environmental samples without dependency on culturing. Characterization included identification of the large tailed spindle virion morphology, determination of the complete 70.8 kb circular ds DNA viral genome content, and identification of its cellular host. Annotation of the ATSV genome revealed a potential three domain gene product containing a N -terminal leucine-rich repeat domain, followed by a likely post-translation regulatory region consisting of a high serine and threonine content and a C terminus an ESCRT-III domain, suggesting interplay with the host ESCRT system. The host of ATSV, which is most closely related to Acidianus hospitalis, was determined by a combination of analysis of cellular CRISPR/Cas loci and dual viral and cellular fluorescent in-situ hybridization (viral FISH) analysis of environmental samples, and confirmed by culture-based infection studies. This work provides an expanded pathway for the discovery, isolation, and characterization of new viruses using culture-independent approaches, and provides a platform for predicting and confirming viral hosts.

    Importance Virus discovery and characterization has been traditionally accomplished using culture-based methods. While a valuable approach, it is limited by the availability of culturable hosts. In this research, we report a virus-centered approach to virus discovery and characterization, linking viral metagenomic sequences to a virus particle, its sequenced genome, and its host directly in environmental samples, without using culture-dependent methods. This approach provides a pathway for the discovery, isolation, and characterization of new viruses. While this study used an acidic hot spring environment to characterize a new archaeal virus, Acidianus tailed spindle virus (ATSV), the approach can be generally applied to any environment to expand knowledge of virus diversity in all three domains of life.

  • Conformational Epitope-Specific Broadly Neutralizing Plasma Antibodies Obtained from an HIV-1 Clade C Infected Elite Neutralizer Mediate Autologous Virus Escape through Mutations in V1 Loop [PublishAheadOfPrint]

  • Broadly neutralizing antibodies isolated from infected patients who are elite neutralizers have identified targets on HIV-1 envelope (Env) glycoprotein that are vulnerable to antibody neutralization; however, it is not known whether infection established by majority of the circulating clade C strains in Indian patients elicit neutralizing antibody responses against any of the known targets. In the present study, we examined the specificity of a broad and potent cross neutralizing plasma obtained from an Indian elite neutralizer infected with HIV-1 clade C. This plasma neutralized 53/57 (93%) HIV pseudoviruses prepared with Env from distinct HIV clades of different geographical origin. Mapping studies using gp120 core protein, single residue knockout mutants and chimeric viruses revealed that G37080 BCN plasma lacks specificities to the CD4 binding site, gp41 membrane proximal external region, N160, N332 glycans as well as R166 and K169 in V1-V3 region and are known predominant targets for BCN antibodies. Depletion of G37080 plasma with soluble trimeric BG505-SOSIP.664 Env (but neither with monomeric gp120 nor with clade C MPER peptides), resulted in significant reduction of virus neutralization, suggesting that G37080 BCN antibodies mainly target epitopes on cleaved trimeric Env. Further examination of autologous circulating Envs revealed association of mutation of residues in V1 loop that contributed in neutralization resistance. In summary, we report identification of plasma antibodies from a clade C infected elite neutralizer that mediates neutralization breadth via epitopes on trimeric gp120 not yet reported and confer autologous neutralization escape via mutation of residues in V1 loop.

    Importance A preventive vaccine to protect against HIV-1 is urgently needed. HIV-1 envelope glycoproteins are targets of neutralizing antibodies and represent a key component for immunogen design. Mapping of epitopes on viral envelopes vulnerable for immune evasion will aid in defining targets of vaccine immunogens. We identified novel conformational epitopes on viral envelope targeted by broadly cross neutralizing antibodies elicited in natural infection in an elite neutralizer infected with HIV-1 clade C. Our data extend our knowledge on neutralizing epitopes associated with virus escape and would potentially contribute in immunogen design and antibody based prophylactic therapy.

  • Influenza A viruses expressing intra- or inter-group chimeric hemagglutinins [PublishAheadOfPrint]

  • A panel of influenza A viruses expressing chimeric hemagglutinins (cHA) with intragroup or intergroup head/stalk combinations was generated. Viruses were characterized for growth kinetics and preservation of stalk epitopes. With few notable exceptions, cHA viruses behaved similar to wild type viruses and maintained stalk epitopes, which indicated their potential as vaccine candidates to induce stalk-specific antibodies.

  • TRIM5{alpha} degradation via autophagy is not required for retroviral restriction [PublishAheadOfPrint]

  • TRIM5aalpha; is an interferon-inducible retroviral restriction factor which prevents infection by inducing the abortive disassembly of capsid cores recognized by its C-terminal PRY/SPRY domain. The mechanism by which TRIM5aalpha; mediates the disassembly of viral cores is poorly understood. Previous studies have demonstrated that proteasome inhibitors abrogate the ability of TRIM5aalpha; to induce premature core disassembly and prevent reverse transcription; however, viral infection is still inhibited, indicating that the proteasome is partially involved in the restriction process. Alternatively, we and others have observed that TRIM5aalpha; associates with proteins involved in autophagic degradation pathways, and one recent study found that autophagic degradation is required for the restriction of retroviruses by TRIM5aalpha;. Here, we show that TRIM5aalpha; is basally degraded via autophagy in the absence of restriction-sensitive virus. We observe that autophagy markers LC3b and LAMP2A localize to a subset of TRIM5aalpha; cytoplasmic bodies, and inhibition of lysosomal degradation with Bafilomycin A1 increases this association. To test the requirement for macro-autophagy in restriction, we examined the ability of TRIM5aalpha; to restrict retroviral infection in cells depleted of autophagic mediators ATG5, Beclin1, and p62. In all cases, restriction of retroviruses by human TRIM5aalpha;, rhesus macaque TRIM5aalpha;, and owl monkey TRIM-Cyp remained potent in cells depleted of these autophagic effectors by siRNA knockdown or CRISPR/Cas9 genome editing. Collectively, these results are consistent with observations that the turnover of TRIM5aalpha; proteins is sensitive to autophagy inhibition; however the data presented here do not support observations that the inhibition of autophagy abrogates retroviral restriction by TRIM5 proteins.

    Importance Restriction factors are a class of proteins that inhibit viral replication. Following fusion of a retrovirus with a host cell membrane, the retroviral capsid is released into the cytoplasm of the target cell. TRIM5aalpha; inhibits retroviral infection by promoting the abortive disassembly of incoming retroviral capsid cores; as a result, the retroviral genome is unable to traffic to the nucleus, and the viral life cycle is extinguished. In the process of restriction, TRIM5aalpha; itself is degraded by the proteasome. However, in the present study, we have shown that in the absence of a restriction-sensitive virus, TRIM5aalpha; is degraded by both proteasomal and autophagic degradation pathways. Notably, we observed that restriction of retroviruses by TRIM5aalpha; does not require autophagic machinery. These data indicate that the effector functions of TRIM5aalpha; can be separated from its degradation and may have further implications for understanding the mechanisms of other TRIM family members.

  • Five of five VHHs neutralizing poliovirus bind the receptor-binding site [PublishAheadOfPrint]

  • Nanobodiesrreg;, or VHHs, that recognize poliovirus type 1 have previously been selected and characterized as candidates for antiviral agents or reagents for standardization of vaccine quality control. In this study, we present high-resolution cryo-electron microscopy reconstructions of poliovirus with five neutralizing VHHs. All VHHs bind the capsid in the canyon at sites that extensively overlap the poliovirus receptor binding site. In contrast, the interaction involves a unique (and surprisingly extensive) surface for each of the five VHHs. Five regions of the capsid were found to participate in binding with all five the VHHs. Four of these five regions are known to alter during the expansion of the capsid associated with viral entry. Interestingly, binding of one of the VHHs, PVSS21E, resulted in significant changes of the capsid structure and thus seems to trap the virus in an early stage of expansion.

    Importance We describe the cryo-EM structures of complexes of five neutralizing VHHs with the Mahoney strain of type 1 poliovirus at resolutions ranging from 3.8-6.3AAring;. All five VHHs bind deep in the virus canyon at similar sites that overlap extensively with the binding site for the receptor (CD155). The binding surfaces on the VHHs are surprisingly extensive, but despite the similarity of the binding surface on the virus, the binding surface on the VHHs is unique for each VHH. In four of the five complexes the virus remains essentially unchanged, but for the fifth there are significant changes reminiscent of, but smaller in magnitude than, changes associated with cell entry, suggesting that this VHH has trapped the virus in a previously undescribed early intermediate state. The neutralizing mechanisms of the VHHs and their potential use as quality control agents for the end game of poliovirus eradication are discussed.

  • Trichoplusia ni kinesin-1 associates with AcMNPV nucleocapsid proteins and is required for the production of budded virus [PublishAheadOfPrint]

  • The mechanism by which nucleocapsids of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) egress from the nucleus to the plasma membrane leading to the formation of budded virus (BV) is not known. AC141 is a nucleocapsid associated protein required for BV egress and has been previously shown to be associated with bbeta;-tubulin. In addition, AC141 and VP39 were previously shown by FRET-FLIM to interact directly with Drosophila melanogaster kinesin-1 light chain (KLC) tetratricopeptide repeat (TPR) domain. These results suggested that microtubule transport systems may be involved in baculovirus nucleocapsid egress and BV formation. In this study we investigated the role of lepidopteran microtubule transport using co-immunoprecipitation, co-localization, yeast 2-hybrid and siRNA analyses. We show that nucleocapsid AC141 associates with the lepidopteran Trichoplusia ni kinesin-1 KLC and heavy chain (KHC) by co-immunoprecipitation and co-localization. Kinesin-1, AC141 and microtubules co-localized predominately at the plasma membrane. In addition, the nucleocapsid proteins VP39, FP25, and BV/ODV-C42 were also co-immunoprecipitated with T. ni KLC. Direct analysis of the role of T. ni kinesin-1 by down regulation of KLC by siRNA resulted in a significant decrease in BV production. Nucleocapsids labeled with VP39-3xmCherry also co-localized with microtubules. Yeast 2-hybrid analysis showed no evidence of direct interaction between kinesin-1 and AC141 or VP39 suggesting either other nucleocapsid proteins or adaptor proteins may be required. These results further support the conclusion that microtubule transport is required for AcMNPV BV formation.

    IMPORTANCE In two key processes of the replication cycle of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV), nucleocapsids are transported through the cell. These include: a) entry of budded virus (BV) into the host cell and b) egress and budding of newly produced nucleocapsids from the plasma membrane. Prior studies have shown that entry of nucleocapsids involves the polymerization of actin to propel nucleocapsids to nuclear pores and entry into the nucleus. For spread of infection, progeny viruses must rapidly exit the infected cells but the mechanism by which AcMNPV nucleocapsids traverse the cytoplasm is unknown. In this study we examined whether nucleocapsids interact with lepidopteran kinesin-1 motor molecules and are potentially carried as cargo on microtubules to the plasma membrane in AcMNPV infected cells. This study indicates that microtubule transport is utilized for the production of budded virus.

  • Secreted oral epithelial cell membrane vesicles induce Epstein-Barr virus (EBV) reactivation in latently infected B-cells. [PublishAheadOfPrint]

  • In the oral epithelium, peripheral stores of Epstein-Barr virus (EBV) are transmitted from infiltrating B-cells to epithelial cells. Once transmitted to epithelial cells, the highly permissive nature of this cell type for lytic replication allows virus amplification and exchange to other hosts. Since the initial transfer of EBV from B-cells to epithelial cells requires transitioning of the B-cell to a state that induces virus reactivation, we hypothesized that there might be epithelial specific signals that allow the infiltrating B-cells to sense the appropriate environment to initiate reactivation and begin this exchange process. We previously found that the epithelial specific miR-200 family of microRNAs promotes EBV lytic replication. Here we show that there are high levels of miR-200 family members in oral and tonsillar epithelium and in saliva. Analysis of cultured oral epithelial cells (OKF6) showed that they actively secrete membrane vesicles/exosomes that are enriched with miR-200 family members. Co-culturing of EBV(+) B-cells with OKF6 cells induced viral reactivation. Further, treatment of EBV(+) B-cells with OKF6-derived membrane vesicles promoted reactivation. Using a cell system that doesn't naturally express miR-200 family members, we found that enforced expression of a miR-200 family member produced membrane vesicles that were able to induce the lytic cascade in EBV(+) B-cells. We propose that membrane vesicles secreted by oral/tonsillar epithelial cells may serve as a tissue specific environmental cue that initiates reactivation in B-cells, promoting the transfer of virus from peripheral B-cell stores to the oral epithelium to facilitate virus amplification and exchange to other hosts.

    IMPORTANCE Epstein-Barr virus (EBV) is an important human pathogen that is causally associated with several lymphomas and carcinomas. The switch from latency to the lytic cycle is critical for successful host infection and for EBV pathogenesis. Although the EBV lytic cycle can be triggered by certain agents in vitro, the mechanisms that signal reactivation in vivo are poorly understood. We previously reported that endogenously expressed miR-200 family members likely play a role in facilitating the lytic tendencies of EBV in epithelial cells. Here we show that membrane vesicles secreted from oral epithelial cells contain miR-200 family members and that they can be transmitted to proximal EBV(+) B-cells where they trigger reactivation. We propose that this intercellular communication pathway may serve as a sensor mechanism for infiltrating B-cells to recognize an appropriate environment to initiate reactivation, thereby allowing the exchange of virus to the oral epithelium.

  • Mono-nucleated blood cell populations display different abilities to transmit prion disease by the transfusion route [PublishAheadOfPrint]

  • Previous experiments carried out in a sheep scrapie model demonstrated that the transfusion of 200 mmu;L of prion-infected whole blood has an apparent 100% efficacy for disease transmission. These experiments also indicated that, despite their apparent low infectious titer, the intravenous administration of white blood cells (WBC) resulted in an efficient disease transmission.

    In the study presented here, using the same TSE animal model, our aim was to determine the minimal number of white blood cells and the specific abilities of mono-nucleated cell populations to transmit scrapie by the transfusion route. Our results confirmed that the transfusion of 100mmu;L but not 10mmu;L of fresh whole blood collected in asymptomatic scrapie infected donor sheep can transmit the disease. The data also show that the intravenous administration of 105 WBCs are sufficient to cause scrapie in recipient sheep. Cell-sorted CD45R+ (predominantly B lymphocytes), CD4+/CD8+ (T lymphocytes) and CD14+ (monocytes/macrophages) blood cell sub-populations were all shown to contain prion infectivity by bioassays in ovine PrP transgenic mice. However while the intravenous administration of 106 CD45+ or CD4/8+ living cells were able to transmit the disease, similar numbers of CD14+ failed to infect the recipients. These data support the contention that mononucleated blood cell populations display different abilities to transmit TSE by the transfusion route. They also represent an important input for the risk assessment of the blood borne prion disease transmission and refining the target performance of leuko-reduction processes that are currently applied to mitigate the transmission risk in transfusion medicine.


    Inter-individual v-CJD transmission through blood and blood derived products is considered as a major public health issue in transfusion medicine. Over the last decade, TSE in sheep has emerged as a relevant model for assessing the blood borne vCJD transmission risk

    In this study, using a sheep TSE model, we characterized the ability of different peripheral blood mono-nucleated cell populations to infect TSE free recipients by the transfusion route.

    Our results indicate that as little as 105 WBC and 100mmu;L of blood collected in asymptomatic scrapie infected animal can transmit the disease. They also demonstrate unambiguously that PBMC subpopulations display dramatically different abilities to transmit the disease.

    These data represent an important input for the risk assessment of the blood borne prion disease transmission and refining the target performance of leuko-reduction processes that are currently applied to mitigate the transmission risk in transfusion medicine.

  • Involvement of the N-terminal DUB domain of human cytomegalovirus UL48 tegument protein in auto-ubiquitination, virion stability, virus entry [PublishAheadOfPrint]

  • Human cytomegalovirus (HCMV) pUL48 is closely associated with the capsid and has a deubiquitinating protease (DUB) activity in its N-terminal region. Although this DUB activity moderately increases virus replication in cultured fibroblast cells, the requirements of the N-terminal region of pUL48 in the viral replication cycle are not fully understood. In this study, we characterized the recombinant viruses encoding UL48(DUB/NLS), which lacks the DUB domain and the adjacent nuclear localization signal (NLS), UL48(DUB), which lacks only the DUB, and UL48(360-1200), which lacks the internal region downstream of the DUB/NLS. While DUB/NLS and 360-1200 mutant viruses did not grow in fibroblasts, the DUB virus replicated to titers 100-fold lower than those for wild-type virus and showed substantially reduced viral gene expression at low multiplicity of infection. The DUB domain contained ubiquitination sites and DUB activity reduced its own proteasomal degradation in trans. Deletion of the DUB domain did not affect the nuclear and cytoplasmic localization of pUL48, whereas the internal region (360-1200) was necessary for cytoplasmic distribution. In co-immunoprecipitation assays, pUL48 interacted with three tegument proteins (pUL47, pUL45, and pUL88) and two capsid proteins (pUL77 and pUL85), but the DUB domain contributed to only pUL85 binding. Furthermore, we found that the DUB virus showed reduced virion stability and less efficiently delivered its genome into the cell than the wild-type virus. Collectively, our results demonstrate that the N-terminal DUB domain of pUL48 contributes to efficient viral growth by regulating its own stability and promoting virion stabilization and virus entry.

    IMPORTANCE HCMV pUL48 and its herpesvirus homologs play key roles in virus entry, regulation of immune signaling pathways, and virion assembly. The N-terminus of pUL48 contains the DUB domain, which is well-conserved among all herpesviruses. Although studies using the active site mutant viruses revealed that the DUB activity promotes viral growth, the exact role of this region in the viral life cycle is not fully understood. In this study, using the mutant virus lacking the entire DUB domain, we demonstrate that the DUB domain of pUL48 contributes to viral growth by regulating its own stability via auto-deubiquitination and promoting virion stability and virus entry. This report is the first to demonstrate the mutant virus with the entire DUB domain deleted, which, along with information on the functions of this region, is useful in dissecting the functions associated with pUL48.

  • ISG15 is upregulated in respiratory syncytial virus infection and reduces virus growth through protein ISGylation [PublishAheadOfPrint]

  • Human Respiratory Syncytial Virus (RSV), for which neither a vaccine nor an effective therapeutic treatment is currently available, is the leading cause of severe lower respiratory tract infections in children. Interferon stimulated gene 15 (ISG15) is an ubiquitin-like protein that is highly increased during viral infections and has been reported to play an antiviral or a proviral activity, depending on the virus. Previous studies from our laboratory demonstrated a strong ISG15 up-regulation during RSV infection in vitro. In this study, an in depth analysis of the role of ISG15 in RSV infection is presented. ISG15 overexpression and siRNA silencing experiments, along with ISG15 knockout cells (ISG15-/-) revealed an anti-RSV effect of this molecule. Conjugation inhibition assays demonstrated that ISG15 exerts its antiviral activity via protein ISGylation. This antiviral activity requires high levels of ISG15 to be present in the cells before RSV infection. Finally, ISG15 is also up-regulated in human respiratory pseudo-stratified epithelia and in nasopharyngeal washes from infants infected with RSV, pointing to a possible antiviral role of this molecule in vivo. These results advance our understanding of the innate immune response elicited by RSV and open new possibilities to control infections by this virus.

    IMPORTANCE At present no vaccine or effective treatment against human respiratory syncytial virus (RSV) is available. This study shows that interferon-stimulated gene 15 (ISG15) lowers RSV growth through protein ISGylation. In addition, ISG15 accumulation highly correlates with RSV load in nasopharyngeal washes from children, indicating that ISG15 may also have an antiviral role in vivo. These results improve our understanding of the innate immune response against RSV and identify ISG15 as a potential target for virus control.

  • Infectivity and Transmissibility of Avian H9N2 Influenza Viruses in Pigs [PublishAheadOfPrint]

  • The H9N2 influenza viruses that are enzootic in terrestrial poultry in China pose a persistent pandemic threat to humans. To investigate whether the continuous circulation and adaptation of these viruses in terrestrial poultry increased their infectivity to pigs, we conducted a serological survey in pig herds with H9N2 viruses selected from the aquatic avian gene pool (Y439 lineage) and the enzootic terrestrial poultry viruses (G1 and Y280 lineages). We also compared the infectivity and transmissibility of these viruses in pigs. It was found that over 15% of the pigs sampled from 2010 to 2012 in southern China were seropositive to either G1 or Y280 lineage viruses, but none of the sera were positive to the H9 viruses from the Y439 lineage. Viruses of the G1 and Y280 lineages were able to infect experimental pigs, with detectable nasal shedding of the viruses and seroconversion, while viruses of the Y439 lineage did not cause a productive infection in pigs. Thus adaptation and prevalence in terrestrial poultry could lead to interspecies transmission of H9N2 viruses from birds to pigs. Although H9N2 viruses do not appear to be continuously transmissible among pigs, repeated introductions of H9 viruses to pigs naturally increase the risk of generating mammalian-adapted or reassorted variants that are potentially infectious to humans. This study highlights the importance of monitoring the activity of H9N2 viruses in terrestrial poultry and pigs.

    IMPORTANCE H9N2 subtype of influenza viruses has repeatedly been introduced into mammalian hosts, including humans and pigs, so awareness of their activity and evolution is important for influenza pandemic preparedness. However, as H9N2 viruses usually cause mild or even asymptomatic infections in mammalian hosts, they may be overlooked in influenza surveillance. Here we found that the H9N2 viruses established in terrestrial poultry had higher infectivity in pigs than those from aquatic birds, which suggests that adaptation of the H9N2 viruses in terrestrial poultry might have increased the infectivity of the virus to mammals. Therefore, monitoring the prevalence and evolution of H9 viruses prevalent in terrestrial birds and conducting risk assessment of their threat to mammals are critical for evaluating the pandemic potential of this virus.

  • Equine Arteritis Virus Uses Equine CXCL16 (EqCXCL16) as an Entry Receptor [PublishAheadOfPrint]

  • Previous studies in our laboratory have identified equine CXCL16 (EqCXCL16) as a candidate molecule and possible cell entry receptor for equine arteritis virus (EAV). In horses, the CXCL16 gene is located on equine chromosome 11 (ECA11) and encodes a glycosylated, type I transmembrane protein with 247 amino acids. Stable transfection of HEK-293T cells with plasmid DNA encoding EqCXCL16 (HEK-EqCXCL16) increased their permissiveness to EAV infection from llt;3% to almost 100% of the cell population. The increase in permissiveness was blocked either by transfection of HEK-EqCXCL16 cells with siRNAs directed against EqCXCL16 or by pre-treatment with guinea pig polyclonal antiserum against EqCXCL16 protein (Gp aalpha;-EqCXCL16 pAb). Furthermore, using a virus overlay protein-binding assay (VOPBA) in combination with Far-Western blotting, gradient purified EAV particles were shown to bind directly to the EqCXCL16 protein in vitro. The binding of biotinylated EAV virulent Bucyrus strain at 4 ddeg;C was significantly higher in HEK-EqCXCL16 cells compared to non-transfected HEK cells. Finally, the results demonstrated that EAV preferentially infects subpopulations of horse CD14+ monocytes expressing EqCXCL16 and that infection of these cells is significantly reduced by pretreatment with Gp aalpha;-EqCXCL16 pAb. The collective data from this study provides confirmatory evidence that the transmembrane form of EqCXCL16 likely plays a major role in EAV host-cell entry processes possibly acting as a primary receptor molecule for this virus.

    Importance Outbreaks of EVA can be a source of significant economic loss for the equine industry from high rates of abortion in pregnant mares, death in young foals, establishment of the carrier state in stallions and trade restrictions imposed by various countries. Similar to other arteriviruses, EAV primarily targets cells of the monocyte/macrophage lineage which when infected are believed to play a critical role in EVA pathogenesis. To this point, however the host-specified molecules involved in EAV binding and entry into monocytes/macrophages have not been identified. Identification of cellular receptors for EAV may provide insights to design antivirals and better prophylactic reagents. In this study, we have demonstrated that EqCXCL16 acts as an EAV entry receptor in EAV susceptible cells, equine monocytes. These findings represent a significant advance in our understanding of the fundamental mechanisms associated with the entry of EAV into susceptible cells.

  • Morphological, biochemical and functional study of viral replication compartments isolated from adenovirus-infected cells. [PublishAheadOfPrint]

  • Adenovirus Replication Compartments (RC) are nuclear microenvironments where the viral genome is replicated and a coordinated program of late gene expression is established. These virus-induced nuclear sites seem to behave as central hubs for regulation of virus-host cell interactions, since proteins that promote efficient viral replication as well as factors that participate in the antiviral response are co-opted and concentrated there. To gain further insight into the activities of viral RC here we report for the first time the morphology, composition and activities of RC isolated from Ad-infected cells. Morphological analyses of isolated RC particles by super-resolution microscopy showed that they were indistinguishable from RC within infected cells and that they displayed a dynamic compartmentalization. Furthermore, the RC-containing fractions (RCf) proved to be functional as they directed de novo synthesis of viral DNA and RNA, as well as, RNA splicing; activities that are associated to RC in vivo.

    A detailed analysis of the production of viral late mRNA from RCf at different times post-infection revealed that viral mRNA splicing occurs in RC and that the synthesis, post-transcriptional processing and release from RC to the nucleoplasm of individual viral late transcripts are spatio-temporally separate events. The results presented here demonstrate that RCf are a powerful system for detailed study into RC structure, composition and activities, and hence the determination of the molecular mechanisms that induce formation of these viral sites of adenoviruses and other nuclear-replicating viruses.

    Importance RC may represent molecular hubs where many aspects of virus-host cell interaction are controlled. Here we show by super-resolution microscopy that RCf have similar morphology to RC within Ad-infected cells and that they appear to be compartmentalized, as nucleolin and DBP display different localization in the periphery of these viral sites. RCf proved to be functional, as they direct de novo synthesis of viral DNA and mRNA, allowing the detailed study of the regulation of viral genome replication and expression. Furthermore, we show that synthesis and splicing of individual viral late mRNA occurs in RC and that they are subject to different temporal patterns of regulation, from their synthesis to their splicing and release from RC to the nucleoplasm. Hence, RCf represent a novel system to study molecular mechanisms that are orchestrated in viral RC to take control of the infected cell and promote an efficient viral replication cycle.

  • A recombinant adenovirus expressing ovine interferon-tau prevents influenza virus induced lethality in mice [PublishAheadOfPrint]

  • Ovine interferon tau (IFN-) is a unique type I interferon with low toxicity and broad host range in vivo. We report the generation of a non replicative recombinant adenovirus expressing biologically active IFN-. Using the B6.A2G-Mx1 mouse model we show that a single dose intranasal administration of the recombinant Ad5-IFN- can effectively prevent highly virulent hv-PR8 influenza virus induced lethality and disease by activating the interferon response and preventing viral replication.

  • Deep sequencing reveals potential antigenic variants at low frequency in influenza A-infected humans. [PublishAheadOfPrint]

  • Influenza vaccines must be frequently reformulated to account for antigenic changes in the viral envelope protein, hemagglutinin (HA). The rapid evolution of influenza virus under immune pressure is likely enhanced by the virus's genetic diversity within a host, though antigenic change has rarely been investigated on the level of individual infected humans. We used deep sequencing to characterize between- and within-host genetic diversity of influenza viruses in a cohort of patients that included individuals who were vaccinated and then infected in the same season. We characterized influenza HA segments from the predominant circulating influenza A subtypes during the 2012-2013 (H3N2) and 2013-2014 (pandemic H1N1; H1N1pdm) flu seasons. We found that HA consensus sequences were similar in non-vaccinated and vaccinated subjects. In both groups purifying selection was the dominant force shaping HA genetic diversity. Interestingly, viruses from multiple individuals harbored low-frequency mutations encoding amino acid substitutions in HA antigenic sites at or near the receptor-binding domain. These mutations included two substitutions in H1N1pdm viruses, G158K and N159K, which were recently found to confer escape from virus-specific antibodies. These findings raise the possibility that influenza antigenic diversity can be generated within individual human hosts, but may not become fixed in the viral population even when they would be expected to have a strong fitness advantage. Understanding constraints on influenza antigenic evolution within individual hosts may elucidate potential future pathways of antigenic evolution at the population level.

    Importance Influenza vaccines must be frequently reformulated due to the virus's rapid evolution rate. We know that influenza viruses exist within each infected host as a "swarm" of genetically distinct viruses, but the role of this within-host diversity in the antigenic evolution of influenza has been unclear. Here we characterized the genetic and potential antigenic diversity of influenza viruses infecting humans, some of whom became infected despite recent vaccination. Influenza between- and within-host genetic diversity was not significantly different in non-vaccinated and vaccinated humans, suggesting that vaccine-induced immunity does not exert strong selective pressure on viruses replicating in individual people. We found low-frequency mutations, below the detection threshold of traditional surveillance methods, in non-vaccinated and vaccinated humans that were recently associated with antibody escape. Interestingly, these potential antigenic variants did not reach fixation in infected people, suggesting that other evolutionary factors may be hindering their emergence in individual humans.

  • The High Degree Of Sequence Plasticity Of The Arenavirus Non-Coding Intergenic Region (IGR) Enables The Use Of A Non-Viral Universal Synthetic IGR To Attenuate Arenaviruses [PublishAheadOfPrint]

  • Hemorrhagic fever arenaviruses (HFA) pose important public health problems in their endemic regions. Concerns about human pathogenic arenaviruses are exacerbated because of the lack of FDA-licensed arenavirus vaccines and current anti-arenaviral therapy being limited to an off-label use of ribavirin that is only partially effective. We have recently shown that the non-coding intergenic region (IGR) present in each, S and L, arenavirus genome segment plays important roles in control of virus protein expression, and that this knowledge could be harnessed for the development of live-attenuated vaccine strains to combat HFA. In this study, we have further investigated the sequence plasticity of the arenavirus IGR. We demonstrate that recombinants of the prototypic arenavirus LCMV whose S-IGR were replaced by S-IGR of Lassa virus (LASV) or an entirely non-viral S-IGR like sequence (Ssyn) are viable indicating that the function of the S-IGR tolerates a high degree of sequence plasticity. In addition, rLCMVs whose L-IGR were replaced by Ssyn or S-IGR of the very distantly related reptarenavirus Golden Gate virus (GGV) were viable and severely attenuated in vivo but able to elicit protective immunity against a lethal challenge with wild type LCMV. Our findings indicate that replacement of L-IGR by a non-viral Ssyn could serve as a universal molecular determinant of arenavirus attenuation.

    IMPORTANCE Hemorrhagic fever arenaviruses (HFA) cause high morbidity and mortality, and pose important public health problems in their endemic regions. Implementation of live-attenuated vaccines (LAV) will represent a major step to combat HFA. Here we document that the arenavirus non-coding intergenic region has a high degree of plasticity compatible with virus viability. This observation led us to generate recombinant LCM viruses containing non-viral synthetic IGRs. These rLCMVs were severely attenuated in vivo but able to elicit protective immunity against a lethal challenge with wild type LCMV. These non-viral synthetic IGRs can be used as universal molecular determinants of arenavirus attenuation for the rapid development of safe and effective, as well as stable, LAV to combat HFA.

  • Requirement for the E1 helicase C-terminal domain in papillomavirus DNA replication in vivo [PublishAheadOfPrint]

  • The papillomavirus (PV) E1 helicase contains a conserved C-terminal domain (CTD), located next to its ATP-binding site, whose function in vivo is still poorly understood. The CTD is comprised of an alpha helix followed by an acidic region (AR) and a C-terminal extension termed the C-tail. Recent biochemical studies on BPV1 E1 showed that the AR and C-tail regulate the oligomerization of the protein into a double-hexamer at the origin. In this study, we assessed the importance of the CTD of HPV11 E1 in vivo, using a cell-based DNA replication assay. Our results indicate that combined deletion of the AR and C-tail drastically reduces DNA replication, by 85%, and that further truncation into the alpha helical region compromises the structural integrity of the E1 helicase domain and its interaction with E2. Surprisingly, removal of the C-tail alone or mutation of highly conserved residues within this domain still allows significant levels of DNA replication (55%). This is in contrast to the absolute requirement for the C-tail reported for BPV1 E1 in vitro and confirmed here in vivo. Characterization of chimeric proteins in which the AR and C-tail from HPV11 E1 were replaced by those of BPV1 indicated that while the function of the AR is transferable, that of the C-tail is not. Collectively, these findings define the contribution of the three CTD subdomains to the DNA replication activity of E1 in vivo and suggest that the function of the C-tail has evolved in a PV type-specific manner.

    IMPORTANCE While much is known about hexameric DNA helicases from superfamily 3, the papillomavirus E1 helicase contains a unique C-terminal domain (CTD) adjacent to its ATP-binding site. We show here that this CTD is important for the DNA replication activity of HPV11 E1 in vivo and that it can be divided into three functional subdomains that roughly correspond to the three conserved regions of the CTD: an alpha helix needed for the structural integrity of the helicase domain, followed by an acidic region (AR) and a C-terminal tail (C-tail) that were shown to regulate the oligomerization of BPV1 E1 in vitro. Characterization of E1 chimeras revealed that while the function of the AR could be transferred from BPV1 E1 to HPV11 E1 that of the C-tail could not. These results suggest that the E1 CTD performs multiple functions in DNA replication, some of them in a virus type-specific manner.

  • Chaperone-Assisted Protein Folding Is Critical for Yellow Fever Virus NS3/4A Cleavage and Replication [PublishAheadOfPrint]

  • DNAJC14, a heat shock protein 40 (Hsp40) cochaperone, assists Hsp70-mediated protein folding. Overexpressed DNAJC14 is targeted to sites of yellow fever virus (YFV) replication complex (RC) formation, where it interacts with viral nonstructural (NS) proteins and inhibits viral RNA replication. How RCs are assembled and the roles of chaperones in this coordinated process are largely unknown. We hypothesize that chaperones are diverted from their normal cellular protein quality control function to play similar roles during viral infection. Here, we show that DNAJC14 overexpression affects YFV polyprotein processing and alters RC assembly. We monitored YFV NS2A-5 polyprotein processing by the viral NS2B-3 protease in DNAJC14-overexpressing cells. Notably, DNAJC14 mutants that do not inhibit YFV replication had minimal effects on polyprotein processing, while overexpressed wild-type DNAJC14 affected NS3/4A and NS4A/2K cleavage sites, resulting in altered NS3:NS3-4A ratios. This suggests that DNAJC14's folding activity normally modulates NS3/4A/2K cleavage events to liberate appropriate levels of NS3 and NS4A and promote RC formation. We introduced amino acid substitutions at the NS3/4A site to alter the levels of NS3 and NS4A products and examined their effects on YFV replication. Residues with reduced cleavage efficiency did not support viral RNA replication and only revertant viruses, with a restored wild-type arginine or lysine residue at the NS3/4A site, were obtained. We conclude that DNAJC14 inhibition of RC formation upon overexpression is likely due to chaperone dysregulation and that YFV probably utilizes DNAJC14's cochaperone function to modulate processing at the NS3/4A site as a mechanism ensuring virus replication.

    Importance Flaviviruses are single-stranded RNA viruses that cause a wide range of illnesses. Upon host cell entry, the viral genome is translated on ER membranes to produce a single polyprotein, which is cleaved by host and viral proteases to generate viral proteins required for genome replication and virion production. Several studies suggest a role for molecular chaperones during these processes. While the details of chaperone roles have been elusive, in this manuscript we show that overexpression of the ER-resident co-chaperone DNAJC14 affects YFV polyprotein processing at the NS3/4A site. This work reveals DNAJC14 modulation of NS3/4A site processing as an important mechanism to ensure virus replication. Our work highlights the importance of finely regulating flavivirus polyprotein processing. In addition, it suggests future studies addressing similarities and/or differences amongst flaviviruses and to interrogate precise mechanisms employed for polyprotein processing mmdash; a critical step that ultimately can be targeted for novel drug development.

  • Cellular Transcriptional Coactivator RanBP10 and Herpes Simplex Virus 1 ICP0 Interact and Synergistically Promote Viral Gene Expression and Replication [PublishAheadOfPrint]

  • To investigate the molecular mechanism(s) by which herpes simplex virus 1 (HSV-1) regulatory protein ICP0 promotes viral gene expression and replication, we screened cells overexpressing ICP0 for ICP0-binding host cell proteins. Tandem affinity purification of transiently expressed ICP0 coupled with mass spectrometry-based proteomics technology and subsequent analyses showed that ICP0 interacted with cell protein RanBP10, a known transcriptional coactivator, in HSV-1-infected cells. Knockdown of RanBP10 in infected HEp-2 cells resulted in a phenotype similar to that observed with the ICP0-null mutation, including reduction in viral replication and in the accumulation of viral immediate-early (ICP27), early (ICP8), and late (VP16) mRNAs and proteins. In addition, RanBP10 knockdown or the ICP0-null mutation increased the level of histone H3 association with the promoters of these viral genes, which is known to repress transcription. These effects observed in wild-type HSV-1-infected HEp-2 RanBP10 knockdown cells or those observed in ICP0-null mutant virus-infected control HEp-2 cells were remarkably increased in ICP0-null mutant virus-infected HEp-2 RanBP10 knockdown cells. Our results suggested that ICP0 and RanBP10 redundantly and synergistically promoted viral gene expression by regulating chromatin remodeling of the HSV-1 genome for efficient viral replication.

    IMPORTANCE Upon entry of herpesviruses into a cell, viral gene expression is restricted by heterochromatinization of the viral genome. Therefore, HSV-1 has evolved multiple mechanisms to counteract this epigenetic silencing for efficient viral gene expression and replication. HSV-1 ICP0 is one of the viral proteins involved in counteracting epigenetic silencing. Here, we identified RanBP10 as a novel cellular ICP0-binding protein and showed that RanBP10 and ICP0 appeared to act synergistically to promote viral gene expression and replication by modulating viral chromatin remodeling. Our results provide insight into the mechanisms by which HSV-1 regulated viral chromatin remodeling for efficient viral gene expression and replication.

  • Activation of RNase L by murine coronavirus in myeloid cells is dependent on basal Oas gene expression and independent of virus-induced interferon [PublishAheadOfPrint]

  • The oligoadenylate synthetase-ribonuclease L (OAS-RNase L) pathway is a potent interferon (IFN) induced antiviral activity. Upon sensing double stranded RNA, OAS produces 2rrsquo; ,5rrsquo; -oligoadenylates (2-5A), which activate RNase L. Murine coronavirus (MHV) non-structural protein 2 (ns2), is a 2rrsquo; ,5rrsquo; -phosphodiesterase (PDE) that cleaves 2-5A, thereby antagonizing RNase L activation. PDE activity is required for robust replication in myeloid cells as a mutant of MHV (ns2H126R) encoding an inactive PDE fails to antagonize RNase L activation and replicates poorly in bone marrow derived macrophages (BMM) while ns2H126R replicates to high titer in several types of non-myeloid cells as well as in IFN receptor deficient (Ifnar1-/-) BMM. We reported previously that myeloid cells express significantly higher basal levels of Oas transcripts than non-myeloid cells. Here, we investigated the contributions of Oas gene expression, basal IFN signaling and virus-induced IFN to RNase L activation. Infection with ns2H126R activated RNase L in Ifih1-/- BMM to a similar extent as in WT BMM despite the lack of IFN induction in the absence of MDA5 expression. However, ns2H126R failed to induce RNase L activation in BMM treated with IFNAR1 blocking antibody as well as in Ifnar1-/- BMM, both expressing low basal levels of Oas genes. Thus, activation of RNase L does not require virus-induced IFN, but rather correlates with adequate levels of basal Oas gene expression, maintained by basal IFN signaling. Finally, overexpression of RNase L is not sufficient to compensate for inadequate basal OAS levels.

    IMPORTANCE The oligoadenylate-ribonuclease L (OAS-RNase L) pathway is a potent antiviral activity. Activation of RNase L during murine coronavirus, MHV, infection of myeloid cells correlates with high basal Oas gene expression and is independent of virus-induced interferon secretion. Thus, our data suggest that cells with high basal Oas gene expression levels can activate RNase L and thereby inhibit virus replication early in infection upon exposure to viral dsRNA, before the induction of interferon and prior to transcription of interferon stimulated antiviral genes. These findings challenge the notion that activation of the OAS/RNase L pathway requires virus to induce type I IFN which in turn upregulates OAS gene expression as well as to provide dsRNA to activate OAS. Our data further suggest that myeloid cells may serve as sentinels to restrict viral replication thus protecting other cell types from infection.

  • {beta}-catenin, a transcription factor activated by canonical Wnt signaling, is expressed in sensory neurons of calves latently infected with bovine herpesvirus 1. [PublishAheadOfPrint]

  • Like many aalpha;-herpesvirinae subfamily members, bovine herpes virus 1 (BoHV-1) expresses an abundant transcript in latently infected sensory neurons: the latency-related (LR)-RNA. LR-RNA encodes a protein (ORF2) that inhibits apoptosis, interacts with Notch family members, interferes with Notch mediated transcription, and stimulates neurite formation in cells expressing Notch. A LR mutant virus containing stop codons at the amino-terminus of ORF2 does not reactivate from latency or replicate efficiently in certain tissues indicating LR gene products are important. In this study, bbeta;-catenin, a transcription factor activated by the canonical Wnt signaling pathway was frequently detected in ORF2+ trigeminal ganglionic neurons of latently infected, but not mock-infected calves. Conversely, the lytic cycle regulatory protein (bICP0) was not frequently detected in bbeta;-catenin+ neurons in latently infected calves. During dexamethasone-induced reactivation from latency, mRNA expression levels of two Wnt antagonists, dickkopf1 (DKK1) and secreted frizzled protein 2 (SFRP2), were induced in bovine TG, which correlated with reduced bbeta;-catenin protein expression in TG neurons six hours after dexamethasone treatment. ORF2 and a coactivator of bbeta;-catenin, mastermind like protein 1 (MAML1), stabilized bbeta;-catenin protein levels and stimulated bbeta;-catenin dependent transcription in mouse neuroblastoma cells more effectively than MAML1 or ORF2 alone. Neuroblastoma cells expressing ORF2, MAML1, and bbeta;-catenin were highly resistant to cell death following serum withdrawal, whereas most cells transfected with only one of these genes died. The Wnt signaling pathway interferes with neuro-degeneration but promotes neuronal differentiation suggesting that stabilization of bbeta;-catenin expression by ORF2 promotes neuronal survival and differentiation.

    IMPORTANCE Bovine herpesvirus 1 (BoHV-1) is an important pathogen of cattle and like many aalpha;-herpesvirinae subfamily members establish latency in sensory neurons. Life-long latency and the ability to reactivate from latency are crucial for virus transmission. Maintaining the survival and normal functions of terminally differentiated neurons is crucial for life-long latency. Our studies revealed BoHV-1 gene products expressed during latency stabilize expression of the transcription factors, bbeta;-catenin, and perhaps its cofactor mastermind like 1 (MAML1). In contrast to latency, bbeta;-catenin expression in sensory neurons is not detectable following treatment of latently infected calves with the synthetic corticosteroid dexamethasone to initiate reactivation from latency. A viral protein (ORF2) expressed in a subset of latently infected neurons stabilized bbeta;-catenin and MAML1 in transfected cells. ORF2, bbeta;-catenin, and MAML1 also enhanced cell survival when growth factors were withdrawn suggesting these genes enhance survival of latently infected neurons.

  • Human cytomegalovirus stimulates the synthesis of select Akt-dependent antiapoptotic proteins during viral entry to promote survival of infected monocytes. [PublishAheadOfPrint]

  • Primary peripheral blood monocytes are responsible for the hematogenous dissemination of human cytomegalovirus (HCMV) following a primary infection. To facilitate viral spread, we have previously shown HCMV to extend the short 48-hour lifespan of monocytes. Mechanistically, HCMV upregulated two specific cellular antiapoptotic proteins, myeloid leukemia sequence 1 (Mcl-1) and heat shock protein 27 (HSP27), to block the two proteolytic cleavages necessary for the formation of fully active caspase 3 and the subsequent initiation of apoptosis. We now show that, compared to normal myeloid growth factors, HCMV more robustly upregulates Mcl-1 and was the only myeloid survival factor to rapidly induce HSP27 prior to the 48-hour cell fate checkpoint. We determined HCMV glycoproteins gB and gH signal through cellular epidermal growth factor receptor (EGFR) and aalpha;vbbeta;3 integrin, respectively, during viral entry in order to drive the increase of Mcl-1 and HSP27 in a Akt-dependent manner. Although Akt is known to regulate protein stability and transcription, we found gB- and gH-initiated signaling preferentially and cooperatively stimulated the synthesis of Mcl-1 and HSP27 through mTOR-mediated translation. Overall, these data suggest that the unique signaling network generated during the viral entry process stimulates the upregulation of select antiapoptotic proteins allowing for the differentiation of short-lived monocyte into long-lived macrophages - a key step in the viral dissemination strategy.

    Importance Human cytomegalovirus (HCMV) infection is endemic within the human population. Although primary infection is generally asymptomatic in immunocompetent individuals, HCMV is a significant cause of morbidity and mortality in the immunocompromised. The multi-organ inflammatory diseases associated with symptomatic HCMV infection is a direct consequence of the monocyte-mediated systemic spread of the virus. In order for peripheral blood monocytes to facilitate viral dissemination, HCMV subverts the short 48-hour lifespan of monocytes by inducing the expression of cellular antiapoptotic proteins Mcl-1 and HSP27. Here, we demonstrate that the rapid and simultaneous upregulation of Mcl-1 and HSP27 to be a distinctive feature of HCMV-induced monocyte survival. Moreover, we decipher the signaling pathways activated during viral entry needed for the robust synthesis of Mcl-1 and HSP27. Identifying the virus-specific mechanisms used to upregulate select cellular factors required for the survival of HCMV-infected monocytes is important to the development of new classes of anti-HCMV drugs.

  • Characterization of Antibody Bipolar Bridging Mediated by the Human Cytomegalovirus Fc receptor gp68 [PublishAheadOfPrint]

  • The human cytomegalovirus glycoprotein gp68 functions as an Fc receptor for host IgGs and can form antibody bipolar bridging (ABB) complexes in which gp68 binds the Fc region of an antigen-bound IgG. Here we show that gp68-mediated endocytosis transports ABB complexes into endosomes, after which the complex is routed to lysosomes, presumably for degradation. These results suggest gp68 contributes to evasion of IgG-mediated immune responses by mediating destruction of host IgG and viral antigens.

  • LIR1 expressing human Natural Killer cell subsets differentially recognize isolates of human cytomegalovirus through the viral MHC Class I homolog UL18 [PublishAheadOfPrint]

  • Immune responses of Natural Killer (NK) cell are controlled by the balance between activating and inhibitory receptors, but the expression of these receptors varies between cells within an individual. Although NK cells are a component of the innate immune system, particular NK cell subsets expressing Ly49H are positively selected and increase in frequency in response to cytomegalovirus infection in mice. Recent evidence suggests that in humans certain NK subsets also have an increased frequency in the blood of HCMV infected individuals. However whether these subsets differ in their capacity of direct control of HCMV infected cells remains unclear. In this study we developed a novel in vitro assay to assess whether human NK cells subsets have differential abilities to inhibit HCMV growth and dissemination. NK cells expressing or lacking NKG2C did not display any differences when controlling viral dissemination. However, when in vitro expanded NK cells were used, cells expressing or lacking the inhibitory receptor Leukocyte Immunoglobulin-like receptor 1 (LIR1) were differentially able to control dissemination. Surprisingly, the ability of LIR1+ NK cells to control virus spread differed between HCMV viral strains, and this phenomenon was dependent on amino acid sequences within the viral ligand UL18. Together, the results here outlined an in vitro technique to compare the long-term immune responses of different human NK cell subsets, and suggest, for the first time, phenotypically defined human NK cell subsets may differentially recognise HCMV infected.

    IMPORTANCE HCMV infection is ubiquitous in most populations, it is not cleared by the host after primary infection but persists for life. The innate and adaptive immune system controls the spread of virus, of which Natural Killer (NK) cells play a pivotal role. NK cells can respond to HCMV infection by rapid, short-term non-specific innate responses, but evidence from murine studies suggested NK cells may display a long-term, memory like responses to murine cytomegalovirus infection. In this study, we developed a new assay that examines human NK cell subsets that have been suggested to play a long-term memory-like response to HCMV infection. We show that changes in a HCMV viral protein that interacts with an NK cell receptor can change the ability of NK cell subsets to control HCMV while the acquisition odf another receptor has no effect on virus control.

  • Antibody response to the hypervariable region-1 interferes with broadly neutralizing antibodies to hepatitis C virus [PublishAheadOfPrint]

  • The hypervariable region-1 (HVR1) (amino acids (aa) 384-410) on the E2 glycoprotein of hepatitis C virus contributes to persistent infection by evolving escape mutations that attenuate binding of inhibitory antibodies and by blocking access of broadly neutralizing antibodies to their epitopes. A third proposed mechanism of immune antagonism is that poorly neutralizing antibodies binding to HVR1 interfere with binding of other superior neutralizing antibodies. Epitope mapping of human monoclonal antibodies (HMAbs) that bind to an adjacent, conserved domain on E2 encompassing aa 412-423 revealed two subsets, designated as HC33 HMAbs. While both subsets have contact residues within aa 412-423, alanine scanning mutagenesis suggested that one subset, which includes HC33.8, has an additional contact residue within HVR1. To test for interference of anti-HVR1 antibodies with binding of antibodies to aa 412-423 and other E2 determinants recognized by broadly neutralizing HMAbs, two murine MAbs against HVR1 (H77.16) and aa 412-423 (H77.39) were studied. As expected, H77.39 inhibited the binding of all HC33 HMAbs. Unexpectedly, H77.16 also inhibited the binding of both subsets of HC33 HMAbs. This inhibition also was observed against other broadly neutralizing HMAbs to epitopes outside of aa 412-423. Combination antibody neutralization studies by the median-effect analysis method with H77.16 and broadly reactive HMAbs revealed antagonism between these antibodies. Structural studies demonstrated conformational flexibility in this antigenic region, which supports the possibility of anti-HVR1 antibodies hindering the binding of broadly neutralizing MAbs. These findings support the hypothesis that anti-HVR1 antibodies can interfere with a protective humoral response against HCV infection.

    Importance. The HVR1 contributes to persistent infection by evolving mutations that escape from neutralizing antibodies to HVR1 and by shielding broadly neutralizing antibodies from their epitopes. This study provides insight into a new immune antagonism mechanism by which the binding of antibodies to HVR1 blocks the binding and activity of broadly neutralizing antibodies to HCV. Immunization strategies that avoid the induction of HVR1 antibodies should increase the inhibitory activity of broadly neutralizing anti-HCV antibodies elicited by candidate vaccines.

  • Type I interferon counteracts antiviral effects of statins in the context of gammaherpesvirus infection. [PublishAheadOfPrint]

  • The cholesterol synthesis pathway is a ubiquitous cellular biosynthetic pathway that is attenuated therapeutically by statins. Importantly, type I interferon (IFN), a major antiviral mediator, also depresses the cholesterol synthesis pathway. Here we demonstrate that attenuation of cholesterol synthesis decreases gammaherpesvirus replication in primary macrophages in vitro and reactivation from peritoneal exudate cells in vivo. Specifically, reduced availability of intermediates required for protein prenylation was responsible for decreased gammaherpesvirus replication in statin-treated primary macrophages. We also demonstrate that statin treatment of a chronically infected host attenuates gammaherpesvirus latency in a route of infection-specific manner. Unexpectedly, we found that the antiviral effects of statins are counteracted by type I IFN. Our studies suggest that type I IFN signaling counteracts the antiviral nature of subdued cholesterol synthesis pathway and offer a novel insight into the utility of statins as antiviral agents.

    Statement of significance. Statins are cholesterol synthesis inhibitors that are therapeutically administered to 12.5% of U.S. population. Statins attenuate replication of diverse viruses in culture; however, this attenuation is not always obvious in an intact animal model. Further, it is not clear whether statins alter parameters of highly prevalent chronic herpesvirus infections. We show that statin treatment attenuated gammaherpesvirus replication in primary immune cells and during chronic infection of an intact host. Further, we demonstrate that type I interferon signaling counteracts antiviral effects of statins. Considering the fact that type I interferon decreases the activity of cholesterol synthesis pathway, it is intriguing to speculate that gammaherpesviruses have evolved to usurp type I interferon pathway to compensate for the decreased cholesterol synthesis activity.

  • The Synthetic Antiviral Drug Arbidol Inhibits Globally Prevalent Pathogenic Viruses [PublishAheadOfPrint]

  • Arbidol (ARB) is a synthetic antiviral originally developed to combat influenza viruses. ARB is currently used clinically in several countries but not in North America. We have previously shown that ARB inhibits in vitro hepatitis C virus (HCV) by blocking HCV entry and replication. In this report, we expand the list of viruses that are inhibited by ARB and demonstrate that ARB suppresses in vitro infection of mammalian cells with Ebola virus (EBOV), Tacaribe arenavirus, and human herpes virus 8 (HHV-8). We also confirm suppression of hepatitis B virus and poliovirus by ARB. ARB inhibited EBOV Zaire Kikwit infection when added before or at the same time as virus infection, and was less effective when added 24 hours post-EBOV infection. Experiments with recombinant vesicular stomatitis virus (VSV) expressing the EBOV Zaire glycoprotein showed that infection was inhibited by ARB at early stages, most likely at the level of viral entry into host cells. ARB inhibited HHV-8 replication to a similar degree as cidofovir. Our data broaden the spectrum of antiviral efficacy of ARB to include globally prevalent viruses that cause significant morbidity and mortality.

    Importance There are many globally prevalent viruses for which there are no licensed vaccines or antiviral medicines. Some of these viruses, like Ebola virus or members of the arenavirus family, rapidly cause severe hemorrhagic diseases that can be fatal. Other viruses like hepatitis B virus or human herpes virus 8 (HHV-8) establish persistent infections that cause chronic illnesses including cancer. Thus, finding an affordable, effective, and safe drug that blocks many viruses remains an unmet medical need. The antiviral drug Arbidol (ARB), already in clinical use in several countries as an anti-influenza treatment, has been previously shown to suppress the growth of many viruses. In this report we expand the list of viruses that are blocked by ARB in a laboratory setting to include Ebola virus, Tacaribe arenavirus, and HHV-8, and propose ARB as a broad-spectrum antiviral drug that may be useful against hemorrhagic viruses.

  • Prolactin regulatory element binding protein is involved in hepatitis C virus replication compartment by interacting with NS4B [PublishAheadOfPrint]

  • It has been proposed that hepatitis C virus (HCV) NS4B protein triggers the membranous HCV replication compartment, but the underlying molecular mechanism is not fully understood. Here, we screened for NS4B-associated membrane proteins by tandem affinity purification and proteome analysis and identified 202 host proteins. Subsequent small interfering RNA screening in replicon cells identified prolactin regulatory element binding (PREB) as a novel HCV host cofactor. The interaction between PREB and NS4B was confirmed by immunoprecipitation, immunofluorescence and proximity ligation assays. PREB colocalized with double-stranded RNA and the newly synthesized HCV RNA labeled by bromouridine triphosphate in HCV replicon cells. Furthermore, PREB shifted to detergent-resistant membranes (DRM), where HCV replication complexes reside, in the presence of NS4B expression in Huh7 cells. However, a PREB mutant lacking the NS4B binding region (PREBd3) could not colocalize with double-stranded RNA and did not shift to the DRM in the presence of NS4B. These results indicate that PREB locates at the HCV replication complex by interacting with NS4B. PREB silencing inhibited the formation of membranous HCV replication compartment and increased the protease and nuclease sensitivity of HCV replicase proteins and RNA in DRM, respectively. Collectively, these data indicate that PREB promotes HCV RNA replication by participating in the formation of the membranous replication compartment and by maintaining its proper structure by interacting with NS4B. Furthermore, PREB was induced by HCV infection in vitro and in vivo. Our findings provide new insights into HCV host cofactors.

    IMPORTANCE The hepatitis C virus (HCV) protein, NS4B, can induce alteration of the endoplasmic reticulum and formation of a llsquo;membranous web' structure, which provides a platform for the HCV replication complex. The molecular mechanism by which NS4B induces membranous HCV replication compartment is not understood. We screened for NS4B-associated membrane proteins by tandem affinity purification and proteome analysis, followed by small interfering RNA screening. We identified prolactin regulatory element binding (PREB) as a novel HCV host cofactor. PREB is induced by HCV infection and recruited into the replication complex by interaction with NS4B. Recruited PREB promotes HCV RNA replication by participating in the formation of membranous HCV replication compartment. To our knowledge, this is the novel report to describe the effect of NS4B-binding protein on the formation of membranous HCV replication compartment. Our findings are expected to provide new insights into HCV host cofactors.

  • Investigation of the Lipid Binding Properties of the Marburg Virus Matrix Protein VP40 [PublishAheadOfPrint]

  • Marburg virus (MARV), which belongs to the Filoviridae family of viruses, causes hemorrhagic fever in humans and non-human primates that is often fatal. MARV is a lipid-enveloped virus that during the replication process extracts its lipid coat from the plasma membrane of the host cell it infects. MARV encodes seven genes, one of which is its matrix protein VP40 (mVP40), which regulates assembly and budding of the virions. To date, a dearth of information is available on mVP40 lipid binding properties. Here we have investigated the in vitro and cellular mechanisms by which mVP40 associates with lipid membranes. mVP40 associates with anionic membranes in a nonspecific manner that was dependent upon the anionic charge density of the membrane. These results are consistent with recent structural determination of mVP40, which elucidated an mVP40 dimer with a flat and extensive cationic lipid-binding interface.

    Importance Marburg virus (MARV) is a lipid-enveloped filamentous virus from the Filoviridae family. MARV was discovered in 1967 and yet little is known about how its seven genes are used to assemble and form a new viral particle in the host cell it infects. The MARV matrix protein VP40 (mVP40), underlies the inner leaflet of the virus and regulates budding from the host cell plasma membrane. In vitro and cellular assays in this study investigated the mechanism by which mVP40 associates with lipids. Results demonstrate that mVP40 interactions with lipid vesicles or the inner leaflet of the plasma membrane are electrostatic but nonspecific in nature and are dependent on the anionic charge density of the membrane surface. Small molecules that can disrupt lipid trafficking or reduce the anionic charge of the plasma membrane interface may be useful in inhibiting assembly and budding of MARV.

  • Analysis of assembly and budding of Lujo virus [PublishAheadOfPrint]

  • The recently identified arenavirus, Lujo virus (LUJV), causes fatal hemorrhagic fever in humans. We analyzed its mechanism of viral release driven by matrix protein (Z) and glycoprotein (GPC). The L-domains in Z are required for efficient virus-like particle release, but Tsg101, ALIX/AIP1, and Vps4A/B are unnecessary for budding. LUJV GPC is cleaved by site 1 protease (S1P) at the RKLM motif, and treatment with the S1P inhibitor, PF-429242, reduced LUJV production.

  • MxB is not responsible for the block to HIV-1 observed in IFN-{alpha}-treated cells [PublishAheadOfPrint]

  • MxB restricts HIV-1 infection by directly interacting with the HIV-1 core, which is form of capsid; however, the contribution of MxB to the HIV-1 restriction observed in IFN-aalpha;-treated human cells is unknown. To understand this contribution, we used HIV-1 viruses bearing the capsid mutant G208R (HIV-1-G208R), which overcomes the restriction imposed by cells expressing MxB. Here we showed that the reason that MxB does not block HIV-1-G208R is that MxB does not interact with HIV-1 cores bearing the mutation G208R. To understand whether MxB contributes to the HIV-1 restriction imposed by IFN-aalpha;-treated human cells, we challenged IFN-aalpha;-treated cells with HIV-G208R, and found that MxB does not contribute to the restriction imposed by IFN-aalpha;-treated cells. To more directly test the contribution of MxB, we challenged IFN-aalpha;-treated human cells that are knocked out for the expression of MxB with HIV-1 viruses. These experiments suggested that MxB does not contribute to the HIV-1 restriction observed in IFN-aalpha;-treated human cells.

    IMPORTANCE MxB is a restriction factor that blocks HIV-1 infection in human cells. Although it has been postulated that MxB is the factor that blocks HIV-1 infection in IFN-aalpha;-treated cells, this is a hard concept to grasp due to the great number of genes that are induced by IFN-aalpha; in cells from the immune system. The work presented here elegantly demonstrates that MxB has minimal or no contribution to the ability of IFN-aalpha;-treated human cells to block HIV-1 infection. Furthermore, this work suggests the presence of novel restriction factors in the IFN-aalpha;-treated human cells that block HIV-1 infection.

  • Subviral hepatitis B virus filaments are released like infectious viral particles via multivesicular bodies [PublishAheadOfPrint]

  • In addition to infectious viral particles, hepatitis B virus-replicating cells secrete high amounts of subviral particles assembled by the surface proteins, but lacking any capsid and genome. Subviral particles form spheres (22 nm particles) and filaments. Filaments contain a much higher amount of the large surface protein (LHBs) as compared to spheres. Spheres are released via the constitutive secretory pathway, while viral particles are ESCRT-dependently released via multivesicular bodies (MVBs). The interaction of virions with the ESCRT machinery is mediated by aalpha;-taxilin that connects the viral surface protein LHBs with the ESCRT-component tsg101. Since filaments in contrast to spheres contain a significant amount of LHBs, it is unclear whether filaments are released like spheres or like virions. To study the release of subviral particles in absence of virion formation, a core-deficient HBV mutant was generated. Confocal microscopy, immune electron microscopy of ultrathin sections and isolation of MVBs revealed that filaments enter MVBs. Inhibition of MVB biogenesis by the small molecule inhibitior U18666A or inhibition of ESCRT-functionality by coexpression of transdominant negative mutants (Vps4A, Vps4B, CHMP3) abolishs the release of filaments while secretion of spheres is not affected. These data indicate that in contrast to spheres which are secreted via the secretory pathway, filaments are released via ESCRT/MVB pathway like infectious viral particles.

    Importance The study revises the current model describing the release of subviral particles by showing that in contrast to spheres, which are secreted via the secretory pathway, filaments are released via the ESCRT/MVB pathway like infectious viral particles. These data significantly contribute to a better understanding of the viral morphogenesis and might be helpful for the design of novel antiviral strategies.

  • Identification of a human respiratory syncytial virus (hRSV) cell entry inhibitor by using a novel lentiviral pseudotype (hRSVpp) system [PublishAheadOfPrint]

  • Lentiviral budding is governed by Group-specific antigen (Gag-proteins) and proceeds in the absence of cognate viral envelope proteins, which has been exploited to create pseudotypes incorporating envelope proteins from non-lentiviral families. Here, we report the generation of infectious lentiviral pseudo-particles incorporating human respiratory syncytial virus F protein alone (hRSV-Fpp) or carrying SH, G and F proteins (hRSV-SH/G/Fpp). These particles recapitulate key infection steps of authentic hRSV particles including utilization of glycosaminoglycans and low pH independent cell entry. Moreover, hRSVpp can faithfully reproduce phenotypic resistance to a small molecule fusion inhibitor in clinical development (BMS-433771) and a licensed therapeutic F protein targeting antibody (palivizumab). Inoculation of several human cell lines from lung and liver revealed more than 30-fold differences in susceptibility to hRSVpp infection, suggesting differential expression of hRSV entry co-factors and/or restriction factors between these cell types. Moreover, we observed functional differences between hRSVpp carrying solely F protein or SH, G and F proteins with regard to utilization of glycosaminoglycans in a cell type-dependent manner. Using hRSVpp we identified penta-O-galloyl-bbeta;-D-glucose (PGG) as a novel hRSV cell entry inhibitor. Moreover, we show that PGG also inhibits cell entry of hRSVpp carrying F-proteins resistant to BMS-433771 or palivizumab. This work sheds new light onto the mechanisms of hRSV cell entry including possible strategies for antiviral intervention. Moreover, hRSVpp should prove valuable to dissect hRSV envelope protein functions including the interaction with cell entry factors.

    IMPORTANCE Lentiviral pseudotypes are highly useful to specifically dissect the function of viral and host factors in cell entry, which has been exploited for numerous viruses. Here we successfully created hRSVpp and show that they faithfully recapitulate key characteristics of parental hRSV cell entry. Importantly, hRSVpp accurately mirror hRSV resistance to small molecule fusion inhibitors and clinically approved therapeutic antibodies. Moreover, we observed highly different susceptibility of cell lines to hRSVpp infection and also between cell entry of hRSVpp types (with F protein alone or with SH, G and F proteins). This indicates differential expression of host factors determining hRSV cell entry between these cell lines and highlights that the hRSVpp system is useful to explore the functional properties of hRSV en velope protein combinations. Therefore, this system will be highly useful to study hRSV cell entry and host factor usage and to explore antiviral strategies targeting hRSV cell entry.

  • Human mucosal mast cells capture HIV-1 and mediate viral trans-infection of CD4+ T cells [PublishAheadOfPrint]

  • The gastrointestinal mucosa is the primary site at where HIV-1 invades, amplifies and becomes persistently established, and cell to cell transmission of HIV-1 infection plays a pivotal role in mucosal viral dissemination. Mast cells are widely distributed in the gastrointestinal tract and are early targets for invasive pathogens, and have been showed increased density in HIV-infected women genital mucosa. Intestinal mast cells express numerous pathogen-associated molecular patterns (PAMPs) and have been shown to combat various viral, parasitic and bacterial infections. However the role of mast cells in HIV-1 infection is poorly defined. In this study, we investigated their potential contributions to HIV-1 transmission. Mast cells isolated from gut mucosal tissues were found to express a variety of HIV-1 attachment factors (HAFs) DC-SIGN, HSPG, aalpha;4bbeta;7 integrin, and mediated capture of HIV-1 on the cell surface. Intriguingly, following co-cultured with CD4+ T cells, mast cell surface-bound viruses were efficiently transferred to target T cells. The prior-blocking with anti-HAFs antibody or manan before co-culture impaired viral trans-infection. Cell-cell conjunctions formed between mast cells and T cells, to which viral particles were recruited, and were required for efficient cell to cell HIV-1 transmission. Our results revealed a potential function of gut mucosal mast cells in HIV-1 dissemination in tissues. Strategies aimed at preventing viral capture and transfer mediated by mast cells could be beneficial in combating primary HIV-1 infection.

    IMPORTANCE In this study, we demonstrate the role of human mast cells isolated from mucosal tissues in mediating HIV-1 trans-infection of CD4+ T cells. The finding facilitates understanding of HIV-1 mucosal infection and benefits the strategy development to combating primary HIV-1 dissemination.

  • Network analysis of the chronic Hepatitis C virome defines HVR1 evolutionary phenotypes in the context of humoral immune responses [PublishAheadOfPrint]

  • The hypervariable region 1 (HVR1) of Hepatitis C virus (HCV) comprises the first 27 N-terminal amino acid residues of E2. It is classically seen as the most heterogeneous region of the HCV genome. In this study, we assessed HVR1 evolution using ultradeep pyrosequencing in a cohort of treatment naïve chronically infected patients over a short 16 week period. Organization of the sequence set into connected components, that represented single nucleotide substitution events, revealed a network dominated by highly connected, centrally positioned master sequences. HVR1 phenotypes were observed to be under strong purifying (stationary) and strong positive (antigenic drift) selection pressures which were co-incident with advancing patient age and cirrhosis of the liver. It followed that stationary viromes were dominated by a single HVR1 variant surrounded by minor variants comprised from conservative single amino acid substitutions events. We present evidence to suggest that neutralization antibody efficacy was diminished for stationary virome HVR1 variants. Our results identify HVR1 network structures, in chronic infection, as the preferential dominance of a single variant within a narrow sequence space.

    IMPORTANCE HCV infection is often asymptomatic and chronic infection is generally well established in advance of initial diagnosis and subsequent treatment. HVR1 can undergo rapid sequence evolution during acute infection and the variant pool is typically seen to diverge away from ancestral sequences as infection progresses from acute into chronic phase. In this report we describe HVR1 viromes in chronically infected patients that are defined by a dominant epitope located centrally within a narrow variant pool. Our findings suggest that weakened humoral immune activity, as a consequence of persistent chronic infection, allows for the acquisition and maintenance of host specific adaptive mutations at the HVR1 that reflect virus fitness.

  • A new mechanism of hepatic fibrogenesis: hepatitis C virus infection induces TGF-{beta}1 production through glucose-regulated protein 94 [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) is one of the leading causes of chronic liver inflammatory disease (hepatitis), which often leads to more severe diseases such as liver fibrosis, cirrhosis, and hepatocellular carcinoma. Liver fibrosis, in particular, is a major pathogenic consequence of HCV infection, and transforming growth factor-bbeta;1 (TGF-bbeta;1) plays a key role in its pathogenesis. Several HCV proteins have been suggested to either augment or suppress the expression of TGF-bbeta;1 by HCV-infected cells. Here, we report that TGF-bbeta;1 levels are elevated in HCV-infected hepatocytes cultured in vitro and in liver tissue of HCV patients. Notably, the level of TGF-bbeta;1 in media from in vitro-cultured HCV-infected hepatocytes was high enough to activate primary hepatic stellate cells isolated from rats. This indicates that TGF-bbeta;1 secreted by HCV-infected hepatocytes is likely to play a key role in the liver fibrosis observed in HCV patients. Moreover, we showed that HCV E2 protein triggers the production of TGF-bbeta;1 by HCV-infected cells through overproduction of glucose-regulated protein 94 (GRP94).

    Importance Hepatic fibrosis is a critical step in liver cirrhosis caused by hepatitis C virus infection. It is already known that immune cells, including Kupffer cells, mediate liver fibrosis. Recently, several papers have suggested that HCV-infected hepatocytes also significantly produce TGF-bbeta;1. Here, we provide the first examination of TGF-bbeta;1 levels in the hepatocytes of HCV patients. And using HCV culture system, we showed that HCV infection increases TGF-bbeta;1 production in hepatocytes. Furthermore, we confirmed that the amount of TGF-bbeta;1 secreted by HCV-infected hepatocytes was sufficient to activate primary hepatic stellate cells.

    To understand the molecular basis of TGF-bbeta;1 production in HCV-infected hepatocytes, we used HCV replicons and various stable cell lines. Finally, we elucidated that HCV E2 triggered TGF-bbeta;1 secretion via GRP94 mediated NF-B activation. This study contributes to the understanding of liver fibrosis by HCV, and suggests a new potential target (GRP94) for blocking liver cirrhosis in HCV patients.

  • Nef proteins from HIV-1 elite controllers are inefficient at preventing ADCC [PublishAheadOfPrint]

  • Impairment of Nef function, including reduced CD4 downregulation, was described in a subset of HIV-1-infected individuals that control viral replication without antiretroviral treatment (elite controllers, EC). Elimination of HIV-1 infected cells by antibody-dependent cellular cytotoxicity (ADCC) requires the presence of envelope glycoproteins (Env) in the CD4-bound conformation, raising the possibility that accumulating CD4 at the surface of virus-infected cells in EC could interact with Env and thereby sensitize these cells to ADCC. We observed a significant increase in the exposure of Env epitopes targeted by ADCC-mediating antibodies at the surface of cells expressing Nef isolates from EC; this correlated with enhanced susceptibility to ADCC. Altogether, our results suggest that enhanced susceptibility of HIV-1-infected cells to ADCC may contribute to the EC phenotype.

    Importance Nef clones derived from elite controllers (EC) have been shown to be attenuated for CD4-downregulation; how this contributes to the non-progressor phenotype of these infected individuals remains uncertain. Increasing evidence supports a role for HIV-specific antibody dependent cellular cytotoxicity (ADCC) in controlling viral infection and replication. Here we show that residual CD4 left at the surface of cells expressing Nef proteins isolated from ECs are sufficient to allow Env-CD4 interaction, leading to increased exposure of Env CD4-induced epitopes and increased susceptibility of infected cells to ADCC. Our results suggest that ADCC might be an active immune mechanism in EC that helps to maintain durable suppression of viral replication and low plasma viremia level in this rare subset of infected individuals. Therefore, targeting Nef's ability to downregulate CD4 could render HIV-1-infected cells susceptible to ADCC and thus have therapeutic utility.

  • Alpha-synuclein expression restricts RNA viral infections in the brain [PublishAheadOfPrint]

  • We have discovered that native, neuronal expression of alpha-synuclein (Asyn) inhibits viral infection, injury, and disease in the central nervous system. Enveloped RNA viruses such as West Nile virus (WNV) invade the central nervous system (CNS) and cause encephalitis; yet, little is known about the innate neuron-specific inhibitors of viral infections in the CNS. Following WNV-infection of primary neurons, we found that Asyn protein expression is increased. Infectious viral titer of WNV and Venezuelan equine encephalitis virus (VEEV) TC83 in the brains of Asyn knockout mice exhibited a mean increase of 104.5 infectious viral particles compared to wild-type and heterozygote littermates. Asyn knockout mice also exhibited significantly increased virus-induced mortality compared to Asyn heterozygote or homozygote control mice. Viral-induced Asyn localized to perinuclear, neuronal regions expressing viral envelope protein and the ER-associated trafficking protein, Rab1. In Asyn knockout primary neuronal cultures, ER signaling pathways known to support WNV replication are significantly elevated before and during viral infection compared to neurons expressing Asyn. We propose a model in which virus-induced Asyn localizes to ER-derived membranes, modulates virus-induced ER-stress signaling, and inhibits viral replication, growth, and injury in the CNS. These data provide a novel and important functional role for native alpha-synuclein expression, a protein that is closely associated with the development of Parkinson's disease.

    Importance Neuroinvasive viruses such as West Nile virus are able to infect neurons and cause severe disease such as encephalitis, infection of brain tissue. Following viral infection in the central nervous system only select neurons are infected, implying that neurons exhibit innate resistance to viral infections. We discovered that native neuronal expression of alpha-synuclein inhibited viral infection in the central nervous system. When the gene for alpha-synuclein was deleted, mice exhibited significantly decreased survival, markedly increased viral growth in the brain, and evidence of increased neuron injury. Virus-induced alpha-synuclein localized to intracellular neuron membranes, and in the absence of alpha-synuclein expression, specific endoplasmic reticulum stress signaling events were significantly increased. We describe a new neuron-specific inhibitor of viral infections in the central nervous system. Given the importance of alpha-synuclein as a cause of Parkinson's disease, these data also ascribe a novel functional role for native expression of alpha-synuclein in the CNS.

  • Animal model of sensorineural hearing loss associated with Lassa virus infection [PublishAheadOfPrint]

  • Approximately one third of Lassa virus (LASV) infected patients develop sensorineural hearing loss (SNHL) in the late stages of acute disease or in early convalescence. With 500,000 annual cases of Lassa Fever (LF), LASV is a major cause of hearing loss in endemic regions of West Africa. To date, no animal models exist that depict the human pathology of LF with associated hearing loss. Here, we aimed to develop an animal model to study LASV-induced hearing loss using human isolates from a 2012 Sierra Leone outbreak. We have recently established a murine model for LF that closely mimics many features of human disease. In this model, LASV isolated from a lethal human case was highly virulent while the virus isolated from a non-lethal case elicited mostly mild disease with moderate mortality. More importantly, both viruses were able to induce SNHL in surviving animals. However, utilization of the non-lethal, human LASV isolate allowed us to consistently produce large numbers of survivors with hearing loss. Surviving mice developed permanent hearing loss associated with mild damage to the cochlear hair cells and, strikingly, significant degeneration of the spiral ganglion cells of the auditory nerve. Therefore, the pathological changes in the inner ear of the mice with SNHL supported the phenotypic loss of hearing and provided further insights into the mechanistic cause of LF-associated hearing loss.

    Importance Sensorineural hearing loss is a major complication for LF survivors. The development of a small animal model of LASV infection that replicates hearing loss and the clinical and pathological features of LF will significantly increase knowledge of pathogenesis and vaccine studies. In addition, such a model will permit detailed characterization of the hearing loss mechanism and allow for the development of appropriate diagnostic approaches and medical care for LF patients with hearing impairment.

  • Detection of Quiescent Infections with Multiple Elephant Endotheliotropic Herpesviruses EEHV2, EEHV3, EEHV6 and EEHV7 within Lymphoid Lung Nodules or Lung and Spleen Tissue Samples from Five Asymptomatic Adult African Elephants. [PublishAheadOfPrint]

  • Summary: More than 80 cases of lethal hemorrhagic disease associated with Elephant Endotheliotropic Herpesviruses (EEHV) have been identified in young Asian elephants worldwide. Diagnostic PCR tests detected six types of EEHV in blood of acute disease cases, although EEHV1A is the predominant pathogenic type. Previously, the presence of herpesvirus virions within benign lung and skin nodules from healthy African elephants led to suggestions that African elephants may be the source of EEHV disease in Asian elephants. Here, we used direct PCR DNA sequencing to detect EEHV genomes in necropsy tissue from five healthy adult African elephants. Two large lung nodules collected from culled wild South African elephants contained high levels of either EEHV3 alone or of both EEHV2 and EEHV3. Similarly, a euthanized USA elephant proved to harbor multiple EEHV types distributed non-uniformly across four small lung nodules, including high levels of EEHV6, lower levels of EEHV3 and EEHV2, and a new GC-rich branch type EEHV7. Several of the same EEHV types were also detected in random lung and spleen samples from two other elephants. Sanger PCR DNA sequence data totaling 100-kb was obtained amongst the total of 15 different strains identified with (except for a few hypervariable genes) the EEHV2, EEHV3, EEHV6 strains all being closely related to known genotypes from acute disease cases, whereas the seven loci (4.0-kb) obtained from EEHV7 averaged 18% divergence from their nearest relative EEHV3. Overall, we conclude that these four EEHV species, but probably not EEHV1, occur commonly as quiescent infections in African elephants.

    Importance: Acute hemorrhagic disease characterized by high level viremia with members of the Proboscivirus genus threatens the future breeding success of endangered Asian elephants worldwide. Although the genomes of six EEHV types from acute cases have been partially or fully characterized, lethal disease predominantly involves a variety of strains of EEHV1 whose natural host has been unclear. Here we carried out partial PCR sequencing genotype analyses on necropsy tissue from five asymptomatic African elephants and identified multiple simultaneous infections by several different EEHV types, including high concentrations in lymphoid lung nodules. Overall, the results provide strong evidence that EEHV2, EEHV3, EEHV6 and EEHV7 represent natural ubiquitous infections in African elephants, whereas Asian elephants harbor EEHV1A, EEHV1B, EEHV4 and EEHV5. Although a single case of fatal cross-species infection by EEHV3 is known, the results do not support a previous concept that highly pathogenic EEHV1A crossed from African to Asian elephants in zoos.

  • Neuropeptide Y negatively influences monocyte recruitment to the CNS during retrovirus infection [PublishAheadOfPrint]

  • Monocyte infiltration into the CNS is a hallmark of several viral infections of the CNS, including retrovirus infection. Understanding the factors that mediate monocyte migration in the CNS is essential for the development of therapeutics that can alter the disease process. In the current study, we found that neuropeptide Y (NPY) suppressed monocyte recruitment to the CNS in a mouse model of polytropic retrovirus infection. NPY-/- mice had increased incidence and kinetics of retrovirus-induced neurological disease, which correlated with a significant increase in monocytes in the CNS compared to wildtype mice. Both Ly6Chi inflammatory and Ly6Clo alternatively activated monocytes were increased in the CNS of NPY-/- mice following virus infection, suggesting that NPY suppresses the infiltration of both cell types. Ex vivo analysis of myeloid cells from brain tissue demonstrated that infiltrating monocytes expressed high levels of the NPY receptor, Y2R. Correlating with the expression of Y2R on monocytes, treatment of NPY-/- mice with a truncated, Y2R-specific NPY peptide suppressed the incidence of retrovirus-induced neurological disease. These data demonstrate a clear role for NPY as a negative regulator of monocyte recruitment into the CNS and provides a new mechanism for suppression of retrovirus-induced neurological disease.

    IMPORTANCE: Monocyte recruitment to the brain is associated with multiple neurological diseases. However, the factors that influence the recruitment of these cells to the brain are still not well understood. In the current study, we found that neuropeptide Y, a protein produced by neurons, affected monocyte recruitment to the brain during retrovirus infection. We show that mice deficient in NPY have increased influx of monocytes into the brain and that this increase in monocytes correlates with neurological disease development. These studies provide a mechanism by which the nervous system, through the production of NPY, can suppress monocyte trafficking to the brain and reduce retrovirus-induced neurological disease.

  • Regulation of norovirus virulence by the VP1 protruding domain correlates with B cell infection efficiency [PublishAheadOfPrint]

  • Human noroviruses are a leading cause of gastroenteritis across the globe but the pathogenic mechanisms responsible for disease are not well-established. The availability of a murine norovirus model system provides the opportunity to elucidate viral and host determinants of virulence in a natural host. For example, previous studies have revealed that the protruding domain of the murine norovirus capsid protein VP1, and specifically residue 296 of VP1, regulates virulent infection. We identified a panel of nonsynonymous mutations in the ORF2 gene encoding VP1 that arose in persistently infected mice and tested whether these mutations conferred phenotypic changes to viral replication and virulence. Consistent with previous studies, we demonstrate that a glutamic acid at position 296 results in attenuation. For the first time, we also demonstrate that a lysine at this position is sufficient to confer virulence to an otherwise attenuated murine norovirus strain. Moreover, our studies reveal a direct correlation between the efficiency of viral replication in B cells and virulence. These data are especially striking because mutations causing reduced B cell replication and attenuation had minimal effect on the ability of the virus to replicate in macrophages. Thus, norovirus infection of B cells may directly contribute to disease outcome.

    IMPORTANCE Human noroviruses are a major global cause of disease yet we know very little about their pathogenic mechanisms. The availability of a murine norovirus model system facilitates investigation of noroviruses in a natural host organism and the identification of viral and host determinants of pathogenesis. We have identified a panel of mutations arising in the viral capsid protein VP1 during persistent infection of mice. Our data reveal that the protruding domain of VP1 regulates the ability of the virus to replicate in B cells and this directly correlates with virulence. Importantly, mutations impairing B cell infection had minimal effect on macrophage infection, revealing a potentially critical role for B cell infection in norovirus pathogenesis.

  • Crystal Structure of the Measles Virus Nucleoprotein Core in complex with an N-terminal Region of Phosphoprotein [PublishAheadOfPrint]

  • The enveloped negative-stranded RNA virus, measles virus (MeV) is an important human pathogen. The nucleoprotein (N0) assembles with the viral RNA into helical ribonucleocapsids (NC) which are in turn coated by a helical layer of the matrix protein. The viral polymerase complex uses the NC as its template. The N0 assembly onto the NC and the activity of the polymerase are regulated by the viral phosphoprotein (P). Here, we pulled down an N01-408 fragment lacking most of its C-terminal tail domain by several affinity-tagged, N-terminal, P fragments to map the N0-binding region of P to the first 48 amino acids. We showed biochemically and using P mutants the importance of the hydrophobic interactions for the binding. We fused an N0 binding peptide, P1-48, to the C-terminus of an N021-408 fragmentlacking both the N-terminal peptide and the C-terminal tail of N protein to reconstitute and crystallize the N0-P complex. We solved the X-ray structure of the resulting N0-P chimeric protein at 2.7 AAring; resolution. The structure reveals the molecular details of the conserved N0-P interface and explains how P chaperones N0 preventing both self-assembly of N0 and its binding to RNA. Finally, we propose a model for a pre-initiation complex for RNA polymerization.

    Importance Measles virus is an important, highly contagious, human pathogen. The nucleoprotein N binds only to viral genomic RNA and forms the helical ribonucleocapsid that serves as a template for viral replication. We address how N is regulated by another protein, the phosphoprotein, P, to prevent newly synthesized N from binding to cellular RNA. We describe the atomic model of an N-P complex and compare it to helical ribonucleocapsid. We thus provide insight into how P chaperones N and helps to start viral RNA synthesis. Our results provide a new insight into mechanisms of paramyxovirus replication. New data on the mechanisms of phosphoprotein chaperone action allows better understanding of the virus genome replication and nucleocapsid assembly. We describe a conserved structural interface for the N-P interaction which could be a target for drug development not only to treat measles but also potentially other paramyxovirus diseases.

  • Naturally-Occurring Major and Minor Capsid Protein Variants of HPV45: Differential Recognition by Cross-Neutralizing Antibodies Generated by the HPV Vaccines [PublishAheadOfPrint]

  • We investigated naturally occurring variation within the major (L1) and minor (L2) capsid proteins of human papillomavirus (HPV) genotype 45. Pseudoviruses (PsV) representing HPV45 sub-lineages A1, A2, A3, B1 and B2 exhibited comparable particle-to-infectivity ratios and morphology but demonstrated both increased (A2, A3 and B1) and decreased (B2) sensitivity to cross-neutralization by HPV vaccine antibodies compared to the A1 sub-lineage. Mutant PsV identified HI loop residue 357 as being critical for conferring this differential sensitivity.

  • Alveolar macrophages are a prominent but non-essential target for Murine cytomegalovirus infecting the lungs [PublishAheadOfPrint]

  • Cytomegaloviruses (CMVs) infect the lungs and cause pathological damage there in immunocompromised hosts. How lung infection starts is unknown. Inhaled murine CMV (MCMV) directly infected alveolar macrophages (AMs) and type 2 alveolar epithelial cells (AEC2s), but not type 1 AECs (AEC1s). By contrast Herpes simplex virus type 1 infected AEC1s, and Murid Herpesvirus-4 (MuHV-4) infected AEC1s via AMs. MCMV-infected AMs prominently expressed viral reporter genes from an HCMV IE1 promoter; but most IE1+ cells were AEC2s, and CD11c-cre mice, which express cre in AMs, switched llt;5% of floxed MCMV in the lungs. By contrast they switched ggt;90% of floxed MuHV-4 in the lungs and 50% of floxed MCMV in the blood. AM depletion also increased acute MCMV lung titers. Thus, their influence was more restrictive than permissive. Circulating monocytes entered infected lungs in large numbers and became infected, but not directly - this occurred mainly via AEC2s. MCMV lacking its m131/m129 chemokine homolog, which promotes macrophage infection, showed a modest difference in lung infiltration by Gr-1+ cells and no defect in lung infection. These results were consistent with myeloid cells mainly disseminating MCMV from the lungs, whereas AEC2s provided local amplification.

    Importance Cytomegaloviruses (CMVs) chronically and systemically infect most mammals. Human CMV infection is usually asymptomatic, but causes lung disease in people with poor immune function. As human infection is hard to analyse, related animal viruses provide important insights. We show that Murine CMV has two targets in the lungs: macrophages and surfactant-secreting epithelial cells. Acute virus replication occurred largely in the epithelial cells. Macrophages had an important defensive role, as removing them increased infection. These results establish the dual nature of lung infection, with local virus replication in epithelial cells, and spread via quiescently infected macrophages. Distinct therapies may be needed to target these contrasting events.

  • Cucumber necrosis virus recruits cellular heat shock protein 70 homologs at several stages of infection. [PublishAheadOfPrint]

  • RNA viruses often depend on host factors for multiplication inside cells due to the constraints of their small genome size and limited coding capacity. One such factor that has been exploited by several plant and animal viruses is heat shock protein 70 (HSP70) family homologs which have been shown to play roles for different viruses in viral RNA replication, viral assembly, disassembly and cell-to-cell movement. Using next generation sequence analysis we reveal that several isoforms of Hsp70 and Hsc70 transcripts are induced to very high levels during cucumber necrosis virus (CNV) infection of Nicotiana benthamiana and that HSP70 proteins are also induced by at least 10 fold. We show that HSP70 family protein homologs are co-opted by CNV at several stages of infection. We have found that overexpression of Hsp70 or Hsc70 leads to enhanced CNV genomic RNA, coat protein and virion accumulation whereas, downregulation leads to a corresponding decrease. Hsc70-2 was found to increase solubility of CNV CP in vitro and to increase accumulation of CNV CP independently of viral RNA replication during co-agroinfiltration in N. benthamiana. Additionally, virus particle assembly into virus-like particles in CP agroinfiltrated plants was increased in the presence of Hsc70-2. HSc70-2 was found to increase targeting of CNV CP to chloroplasts during infection reinforcing the role of HSP70 in chloroplast targeting of host proteins. Hence, our findings have led to the discovery of a highly induced host factor that has been co-opted to play multiple roles during several stages of the CNV infection cycle.

    Importance Because of the small size of its RNA genome, CNV is dependent on interaction with host cellular components to successfully complete its multiplication cycle. We have found that CNV induces HSP70 family homologs to a high level during infection, possibly as a result of the host response to the high levels of CNV proteins that accumulate during infection. Moreover, we have found that CNV co-opts HSP70 family homologs to facilitate several aspects of the CNV infection process such as viral RNA, coat protein and virus accumulation. Chloroplast targeting of the CNV CP is also facilitated, which may aid in CNV suppression of host defense responses. Several viruses have been shown to induce HSP70 during infection and others to utilize HSP70 for specific aspects of infection such as replication, assembly and disassembly. We speculate that HSP70 may play multiple roles in the infection processes of many viruses.

  • Characterisation of the localised immune response in the respiratory tract of ferrets following infection with influenza A and B viruses [PublishAheadOfPrint]

  • The burden of infection with seasonal influenza viruses is significant. Each year is typically characterised by the dominance of one (sub)type or lineage of influenza A or B virus, respectively. The incidence of disease varies annually and whilst this may be attributed to a particular virus strain or subtype, the impacts of prior immunity, population differences and variation in clinical assessment are also important. To improve our understanding of the impact of seasonal influenza viruses, we directly compared clinical symptoms, virus shedding and expression of cytokines, chemokines and immune mediators in the upper respiratory tract (URT) of ferrets infected with contemporary A(H1N1)pdm09, A(H3N2) or influenza B viruses. Gene expression was also assessed in the lower respiratory tract (LRT). Clinical symptoms were minimal. Overall cytokine/chemokine profiles in the URT were consistent in pattern and magnitude between animals infected with influenza A and B viruses and peak expression of IL1aalpha;, IL1bbeta;, IL6, IL12p40, IFNaalpha;, IFNbbeta; and TNFaalpha; mRNAs correlated with peak levels of viral shedding. MCP1 and IFN were expressed after the virus peak. Granzymes A and B and IL10 reached peak expression as virus cleared and seroconversion was detected. Cytokine/chemokine gene expression in the LRT following A(H1N1)pdm09 virus infection reflected the observations of URT, but were delayed by two-three days, as was virus replication. These data indicate that disease and localised immune responses following infection with seasonal influenza A and B viruses are similar, suggesting that other factors are likely to modulate the incidence and the impact of seasonal influenza.

    Importance Both influenza A and B viruses co-circulate in the human population and annual influenza seasons are typically dominated by an influenza A subtype or an influenza B lineage. Surveillance data indicates that the burden of disease is higher in some seasons, yet it is unclear whether this is due to specific virus strains or to other factors, such as cross reactive immunity or clinical definitions of influenza. We directly compared disease and the localised inflammatory response to different seasonal influenza strains, including the 2009 pandemic strain, in healthy naïve ferrets. We have found that disease, as well as the cytokine and chemokine responses, were similar irrespective of the seasonal strain or the location of the infection in the respiratory tract. This suggests that factors other than the immune response to a particular virus (sub)type contribute to the variable impact of influenza infection in a population.

  • Identification of Stabilizing Mutations in an H5 HA Influenza Virus Protein [PublishAheadOfPrint]

  • Highly pathogenic avian influenza viruses of the H5N1 subtype continue to circulate in poultry in Asia, Africa, and the Middle East. Recently, outbreaks of novel reassortant H5 viruses have also occurred in North America. Although the number of human infections with highly pathogenic H5N1 influenza viruses continues to rise, these viruses remain unable to efficiently transmit between humans. However, we and others have identified H5 viruses capable of respiratory droplet transmission in ferrets. Two experimentally introduced mutations in the viral hemagglutinin (HA) receptor-binding domain conferred binding to human-type receptors but reduced HA stability. Compensatory mutations in HA (acquired during virus replication in ferrets) were essential to restore HA stability. These stabilizing mutations in HA also affected the pH at which HA undergoes an irreversible switch to its fusogenic form in host endosomes, a crucial step for virus infectivity. To identify additional stabilizing mutations in an H5 HA, here we subjected a virus library possessing random mutations in the ectodomain of an H5 HA (altered to bind human-type receptors) to three rounds of treatment at 50 ddeg;C. We isolated several mutants that maintained their human-type receptor-binding preference, but acquired an appreciable increase in heat stability and underwent membrane fusion at a lower pH; collectively, these properties may aid H5 virus respiratory droplet transmission in mammals.

    Importance We have identified mutations in HA that increase its heat stability and affect the pH that triggers an irreversible conformational change (a prerequisite for virus infectivity). These mutations were identified in the genetic background of an H5 HA protein that was mutated to bind to human cells. The ability to bind to human-type receptors, together with physical stability and an altered pH threshold for HA conformational change, may facilitate avian influenza virus transmission via respiratory droplets in mammals.

  • Tumor suppressor IRF-1 counteracts germinal center reaction driven by a cancer-associated gammaherpesvirus. [PublishAheadOfPrint]

  • Gammaherpesviruses are ubiquitous pathogens that are associated with the development of B cell lymphomas. Gammaherpesviruses employ multiple mechanisms to transiently stimulate a broad, polyclonal germinal center reaction, an inherently mutagenic stage of B cell differentiation that is thought to be the primary target of malignant transformation in virus-driven lymphomagenesis. We found that this gammaherpesvirus-driven germinal center expansion was exaggerated and had lost its transient nature in the absence of interferon regulatory factor-1 (IRF-1), a transcription factor with antiviral and tumor suppressor functions. Uncontrolled and persistent expansion of germinal center B cells led to pathologic changes in the spleens of chronically infected IRF-1 deficient animals. Additionally, we found decreased IRF-1 expression in human post-transplant lymphoproliferative disorder, a malignant condition associated with gammaherpesvirus infection. The results of our study define an unappreciated role for IRF-1 in B cell biology and provide insight into the potential mechanism of gammaherpesvirus-driven lymphomagenesis.

    Significance. Gammaherpesviruses establish life-long infection in most adults and are associated with B cell lymphomas. While the infection is asymptomatic in many hosts, it is critical to identify individuals who may be at an increased risk of viral cancer. Such identification is currently impossible as the host risk factors that predispose towards viral lymphomagenesis are poorly understood. The current study identifies Interferon Regulatory Factor 1 (IRF-1) as one of such candidate host factors. Specifically, we found that IRF-1 enforces long-term suppression of an inherently mutagenic stage of B cell differentiation that gammaherpesviruses are thought to target for transformation. Correspondingly, in the absence of IRF-1, chronic gammaherpesvirus infection induced pathological changes in the spleens of infected animals. Further, we found decreased IRF-1 expression in human gammaherpesvirus-induced B cell malignancies.

  • EBNA3 proteins regulate EBNA2 binding to distinct RBPJ genomic sites [PublishAheadOfPrint]

  • Latent infection of B lymphocytes by Epstein-Barr virus (EBV) in vitro results in their immortalization into lymphoblastoid cell lines (LCLs); this latency program is controlled by the EBNA2 viral transcriptional activator that targets promoters via RBPJ, a DNA binding protein in the Notch signaling pathway. Three other EBNA3 proteins (EBNA3A, EBNA3B, and EBNA3C) interact with RBPJ to regulate cell gene expression. The mechanism by which EBNAs regulate different genes via RBPJ remains unclear. Our ChIP-seq analysis of the EBNA3 proteins analyzed in concert with prior EBNA2 and RBPJ data demonstrate that EBNA3A, EBNA3B and EBNA3C bind to distinct, partially overlapping genomic locations. Although RBPJ interaction is critical for EBNA3A and EBNA3C growth effects, only 30-40% of EBNA3 bound sites co-localize with RBPJ. Using LCLs conditional for EBNA3A or EBNA3C activity, we demonstrate that EBNA2 binding at sites near EBNA3A or EBNA3C regulated genes is specifically regulated by the respective EBNA3. To investigate EBNA3 binding specificity, we identified sequences and transcription factors enriched at EBNA3A, EBNA3B, and EBNA3C bound sites. This confirmed the prior observation that IRF4 is enriched at EBNA3A and EBNA3C bound sites and revealed IRF4 enrichment at EBNA3B bound sites. Using the IRF4 negative BJAB cell, we demonstrate that IRF4 is essential for EBNA3C, but not EBNA3A or EBNA3B binding to specific sites. These results support a model in which EBNA2 and EBNA3s compete for distinct subsets of RBPJ sites to regulate cell genes and where EBNA3 subset specificity is determined by interactions with other cell transcription factors.

    IMPORTANCE Epstein-Barr virus (EBV) latent gene products cause human cancers and transform B-lymphocytes into immortalized lymphoblastoid cell lines in vitro. EBV nuclear antigens (EBNAs) and membrane proteins constitutively activate pathways important for lymphocyte growth and survival. An important unresolved question is how four different EBNAs (2, 3A, 3B, and 3C) exert unique effects via a single transcription factor, RBPJ. Here we report that each EBNA binds to distinct but partially overlapping sets of genomic sites. EBNA3A and EBNA3C specifically regulate EBNA2's access to different RBPJ sites, providing a mechanism by which each EBNA can regulate distinct cell genes. We show IRF4, an essential regulator of B cell differentiation, is critical for EBNA3C binding specificity; EBNA3A and EBNA3B specificity is likely due to interactions with other cell transcription factors. EBNA3 titration of EBNA2 transcriptional function at distinct sites likely limits cell defenses that would be triggered by unchecked EBNA2 pro-oncogenic activity.

  • Determination of sequences required for HERV-K transduction and its recognition by foreign retroviral virions. [PublishAheadOfPrint]

  • Sequences necessary for transduction of HERV-Kcon, a consensus of the HERV-K(HML-2) family were analysed and found to reside in the leader/gag region. They act in an orientation-dependent way and consist of at least two sites working together. Having defined these sequences, we exploited this information to produce a simple system to investigate to what extent virions of HERV-Kcon, MLV and HIV-1 have the ability to transduce each other's genomes, leading to potential contamination of gene therapy vectors.

  • Functional Comparison of the Molluscum Contagiosum Virus vFLIP MC159 with the Murine Cytomegalovirus M36/vICA and M45/vIRA Proteins [PublishAheadOfPrint]

  • Molluscum contagiosum virus (MCV) gene MC159 encodes a viral FLICE inhibitory protein (vFLIP) that inhibits caspase-8-mediated apoptosis. The MC159 protein was also reported to inhibit programmed necrosis (necroptosis) and modulate NF-B activation by interacting with RIP1 and NEMO. The importance of MC159 during MCV infection has remained unknown as there is no system for propagation and genetic manipulation of this virus. Here we investigated the functions of MC159 during viral infection using murine cytomegalovirus (MCMV) as a surrogate virus. MC159 was inserted into the MCMV genome replacing M36 or M45, two MCMV genes with functions similar to those reported for MC159. M36 encodes a viral inhibitor of caspase-8-induced apoptosis (vICA) and M45 a viral inhibitor of RIP activation (vIRA), which inhibits RIP1/RIP3-mediated necroptosis. The M45 protein also blocks NF-B activation by interacting with NEMO. When expressed by MCMV, MC159 blocked TNFaalpha;-induced apoptosis of infected cells and partially restored MCMV replication in macrophages. However, MC159 did not fully replace M45 as it did not inhibit necroptosis in murine cells, but it reduced TNFaalpha;-induced necroptosis in MCMV-infected human HT-29 cells. MC159 also differed from M45 in its effect on NF-B. While MCMV-encoded M45 blocked NF-B activation by TNFaalpha; and IL-1bbeta;, MC159 inhibited TNFaalpha;- but not IL-1bbeta;-induced NF-B activation in infected mouse fibroblasts. These results indicate that the spectrum of MC159's functions differs depending on cell type and expression system and that a cell culture system for the propagation of MCV is needed to determine the biological relevance of presumed viral gene functions.

    Importance MCV is a human pathogenic poxvirus that cannot be propagated in cell culture or laboratory animals. Therefore, MCV gene products have been studied predominantly in cells expressing individual viral genes. In this study we analyzed the function of the MCV gene MC159 by expressing it from a different virus and comparing its functions to those of two well-characterized MCMV genes. In this system, MC159 displayed some but not all of the previously described functions, suggesting that functions of a viral gene depend on conditions under which it is expressed. Until a cell culture system for the analysis of MCV becomes available, it might be necessary to analyze MCV genes in several different systems to extrapolate their biological importance.

  • Structure-guided redesign increases the propensity of HIV Env to generate highly stable soluble trimers [PublishAheadOfPrint]

  • Due to high viral diversity, an effective HIV-1 vaccine will likely require Envs derived from multiple subtypes to generate broadly neutralizing antibodies (bNAbs). Soluble Env mimetics, like the NFL (native flexibly linked) and SOSIP trimers, derived from the subtype A BG505 Env, form homogeneous, stable native-like trimers. However, other Env sequences, such as JRFL and 16055 from subtypes B and C, do so to a lesser degree. The high-resolution BG505 SOSIP crystal structures permit the identification and redesign of Env elements involved in trimer stability. Here, we identified structure trimer-derived (TD) residues that increased the propensity of the subtype B JRFL and subtype C 16055-Env sequences to form well-ordered, homogenous and highly stable soluble trimers. The generation of these spike mimetics no longer required antibody-based selection, positive or negative. Using the redesigned subtype B and C trimer representatives as respective foundations, we further stabilized the NFL TD trimers by engineering an intra-protomer disulfide linkage in the pre-bridging sheet, I201C-A433C (CC) that locks the gp120 in the receptor non-triggered state. We demonstrated that this disulfide pair prevented CD4 induced-conformational rearrangements in NFL trimers derived from that prototypic subtype A, B and C representatives. Coupling the TD-based design with the engineered disulfide linkage, CC, increased the propensity of Env to form soluble highly stable spike mimetics that are resistant to CD4-induced changes. These advances will allow testing the hypothesis that such stabilized immunogens will more efficiently elicit neutralizing antibodies in small animal models and primates.

    Importance HIV-1 displays unprecedented global diversity circulating in the human population. Since the envelope glycoprotein (Env) is the target of neutralizing antibodies, Env-based vaccine candidates that address such diversity are needed. Soluble well-ordered Env mimetics, typified by NFL and SOSIP trimers are attractive vaccine candidates. However, the current designs do not allow most Envs to form well-ordered trimers. Here, we made design modifications to increase the propensity of representatives from two of the major HIV subtypes to form highly stable trimers. This approach should be applicable to other viral Envs, permitting the generation of a repertoire of homogeneous highly stable trimers. The availability of such an array will allow us to assess if sequential or cocktail immune strategies can overcome some of the vaccine challenges presented by HIV diversity.

  • Membrane perturbation-associated Ca2+ signalling and incoming genome sensing are required for the host response to low-level enveloped virus particle entry [PublishAheadOfPrint]

  • The type-I interferon (IFN) response is an important aspect of innate antiviral defense and the transcription factor IRF3 plays an important role in its induction. Membrane perturbation during fusion, a necessary step for enveloped virus particle entry, appears sufficient to induce transcription of a subset of IFN-stimulated genes (ISGs) in an IRF3-dependent, IFN-independent fashion. IRF3 is emerging as a central node in host cell stress responses, although it remains unclear how different forms of stress activate IRF3. Here we investigated the minimum number of Sendai virus (SeV) and human cytomegalovirus (HCMV) particles required to activate IRF3 and trigger an antiviral response. We found that Ca2+ signalling associated with membrane perturbation and recognition of incoming viral genomes by cytosolic nucleic acid receptors are required to activate IRF3 in response to fewer than 13 particles of SeV and 84 particles of HCMV per cell. Moreover, it appears that Ca2+ signalling is important for activation of STING and IRF3 following HCMV particle entry, suggesting that Ca2+ signalling sensitizes cells to recognize genomes within incoming virus particles. To our knowledge, this is the first evidence that cytosolic nucleic acid sensors recognize genomes within incoming virus particles prior to virus replication. These studies highlight the exquisite sensitivity of the cellular response to low level stimuli and suggest that virus particle entry is sensed as a stress signal.

    Importance The mechanism by which replicating viruses trigger IRF3 activation and type I IFN induction through the generation and accumulation of viral pathogen associated molecular patterns has been well characterized. However, the mechanism by which enveloped virus particle entry mediates a stress response, leading to IRF3 activation and the IFN-independent response, remained elusive. Here, we find that Ca2+ signalling associated with membrane perturbation appears to sensitize cells to recognize genomes within incoming virus particles. To our knowledge, this is the first study to show that cytosolic receptors recognize genomes within incoming virus particles prior to virus replication. These findings not only highlight the sensitivity of cellular responses to low level virus particle stimulation, but provide important insights as to how non-replicating virus vectors or synthetic lipid-based carriers used as clinical delivery vehicles activate innate immune responses.

  • Defining the microglia response during the time course of chronic neurodegeneration [PublishAheadOfPrint]

  • Inflammation has been proposed as a major component of neurodegenerative diseases although the precise role it plays has yet to be defined. We have examined the role of key contributors to this inflammatory process, microglia, the major resident immune cell population of the brain, in a prion disease model of chronic neurodegeneration. Initially, we performed an extensive reanalysis of a large study of prion disease, where the transcriptome of mouse brains had been monitored throughout the time-course of disease. Our analysis has provided a detailed classification of the disease-associated genes based on cell type of origin and gene function. This revealed that the genes up-regulated during disease, regardless of the strain of mouse or prion protein, are expressed predominately by activated microglia. In order to study the microglia contribution more specifically we established a mouse model of prion disease in which the 79A murine prion strain was introduced by an intraperitoneal route into BALB/cJFms-EGFP/- mice, which express Enhanced Green Fluorescent Protein (EGFP) under control of the c-fms operon. Samples were taken at time points during disease progression and histological analysis of the brain and transcriptional analysis of isolated microglia was carried out. The analysis of isolated microglia revealed a disease specific, highly pro-inflammatory signature in addition to an up-regulation of genes associated with metabolism and respiratory stress. This study strongly supports the growing recognition of the importance of microglia within the prion disease process and identifies the nature of the response through gene expression analysis of isolated microglia.

    Importance Inflammation has been proposed as a major component of neurodegenerative diseases. We have examined the role of key contributors to this inflammatory process, microglia, the major resident immune cell population of the brain, in a murine prion disease model of chronic neurodegeneration. Our study demonstrates that genes up-regulated throughout the disease process, are expressed predominately by microglia. A disease specific highly pro-inflammatory signature was observed in addition to an up-regulation of genes associated with metabolism and respiratory stress. This study strongly supports the growing recognition of the important contribution of microglia to a chronic neurodegenerative disease process.

  • Isolation and characterization of a novel bat coronavirus closely related to the direct progenitor of SARS coronavirus [PublishAheadOfPrint]

  • We report the isolation and characterization of a novel bat coronavirus which is much closer to the SARS coronavirus (SARS-CoV) in genomic sequence than others previously reported, particularly in the S gene. Cell entry and susceptibility studies indicated that this virus can use ACE2 as receptor and infect animal and human cell lines. Our results provide further evidence of bat origin of the SARS-CoV and highlight the likelihood of future bat coronavirus emergence in humans.

  • Plasmacytoid dendritic cells require direct infection to sustain the pulmonary influenza A virus-specific CD8 T cell response [PublishAheadOfPrint]

  • Following influenza A virus infection (IAV) development of a robust IAV-specific CD8 T cell response is required for clearance of primary infection and enhances memory protection. Following IAV infection, plasmacytoid dendritic cells (pDC) or CD8aalpha;+ DC regulated pulmonary effector CD8 T cell responses within the lung. Without this DC: T cell interaction, insufficient effector CD8 T cells are maintained in the lungs, leading to enhanced morbidity and mortality. Previous studies have demonstrated pDC are capable of classical- or cross-presentation of IAV antigens and could potentially regulate IAV-specific CD8 T cell responses through either mechanism. Our results demonstrate pDC from the lungs of donor mice infected with an IAV that is not able to replicate in hematopoietic cells (142t-IAV), unlike donor pDC isolated from the lungs of control infected mice, are not able to rescue the host IAV-specific CD8 T cell response from apoptosis. This indicates pDC must utilize the direct presentation pathway for this rescue. This inability to of pDC from 142t-IAV donors to rescue the IAV-specific CD8 T cell response is not due to differences in the overall ability of the 142t-IAV to replicate within the lungs, generate defective viral genomes or differences in levels of costimulatory molecules required for this interaction. We further demonstrate that bypassing the antigen presentation pathway by coating the 142t-IAV pDC with IAV peptide epitopes restores their ability to rescue the IAV-specific CD8 T cell response.

    Importance IAV continues to be a global health burden that infects 5-20% of the global population annual. Continued investigation into the mechanisms that mediate protective immune responses against IAV is important to improving current vaccination and antiviral strategies antagonistic towards IAV. Our findings presented in this manuscript demonstrate a key requirement for pDC promotion of effector CD8 T cell survival: that rather than utilize cross-presentation, pDC must be infected and utilize the endogenous pathway for presentation of antigens to CD8 T cells during in vivo IAV infections. This suggests that targeting presentation via the endogenous pathway in pDC could be important for the development of unique antiviral cellular therapies.

  • The role of cysteines in stabilizing the randomized receptor binding domains within feline leukemia virus Envelope proteins. [PublishAheadOfPrint]

  • Retargeting of gammaretroviral envelope proteins has shown promising results in the isolation of novel isolates with therapeutic potential. However, the optimal conditions required to obtain high-affinity retargeted envelope proteins with narrow tropism is not understood. This study highlights the advantage of constrained peptides within receptor-binding domains and validates the random library screening technique of obtaining novel retargeted Env proteins. Using a modified vector backbone to screen the Envelope libraries on 143B osteosarcoma cells, three novel and unique retargeted Envelopes were isolated. The use of complex disulfide bonds within variable regions required for receptor binding is found within natural gammaretroviral Envelope isolates. Interestingly, two of the isolates, named AII and BV2, have a pair of cysteines located within the randomized region of 11 amino acids similar to that identified within the CP Env, an isolate identified in a previous Env library screen on the human renal carcinoma Caki-1 cell line. The amino acids within the randomized region of AII and BV2 Envelopes that are essential for viral infection have been identified here and include these cysteine residues. Through mutagenesis studies, the putative disulfide bond pairs including and beyond the randomized region were examined. In parallel, the disulfide bonds of CP Env were identified using mass spectrometry. The results indicate that this pair of cysteines creates the structural context to position key hydrophobic (F, W) and basic (K, H) residues critical for viral titer and suggest that AII, BV2 and CP internal cysteines are bonding together in distinct ways.

    Importance Retargeted gammaretroviral particles have broad applications for therapeutic use. Although great advances have been achieved in identifying new Env:host cell receptor pairs, the rules for designing optimal Env libraries are still unclear. We have found that isolates with an additional pair of cysteines within the randomized region have the highest transduction efficiencies. This emphasizes the importance of considering cysteine pairs in the design of new libraries. Furthermore, our data clearly indicates that these cysteines are essential for viral infectivity by presenting essential residues to the host-cell receptor. These studies facilitate the screening of Env libraries for functional entry into target cells, allowing the identification of novel gammaretroviral Envs targeting alternative host-cell receptors for gene and protein delivery.

  • HCMV US28 is important for latent infection of hematopoietic progenitor cells [PublishAheadOfPrint]

  • Human cytomegalovirus (HCMV) resides latently in hematopoietic progenitor cells (HPCs). During latency, only a subset of HCMV genes is transcribed, including one of the four viral-encoded G protein-coupled receptors (GPCRs), US28. Although US28 is a multifunctional lytic protein, its function during latency remained undefined. We generated a panel of US28 recombinant viruses in the bacterial artificial (BAC)-derived clinical HCMV strain, TB40/E-mCherry. We deleted the entire US28 open reading frame (ORF), deleted all four of the viral GPCR ORFs, or deleted three of the HCMV GPCRs, while leaving US28 wild type. Using these recombinant viruses, we assessed the requirement for US28 during latency in the Kasumi-3 in vitro latency model system and in primary ex vivo cultured CD34+ HPCs. Our data suggests that US28 is required for latency, as infection with viruses lacking the US28 ORF alone or in combination with the remaining HCMV-encoded GPCR results in transcription from the major immediate early promoter, the production of extracellular virions, and the production of infectious virus capable of infecting naïve fibroblasts. The other HCMV GPCRs are not required for this phenotype, as a virus expressing only US28 but not the remaining viral-encoded GPCRs is phenotypically similar to that of wild type latent infection. Finally, we found that US28 co-purifies with mature virions and is expressed in HPCs upon virus entry, although its expression at the time of infection does not complement the US28-deletion latency phenotype. This work suggests that US28 protein functions to promote a latent state within hematopoietic progenitor cells.

    IMPORTANCE Human cytomegalovirus (HCMV) is a widespread pathogen that once acquired, remains with its host for life. HCMV remains latent, or quiescent, in cells of the hematopoietic compartment and upon immune challenge, can reactivate to cause disease. HCMV-encoded US28 is one of several genes expressed during latency, although its biological function during this phase of infection remained undefined. Herein, we show that US28 aids in promoting experimental latency in tissue culture.

  • PIV-3 blocks antiviral mediators downstream of the IFN-{lambda}R by modulating Stat1 phosphorylation [PublishAheadOfPrint]

  • Parainfluenza viruses are known to inhibit type I interferon (IFN) production, however there is a lack of information regarding the type III IFN response during infection. Type III IFNs signal through a unique heterodimeric receptor, the IFN-R1/IL-10R2, which is primarily expressed by epithelial cells. Parainfluenza virus type 3 (PIV-3) infection is highly restricted to the airway epithelium. We therefore sought to examine type III IFN signaling pathways during PIV-3 infection of epithelial cells. We used three strains of PIV-3: human PIV-3 (HPIV-3), bovine PIV-3 (BPIV-3), and dolphin PIV-1 (TtPIV-1). Here we show that message levels of IL-29 are significantly increased during PIV-3 infection, yet downstream antiviral signaling molecules are not upregulated to levels similar to the positive control. Furthermore, in Vero cells infected with PIV-3, stimulation with recombinant IL-29/28A/28B does not cause upregulation of downstream antiviral molecules, suggesting that PIV-3 interferes with the JAK/STAT pathway downstream of the IFN-R1/IL-10R2 receptor. We used western blotting to examine the phosphorylation of Stat1 and Stat2 in Vero and the bronchial epithelial cell line BEAS-2B. In Vero cells, we observed reduced phosphorylation of the serine 727 (S727) site on Stat1, while in BEAS-2B cells Stat1 phosphorylation was decreased at the tyrosine 701 (Y701) site during PIV-3 infection. PIV-3 therefore interferes with the phosphorylation of Stat1 downstream of the type III IFN receptor. These data provide new evidence regarding strategies employed by parainfluenza viruses to effectively circumvent respiratory epithelial cell-specific antiviral immunity.

    Importance Parainfluenza virus (PIV) in humans is associated with bronchiolitis and pneumonia and can be especially problematic in infants and the elderly. Also seen in cattle, bovine PIV-3 causes respiratory infections in young calves. In addition, PIV-3 is one of a number of pathogens that contributes to the bovine respiratory disease complex (BRDC). As their name suggests, interferons (IFNs) are produced by cells to interfere with viral replication. Paramyxoviruses have previously been shown to block production and downstream signaling of type I IFNs. For the first time, it is shown here that PIV-3 can induce protective type III IFNs in epithelial cells, the primary site of PIV-3 infection. However, we found that PIV-3 modulates signaling pathways downstream of the type III IFN receptor to block production of several specific molecules that aid in a productive antiviral response. Importantly, this work expands our understanding of how PIV-3 effectively evades host innate immunity.

  • Nonstructural 5A Protein of Hepatitis C Virus Regulates Soluble Resistance-related Calcium-binding Protein Activity for Viral Propagation [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) is a major cause of chronic liver disease and is highly dependent on cellular proteins for virus propagation. To identify the cellular factors involved in HCV propagation, we recently performed protein microarray assays using the HCV nonstructural 5A (NS5A) protein as a probe. Of 90 cellular protein candidates, we selected soluble resistance-related calcium-binding protein (sorcin) for further characterization. Sorcin is a calcium binding protein and is highly expressed in certain cancer cells. We verified that NS5A interacted with sorcin through domain I of NS5A and phosphorylation of the threonine residue 155 of sorcin played a crucial role in protein interaction. siRNA-mediated knockdown of sorcin impaired HCV propagation. Silencing of sorcin expression resulted in decrease of HCV assembly without affecting HCV RNA and protein levels. We further demonstrated that polo-like kinase 1 (PLK1)-mediated phosphorylation of sorcin was increased by NS5A. We showed that both phosphorylation and calcium binding activity of sorcin was required for HCV propagation. These data indicate that HCV modulates sorcin activity via NS5A protein for its own propagation.

    IMPORTANCE Sorcin is a calcium binding protein and regulates intracellular calcium homeostasis. HCV NS5A interacts with sorcin and phosphorylation of sorcin is required for protein interaction. Gene silencing of sorcin impaired HCV propagation at the assembly step of the HCV life cycle. Sorcin is phosphorylated by PLK1 via protein interplay. We showed that sorcin interacted with both NS5A and PLK1, and PLK1-mediated phosphorylation of sorcin was increased by NS5A. Moreover, calcium-binding activity of sorcin played a crucial in HCV propagation. These data provide evidence that HCV regulates host calcium metabolism for virus propagation and thus manipulation of sorcin activity may represent a novel therapeutic target for HCV.

  • Hemagglutinin stalk immunity reduces influenza virus replication and transmission in ferrets [PublishAheadOfPrint]

  • We assessed in ferrets whether influenza virus hemagglutinin stalk-based immunity provided protection against aerosol-transmitted H1N1 infection. Immunization of ferrets with a universal influenza virus vaccine strategy based on viral vectors expressing chimeric hemagglutinin constructs induced stalk-specific antibody responses. Stalk-immunized ferrets were co-housed with H1N1-infected ferrets under conditions that permitted virus transmission. Hemagglutinin stalk-immunized ferrets had lower viral titers, delayed or no virus replication at all following natural exposure to influenza virus.

  • Functional Genomics Reveals Linkers Critical for Influenza Polymerase [PublishAheadOfPrint]

  • Influenza virus mRNA synthesis by the RNA-dependent RNA polymerase involves binding and cleavage of capped cellular mRNA by the PB2 and PA subunits, respectively, and extension of viral mRNA by PB1. However, the mechanism for such a dynamic process is unclear. Using high-throughput mutagenesis and sequencing analysis, we have not only generated a comprehensive functional map for the microdomains of individual subunits, but have also revealed the PA linker to be critical for polymerase activity. This PA linker binds to PB1 and also forms ionic interactions with the PA C-terminal channel. Nearly all mutants with five amino acid insertions in the linker were non-viable. Our model further suggests that the PA linker may play an important role in the conformational changes that occur between stages that favor capped mRNA binding and cleavage and those associated with viral mRNA synthesis.

    Importance The RNA-dependent RNA polymerase of influenza virus consists of the PB1, PB2 and PA subunits. By combining genome-wide mutagenesis analysis with the recently discovered crystal structure of the influenza polymerase heterotrimer, we generated a comprehensive functional map of the entire influenza polymerase complex. We identified the microdomains of individual subunits, including the catalytic domains, the interaction interfaces between subunits, and nine linkers interconnecting different domains. Interestingly, we found that mutants with five amino acid insertions in individual linkers were non-viable, suggesting the critical roles these linkers play in coordinating spatial relationships between the subunits. We furthermore identified an extended PA linker that binds to PB1 and also forms ionic interactions with the PA C-terminal channel.

  • Immunogenicity of a Prefusion HIV-1-Envelope Trimer in Complex with a Quaternary-Specific Antibody [PublishAheadOfPrint]

  • The HIV-1 envelope trimer (Env) is the target of broadly neutralizing antibodies and is being explored as a vaccine candidate to elicit protective antibodies. One of the most promising antigenic and structural mimics of HIV-1 Env is the SOSIP.664-stabilized soluble trimer from the clade A strain BG505, which is preferentially recognized by broadly neutralizing antibodies. Trimer immunization elicits high titer neutralization to the autologous tier 2-BG505 strain; however breadth is limited, and substantial interest has focused on understanding and improving trimer immunogenicity. We sought to improve the antigenic specificity of BG505 SOSIP.664 by reducing recognition of the variable loop 3 (V3) region, which elicits only weakly neutralizing antibodies. To stabilize the trimer in its prefusion closed conformation, we complexed trimeric BG505 SOSIP.664 with the antigen-binding fragment (Fab) of PGT145, a broadly neutralizing quaternary-specific antibody. Compared to the ligand-free trimer, the PGT145 Fab-BG505 SOSIP.664 complex displayed increased melting temperature stability and reduced V3 recognition. In guinea pigs, immunization with the PGT145 Fab-BG505 SOSIP.664 complex elicited ~100-fold lower V3-directed binding and neutralization titers compared to ligand-free BG505 SOSIP.664. Both complexed and ligand-free BG505 SOSIP.664 elicited comparable neutralization of the autologous BG505 virus and in both cases BG505 neutralization mapped to the outer domain of gp120 in some guinea pigs. Our results indicate that it is possible to reduce immune recognition of the V3-region of the trimer while maintaining the antigenic profile needed to induce autologous neutralizing antibodies. These data suggest that appropriate modifications of trimer immunogens could further focus the immune response on key neutralization epitopes.

    Importance HIV-1-Env trimers have been proposed as preferred HIV-1 vaccine immunogens. One version, BG505 SOSIP.664, a soluble stabilized trimer, has recently been shown to elicit high titer autologous neutralizing antibodies (NAbs) in rabbits. Here we compared two immunogens: the ligand-free BG505 SOSIP.664 trimer and this same trimer bound to the antigen-binding fragment (Fab) of antibody PGT145, a broadly neutralizing antibody which recognizes the trimer at its membrane-distal apex. We hypothesized that the Fab-bound complex would stabilize BG505 SOSIP.664 in its prefusion closed conformation and limit reactivity to weakly neutralizing antibodies targeting the variable loop 3 (V3) region. In guinea pigs, the Fab-complexed trimer induced 100-fold lower responses to the V3 region, while both ligand-free and Fab-complexed trimer elicited similar levels of autologous NAbs. Our findings demonstrate the potential to reduce "off-target" immunogenicity while maintaining the capacity to generate autologous NAbs.

  • Native conformation and canonical disulfide bond formation are interlinked properties of HIV-1 Env glycoproteins [PublishAheadOfPrint]

  • We investigated whether there is any association between native-like conformation and the presence of only the canonical (i.e., native) disulfide bonds in the gp120 subunits of a soluble, recombinant human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein. We used a mass spectrometry (MS)-based method to map the disulfide bonds present in non-native uncleaved gp140s and native-like SOSIP.664 trimers based on the BG505 env gene. Our results show that uncleaved gp140 proteins were not homogenous, in that substantial sub-populations (20-80%) contained aberrant disulfide bonds. In contrast, the gp120 subunits of the native-like SOSIP.664 trimer almost exclusively retained the canonical disulfide bond pattern. We also observed that the purification method could influence the proportion of an Env protein population that contained aberrant disulfide bonds. We infer that gp140 proteins may always contain a variable but substantial proportion of aberrant disulfide bonds, but that the impact of this problem can be minimized via design and/or purification strategies that yield native-like trimers. The same factors may also be relevant to the production and purification of monomeric gp120 proteins that are free of aberrant disulfide bonds.

    Importance It is widely thought that a successful HIV-1 vaccine will include a recombinant form of the Env protein, a trimer located on the virion surface. To increase yield and simplify purification, Env proteins are often made in truncated, soluble forms. A consequence, however, can be the loss of the native conformation associated with the virion-associated trimer. Moreover, some soluble recombinant Env proteins contain aberrant disulfide bonds that are not expected to be present in the native trimer. To assess whether these observations are linked, to determine the extent of disulfide bond scrambling, and to understand why scrambling occurs, we determined the disulfide bond profiles of two different soluble Env protein designs that are being assessed as vaccine candidates. We found that uncleaved gp140 forms heterogeneous mixtures in which aberrant disulfide bonds abound. In contrast, BG505 SOSIP.664 trimers are more homogenous, native-like entities that contain predominantly the native disulfide bond profile.

  • Polyprotein driven formation of two interdependent sets of complexes supporting hepatitis C virus genome replication [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) requires proteins from the NS3-NS5B polyprotein to create a replicase unit for replication of its genome. The replicase proteins form membranous compartments in cells to facilitate replication, but little is known about their functional organization within these structures. We recently reported on intragenomic replicons, bicistronic viral transcripts expressing an authentic replicase from ORF2 and a second duplicate NS polyprotein from ORF1. Using these constructs and other methods, we have assessed polyprotein requirements needed for rescue of different lethal point mutations across NS3-5B. Mutations readily tractable to rescue broadly fell into two groupings; those requiring expression of a minimum NS3-5A and those requiring expression of a minimum NS3-5B polyprotein. A cis-acting mutation that blocked NS3 helicase activity, T1299A, was tolerated when introduced into either ORF within the intragenomic replicon, but unlike many other mutations required the other ORF to express a functional NS3-5B. Three mutations were identified as more refractile to rescue; one that blocked cleavage of the NS4B5A boundary (S1977P), another in the NS3 helicase (K1240N) and a third in NS4A (V1665G). Introduced into ORF1, these exhibited a dominant negative phenotype, but with K1240N inhibiting replication as a minimum NS3-5A polyprotein whereas V1665G and S1977P only impaired replication as a NS3-5B polyprotein. Furthermore, a S1977P mutated NS3-5A polyprotein complemented other defects shown to be dependent on NS3-5A for rescue. Overall, our findings suggest the existence of two inter-dependent sets of protein complexes supporting RNA replication, distinguishable by the minimum polyprotein requirement needed for their formation.

    Importance Positive strand RNA viruses reshape the intracellular membranes of cells to form a compartment within which to replicate their genome, but little is known about functional organization of viral proteins within this structure. We have complemented protein-encoded defects in HCV by constructing sub-genomic HCV transcripts capable of simultaneously expressing both a mutated and functional polyprotein precursor needed for RNA genome replication (intragenomic replicons). Our results reveal that HCV relies on two interdependent sets of protein complexes to support viral replication. They also show that the intragenomic replicon offers a unique way to study replication complex assembly as it enables improved composite polyprotein complex formation compared to traditional trans-complementation systems. Finally, the differential behaviour of distinct NS3 helicase knock-out mutations hints that certain conformations of this enzyme might be particularly deleterious for replication.

  • Structural evolution of the emerging 2014/15 GII.17 noroviruses [PublishAheadOfPrint]

  • Recent reports suggest that human GII.17 noroviruses are increasing in prevalence. We analyzed the evolutionary changes of three GII.17 capsid protruding (P) domains. We found that the GII.17 P domains had little cross-reactivity with antisera raised against the dominant GII.4 strains. X-ray structural analysis of GII.17 P domains from 2002 to 2014/15 suggested that surface exposed substitutions on the uppermost part of the P domain might have generated a novel 2014/15 GII.17 variant.

  • European academies advise on Gain of Function studies in influenza virus research [PublishAheadOfPrint]

  • Gain of Function (GoF) studies to understand factors affecting transmissibility of potentially pandemic pathogens are controversial. EASAC recently published consensus recommendations relating to GoF research review and management on: self-regulation and harmonisation; bioethical considerations; benefit-risk assessment; biosafety and biosecurity advisory options; and publication of sensitive information. A layered approach to integration of responsibilities must include conforming to the stringent rules and guidance already existing. Further commitment is essential to extend the debate on issues worldwide.

  • Dual role of HSV-1 pUS9 in virus anterograde axonal transport and final assembly in growth cones in distal axons [PublishAheadOfPrint]

  • The herpes simplex virus type 1 (HSV-1) envelope protein pUS9 plays an important role in virus anterograde axonal transport and spread from neuronal axons. In this study, we have used both confocal microscopy and transmission electron microscopy (TEM) to examine the role of pUS9 on the anterograde transport and assembly of HSV-1 in the distal axon of human and rat DRG neurons using US9 deletion, repair and wild-type (F, 17 and KOS strain) viruses. Using confocal microscopy and single and tri-chamber culture systems, we observed a reduction but not complete block in the anterograde axonal transport of capsids to distal axons but a marked (~90%) reduction in virus spread from axons to Vero cells with the US9 deletion viruses. Axonal transport of glycoproteins (gC, gD and gE) was unaffected. Using TEM, there was a marked reduction or absence of enveloped capsids, in varicosities and growth cones, in KOS strain and US9 deletion viruses, respectively. Capsids (40-75%) in varicosities and growth cones infected with strain 17, F and US9 repair viruses were fully enveloped compared to less than 5% of capsids found in distal axons infected with KOS strain (which also lacks pUS9) and still lower (llt;2%) with the US9 deletion viruses. Hence, there was a secondary defect in virus assembly in distal axons in the absence of pUS9 despite the presence of key envelope proteins. Overall, our study supports a dual role for pUS9, firstly in anterograde axonal transport and secondly in virus assembly in growth cones in distal axons.

    IMPORTANCE HSV-1 has evolved mechanisms for its efficient transport along sensory axons and subsequent spread from axons to epithelial cells after reactivation. In this study, we show that deletion of the envelope protein pUS9 leads to defects in virus transport along axons (partial defect) and in virus assembly and egress from growth cones (marked defect). Virus assembly and exit in the neuronal cell body is not impaired in the absence of pUS9. Thus, our findings indicate that pUS9 contributes to the overall HSV-1 anterograde axonal transport, including a major role in virus assembly at the axon terminus, which is not essential in the neuronal cell body. Overall, our data suggests that the process of virus assembly at the growth cones differs from that in the neuronal cell body and that HSV-1 has evolved different mechanisms for virus assembly and exit from different cellular compartments.

  • Lipid membranes facilitate conformational changes required for reovirus cell entry [PublishAheadOfPrint]

  • Cellular entry of nonenveloped and enveloped viruses is often accompanied by dramatic conformational changes within viral structural proteins. These rearrangements are triggered by a variety of mechanisms, such as low pH, virus-receptor interactions, and virus-host chaperone interactions. Reoviruses, a model system for entry of nonenveloped viruses, undergo a series of disassembly steps within the host endosome. One of these steps, ISVP-to-ISVP* conversion, is necessary for delivering the genome-containing viral core into host cells, but the physiological trigger that mediates ISVP-to-ISVP* conversion during cell entry is unknown. Structural studies of the reovirus membrane penetration protein, mmu;1, predict that interactions between mmu;1 and negatively charged lipid head groups may promote ISVP* formation; however, experimental evidence for this idea is lacking. Here, we show that the presence of polyanions (SO42- and HPO42-) or lipids in the form of liposomes, facilitate ISVP-to-ISVP* conversion. The requirement for charged lipids appears to be selective since phosphatidylcholine and phosphatidylethanolamine promote ISVP* formation, whereas other lipids, such as sphingomyelin and sulfatide, either did not affect ISVP* formation or prevented ISVP* formation. Thus, our work provides evidence that interactions with membranes can function as a trigger for a nonenveloped virus to gain entry into host cells.

    IMPORTANCE Cell entry, a critical stage in the virus life cycle, concludes with delivery of the viral genetic material across host membranes. Regulated structural transitions within nonenveloped and enveloped viruses are necessary for accomplishing this step; these conformational changes are predominantly triggered by low pH and/or interactions with host proteins. In this work, we describe a previously unknown trigger, interactions with lipid membranes, which can induce structural rearrangements required for cell entry. This mechanism operates during entry of mammalian orthoreoviruses. We show that interactions between reovirus entry intermediates and lipid membranes devoid of host proteins promote conformational changes within the viral outer capsid that lead to membrane penetration. Thus, this work illustrates a novel strategy that nonenveloped viruses can use to gain access into cells, and how viruses usurp disparate host factors to initiate infection.

  • Antigenic and cryo-electron microscopy structure analysis of a chimeric sapovirus capsid [PublishAheadOfPrint]

  • The capsid protein (VP1) of all caliciviruses forms an icosahedral particle with two principal domains, shell (S) and protruding (P) domains, which are connected via a flexible hinge region. The S domain forms a scaffold surrounding the nucleic acid, while the P domains form a homodimer that interacts with receptors. The P domain is further subdivided into two subdomains, termed P1 and P2. The P2 subdomain is likely an insert in the P1 subdomain; consequently, the P domain is divided into the P1-1, P2, and P1-2. In order to investigate capsid antigenicity, the N-term/S/P1-1 and P2/P1-2 were switched between two sapovirus genotypes GI.1 and GI.5. The chimeric VP1 constructs were expressed in insect cells and these were shown to self-assemble into virus-like particles (VLPs) morphologically similar to the parental VLPs. Interestingly, the chimeric VLPs had higher levels of cross-reactivities against heterogeneous antisera when compared to the parental VLPs. In order to better understand the antigenicity from a structural perspective, we determined an intermediate-resolution (8.5 AAring;) cryo-EM structure of a chimeric VLP and developed a VP1 homology model. The cryo-EM structure revealed that the P domain dimers were slightly raised (~5 AAring;) above the S domain. The VP1 homology model allowed us predict the S domain (67-229) and P1-1 (229-280), P2 (281-447), and P1-2 (448-567) subdomains. Our results suggested that the raised P dimers might expose immuno-reactive S/P1-1 subdomain epitopes. Consequently, the higher levels of cross-reactivities with the chimeric VLPs resulted from a combination of GI.1 and GI.5 epitopes.

    IMPORTANCE We developed sapovirus chimeric VP1 constructs and produced the chimeric VLPs in insect cells. We found that both chimeric VLPs had a higher level of cross-reactivity against heterogeneous VLP antisera when compared to the parental VLPs. The cryo-EM structure of one chimeric VLP (Yokote/Mc114) was solved to 8.5 AAring; resolution. A homology model of the VP1 indicated for the first time the putative S and P (P1-1, P2, and P1-2) domains. Yokote/Mc114 overall structure contained common features among other caliciviruses. We showed that the P2 subdomain was mainly involved in the homodimeric interface, whereas a large gap between the P1 subdomains had less interactions.

  • Generation of recombinant Oropouche orthobunyaviruses lacking the non-structural proteins NSm or NSs [PublishAheadOfPrint]

  • Oropouche virus (OROV) is a midge-borne human pathogen with a geographic distribution in South America. OROV was first isolated in 1955 and since then is known to cause recurring outbreaks of a dengue-like illness in the Amazonian regions of Brazil. OROV however, remains amongst one of the poorly understood emerging viral zoonosis. Here we describe the successful rescue of infectious OROV entirely from cDNA copies of its genome and generation of OROV mutant viruses lacking either the NSm or the NSs coding regions. Characterisation of the recombinant viruses carried out in vitro demonstrated that the NSs protein of OROV is an IFN antagonist as in other NSs-encoding bunyaviruses. Additionally, we demonstrate the importance of the nine C-terminal amino acids of OROV NSs in IFN antagonistic activity. OROV was also found to be sensitive to IFN-aalpha; when cells were pre-treated, however the virus was still capable of replicating at doses as high as 10,000 U/ml of IFN-aalpha; in contrast to the family prototype BUNV. We found that the OROV lacking a complete NSm protein displayed characteristics similar to the wild-type virus, suggesting that the NSm protein is dispensable for virus replication in mammalian and mosquito cell-lines that were tested.

    IMPORTANCE Oropouche virus (OROV) is a public health threat in Central and South America where it causes periodic outbreaks of dengue-like illness. In Brazil, OROV is the second most frequent cause of arboviral febrile illness after dengue virus and with the current rates of urban expansion more cases of this emerging viral zoonosis could occur. To better understand the molecular biology of OROV we have successfully rescued the virus along with mutants. We have established that the C-terminus of the NSs protein is important in interferon antagonism and that the NSm protein is dispensable for virus replication in cell-culture. The tools described in this paper are important in terms of understanding this important yet neglected human pathogen.

  • A diverse panel of Hepatitis C Virus glycoproteins for use in vaccine research reveals extremes of monoclonal antibody neutralization resistance [PublishAheadOfPrint]

  • Despite significant advances in the treatment of hepatitis C virus (HCV) infection, the need to develop preventative vaccines remains. Identification of the best vaccine candidates and evaluation of their performance in pre-clinical and clinical development will require appropriate neutralisation assays utilizing diverse HCV isolates. We aimed to generate and characterise a panel of HCV E1E2 glycoproteins suitable for subsequent use in vaccine and therapeutic antibody testing. Full-length E1E2 clones were PCR amplified from patient-derived serum samples, cloned into an expression vector and used to generate viral pseudoparticles (HCVpp). In addition, some of these clones were used to generate cell culture infectious (HCVcc) chimeras. Infectivity and the neutralisation sensitivity of these viruses were then determined. Bioinformatic and HCVpp infectivity screening of approximately 900 E1E2 clones resulted in the assembly of a panel of 78 functional E1E2s, representing distinct HCV genotypes and different stages of infection. These HCV glycoproteins differed markedly in their sensitivity to neutralising antibodies. We used this panel to predict antibody efficacy against circulating HCV strains, highlighting the likely reason why some monoclonal antibodies failed in previous clinical trials. This study provides the first objective categorization of cross-genotype patient-derived HCV E1E2 clones according to their sensitivity to antibody neutralization. It has shown that HCV isolates have clearly distinguishable neutralization-sensitive, -resistant or -intermediate phenotypes, which are independent of genotype. This panel provides a systematic means for characterisation of the neutralizing response elicited by candidate vaccines and for defining the therapeutic potential of monoclonal antibodies.

    IMPORTANCE Hepatitis C virus (HCV) has a global burden of more than 170 million people, many of whom cannot attain the new, expensive, direct-acting antiviral therapies. A safe and effective vaccine, which generates both T cell responses and neutralizing antibodies, is required to eradicate the disease. Regions within the HCV surface glycoproteins E1 and E2 are essential for virus entry and are targets for neutralizing antibodies. Screening of vaccine candidates requires suitable panels of glycoproteins that represent breadth of neutralization resistance. Use of a standard reference panel for vaccine studies will ensure comparability of datasets, as has become routine for HIV-1. Here, we describe a large panel of patient-derived HCV glycoproteins with an assessment of their neutralization to defined monoclonal antibodies, which has enabled us to predict their likely efficacy in the wider HCV-infected population. This panel could also be important for future selection of additional therapeutic antibodies and vaccine design.

  • Homologous Recombination Repair Factors, Rad51 and BRCA1, are Necessary for Productive Replication of Human Papillomavirus 31 [PublishAheadOfPrint]

  • High-risk HPV31 positive cells exhibit constitutive activation of the ATM-dependent DNA damage response (DDR), which is necessary for productive viral replication. In response to double-strand DNA breaks (DSBs), ATM activation leads to DNA repair through homologous recombination (HR), which requires the principal recombinase protein Rad51, as well as BRCA1. Previous studies from our lab demonstrated that Rad51 and BRCA1 are expressed at high levels in HPV31 positive cells and localize to sites of viral replication. These results suggest that HPV may utilize ATM activity to increase HR activity as a means to facilitate viral replication. In this study, we demonstrate that high-risk HPV E7 expression alone is sufficient for the increase in Rad51 and BRCA1 protein levels. We have found this increase occurs, at least in part, at the level of transcription. Studies analyzing protein stability indicate that HPV may also protect Rad51 and BRCA1 from turnover, contributing to the overall increase in cellular levels. We also demonstrate that Rad51 is bound to HPV31 genomes, with binding increasing per viral genome upon productive replication. We have found that depletion of Rad51 and BRCA1, as well as inhibition of Rad51's recombinase activity abrogates productive viral replication upon differentiation. Overall, these results indicate that Rad51 and BRCA1 are required for the process of HPV31 genome amplification, and suggest that productive replication occurs in a manner dependent upon recombination.

    IMPORTANCE: Productive replication of HPV31 requires activation of an ATM-dependent DNA damage response, though how ATM activity contributes to replication is unclear. Rad51 and BRCA1 play essential roles in repair of double-strand breaks, as well as the restart of stalled replication forks through homologous recombination (HR). Given that ATM activity is required to initiate HR repair, coupled with the requirement of Rad51 and BRCA1 for productive viral replication, our findings suggest that HPV may utilize ATM activity to ensure localization of recombination factors to productively replicating viral genomes. The finding that E7 increases the levels of Rad51 and BRCA1 suggests E7 contributes to productive replication by providing DNA repair factors required for viral DNA synthesis. Our studies not only implicate a role for recombination in the regulation of productive HPV replication, but provide further insight into how HPV manipulates the DDR to facilitate the productive phase of the viral life cycle.

  • The Interferon-stimulated gene Ifi27l2a restricts West Nile virus infection and pathogenesis in a cell-type and region-specific manner [PublishAheadOfPrint]

  • The mammalian host responds to viral infections by inducing expression of hundreds of interferon-stimulated genes (ISGs). While the functional significance of many ISGs has yet to be determined, their cell-type and temporal nature of expression suggests unique activities against specific pathogens. Using a combination of ectopic expression and gene silencing approaches in cell culture, we previously identified Ifi27l2a as a candidate antiviral ISG within neuronal subsets of the central nervous system (CNS) that restricts West Nile virus (WNV) infection, an encephalitic flavivirus of global concern. To investigate the physiological relevance of Ifi27l2a in the context of viral infection, we generated Ifi27l2a-/- mice. Although adult mice lacking Ifi27l2a were more vulnerable to lethal WNV infection, viral burden was greater only within the CNS, particularly in the brain stem, cerebellum, and spinal cord. Within neurons of the cerebellum and brain stem, in the context of WNV infection, a deficiency of Ifi27l2a was associated with less cell death, which likely contributed to sustained viral replication and higher titers in these regions. Infection studies in primary cell culture revealed that Ifi27l2a-/- cerebellar granule cell neurons and macrophages but not cerebral cortical neurons, embryonic fibroblasts, or dendritic cells sustained higher WNV infection compared to wild-type cells, and this difference was greater under conditions of IFN-bbeta; pretreatment. Collectively, these findings suggest that Ifi27l2a has an antiviral phenotype in subsets of cells, and that at least some ISGs have specific inhibitory functions in restricted tissues.

    IMPORTANCE STATEMENT The interferon-stimulated gene, Ifi27l2a, is expressed differentially within the central nervous system upon interferon stimulation or viral infection. Prior studies in cell culture suggested an antiviral role for Ifi27l2a during infection by West Nile virus (WNV). To characterize its antiviral activity in vivo, we generated mice with a targeted gene deletion of Ifi27l2a. Based on extensive virological analyses, we determined that Ifi27l2a protects mice from WNV-induced mortality by contributing to the control of infection of the hindbrain and spinal cord, possibly by regulating cell death of neurons. This antiviral activity was validated in granule cell neurons derived from the cerebellum and in macrophages but was not observed in other cell types. Collectively, these data suggest Ifi27l2a contributes to innate immune restriction of WNV in a cell-type and tissue-specific manner.

  • Structure-based mutagenesis of STIV B204 reveals essential residues in the virion-associated DNA packaging ATPase [PublishAheadOfPrint]

  • Sulfolobus turreted icosahedral virus (STIV), an archaeal virus that infects the hyperthermoacidophile, Sulfolobus solfataricus, is one of the most well studied viruses of the domain Archaea. STIV shares structural, morphological, and sequence similarities with viruses from other domains of life, all of which are thought to belong to the same viral lineage. Several of these common features include a conserved coat protein fold, an internal lipid membrane, and a DNA-packaging ATPase. B204 is the ATPase encoded by STIV and is thought to drive packaging of viral DNA during the replication process. Here, we report the crystal structure of B204 along with the biochemical analysis of B204 mutants chosen based on structural information and sequence conservation patterns observed among members of the same viral lineage and the larger FtsK/HerA Superfamily to which B204 belongs. Both in vitro ATPase activity assays and transfection assays with mutant forms of B204 confirmed the essentiality of conserved and non-conserved positions. We have also identified two distinct particle morphologies during a STIV infection that differ in their presence of the B204 protein. The biochemical and structural data presented herein is informative for not only the STIV replication process, but can also be useful in deciphering DNA-packaging mechanisms for other viruses belonging to this lineage.

    IMPORTANCE Sulfolobus turreted icosahedral virus (STIV) is a virus that infects a host from the domain Archaea that replicates in high temperature, acidic environments. While STIV has many unique features, there exist several striking similarities between this virus and others that replicate in different environments and infect a broad range of hosts from Bacteria and Eukarya. Aside from structural features shared by viruses from this lineage, there exists a significant level of sequence similarity between the ATPase genes encoded by these different viruses; this gene encodes an enzyme thought to provide energy that drives DNA-packaging into the virion during infection. The experiments described here highlight the elements of this enzyme that are essential for proper function and also provide supporting evidence that B204 is present in the mature STIV virion.

  • Deletion of the Murid herpesvirus 4 ORF63 affects the trafficking of incoming capsids towards the nucleus. [PublishAheadOfPrint]

  • Gammaherpesviruses are important human and animal pathogens. Despite the fact that they display the classical architecture of herpesviruses, the function of most of their structural proteins is still poorly defined. This is especially true for tegument proteins. Interestingly, a potential role in immune evasion has recently been proposed for the tegument protein encoded by Kaposi's Sarcoma-associated Herpesvirus ORF63. To gain insight about the roles of ORF63 in the lifecycle of a gammaherpesvirus, we generated null mutations in the ORF63 gene of Murid Herpesvirus 4 (MuHV-4). We showed that disruption of ORF63 was associated with a severe MuHV-4 growth deficit both in vitro and in vivo. The latter deficit was mainly associated with a defect of replication in the lung but did not affect the establishment of latency in the spleen. On a functional point of view, inhibition of caspase-1 or inflammasome did not restore the growth of the ORF63 deficient mutant suggesting that the observed deficit was not associated with the immune evasion mechanism identified previously. Moreover, this growth deficit was also not associated with a defect in virion egress from the infected cells. In contrast, it appeared that MuHV-4 ORF63 deficient mutants failed to address most of their capsids to the nucleus during entry into the host cell, suggesting that ORF63 plays a role in capsid movement. In the future, ORF63 could therefore be considered as a target to block gammaherpesvirus infection at a very early stage of the infection.

    IMPORTANCE The important diseases caused by gammaherpesviruses in human and animal populations justify a better understanding of their lifecycle. Especially, the role of most of their tegument proteins is still largely unknown. In this study, we used Murid herpesvirus 4, a gammaherpesvirus infecting mouse, to decipher the role of the protein encoded by the viral ORF63 gene. We showed that the absence of this protein is associated with a severe growth deficit both in vitro and in vivo that was mainly due to impaired migration of viral capsids towards the nucleus during entry. Altogether, these results provide new insights about the lifecycle of gammaherpesviruses and could allow the development of new antiviral strategies aiming at blocking gammaherpesvirus infection at the very early stages.

  • Low-fidelity polymerases of alphaviruses recombine at higher rates to overproduce defective interfering particles [PublishAheadOfPrint]

  • Low-fidelity RNA-dependent RNA polymerases for many RNA virus mutators have been shown to confer attenuated phenotypes, presumably due to increased mutation rates. Additionally, for many RNA viruses, replication to high titers results in the production of defective interfering particles (DIs) that also attenuate infection. We hypothesized that fidelity, recombination and DI production are tightly linked. We show that a Sindbis virus mutator replicating at high multiplicity of infection manifests an earlier and greater accumulation of DIs compared to its wildtype counterpart. The isolated DIs interfere with the replication of full-length virus in a dose-dependent manner. Importantly, the ability of the mutator virus to overproduce DIs could be linked to an increased recombination frequency. These data confirm that RNA-dependent RNA polymerase fidelity and recombination are inversely correlated for this mutator. Our findings suggest that defective interference resulting from higher recombination rates may be more detrimental to RNA virus mutators than the increase in mutational burden.

    IMPORTANCE Replication, adaptation and evolution of RNA viruses rely in large part on their low-fidelity RNA-dependent RNA polymerase. Viruses artificially modified in their polymerase to decrease fidelity (mutator viruses) are attenuated in vivo, demonstrating the important role of fidelity in viral fitness. However, attenuation was solely attributed to the modification of the viral mutation rate and the accumulation of detrimental point mutations. In this work, we described an additional phenotype of mutator viruses: an increased recombination rate leading to defective interfering particle (DI) overproduction. Because DIs are known for their inhibitory effect on viral replication, our work suggests that fidelity variants may be attenuated in vivo via several mechanisms. This has important implications in the development of fidelity variants as live-attenuated vaccine strains.

  • Nipah virus matrix protein influences fusogenicity and is essential for particle infectivity and stability [PublishAheadOfPrint]

  • Nipah virus (NiV) causes fatal encephalitic infections in humans. To characterize the role of the matrix (M) protein in the viral life cycle, we generated a reverse genetics system based on the NiV Malaysia strain. Using an eGFP-expressing matrix (M) protein-deleted NiV, we observed a slightly increased cell-cell fusion, slower replication kinetics and significantly reduced peak titers compared to the parental virus. While increased amounts of viral proteins were found in the supernatant of cells infected with M-deleted NiV, the infectivity-to-particle ratio was more than 100-fold reduced, and the particles were less thermostable and of more irregular morphology. Taken together, our data demonstrate that the M protein is not absolutely required for the production of cell-free NiV, but is necessary for proper assembly and release of stable infectious NiV particles.

    IMPORTANCE Henipaviruses cause a severe disease with high mortality in human patients. Therefore, these viruses can only be studied in BSL-4 laboratories, making it more challenging to characterize their life cycle. Here we investigated the role of the Nipah virus matrix protein in virus-mediated cell-cell fusion and in the formation and release of newly produced particles. We found that even though low levels of infectious viruses are produced in the absence of the matrix protein, it is required for the release of highly infectious and stable particles. Fusogenicity of matrix-less viruses was slightly enhanced, further demonstrating the critical role of this protein in different steps of Nipah virus spread.

  • Peripheral T Follicular Helper Cells Are the Major HIV Reservoir Within Central Memory CD4 T Cells in Peripheral Blood from chronic HIV infected individuals on cART [PublishAheadOfPrint]

  • Objectives: In this study, we examined the peripheral blood (PB) TCM subsets designated as peripheral T follicular helper cells (pTfh) and non-pTfh cells to assess HIV permissiveness and persistence.

    Methods: Purified pTfh and non-pTfh cells from healthy HIV negative donors were tested for HIV permissiveness using GFP expressing HIV-1NL4-3/Ba-L followed by viral reactivation with antiCD3/CD28 stimulation. The role of pTfh cells in HIV persistence was analyzed in 12 chronically HIV-1 infected patients before and 48 weeks after initiation of Raltegravir containing combination antiretroviral therapy (cART). Total cellular HIV-1 DNA and 2LTR circles were analyzed in using droplet digital PCR in the purified pTfh and non-pTfh cells. Activation-inducible HIVp24 expression was determined by flow cytometry.

    Results: Results indicate that pTfh, in particular PD1+pTfh showed greater permissiveness for HIV infection than non-pTfh cells. At week 48 on cART, HIV DNA levels were unchanged from pre-cART although a significant decrease in 2LTR circles was observed in both cell subsets. Inducible HIVp24 expression was higher in pTfh compared to non-pTfh, with highest frequencies in the PD1+CXCR3- pTfh subset. Frequencies of HLADR+CD38+ activated CD4 T cells correlated with 2 LTR circles in pTfh and non-pTfh cells at both time points and with p24+ cells at entry.

    Conclusion: Among CD4 TCM cells in PB of aviremic patients on cART, pTfh cells, in particular the PD-1+CXCR3- subset constitute a major HIV reservoir that is sustained by ongoing residual immune activation. The inducible HIV p24 assay is useful for monitoring HIV reservoirs in defined CD4 T cell subsets.

    IMPORTANCE Identification of the type and nature of the cellular compartments of circulating HIV reservoirs is important for targeting of HIV cure strategies. In lymph nodes (LN) a subset of CD4 T cells called T follicular helper (Tfh) cells are preferentially infected by HIV. Central memory (TCM) CD4 T cells are the major cellular reservoir for HIV in peripheral blood and contain a subset of CD4 TCM cells expressing chemokine receptor CXCR5 similar in function to LN Tfh termed peripheral Tfh (pTfh) cells. We found that the circulating pTfh cells are highly susceptible for HIV infection and that in HIV infected patients, HIV persists in these cells following plasma virus suppression with potent cART. These pTfh cells that constitute a subsets of TCM CD4 T cells can be readily monitored in peripheral blood to assess HIV persistence.

  • Murine gammaherpesvirus-68 LANA and SOX homologs counteract ATM-driven p53 activity during lytic viral replication [PublishAheadOfPrint]

  • Tumor suppressor p53 is activated in response to numerous cellular stresses, including viral infection. However, whether murine gammaherpesvirus-68 (MHV68) provokes p53 during the lytic replication cycle has not been extensively evaluated. Here we demonstrate that MHV68 lytic infection induces p53 phosphorylation and stabilization in a manner that is dependent on DNA damage response kinase ataxia telangiectasia mutated (ATM). Induction of p53 during MHV68 infection occurred in multiple cell-types, including splenocytes of infected mice. ATM and p53 activation required early viral gene expression, but occurred independently of viral DNA replication. At early timepoints during infection, p53-responsive cellular genes were induced, coinciding with p53 stabilization and phosphorylation. However, p53-related gene expression subsided as infection progressed, even though p53 remained stable and phosphorylated. Infected cells also failed to initiate p53-dependent gene expression and undergo apoptosis in response to treatment with exogenous p53 agonists. Inhibition of p53 responses during infection required expression of the MHV68 homologs of the shut-off and exonuclease protein (muSOX) and latency-associated nuclear antigen (mLANA). Interestingly, mLANA, but not muSOX, was necessary to prevent p53-mediated death in MHV68-infected cells under the conditions tested. This suggests the possibility that muSOX and mLANA are differentially required for inhibiting p53 in specific settings. These data reveal that DDR responses triggered by MHV68 infection promote p53 activation. However, MHV68 encodes at least two proteins capable of limiting the potential consequences of p53 function.

    IMPORTANCE Gammaherpesviruses are oncogenic herpesviruses that establish lifelong chronic infections. Defining how gammaherpesviruses overcome host responses to infection is important for understanding how these viruses infect and cause disease. Here we establish that murine gammaherpesvirus-68 induces activation of tumor suppressor p53. p53 activation was dependent on the DNA damage response kinase ataxia telangiectasia mutated. Though active early after infection, p53 became dominantly inhibited as the infection cycle progressed. Viral inhibition of p53 was mediated by the murine gammaherpesvirus-68 homologs of the shut-off and exonuclease protein (muSOX) and latency-associated nuclear antigen (mLANA). Inhibition of the p53 pathway enabled infected cells to evade p53-mediated cell death responses. These data demonstrate that a gammaherpesvirus encodes multiple proteins to limit p53-mediated responses to productive viral infection, which likely benefits acute viral replication and establishment of chronic infection.

  • ICP8 Filament Formation is Essential for Replication Compartment Formation During Herpes Simplex Virus Infection [PublishAheadOfPrint]

  • HSV dramatically reorganizes the infected cell nucleus leading to the formation of prereplicative sites and replication compartments. This process is driven by the essential viral ssDNA binding protein, ICP8, which can form double helical filaments in the absence of DNA. In this paper we show that two conserved regions FNF (F1142, N1143, and F1144), and FW (F843 and W844) are essential for ICP8 self-interactions, and we propose that the FNF motif docks into the FW region during filament formation. Mammalian expression plasmids bearing mutations in these motifs (FNF and FW) were unable to complement an ICP8 null mutant for growth and replication compartment formation. Furthermore, FNF and FW mutants were able to inhibit wild type (WT) virus plaque formation and filament formation; whereas, a double mutant (FNF-FW) was not. These results suggest that single mutant proteins are incorporated into non-productive ICP8 filaments, while the double mutant is unable to interact with WT ICP8 and does not interfere with WT growth. Cells transfected with WT ICP8 and helicase/primase (H/P) complex exhibited punctate nuclear structures that resemble prereplicative sites; however, the FNF and FW mutants failed to do so. Taken together these results suggest that the FNF and FW motifs are required for ICP8 self-interactions and that these interactions may be important for the formation of prereplicative sites and replication compartments. We propose that filaments or other higher order structures of ICP8 may provide a scaffold onto which other proteins can be recruited to form prereplicative sites and replication compartments.

    IMPORTANCE For nuclear viruses such as HSV, efficient DNA replication requires the formation of discrete compartments within the infected cell nucleus in which replication proteins are concentrated and assembled into the HSV replisome. In this paper, we characterize the role of filament formation by the single strand DNA binding protein ICP8 in the formation of prereplicative sites and replication compartments. We propose that ICP8 protein filaments generates a protein scaffold for other cellular and viral proteins resulting in a structure that concentrates both viral DNA and replication proteins. Replication compartments may be similar to other types of cellular membrane-less compartments thought to be formed by phase separations caused by low-affinity, multivalent interactions involving proteins and nucleic acids within cells. ICP8 scaffolds could facilitate replication compartment formation by mediating interactions with other components of the replication machinery.

  • Early activation of primary brain microvascular endothelial cells by Nipah virus glycoprotein containing particles [PublishAheadOfPrint]

  • Nipah virus (NiV) is a highly pathogenic paramyxovirus that causes pronounced infection of brain endothelia and CNS inflammation. Using primary porcine brain microvascular endothelial cells, we show that upregulation of E-selectin precedes cytokine induction and is not only induced by infectious NiV but also by NiV-glycoprotein containing virus-like particles. This demonstrates that very early events in NiV brain endothelial infection do not depend on NiV replication but can be triggered by the NiV glycoproteins alone.

  • A diverse array of new viral sequences identified in worldwide populations of the Asian citrus psyllid (Diaphorina citri) using viral metagenomics [PublishAheadOfPrint]

  • The Asian citrus psyllid, Diaphorina citri, is the natural vector of the causal agent of Huanglongbing (HLB), or citrus greening disease. Together; HLB and D. citri represent a major threat to world citrus production. As there is no cure for HLB, insect vector management is considered to be one strategy to help control the disease, and D. citri viruses might be useful. Here, we used a metagenomic approach to analyze viral sequences associated with the global population of D. citri. By sequencing small RNAs and the transcriptome coupled with bioinformatics analysis, we show that the virus-like sequences of D. citri are diverse. We identified novel viral sequences belonging to the Picornavirus super family, and the Reoviridae, Parvoviridae and Bunyaviridae families, and an unclassified positive-sense single-stranded RNA virus. Moreover, a Wolbachia prophage-related sequence was identified. This is the first comprehensive survey to assess the viral community from worldwide populations of an agricultural insect pest. Our results provide valuable information on new putative viruses, some of which may have the potential to be used as biocontrol agents.

    IMPORTANCE Insects have the most species of all animals, and are hosts to, and vectors of, a great variety of known and unknown viruses. Some of these most likely have the potential to be important fundamental and/or practical resources. In this study, we used high-throughput next generation sequencing (NGS) technology and bioinformatics analysis to identify putative viruses associated with Diaphorina citri, the Asian citrus psyllid. D. citri is the vector of the bacterium causing Huanglongbing (HLB), currently the most serious threat to citrus worldwide. Here, we report several novel viral sequences associated with D. citri.

  • Dissecting the role of E2 protein domains on alphavirus pathogenicity [PublishAheadOfPrint]

  • Alphaviruses represent a diverse set of arboviruses, many of which are important pathogens. Chikungunya virus (CHIKV), an arthritis-inducing alphavirus, is the cause of a massive ongoing outbreak in the Caribbean and South America. In contrast to CHIKV, other related alphaviruses such as Venezuelan equine encephalitis virus (VEEV) and Semliki Forest virus (SFV) can cause encephalitic disease. E2, the receptor binding protein, has been implicated as a determinant in cell tropism, host range, pathogenicity, and immunogenicity. Previous reports also have demonstrated that E2 contains residues important for host range expansions and monoclonal antibody binding; however, little is known about what role each protein domain (e.g., A, B, and C) of E2 plays on these factors. Therefore, we constructed chimeric cDNA clones between CHIKV and VEEV or SFV to probe the effect of each domain on pathogenicity in vitro and in vivo. CHIKV chimeras containing each of the domains of the E2 (DomA/DomB/DomC) from SFV, but not VEEV, were successfully rescued. Interestingly, while all chimeric viruses were attenuated as compared to CHIKV in mice, DomB virus showed similar rates of infection and dissemination in Aedes aegypti mosquitoes, suggesting differing roles for the E2 protein in different hosts. In contrast to CHIKV; DomB, and to a lesser extent DomA, caused neuron degeneration and demyelination in mice infected intracranially, suggesting a shift towards a phenotype similar to SFV. Thus, chimeric CHIKV/SFV provide insights on the role the alphavirus E2 protein plays on pathogenesis.

    IMPORTANCE Chikungunya virus (CHIKV) has caused large outbreaks of acute and chronic arthritis throughout Africa and Southeast Asia, and has now become a massive public health threat in the Americas, causing an estimated 1.2 million human cases in just over a year. No approved vaccines or antivirals exist for human use against CHIKV, or any other alphavirus. Despite the threat, little is known about the role the receptor binding protein (E2) plays on disease outcome in an infected host. To study this, our laboratory generated chimeric CHIKV containing corresponding regions of the Semliki Forest virus (SFV) E2 (Domains A, B and C) substituted into the CHIKV genome. Our results demonstrate that each domain of E2 likely plays a critical, but dissimilar role in the viral life cycle. Our experiments show that manipulation of E2 domains can be useful for studies on viral pathogenesis and potentially the production of vaccines/antivirals.

  • Hemagglutinin of influenza A virus antagonizes type I IFN responses by inducing degradation of type I IFN receptor 1 [PublishAheadOfPrint]

  • Influenza A virus (IAV) employs diverse strategies to circumvent type I interferon (IFN) responses particularly by inhibiting the synthesis of type I IFNs. However, it is poorly understood if and how IAV regulates type I IFN receptor (IFNAR)-mediated signaling mode. In this study, we demonstrate that IAV induces degradation of IFNAR subunit 1 (IFNAR1) to attenuate the type I IFN-induced anti-viral signaling pathway. Following infection, the level of IFNAR1 protein, but not mRNA, has decreased. Indeed, IFNAR1 was phosphorylated and ubiquitinated by IAV infection, which resulted in IFNAR1 elimination. The transiently overexpressed IFNAR1 displayed anti-viral activity by inhibiting virus replication. Importantly, the hemagglutinin (HA) protein of IAV was proved to trigger the ubiquitination of IFNAR1, diminishing the levels of IFNAR1. Further, influenza A viral HA1 subunit, but not HA2 subunit, downregulated IFNAR1. However, viral HA-mediated degradation of IFNAR1 was not caused by the ER stress response. IAV HA robustly reduced cellular sensitivity to type I IFNs, suppressing the activation of STAT1/STAT2 and induction of IFN-stimulated anti-viral proteins. Taken together, our findings suggest that IAV HA causes IFNAR1 degradation, which in turn helps the virus escape the powerful innate immune system. Thus, the research elucidated an influenza viral mechanism for eluding the IFNAR signaling pathway, which could provide new insights into the interplay between influenza virus and host innate immunity.

    IMPORTANCE Influenza A virus (IAV) infection causes significant morbidity and mortality worldwide and remains a major health concern. When triggered by influenza viral infection, host cells produce type I interferon (IFN) to block viral replication. Although IAV was shown to have diverse strategies to evade this powerful, IFN-mediated antiviral response, it is not well-defined if IAV manipulates the IFN receptor-mediated signaling pathway. Here, we uncovered that influenza viral hemagglutinin (HA) protein causes the degradation of type I IFN receptor subunit 1 (IFNAR1). HA promoted phosphorylation and poly-ubiquitination of IFNAR1, which facilitated the degradation of this receptor. The HA-mediated elimination of IFNAR1 notably decreased the cells' sensitivities to type I IFNs, as demonstrated by the diminished expression of IFN-induced antiviral genes. This discovery could help us understand how IAV regulates host innate immune response to create an environment optimized for viral survival in host cells.

  • Pharmacologic Inhibition of Nedd8 Activation Enzyme Exposes CD4-induced Epitopes within Env on Cells Expressing HIV-1 [PublishAheadOfPrint]

  • HIV-1 Vpu decreases the exposure of epitopes within the viral envelope glycoprotein (Env) on the surface of infected cells by downregulating both BST2 and CD4. To test the hypothesis that inhibiting Vpu-activity would increase the exposure of these epitopes and sensitize infected cells to antibody dependent cellular cytotoxicity (ADCC), we treated cells with the Nedd8-activation-enzyme (NAE) inhibitor MLN4924, which inhibits the cullin1-based ubiquitin ligase complex co-opted by Vpu to degrade cellular targets. Treatment of HeLa cells with MLN4924, or expression of a dominant negative mutant of cullin1, inhibited the Vpu-mediated downregulation of CD4 but not the downregulation of BST2. NAE-inhibition also increased the surface-exposure of CD4-induced epitopes within Env on HEK293 cells containing an inducible HIV-genome, on infected CEM T cells, and on infected primary T cells. In contrast, the Vpu-mediated downregulation of BST2 was substantially inhibited by MLN4924 only when T cells were treated with interferon-aalpha; (IFN) to induce high levels of BST2-expression. As reported previously, the absence of vpu or nef, and even more so the combined absence of both genes, sensitized infected cells to ADCC. However, NAE-inhibition affected ADCC minimally. Paradoxically, even in infected, IFN-treated cells in which NAE-inhibition substantially rescued the surface level of BST2, the surface level of Env detected with an antibody recognizing a CD4-independent epitope (2G12) was minimally increased. Mutation of the C-terminal Vpu-residue W76, which supports the ability of Vpu to stimulate virion-release by displacing BST2 from assembly sites on the plasma membrane by a cullin1-independent mechanism, increased the exposure of Env detected by 2G12 on infected T cells. Thus, inhibiting the displacement function of Vpu together with its ability to degrade CD4 and BST2 may be required to sensitize infected cells to ADCC.

    IMPORTANCE Pathogenic viruses encode gene products that enable evasion of host immune surveillance mechanisms. One such mechanism is antibody dependent cellular cytotoxicity (ADCC), wherein host-antibodies bind envelope glycoproteins of the virus that are inserted into the cellular membrane and direct the destruction of infected cells. Targeting pharmacologically the activity of HIV-1 Vpu, which contributes to evasion of ADCC, could potentially sensitize infected cells to this immune surveillance mechanism, an outcome that would have therapeutic implications with respect to the goal of curing HIV-1 infection. The Nedd8 activation enzyme inhibitor MLN4924 blocks the activity of the host ubiquitin ligase that Vpu co-opts to direct the degradation of CD4 and BST2. We observed that while MLN4924 partially reverses the activity of Vpu and could become part of a therapeutic approach by virtue of CD4-induced epitope-exposure, sufficient Vpu-activity as an antagonist of BST2 persists despite this drug to allow escape from ADCC.

  • Membrane binding of the Rous sarcoma virus Gag protein is cooperative and dependent on the SPA domain. [PublishAheadOfPrint]

  • The principles underlying membrane binding and assembly of retroviral Gag proteins into a lattice are understood. However, little is known about how these processes are related. Using purified Rous sarcoma virus Gag and Gag truncations, we studied the interrelation of Gag-Gag interaction and Gag-membrane interaction. Both by liposome binding and by surface plasmon resonance on a supported bilayer, Gag bound to membranes much more tightly than did MA, the isolated membrane-binding domain. In principle this difference could be explained either by protein-protein interactions leading to cooperativity in membrane binding, or by the simultaneous interaction of the N-terminal MA and the C-terminal NC of Gag with the bilayer, since both are highly basic. However, we found that NC was not required for strong membrane binding. Instead the spacer peptide assembly domain (SPA), a putative 24 residue helical sequence comprising the 12-residue SP segment of Gag and overlapping the CA C-terminus and the NC N-terminus, was required. SPA is known to be critical for proper assembly of the immature Gag lattice. A single amino acid mutation in SPA that abrogates assembly in vitro dramatically reduced binding of Gag to liposomes. In vivo, plasma membrane localization was dependent on SPA. Disulfide crosslinking based on ectopic Cys residues showed that the contacts between Gag proteins on the membrane are similar to the known contacts in virus-like particles. Taken together, we interpret these results to mean that Gag membrane interaction is cooperative in that it depends on the ability of Gag to multimerize.

    IMPORTANCE The retroviral structural protein Gag has three major domains. The N-terminal MA domain interacts directly with the plasma membrane (PM) of cells. The central CA domain, together with immediately adjoining sequences, facilitates the assembly of thousands of Gag molecules into a lattice. The C-terminal NC domain interacts with the genome resulting in packaging of viral RNA. For assembly in vitro with purified Gag, in the absence of membranes, binding of NC to nucleic acid somehow facilitates further Gag-Gag interactions that lead to formation of the Gag lattice. The contributions of MA-mediated membrane binding to virus particle assembly are not well understood. Here we report that in the absence of nucleic acid, membranes provide a platform that facilitates Gag-Gag interactions. This study demonstrates that the binding of Gag, but not of MA, to membranes is cooperative, and identifies SPA as a major factor that controls this cooperativity.

  • A single mutation at PB1 residue 319 dramatically increases the safety of PR8 LAIV in a murine model without compromising vaccine efficacy. [PublishAheadOfPrint]

  • The live attenuated influenza vaccine (LAIV) is preferentially recommended for use in most children yet remains unsafe for the most at risk groups. Here we have improved the safety of a mouse adapted live attenuated influenza vaccine containing the same attenuating amino acid mutations as human LAIV, by adding an additional mutation at PB1 residue 319. This results in a vaccine with a 20 fold decrease in protective efficacy and a 10,000-fold increase in safety.

  • A novel live Pichinde virus-based vaccine vector induces enhanced humoral and cellular immunity upon a booster dose [PublishAheadOfPrint]

  • Pichinde virus (PICV) is a bi-segmented enveloped RNA virus that targets macrophages and dendritic cells (DCs) early in the infection and induces strong innate and adaptive immunity in mice. We have developed a reverse genetics system to produce live recombinant PICV viruses (P18 strain) with a tri-segmented RNA genome (rP18tri), which encodes all four PICV gene products and up to two foreign genes. We have engineered the vector to express the GFP reporter gene (abbreviated as G) and either the hemagglutination (HA or H) or the nucleoprotein (NP or P) gene of the influenza A virus A/PR8. The tri-segmented viruses, rP18tri-G/H and rP18tri-G/P, showed a slightly reduced growth in vitro and expressed HA and NP, respectively. Mice immunized with rP18tri-G/H were completely protected against lethal influenza viral challenge even after 120 days of immunization. These rP18tri-based vectors could efficiently induce both neutralizing antibodies and antigen-specific T cell responses via different immunization routes. Interestingly, the immune responses were significantly increased upon a booster dose and remained at high levels even after three booster doses. In summary, we have developed a novel PICV-based live vaccine vector that can express foreign antigens to induce strong humoral and cell-mediated immunity and is ideal for a prime-and-boost vaccination strategy.

    IMPORTANCE We have developed a novel Pichinde virus (PICV)-based live viral vector rP18tri that packages three RNA segments and encodes up to two foreign genes. Using influenza virus HA and NP genes as model antigens, we show that this rP18tri vector can induce strong humoral and cellular immunity via different immunization routes and lead to protection in mice. Interestingly, a booster dose further enhances the immune responses, a feature that is distinct from other known live viral vectors. In summary, our study demonstrates a unique feature of this live rP18tri vector to be used as a novel vaccine platform for a prime-and-boost vaccination strategy.

  • Determination of the Spontaneous Mutation Frequency of Ebola virus and Exploitation of this Therapeutically [PublishAheadOfPrint]

  • Ebola virus (EBOV) is an RNA virus that can cause hemorrhagic fever with high fatality rates and there are no approved vaccines or therapies. Typically, RNA viruses have high spontaneous mutation rates, which permit rapid adaptation to selection pressures and have other important biological consequences. However, it is unknown if filoviruses exhibit high mutation frequencies. Ultra deep sequencing and a recombinant EBOV that carries the gene encoding green fluorescent protein were used to determine the spontaneous mutation frequency of EBOV. The effects of the guanosine analogue ribavirin during EBOV infections were also assessed. Ultra deep sequencing revealed that the mutation frequency for EBOV was high and similar to other RNA viruses. Interestingly, significant genetic diversity was not observed in viable viruses, implying that changes were not well tolerated. We hypothesized that this could be exploited therapeutically. In vitro, presence of ribavirin increased the error rate and the IC50 was 27 mmu;M. In a mouse model of pre-EBOV exposure ribavirin therapy, ribavirin treatment corresponded with a significant delay in time to death and up to 75% survival. In mouse and monkey models of post-EBOV exposure therapy, ribavirin treatment also delayed the time to death and increased survival. These results demonstrate that EBOV has a spontaneous mutation frequency similar to other RNA viruses. These data also suggest a potential for therapeutic use of ribavirin for human EBOV infections. Ebola virus (EBOV) causes severe hemorrhagic disease with high case fatality rates; there are no approved vaccines or therapies. We determined the spontaneous mutation frequency of EBOV, which is relevant to understanding the potential for the virus to adapt. The frequency was similar to other RNA viruses. Significant genetic diversity was not observed in viable viruses, implying that changes were not well tolerated. We hypothesized that this could be exploited therapeutically. Ribavirin is a viral mutagen approved for treatment of several virus infections; it is also cheap and readily available. In cell culture, we showed that ribavirin was effective at reducing production of infectious EBOV. In mouse and monkey models of post-EBOV exposure therapy, ribavirin treatment delayed the time to death and increased survival. These data provide a better understanding of EBOV spontaneous mutation and suggest that ribavirin may have great value in the context of human disease.

  • Infection by Herpes Simplex Virus Type-1 Causes Near-Complete Loss of RNA Polymerase II Occupancy on the Host Cell Genome [PublishAheadOfPrint]

  • Lytic infection by herpes simplex virus type-1 (HSV-1) triggers a change in many host cell programs as the virus strives to express its own genes and replicate. Part of this process is repression of host cell transcription by RNA polymerase II (Pol II), which also transcribes the viral genome. Here we describe a global characterization of Pol II occupancy on the viral and host genomes in response to HSV-1 infection using ChIP-seq. The data reveal a near complete loss of Pol II occupancy throughout host cell mRNA genes, both in their bodies and promoter proximal regions. Increases in Pol II occupancy of host cell genes, which would be consistent with robust transcriptional activation, were not observed. HSV-1 infection induced a more potent and widespread repression of Pol II occupancy compared to heat shock, another cellular stress that widely represses transcription. Concomitant with the loss of host genome Pol II occupancy, we observed Pol II covering the HSV-1 genome, reflecting a high level of viral gene transcription. Interestingly, the positions of the peaks of Pol II occupancy at HSV-1 and host cell promoters were different. The primary peak of Pol II occupancy at HSV-1 genes is ~170 bp upstream of where it is positioned at host cell genes, suggesting that specific steps in transcription are regulated differently at HSV-1 genes compared to host cell mRNA genes.

    IMPORTANCE We investigated the effect of herpes simplex virus type-1 (HSV-1) infection on transcription of host cell and viral genes by RNA polymerase II (Pol II). The approach we used was to determine how levels of genome-bound Pol II changed after HSV-1 infection. We found that HSV-1 caused a profound loss of Pol II occupancy across the host cell genome. Increases in Pol II occupancy were not observed, showing no host genes were activated after infection. By contrast, Pol II occupied the entire HSV-1 genome. Moreover, the pattern of Pol II at HSV-1 genes differed from that on host cell genes, suggesting a unique mode of viral gene transcription. These studies provide new insight into how HSV-1 causes changes in the cellular program of gene expression, and how the virus co-opts host Pol II for its own use.

  • Human miRNA miR-532-5p exhibits antiviral activity against West Nile virus via suppression of host genes SESTD1 and TAB3 required for virus replication [PublishAheadOfPrint]

  • West Nile virus (WNV) is a mosquito-transmitted flavivirus, which naturally circulates between mosquitos and birds, but can also infect humans causing severe neurological disease. The early host response to WNV infection in vertebrates primarily relies on the type I interferon pathway, however, recent studies suggest that micro RNAs (miRNAs) may also play a notable role. In this study, we assessed the role of host miRNAs in response to WNV infection in human cells. We employed RNASeq analysis to determine changes in the expression of host miRNAs in HEK293 cells infected with an Australian strain of WNV, Kunjin, (WNVKUN) and identified a number of host miRNAs differentially expressed in response to infection. Three of these miRNAs were confirmed to be significantly up regulated in infected cells by qRT-PCR and Northern blot analyses and one of them, miR-532-5p, exhibited a significant antiviral effect against WNVKUN infection. We have demonstrated that miR-532-5p targets and down-regulates expression of the host genes, SESTD1 and TAB3 in human cells. siRNA depletion studies showed that both SESTD1 and TAB3 were required for efficient WNVKUN replication. We have also demonstrated up regulation of mir-532-5p expression and corresponding decrease in the expression of its targets, SESTD1 and TAB3 in the brains of WNVKUN-infected mice. Our results show that up-regulation of miR-532-5p and subsequent suppression of SESTD1 and TAB3 genes represents a host antiviral response aimed at limiting WNVKUN infection and highlight the important role of miRNAs in controlling RNA virus infections in mammalian hosts.

    IMPORTANCE West Nile virus (WNV) is a significant viral pathogen, which poses a considerable threat for human health across the globe. There is no specific treatment or licenced vaccine available for WNV and deeper insight into how this virus interacts with the host is required to facilitate their development. In this study we addressed the role of host micro RNAs (miRNA) in antiviral response to WNV in human cells. We identified miR-532-5p as a novel antiviral miRNA and showed that it is up regulated in response to WNV infection and suppresses the expression of host genes TAB3 and SESTD1 required for WNV replication. Our results show that up-regulation of miR-532-5p and subsequent suppression of SESTD1 and TAB3 genes represents an antiviral response aimed at limiting WNV infection and highlights the important role of miRNAs in controlling virus infections in mammalian hosts.

  • Vaccination with VSV-vectored chimeric hemagglutinins protects mice against divergent influenza virus challenge strains [PublishAheadOfPrint]

  • Seasonal influenza virus infections continue to cause significant disease each year, and there is a constant threat of the emergence of reassortant influenza strains causing a new pandemic. Available influenza vaccines are variably effective each season, are of limited scope at protecting against viruses that have undergone significant antigenic drift, and offer low protection against newly emergent pandemic strains. "Universal" influenza vaccine strategies that focus on the development of humoral immunity directed against the stalk domains of the viral hemagglutinin (HA) show promise for protecting against diverse influenza viruses. Here, we describe such a strategy that utilizes vesicular stomatitis virus (VSV) as a vector for chimeric hemagglutinin (cHA) antigens. This vaccination strategy is effective at generating HA stalk-specific, broadly cross-reactive serum antibodies by both the intramuscular and intranasal routes of vaccination. We show that prime-boost vaccination strategies provide protection against both lethal homologous and heterosubtypic influenza challenge, and that protection is significantly improved with intranasal vaccine administration. Additionally, we show that vaccination with VSV-cHAs generates greater stalk-specific and cross-reactive serum antibodies than does vaccination with VSV vectored full-length HAs, confirming that cHA-based vaccination strategies are superior at generating stalk-specific humoral immunity. VSV-vectored influenza vaccines that express chimeric hemagglutinin antigens offer a novel means for protecting against widely diverging influenza viruses.

    IMPORTANCE Universal influenza vaccination strategies should be capable of protecting against a wide array of influenza viruses and we have developed such an approach utilizing a single viral vector system. The potent antibody responses that these vaccines generate are shown to protect mice against lethal influenza challenges with highly divergent viruses. Notably, intranasal vaccination offers significantly better protection than intramuscular vaccination in a lethal virus challenge model. The results described in this study offer insights into the mechanisms by which chimeric hemagglutinin-based vaccines confer immunity, namely that the invariant stalk of cHA antigens is superior to full length HA antigens at inducing cross-reactive humoral immune responses, and that VSV-cHA vaccine-induced protection varies by site of inoculation, and contribute to the further development of universal influenza virus vaccines.

  • Pulmonary C Fibers Modulate MMP-12 Production via PAR2 and Are Involved in the Long-Term Airway Inflammation and Airway Hyperresponsiveness Induced by Respiratory Syncytial Virus Infection [PublishAheadOfPrint]

  • Children with acute RSV infection often develop sequelae of persistent airway inflammation and wheezing. Pulmonary C fibers (PCFs) are involved in generation of airway inflammation and resistance, however, their role in persistent airway diseases after RSV is unexplored. Here, we elucidated pathogenesis of PCFs activation in RSV induced persistent airway disorders. PCFs - degenerated and intact mice were used in the current study. The airway inflammation and airway resistance were evaluated. MMP408 and FSLLRY-NH2 were the selective antagonists for MMP-12 and PAR2, respectively, to investigate the roles of MMP-12 and PAR2 in PCFs nndash; mediating airway diseases. As results, PCFs degeneration significantly reduced following responses to RSV infection: augmented inflammatory cells, especially macrophages, and infiltrating inflammatory cells in lung tissues; sRaw response to methacholine; and upregulation of MMP-12 and PAR2 expression. Moreover, inhibition MMP-12 reduced the number of total cells and macrophages in BALF, as well inflammatory cells infiltrating, and decreased sRaw response to methacholine. In addition, PAR2 was up regulated especially at the later stage of RSV infection. Down nndash; regulation PAR2 ameliorated airway inflammation and resistance following RSV infection, and suppressed level of MMP-12. In all, the results suggest that PCFs involvement long-term airway inflammation and AHR was at least partial via modulating MMP-12, and the activation of PAR2 might be related with PCFs modulated MMP-12 production. Our initial findings indicated that inhibition of PCFs activity would be targeted therapeutically for virus infection-induced long-term airway disorders.

    IMPORTANCE The current study is critical to understand that PCFs was involved in long nndash; term airway inflammation and airway resistance post RSV infection through mediating MMP-12 production via PAR2, indicating that inhibition of PCFs activity would be targeted therapeutically for virus infection-induced long-term airway disorders.

  • Mouse Hepatitis Virus infection remodels Connexin43 mediated gap junction intercellular communication both in vitro and in vivo [PublishAheadOfPrint]

  • Gap junctions (GJs) form intercellular channels which directly connect the cytoplasm between neighboring cells to facilitate transfer of ions and small molecules. GJs play a major role in the pathogenesis of infection associated inflammation. Mutations of gap junction proteins, connexins (Cxs), cause dysmyelination and leukoencephalopathy. In multiple sclerosis (MS) patients and its animal model experimental autoimmune encephalitis (EAE), Cx43 was shown to be modulated in the central nervous system (CNS). The mechanism behind Cx43 alteration and its role in MS remains unexplored. Mouse hepatitis virus (MHV) infection induced demyelination is one of the best studied experimental animal models for MS. Our studies demonstrated that MHV infection downregulated Cx43 expression at protein and mRNA levels in vitro in primary astrocytes obtained from neonatal mice brains. After infection, a significant amount of Cx43 was retained in endoplasmic reticulum/endoplasmic reticulum golgi intermediate complex (ER/ERGIC) and GJ plaque formation was impaired at the cell surface, as evidenced by reduction of the Triton X-100 insoluble fraction of Cx43. Altered trafficking and impairment of GJ plaque formation may cause loss of functional channel formation in MHV infected primary astrocytes, as demonstrated by a reduced number of dye-coupled cells after scrape loading Lucifer yellow dye transfer assay. Upon MHV infection, a significant downregulation of Cx43 was observed in the virus-infected mouse brain. This study demonstrates that astrocytic Cx43 expression and function can be modulated due to virus stress and can be an appropriate model to understand the basis of cellular mechanisms involved in alteration of gap junction intercellular communication (GJIC) in CNS neuroinflammation.

    IMPORTANCE We found that MHV infection leads to downregulation of Cx43 in vivo in the CNS. In addition, results show that MHV infection impairs Cx43 expression in addition to gap junction communication in primary astrocytes. After infection, Cx43 did not traffic normally to the membrane to form gap junction plaques, and that could be the basis of reduced functional gap junction coupling between astrocytes. This is an important first step towards understanding how viruses affect Cx43 expression and trafficking at the cellular level. This may provide the basis of understanding how structural alteration of astrocytic gap junctions can disrupt gap junction communication between other CNS cells in CNS environments altered due to infection and inflammation. More specifically, alteration of Cx43 may be the basis of the destabilization of Cx47 in oligodendrocytes seen in and around inflammatory demyelinating plaques in MS patients.

  • Identification of Novel Fusion Inhibitors of Influenza A Virus by Chemical Genetics [PublishAheadOfPrint]

  • A previous screening of more than 50,000 compounds led to the identification of a pool of bioactive small molecules with inhibitory effect on the influenza A virus. One of these compounds, now widely known as Nucleozin, is a small molecule that targets the influenza A nucleoprotein. Herein we identify and characterize two structurally different novel fusion inhibitors of the influenza A group 1 hemagglutinin, FA-583 and FA-617, with low nanomolar activities. Highly resistant escape mutants against each of these compounds were generated and both were found to carry mutations localized in close proximity to the B-loop of the hemagglutinin 2 protein, which plays a crucial role in the virion-host cell fusion process. Recombinant virus, generated through reverse genetics, confirmed the resistant phenotype. In addition, the proposed binding pockets predicted by molecular docking studies are in accordance with the resistance-bearing mutation sites. We show through mechanistic studies that FA-583 and FA-617 act as fusion inhibitors by prohibiting the low pH-induced conformational change of hemagglutinin. Our study has offered concrete biological and mechanistic explorations for the strategic development of novel fusion inhibitors of influenza A viruses.

    IMPORTANCE Here we report two structurally distinctive novel fusion inhibitors of influenza A virus that act by interfering with the structural change of HA at acidic pH, a process necessary for successful entry of the virus. Mutational and molecular docking studies have identified their binding pockets closely situated to the B-loop region of hemagglutinin 2. The reduced sensitivity of FA-583- or FA-617-associated mutants to another compound suggests close proximity and even partially overlapping of their binding sites on hemagglutinin. Amino acid sequence alignments and crystal structures of group 1 and group 2 hemagglutinins have shed light on the possible binding mode of these two compounds. This report offers new lead compounds for the design of fusion inhibitors for influenza A viruses and further shows that forward chemical genetics is a highly effective approach for the identification of novel compounds that can perturb the infectivity of viruses and to probe new druggable targets or druggable domains in various viruses.

  • Targeting innate immunity for antiviral therapy through small molecule agonists of the RLR pathway [PublishAheadOfPrint]

  • The cellular response to virus infection is initiated when pathogen recognition receptors (PRR) engage viral pathogen associated molecular patterns (PAMPs). This process results in induction of downstream signaling pathways that activate the transcription factor IRF3. IRF3 plays a critical role in antiviral immunity to drive the expression of innate immune response genes, including those encoding antiviral factors, type 1 interferon, and immune modulatory cytokines that act in concert to restrict virus replication. Thus, small molecule agonists that can promote IRF3 activation and induce innate immune gene expression could serve as antivirals to induce tissue-wide innate immunity for effective control of virus infection. We identified small molecule compounds that activate IRF3 to differentially induce discrete subsets of antiviral genes. We tested a lead compound and derivatives for the ability to suppress infection by a broad range of RNA viruses. Compound administration significantly decreased the viral RNA load in cultured cells that were infected with the family Flaviviridae, including West Nile virus, dengue virus and hepatitis C virus as well as viruses of the families Filoviridae (Ebola virus), Orthomyxoviridae (influenza A virus), Arenaviridae (Lassa virus) and Paramyxoviridae (respiratory syncytial virus, Nipah virus) to suppress infectious virus production. Knockdown studies mapped this response to the RIG-I-like receptor pathway. This work identifies a novel class of host-directed immune modulatory molecules that activate IRF3 to promote host antiviral responses to broadly suppress infection by RNA viruses of distinct genera.

    IMPORTANCE Incidences of emerging and re-emerging RNA viruses highlight a desperate need for broad-spectrum antiviral agents that can effectively control infection by viruses of distinct genera. We identified small molecule compounds that can selectively activate IRF3 for the purpose of identifying drug-like molecules that can be developed for the treatment of viral infections. Here, we report the discovery of a hydroxyquinoline family of small molecules that can activate IRF3 to promote cellular antiviral responses. These molecules can prophylactically or therapeutically control infection in cell culture by pathogenic RNA viruses including West Nile virus, dengue virus, hepatitis C virus, influenza A virus, respiratory syncytial virus, Nipah virus, Lassa virus and Ebola virus. Our study thus identifies a class of small molecules with novel mechanism to enhance host immune responses for antiviral activity against a variety of RNA viruses that pose a significant healthcare burden and/or with known high case fatality rates.

  • HCMV Promotes Survival of Infected Monocytes via a Distinct Temporal Regulation of Cellular Bcl-2 Family Proteins [PublishAheadOfPrint]

  • Monocytes play a key role in the hematogenous dissemination of HCMV to target organ systems. To infect monocytes and reprogram them to deliver infectious virus, HCMV must overcome biological obstacles, including the short lifespan of monocytes, and their anti-viral pro-apoptotic response to infection. We have shown that virally-induced up-regulation of cellular Mcl-1 promotes early survival of HCMV-infected monocytes, allowing cells to overcome an early apoptotic checkpoint at around 48 hours post infection (hpi). Here, we demonstrate an HCMV-dependent shift from Mcl-1 as the primary anti-apoptotic player to the related protein, Bcl-2, later during infection. Bcl-2 was up-regulated in HCMV-infected monocytes beginning at 48 hpi. Treatment with the Bcl-2 antagonist, ABT-199, only reduced the pro-survival effects of HCMV in target monocytes beginning at 48 hpi, suggesting that Mcl-1 controls survival prior to 48 hpi, while Bcl-2 promotes survival after 48 hpi. Although Bcl-2 was up-regulated following viral binding/signaling through cellular integrins (compared to Mcl-1, which is up-regulated through binding/activation of EGFR), it functioned similarly to Mcl-1, adopting the early role of Mcl-1 in preventing caspase-3 cleavage/activation. This distinct, HCMV-induced shift from Mcl-1 to Bcl-2 occurs in response to a cellular up-regulation of pro-apoptotic Bax, as siRNA-mediated knockdown of Bax reduced the up-regulation of Bcl-2 in infected monocytes and rescued the cells from the apoptotic effects of Bcl-2 inhibition. Our data demonstrate a distinct survival strategy whereby HCMV induces a bi-phasic regulation of cellular Bcl-2 proteins to promote host cell survival, leading to viral dissemination and the establishment of persistent HCMV infection.

    IMPORTANCE Hematogenous dissemination of HCMV via infected monocytes is a crucial component of the viral survival strategy, and is required for the establishment of persistent infection and for viral spread to additional hosts. Our system of infected primary human blood monocytes provides us with an opportunity to answer specific questions about viral spread and persistence in in vivo-relevant myeloid cells that cannot be addressed with the more traditionally used replication-permissive cells. Our goal in examining the mechanisms whereby HCMV reprograms infected monocytes to promote viral dissemination is to uncover new targets for therapeutic intervention that would disrupt key viral survival and persistence strategies. Because of this important role in maintaining survival of HCMV-infected monocytes, our new data on the role of Bcl-2 regulation during viral infection represents a promising molecular target for mitigating viral spread and persistence.

  • Infection of a single cell line with distinct strains of HCMV can result in large variations in virion production and facilitate efficient screening of virus protein function [PublishAheadOfPrint]

  • Previously, we reported that the absence of the ataxia telangiectasia mutated (ATM) kinase, a critical DDR signaling component for double-strand breaks, caused no change in HCMV Towne virion production. Later, others reported decreased AD169 viral titers in the absence of ATM. To address this discrepancy human foreskin fibroblasts (HFF) and three ATM- lines (GM02530, GM05823, GM03395) were infected with both Towne and AD169. Two additional ATM- lines (GM02052 and GM03487) were infected with Towne. Remarkably, both previous studies' results were confirmed. However, the increased number of cell lines and infections with both lab-adapted strains confirmed that ATM was not necessary to produce wildtype-level titers in fibroblasts. Instead, interactions between individual virus strains and the cellular microenvironment of the individual ATM- line determined efficiency of virion production. Surprisingly, these two commonly used lab-adapted strains produced drastically different titers in one ATM- line, GM05823s. The differences in titer suggested a rapid method for identifying genes involved in differential virion production. In silico comparison of the Towne and AD169 genomes determined a list of 28 probable candidates responsible for the difference. Using serial iterations of an experiment involving virion entry and input genome nuclear trafficking with a panel of related strains, we reduced this list to four (UL129, UL145, UL147, UL148). As a proof of principal, reintroduction of UL148 largely rescued genome trafficking. Therefore, use of a battery of related strains offers an efficient method to narrow lists of candidate genes affecting various virus lifecycle checkpoints.

    IMPORTANCE Human Cytomegalovirus (HCMV) infection of multiple cell lines lacking Ataxia telangiectasia mutated (ATM) protein produced wildtype-levels of infectious virus. Interactions between virus strains and the microenvironment of individual ATM- lines determined the efficiency of virion production. Infection of one ATM- cell line, GM05823, produced large titer differentials dependent on the strain used, Towne or AD169. This discrepancy resolved a disagreement in the literature of a requirement for ATM expression and HCMV reproduction. The titer differentials in GM08523 cells were due, in part, to a decreased capacity of AD169 virions to enter the cell and traffic genomes to the nucleus. In silico comparison of the Towne, AD169, and related variant strains' genomes was coupled with serial iterations of a virus entry experiment, narrowing 28 candidate proteins responsible for the phenotype to four. Reintroduction of UL148 significantly rescued genome trafficking. Differential behavior of virus strains can be exploited to elucidate gene function.

  • Comparative mutagenesis of Pseudorabies and Epstein-Barr virus gH identifies a structural determinant within domain III of gH required for surface expression and entry function. [PublishAheadOfPrint]

  • Herpesviruses infect cells using the conserved core fusion machinery composed of glycoproteins B (gB) and gH/gL. The gH/gL complex plays an essential but still poorly characterized role in membrane fusion and cell tropism. Our previous studies demonstrated that the conserved disulfide bond (DB) C278/C335 of domain II (D-II) of Epstein-Barr virus (EBV) gH has an epithelial cell-specific function, whereas the interface of D-II/D-III is involved in formation of the B cell entry complex by binding to gp42. To extend these studies, we compared gH of the alphaherpesvirus Pseudorabies virus (PrV) with gH of the gammaherpesvirus EBV to identify functionally equivalent regions critical for gH function during entry. We identified several conserved amino acids surrounding the conserved DB that connects three central helices of D-III of PrV and EBV gH. The present study verified that the conserved DB and several contacting amino acids in D-III modulate cell surface expression, and thereby contribute to gH function. In line with this finding, we found that DB C404/C439 and T401 are important for cell-to-cell spread and efficient entry of PrV. This parallel comparison between PrV and EBV gH function brings new insights into how gH structure impacts fusion function during herpesvirus entry.

    IMPORTANCE The alphaherpesvirus PrV is known for its neuroinvasion, whereas the gammaherpesvirus EBV is associated with cancer of epithelial and B cell origin. Despite low amino acid conservation, PrV and EBV gH show a strikingly similar structure. Interestingly, both PrV and EBV gH contain a structural motif composed of a DB and supporting amino acids which is highly conserved within the Herpesviridae. Our study verified that PrV gH uses a minimal motif with the DB as core, whereas the DB of EBV gH forms extensive connections through hydrogen bonds to surrounding amino acids ensuring the cell surface expression of gH/gL. Our study verifies that the comparative analysis of distantly-related herpesviruses such as PrV and EBV allows the identification of common gH functions. In addition, we provide an understanding how functional domains can evolve over time resulting in subtle differences in domain structure and function.

  • The 1918 PB2 protein enhances virulence through the disruption of inflammatory and Wnt-mediated signaling in mice. [PublishAheadOfPrint]

  • The 1918-1919 influenza pandemic remains the single greatest infectious disease outbreak in the past century. Mouse and nonhuman primate infection models have shown that the 1918 virus induces overly aggressive innate and pro-inflammatory responses. To understand the response to viral infection and the role of individual 1918 genes on the host response to the 1918 virus, we examined reassortant avian viruses nearly identical to the pandemic 1918 virus (1918-like avian virus) carrying either the 1918 HA or PB2 gene. In mice, both genes enhanced 1918-like avian virus replication, but only the mammalian host-adaptation of the 1918-like avian virus through reassortment of the 1918 PB2 led to increased lethality. Through the combination of viral genetics and host transcriptional profiling, we provide a multidimensional view of the molecular mechanisms by which the 1918 PB2 gene drives viral pathogenicity. We demonstrate that 1918 PB2 enhances immune and inflammatory responses concomitant with increased cellular infiltration in the lung. We also show for the first time, that 1918 PB2 expression results in the repression of both canonical and non-canonical Wnt signaling pathways, which are crucial for inflammation-mediated lung regeneration and repair. Finally, we utilize regulatory enrichment and network analysis to define the molecular regulators of inflammation, epithelial regeneration, and lung immunopathology that are dysregulated during influenza virus infection. Taken together, our data suggest that while both HA and PB2 are important for viral replication, only 1918 PB2 exacerbates lung damage in mice infected with a reassortant 1918-like avian virus.

    IMPORTANCE As viral pathogenesis is determined in part by the host response, understanding the key host molecular driver(s) of virus-mediated disease, in relation to individual viral genes, is a promising approach to host-oriented drug efforts in preventing disease. Previous studies have demonstrated the importance of host adaptive genes, HA and PB2, in mediating disease although the mechanisms by which they do so are still poorly understood. Here, we combine viral genetics and host transcriptional profiling to show that although both 1918 HA and 1918 PB2 are important mediators of efficient viral replication, only 1918 PB2 impacts the pathogenicity of an avian influenza virus sharing high homology to the 1918 pandemic influenza virus. We demonstrate that 1918 PB2 enhances deleterious inflammatory responses and the inhibition of regeneration and repair functions coordinated by Wnt signaling in the lungs of infected mice, thereby promoting virus-associated disease.

  • HMGB1 promotes hepatitis C virus replication by interaction with the stem-loop 4 in viral 5 untranslated region [PublishAheadOfPrint]

  • High-mobility group box 1 (HMGB1) protein is a highly conserved nuclear protein and involved in multiple human diseases including infectious diseases, immune disorders, metabolic disorders, and cancer. HMGB1 is comprised of two tandem HMG boxes (A box and B box) containing DNA-binding domains, and an acidic C-terminal peptide. It's reported that HMGB1 enhances viral replication by binding to viral proteins. However, its role in hepatitis C virus (HCV) replication is unknown. Here, we showed that HMGB1 promoted HCV replication but had no effect on HCV translation. RNA immunoprecipitation experiments indicated that the positive-strand, not negative-strand, of HCV RNA interacted with HMGB1. HCV infection triggered HMGB1 protein translocation from the nucleus to the cytoplasm, in which it interacted with HCV genome. Moreover, the A box of HMGB1 is the pivotal domain to interact with the stem-loop 4 (SL4) of HCV 5rrsquo; untranslated region (5rrsquo; UTR). Deletion of HMGB1 A box abrogated the enhancement of HCV replication by HMGB1. Our data suggested that HMGB1 serves as a proviral factor of HCV to facilitate viral replication in the hepatocytes by interaction with HCV genome.

    IMPORTANCE Hepatitis C virus (HCV) is a major global health threat with more than 170 million people infection worldwide. These patients are at high risk of developing severe liver diseases such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Currently, no vaccine is available. Many host factors may be implicated in the pathogenesis of HCV-related diseases. In this study, we found a novel HCV RNA-binding protein HMGB1 that promotes HCV RNA replication. Moreover, the SL4 in 5rrsquo; UTR of HCV genome is the key region for HMGB1 binding and the A box of HMGB1 protein is the functional domain to interact with HCV RNA and enhance viral replication. HMGB1 appears to play an important role in HCV-related diseases and further investigation is highly encouraged to elucidate the specific actions of HMGB1 in HCV pathogenesis.

  • Intranasal introduction of Fc-fused interleukin-7 provides long-lasting prophylaxis from lethal influenza infection [PublishAheadOfPrint]

  • Influenza A virus (IAV) infection frequently causes hospitalization and mortality due to severe immunopathology. Annual vaccination and antiviral drugs are the current countermeasures against IAV infection, but have a limited efficacy against new IAV variants. Here, we show that intranasal pretreatment of Fc-fused interleukin-7 (IL-7-mFc) protect mice from lethal IAV infections. Protective activity of IL-7-mFc relies on transcytosis via neonatal Fc receptor (FcRn) in the lung and lasts for several weeks. Introduction of IL-7-mFc alters pulmonary immune environments, leading to recruitment of T cells from circulation and their subsequent residency as the tissue-resident memory-like T (TRM-like) cells. IL-7-mFc-primed pulmonary TRM-like cells contribute to protection upon IAV infection by dual modes. Firstly, TRM-like cells, although not antigen specific but polyclonal, attenuate viral replication at the early phase of IAV infection. Secondly, TRM-like cells augment expansion of IAV-specific CTLs, in particular at the late phase of infection, which directly control viruses. Thus, accelerated viral clearance facilitated by pulmonary T cells, which are either antigen-specific or not, alleviates immunopathology in the lung and mortality from IAV infection. Depleting a subset of pulmonary T cells indicates that both CD4 and CD8 T cells contribute to protection from IAV, although IL-7-primed CD4 T cells have a more prominent role. Collectively, we propose intranasal IL-7-mFc pretreatment as an effective means for generating protective immunity against IAV infections, which could be applied to a potential prophylaxis for influenza pandemics in future.

    IMPORTANCE The major consequence of a highly-pathogenic IAV infection is severe pulmonary inflammation, which can result in organ failure and death at worst. Although vaccines for seasonal IAVs are effective, frequent variation of surface viral proteins hampers development of protective immunity. In this study, we demonstrated that intranasal IL-7-mFc pretreatment protected immunologically naïve mice from lethal IAV infections. Intranasal pretreatment of IL-7-mFc induced an infiltration of T cells in the lung, which reside as effector/memory T cells with lung-retentive markers. Those IL-7-primed pulmonary T cells contributed to development of protective immunity upon IAV infection, reducing pulmonary immunopathology, while increasing IAV-specific cytotoxic T lymphocytes. Since a single treatment of IL-7-mFc was effective in the protection against multiple strains of IAV for an extended period of time, our findings suggest a possibility that IL-7-mFc treatment, as a potential prophylaxis, can be developed for controlling highly pathogenic IAV infections.

  • Mutations in pseudorabies virus glycoproteins gB, gD and gH functionally compensate for absence of gL [PublishAheadOfPrint]

  • Entry of herpesviruses depends on the combined action of viral glycoproteins (g)B and the heterodimeric gH/gL complex which are activated by binding of the virion to specific cellular receptors. Although gB carries signatures of a bona fide fusion protein, efficient membrane fusion requires gH/gL. However, although gB and gH/gL are essential for entry, the alphaherpesvirus Pseudorabies Virus (PrV) is capable of limited cell-to-cell spread in the absence of gL. To understand gH/gL function in more detail, the limited spread of PrV-gL was used for reversion analyses by serial cell culture passages. In a first experiment an infectious gL-negative mutant was isolated in which gL function was substituted by generation of a gD-gH hybrid protein (Klupp and Mettenleiter, 1999). In a second, independent experiment PrV-gLPassB4.1 was isolated, which also replicated productively without gL. Sequence analysis revealed mutations in gH, but also in gB and gD. In a transfection based fusion assay two amino acid substitutions in the N-terminal part of gHB4.1 (L70P; W103R) were found to be sufficient to compensate for lack of gL, while mutations present in gBB4.1 enhanced fusogenicity. Co-expression of gBB4.1 with the homologous gHB4.1 resulted in strongly enhanced syncytium formation, which was further augmented by truncation of the gBB4.1 C-terminal 29 amino acids. Nevertheless, gH was still required for membrane fusion. Surprisingly, co-expression of gDB4.1 blocked syncytium formation in the fusion assays, which could be attributed to a V106A substitution within the ectodomain of gDB4.1.

    IMPORTANCE In contrast to many other enveloped viruses, herpesviruses rely on the concerted action of four viral glycoproteins for membrane fusion during infectious entry. Although the highly conserved gB shows signatures of a fusion protein, for fusion induction it requires the gH/gL complex, whose role is still elusive. Here we demonstrate fusion activation by gH in the absence of gL after reversion analysis of gL-deleted pseudorabies virus. This gL-independent fusion activity depended on single amino acid exchanges affecting the gL-binding domain in gH, increasing fusogenicity in gB and allowing negative fusion regulation by gD. Thus, our results provide novel information on the interplay in the fusion machinery of herpesviruses.

  • Structural, Mechanistic, and Antigenic Characterization of the Human Astrovirus Capsid [PublishAheadOfPrint]

  • Human astroviruses (HAstVs) are non-enveloped, positive-sense, single-stranded RNA viruses and are a leading cause of viral gastroenteritis. HAstV particles display T=3 icosahedral symmetry formed by 180 copies of the capsid protein (CP), which undergoes proteolytic maturation to generate infectious HAstV particles. Little is known about the molecular features that govern HAstV particle assembly, maturation, infectivity, and immunogenicity. Here we report the crystal structures of the two main structural domains of the HAstV CP: the core domain at 2.60-AAring; resolution and the spike domain at 0.95-AAring; resolution. Fitting of these structures into the previously determined 25-AAring; resolution electron cryomicroscopy density maps of HAstV allowed us to characterize the molecular features on the surfaces of immature and mature T=3 HAstV particles. The highly electropositive inner surface of HAstV support a model in which interaction of the HAstV CP core with viral RNA is a driving force in T=3 HAstV particle formation. Additionally, mapping conserved residues onto the HAstV CP core and spike domains in the context of the immature and mature HAstV particles reveals dramatic changes to the exposure of conserved residues during virus maturation. Indeed, we show that antibodies raised against mature HAstV have reactivity to both the HAstV CP core and spike domains, revealing for the first time that the CP core domain is antigenic. Together these data provide new molecular insights into HAstV that have practical applications for the development of vaccines and antiviral therapies.

    IMPORTANCE Astroviruses are a leading cause of viral diarrhea in young children, immunocompromised individuals, and the elderly. Despite the prevalence of astroviruses, little is known at the molecular level how the astrovirus particle assembles and is converted into an infectious, mature virus. In this paper, we describe the high-resolution structures of the two main astrovirus capsid proteins. Fitting these structures into previously determined low-resolution maps of astrovirus has allowed us to characterize the molecular surfaces of the immature and mature astrovirus. Our studies provide the first evidence that astroviruses undergo viral RNA-dependent assembly. We also provide new insight into the molecular mechanisms that lead to astrovirus maturation and infectivity. Finally, we show that both capsid proteins contribute to the adaptive immune response against astrovirus. Together, these studies will help to guide the development of vaccines and antiviral drugs targeting astrovirus.

  • SIVagm efficiently utilizes non-CCR5 entry pathways in African green monkey lymphocytes: Potential role for GPR15 and CXCR6 as Viral Coreceptors [PublishAheadOfPrint]

  • African green monkeys (AGM) are natural hosts of simian immunodeficiency virus (SIV) and infection in these animals is generally non-pathogenic, whereas infection of non-natural hosts such as rhesus macaques (RM) is commonly pathogenic. CCR5 has been described as the primary entry coreceptor for SIV in vivo while human-derived CXCR6 and GPR15 also appear to be used in vitro. However, sooty mangabeys that are genetically deficient in CCR5 due to an out-of-frame deletion are infectable with SIVsmm, indicating that SIVsmm can use alternative coreceptors in vivo. In this study, we examined the CCR5-dependence of SIV strains derived from vervet AGM (SIVagmVer) and the ability of AGM-derived GPR15 and CXCR6 to serve as potential entry coreceptors. We found that SIVagmVer efficiently replicated in AGM and RM PBMC in the presence of the CCR5 antagonist, maraviroc despite the fact that maraviroc was capable of blocking the CCR5-tropic strains SIVmac239, SIVsmE543-3 and SHIV-AD8 in RM PBMC. We also found that AGM-CXCR6, and AGM-GPR15 to a lesser extent, supported entry of pseudotype viruses bearing SIVagm envelopes, including SIVagm transmitted/founder envelopes. Lastly, we found that CCR5, GPR15 and CXCR6 mRNA were detected in AGM and RM memory CD4+ T cells. These results suggest that GPR15 and CXCR6 are expressed on AGM CD4+ T cells and are potential alternative coreceptors for SIVagm use in vivo. These data suggest that the use of non-CCR5 entry pathways may be a common feature of SIV replication in natural host species, with potential to contribute to non-pathogenicity in these animals.

    IMPORTANCE African green monkeys (AGM) are natural hosts of SIV and infection in these animals generally does not cause AIDS, whereas SIV-infected rhesus macaques (RM) typically develop AIDS. Although, it has been reported that SIV generally uses CD4 and CCR5 to enter target cells in vivo, other molecules, such as GPR15 and CXCR6, also function as SIV coreceptors in vitro. In this study, we investigated whether SIV from the vervet AGM can use non-CCR5 entry pathways, as has been observed in sooty mangabeys. We found that SIVagmVer efficiently replicated in AGM and RM peripheral blood mononuclear cells in the presence of CCR5 antagonist, maraviroc, suggesting that non-CCR5 entry pathways can support SIVagm. We found that AGM-derived GPR15 and CXCR6 support SIVagmVer entry in vitro and may serve as entry coreceptors for SIVagm in vivo since their mRNA was detected in AGM memory CD4+ T cells, the preferred target cells of SIV.

  • Deletion of a Predicted {beta}-sheet Domain within the Amino-terminus of Herpes Simplex Virus Glycoprotein K (gK) Conserved among Alphaherpesviruses Prevents Virus Entry into Neuronal Axons [PublishAheadOfPrint]

  • We have shown previously that HSV-1 lacking expression of the entire gK or expressing gK with a 38 amino acid deletion (gK31-68 mutation) failed to infect ganglionic neurons after ocular infection of mice. We constructed a new model for the predicted 3-dimensional structure of gK revealing that the gK31-68 mutation spans a well-defined bbeta;-sheet structure within the amino terminus of gK, which is conserved among alphaherpesviruses. The HSV-1(McKrae) gK31-68 virus was tested for ability to enter into ganglionic neuronal axons in cell culture of explanted rat ganglia using a novel virus entry proximity ligation assay (VEPLA). In this assay, cell-surface bound virions were detected by the colocalization of glycoprotein D (gD) and its cognate receptor nectin-1 on infected neuronal surfaces. Capsids that have entered into the cytoplasm were detected by the colocalization of the virion tegument protein UL37 with dynein required for loading of virion capsids onto microtubules for retrograde transport to the nucleus. HSV-1(McKrae) gK31-68 attached to cell surfaces of Vero cells and ganglionic axons in cell culture as efficiently as wild-type HSV-1(McKrae). However, unlike the wild-type virus, the mutant virus failed to enter into the axoplasm of ganglionic neurons. This work suggests that the amino terminus of gK is a critical determinant for entry into neuronal axons and may serve similar conserved functions for other alphaherpesviruses.

    IMPORTANCE Alphaherpesviruses unlike beta and gamma herpesviruses have the unique ability to infect and establish latency in neurons. Glycoprotein K (gK) and the membrane protein UL20 are conserved among all alphaherpesviruses. We show here that a predicted bbeta;-sheet domain, which is conserved among alphaherpesviruses, functions in HSV-1 entry into neuronal axons suggesting that it may serve similar functions for other herpesviruses. These results are in agreement with our previous observations that deletion of this gK domain prevents the virus from successfully infecting ganglionic neurons after ocular infection of mice.

  • Investigating the influence of ribavirin on human respiratory syncytial virus RNA synthesis using a high-resolution RNAseq approach [PublishAheadOfPrint]

  • Human respiratory syncytial virus (HRSV) is a major cause of serious respiratory tract infection. Treatment options include administration of ribavirin, a purine analog, although the mechanism of its anti-HRSV activity is unknown. We used RNAseq to investigate genome mutation frequency and viral mRNA accumulation in HRSV-infected cells that had been untreated or treated with ribavirin. In the absence of ribavirin, HRSV specific transcripts accounted for up to one third of total RNA reads from the infected cell RNA population. Ribavirin treatment resulted in a greater than 90% reduction in abundance of viral mRNA reads, while at the same time no such reduction was detected for the abundance of cellular transcripts. The presented data revealed that ribavirin significantly increased the frequency of HRSV-specific RNA mutations, suggesting direct influence on the fidelity of the HRSV polymerase. The presented data shows transition and transversion occurs during HRSV replication, and that these changes occurred in llsquo;hot spots' along the HRSV genome. Examination of nucleotide substitution rates in the viral genome indicated an increase in the frequency of transition but not transversion mutations in the presence of ribavirin. In addition, our data indicated that in the continuous cell types used, and at the time points analyzed, the abundance of some HRSV mRNAs did not reflect the order in which the mRNAs were transcribed.

    IMPORTANCE Human respiratory syncytial virus (HRSV) is a major pediatric pathogen. Ribavirin can be used in children who are extremely ill to reduce the amount of virus and lower the burden of disease. Ribavirin is used as an experimental therapy with other viruses. The mechanism of action of ribavirin in HRSV is not well understood, although it is thought to increase the mutation rate of the viral polymerase during replication. To investigate this we used a high-resolution approach that allowed us to determine the genetic sequence of the virus to great depth of coverage. We found that ribavirin did not cause a detectable change in the relative amounts of viral mRNA transcripts. However, we found that ribavirin treatment did indeed cause an increase in the number of mutations, which was associated with a decrease in virus production.

  • Nucleoprotein of Human Parainfluenza Virus Type 3 interaction with Matrix protein Mediates Internal Viral Protein Assembly [PublishAheadOfPrint]

  • Human Parainfluenza virus type 3 (HPIV3) belongs to the Paramyxoviridae family. Its three internal viral proteins, the nucleoprotein (N), the phosphoprotein (P), and the polymerase (L), form the ribonucleoprotein (RNP) complex, which encapsidates the viral genome and associates with M protein for virion assembly. We previously showed that the M protein expressed alone is sufficient to assemble and release virus-like particles (VLPs), and a point mutant of M protein, ML305A, has VLP formation ability similar to that of wild-type M protein. In addition, the recombinant HPIV3 containing the ML305A mutation (rHPIV3-ML305A) could be successfully recovered. Herein, we found that the titer of rHPIV3-ML305A was at least 10-fold lower than the titer of rHPIV3. Using VLP incorporation and co-immunoprecipitation assays, we found that M-VLPs can efficiently incorporate N and P via N-M or P-M interaction, and ML305A-VLPs had a similar ability to incorporate P via P-M interaction but unable to incorporate N and no longer interacted with N. Furthermore, we found that the incorporation of P into ML305A-VLPs, but not M-VLPs, was inhibited in the presence of N. In addition, we provide evidence that the C-terminal region of P is involved in its interaction with both N and M, and N binding to the C-terminal region of P inhibited the incorporation of P into ML305A-VLPs. Our findings provide new molecular details to support the idea that N-M not P-M interaction is critical for packaging N and P into infectious viral particles.

    IMPORTANCE Human parainfluenza virus type 3 (HPIV3) is a nonsegmented, negative-sense, single-stranded RNA virus that belongs to the Paramyxoviridae family and can cause lower respiratory tract infections in infants and young children as well as elderly or immunocompromised individuals. However, no effective vaccine has been developed or licensed. We used virus-like particle (VLP) incorporation and co-immunoprecipitation assays to determine how M protein assembles internal viral proteins. We demonstrated that both nucleoprotein (N) and phosphoprotein (P) can incorporate into M-VLPs, and N inhibited the M-P interaction via the binding of N to the C-terminus of P. We also provide additional evidence that N-M interaction but not P-M interaction is critical for the regulation of HPIV3 assembly. Our studies provide a more complete characterization of HPIV3 virion assembly and substantiation that N interaction with M regulates internal viral organization.

  • High Degree of HIV-1 group M Genetic Diversity within Circulating Recombinant Forms: Insight into the Early Events of HIV-1M Evolution [PublishAheadOfPrint]

  • The existence of various highly divergent HIV-1 lineages and of recombination-derived sequence-tracts of indeterminate origin within established circulating recombinant forms (CRFs) strongly suggests that HIV-1 group M (HIV-1M) diversity is not fully represented under the current classification system. Here we use a fully exploratory screen for recombination on a set of 480 near full-length genomes representing the full known diversity of HIV-1M. We decompose recombinant sequences into their constituent parts and then use maximum likelihood phylogenetic analyses of this mostly recombination-free dataset to identify rare divergent sequence lineages that fall outside the major named HIV-1M taxonomic groupings. We find that many of the sequence fragments occurring within CRFs (including CRF04_cpx, CRF06_cpx, CRF11_cpx, CRF18_cpx, CRF25_cpx, CRF27_cpx and CRF49_cpx) are in fact likely derived from divergent unclassified parental lineages that may pre-date the current subtypes, even though they are presently identified as derived from currently defined HIV-1M subtypes. Our evidence suggests that some of these CRFs are predominantly descended from what were/are major previously unidentified HIV-1M lineages that were likely epidemiologically relevant during the early stages of the HIV-1M epidemic. The restriction of these divergent lineages to the Congo basin suggests that they were either less infectious and/or simply not present at the time and place of the initial migratory wave that triggered the global epidemic.

    IMPORTANCE HIV-1 group M (HIV-1M) likely spread to the rest of the world from the Congo basin in the mid-1900s (Faria N.R. et al., 2014, Science 346:56) and is today the principal cause of the AIDS pandemic. Here, we show that large sequence fragments from several HIV-1M Circulating Recombinant Forms (CRFs) are derived from divergent parental lineages that cannot reasonably be classified within the nine established HIV-1M subtypes. These lineages are likely to have been epidemiologically relevant in the Congo basin at the onset of the epidemic. Nonetheless, they appear not to have undergone the same explosive global spread as other HIV-1M subtypes perhaps because they were less transmissible. Concerted efforts to characterize more of these divergent lineages could allow the accurate inference and chemical synthesis of epidemiologically key ancestral HIV-1M variants so as to directly test competing hypotheses relating to the viral genetic factors that enabled the present pandemic.

  • HIV-1 antibody neutralization breadth is associated with enhanced HIV-specific CD4+ T cell responses [PublishAheadOfPrint]

  • Antigen-specific CD4+ T helper cell responses have long been recognized to be a critical component of effective vaccine immunity. CD4+ T cells are necessary to generate and maintain humoral immune responses by providing help to antigen-specific B cells for the production of antibodies. In HIV infection, CD4+ T cells are thought to be necessary for the induction of Env-specific broadly neutralizing antibodies. However, few studies have investigated the role of HIV-specific CD4+ T cells in association with HIV neutralizing antibody activity in vaccination or natural infection settings. Here we conducted a comprehensive analysis of HIV-specific CD4+ T cell responses in a cohort of 34 untreated HIV-infected viremic controllers matched for viral load, with and without neutralizing antibody breadth to a panel of viral strains. Our results show that the breadth and magnitude of Gag-specific CD4+ T cell responses were significantly higher in individuals with neutralizing antibodies versus those without neutralizing antibodies. The breadth of Gag-specific CD4+ T cell responses was positively correlated with the breadth of neutralizing antibody activity. Furthermore, the breadth and magnitude of gp41-specific, but not gp120-specific, CD4+ T cell responses were significantly elevated in individuals with neutralizing antibodies. Together, these data suggest that robust Gag-specific CD4+ T cells, and to a lesser extent, gp41-specific CD4+ T cells, may provide important intermolecular help to Env-specific B cells that promote the generation or maintenance of Env-specific neutralizing antibodies.

    IMPORTANCE One of the earliest discoveries related to CD4+ T cell function was their provision of help to B cells in the development of antibody responses. Yet, little is known about the role of CD4+ T helper responses in the setting of HIV infection, and no studies to date have evaluated the impact of HIV-specific CD4+ T cells on the generation of antibodies that can neutralize multiple different strains of HIV. Here, we addressed this question by analyzing HIV-specific CD4+ T cell responses in untreated HIV-infected persons with and without neutralizing antibodies. Our results indicate that HIV-infected persons with neutralizing antibodies have significantly more robust CD4+ T cell responses targeting Gag and gp41 proteins when compared with individuals that lack neutralizing antibodies. These associations suggest that Gag- and gp41-specific CD4+ T cell responses may provide robust help to B cells for the generation or maintenance of neutralizing antibodies in natural HIV-infection.

  • Scanning mutagenesis of human cytomegalovirus gH/gL. [PublishAheadOfPrint]

  • The core, conserved function of the herpesvirus gH/gL is to promote gB-mediated membrane fusion during entry, although the mechanism is poorly understood. The HCMV gH/gL can exist as either the gH/gL/gO trimer, or the gH/gL/UL128-131 pentamer. One model suggests that gH/gL/gO provides the core fusion role during entry into all cells within the broad tropism of HCMV, whereas gH/gL/UL128-131 acts at an earlier stage, by a distinct receptor-binding mechanism to enhance infection of select cell types such as epithelial, endothelial and monocyte/macrophages. To further study the distinct functions of these complexes, individual charged cluster-to-alanine (CCTA) mutants of gH and gL were combined to generate a library of 80 mutant gH/gL heterodimers. The majority of the mutant gH/gL complexes were unable to facilitate gB-mediated membrane fusion in transient expression cell-cell fusion experiments. In contrast, these mutants supported the formation of gH/gL/UL128-131 complexes that could block HCMV infection in receptor interference experiments. These results suggest that receptor interactions with gH/gL/UL128-131 involve surfaces contained on the UL128-131 proteins, not on gH/gL. gH/gL/UL128-131 receptor interference could be blocked with anti-gH antibodies, suggesting that interference is a cell surface phenomenon, and that anti-gH antibodies can block gH/gL/UL128-131, distinct from gH/gL/gO.

    IMPORTANCE Interest in the gH/gL complexes of HCMV (especially gH/gL/UL128-131) as vaccine targets has far outpaced our understanding of the mechanism by which they facilitate in entry and contribute to broad cellular tropism. For Epstein-Barr virus (EBV), gH/gL and gH/gL/gp42 are both capable of promoting gB fusion for entry into epithelial or B cells, respectively. In contrast, HCMV gH/gL/gO appears to be the sole fusion cofactor that promotes gB fusion activity, whereas gH/gL/UL128-131 expands cell tropism through a distinct, yet unknown mechanism. This study suggests that surfaces of HCMV gH/gL are critical for promoting gB fusion but are dispensable for gH/gL/UL128-131 receptor interaction. This underscores the importance of gH/gL/gO in HCMV entry into all cell types, and reaffirms the complex as candidate target for vaccine development. The two functionally distinct forms of gH/gL present in HCMV make for a useful model to study the fundamental mechanisms by which herpesvirus gH/gL regulates gB fusion.

  • A single amino-acid substitution in the M protein attenuates Japanese encephalitis virus in mammalian hosts [PublishAheadOfPrint]

  • Japanese Encephalitis virus (JEV) membrane (M) protein plays important structural roles in the processes of fusion and maturation of progeny virus during cellular infection. The M protein is anchored in the viral membrane and its ectodomain is composed of a flexible N-terminal loop and a perimembrane helix. In this study, we performed site-directed mutagenesis on the residue 36 of JEV M protein and showed that the resulting mutation had little or no effect on the entry process but greatly affected virus assembly in mammalian cells. Interestingly, this mutant virus had a host-dependent phenotype, and could develop a wild-type infection in insect cells. Experiments performed on infectious virus as well as in a virus-like particle (VLP) system indicate that the JEV mutant expresses structural proteins but fails to form infectious particles in mammalian cells. Using a mouse model for JEV pathogenesis, we showed that the mutation conferred complete attenuation in vivo. The production of JEV neutralizing antibodies in challenged mice was indicative of the immunogenicity of the mutant virus in vivo. Together, our results indicate that the introduction of a single mutation in the M protein, while being tolerated in insect cells, strongly impacts JEV infection in mammalian hosts.

    IMPORTANCE JEV is a mosquito-transmitted Flavivirus and is a medically important pathogen in Asia. The M protein is thought to be important for accommodating the structural rearrangements undergone by the virion during viral assembly, and may play additional roles in the JEV infectious cycle. In the present study, we show that a sole mutation in the M protein impairs JEV infection cycle in mammalian hosts, but not in mosquito cells. This finding highlights differences in Flavivirus assembly pathways amongst hosts. Moreover, infection of mice indicated that the mutant was completely attenuated and triggered a strong immune response to JEV, thus providing new insights for further development of JEV vaccines.

  • HTLV-1 infection of the three monocyte subsets contributes to viral burden in humans [PublishAheadOfPrint]

  • Because viral DNA burden correlates with disease development, we investigated the contribution of monocyte subsets (classical, intermediate and non-classical) to the total viral burden in 22 HTLV-1 infected individuals by assessing their infectivity status, frequency, as well as chemotactic and phagocytic functions. The three monocyte subsets sorted from HTLV-1 infected individuals were all positive for viral DNA and the frequency of classical monocytes in blood was lower while expression levels of the chemokine receptors CCR5, CXCR3 and CX3CR1 was higher; the percentage of intermediate monocytes and their chemokine receptor expression did not differ. However, the migratory capacity of intermediate monocytes to CCL5, the ligand for CCR5, was higher and there was a higher proportion of non-classical monocytes that expressed CCR1, CXCR3 and CX3CR1. The level of viral DNA in the monocyte subsets correlated with the migration capacity to CCL2, CCL5 and CX3CL1 for classical monocytes, with lower phagocytosis for intermediate monocytes, and with the level of viral DNA in CD8+ and CD4+ T-cells for non-classical monocytes. These data suggest a model whereby HTLV-1 infection augments the number of classical monocytes that migrate to tissues and become infected and the number of infected non-classical monocytes that transmit virus to CD4+ and CD8+ T-cells. These results, together with prior findings in a macaque model of HTLV-1 infection, support the notion that infection of monocytes by HTLV-1 is likely a requisite for viral persistence in humans.

    IMPORTANCE Monocytes have been implicated in immune regulation and disease progression in patients with HTLV-1-associated inflammatory diseases. We detected HTLV-1 viral DNA in all three monocyte subsets and found that infection impacts surface receptor expression, migratory function and subset frequency. The frequency of non-classical patrolling monocytes is increased in HTLV-1-infected individuals and they have increased expression of CCR1, CXCR3 and CX3CR1. The viral DNA level in non-classical monocytes correlated with viral DNA level in CD4+ and CD8+ T-cells. Altogether, these data suggest an increased recruitment of classical monocytes to inflammation sites that may result in virus acquisition and, in turn, facilitates virus dissemination and viral persistence. Our findings thus provide new insight into the importance of monocyte infection in viral spread and suggest targeting monocytes for therapeutic intervention.

  • The C5-substituted Tetrahydropyrano-tetrahydofuran-derived Protease Inhibitors (PIs) Exert Potent Inhibition of the Replication of HIV-1 Variants Highly Resistant to Various PIs Including Darunavir [PublishAheadOfPrint]

  • We identified three non-peptidic HIV-1 protease inhibitors (PIs), GRL-015, -085, and -097 containing tetrahydropyrano-tetrahydrofuran (Tp-THF) with a C5 hydroxyl. The three compounds were potent against a wild-type laboratory HIV-1 strain (HIV-1WT) with 50% effective concentrations (EC50s) of 3.0-49 nM and minimal cytotoxicity with 50% cytotoxic concentrations (CC50) for GRL-015, -085, and -097 of 80, ggt;100, and ggt;100 mmu;M, respectively. All the three compounds potently inhibited the replication of highly PI-resistant HIV-1 variants selected with each of the currently available PIs and recombinant clinical HIV-1 isolates obtained from patients harboring multi-drug resistant HIV-1 variants (HIVMDR). Importantly, darunavir (DRV) was ggt;1,000 times less active against a highly DRV-resistant HIV-1 variant (HIV-1DRVRP51), the three compounds remained active to HIV-1DRVRP51 only with 6.8- to 68-fold reduction. Moreover, the emergence of drug resistant HIV-1s against the three compounds was considerably delayed compared to the case of DRV. Especially, HIV-1 variants resistant to GRL-085 and -097 did not emerge even by using two different highly-DRV-resistant HIV-1s as a starting population. In the structural analyses, Tp-THF of GRL-015, -085, and -097 showed strong hydrogen-bond interactions with the backbone atoms of active-site amino acid residues (Asp29 and Asp30) of HIV-1 protease. A strong hydrogen bonding formation between the hydroxyl moiety of Tp-THF and a carbonyl oxygen atom of Gly48 was newly identified. The present findings warrant that the three compounds be further studied as possible therapeutic agents for treating individuals harboring wild-type and/or HIVMDR.

    IMPORTANCE Darunavir (DRV) inhibits the replication of most existing multidrug-resistant HIV-1s and has a high genetic barrier. However, the emergence of highly DRV-resistant HIV-1s (HIVDRVR) has recently been reported in vivo and in vitro. Here we identified three novel HIV-1 protease inhibitors (PIs) containing a tetrahydropyrano-tetrahydrofuran (Tp-THF) moiety with a C5 hydroxyl (GRL-015, -085, and -097), which potently suppress the replication of HIVDRVR. Moreover, the emergence of drug resistant HIV-1s against the three compounds was considerably delayed compared to the case of DRV. The C5 hydroxyl formed a strong hydrogen bonding interaction with the carbonyl oxygen atom of Gly48 of protease as examined in the structural analyses. Interestingly, a compound with Tp-THF lacking the hydroxyl moiety substantially decreased the activity against HIVDRVRs. The three novel compounds should be further developed as potential drugs for treating individuals harboring wild-type and multi-PI-resistant HIV variants as well as HIVDRVR.

  • A subset of CD4/CD8 double negative T cells expresses HIV proteins in patients on ART [PublishAheadOfPrint]

  • A major goal in HIV eradication research is characterizing the reservoir cells that harbor HIV in the presence of anti-retroviral therapy (ART), which reseed viremia after treatment is stopped. In general it is assumed that the reservoir consists of CD4+ T cells that express no viral proteins. However, recent findings suggest that this may be an overly simplistic view, and that the cells that contribute to the reservoir may be a diverse population that includes both CD4+ and CD4- cells. Here, we directly infected resting CD4+ T cells and used fluorescence-activated cell sorting (FACS) and Fiber-Optic Array Scanning Technology (FAST) to identify and image cells expressing HIV Gag. We found that Gag expression from integrated proviruses occurred in resting cells that lacked surface CD4, likely resulting from Nef and Env-mediated receptor internalization. We also extend our approach to detect cells expressing HIV proteins in patients suppressed on ART. We found evidence that rare Gag+ cells persist during ART and that these cells are often negative for CD4. We propose that these double negative aalpha;/bbeta; T cells that express HIV protein may be a component of the long-lived reservoir.

    IMPORTANCE A reservoir of infected cells persists in HIV-infected patients during anti-retroviral therapy (ART) that leads to rebound of virus if treatment is stopped. Here, we use flow cytometry and cell imaging to characterize protein expression in HIV infected resting cells. HIV Gag protein can be directly detected in infected resting cells and occurs with simultaneous loss of CD4, consistent with the expression of additional viral proteins such as Env and Nef. Gag+CD4- cells can also be detected in suppressed patients, suggesting that a subset of infected cells express proteins during ART. Understanding the regulation of viral protein expression during ART will be key to designing effective strategies to eradicate HIV reservoirs.

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