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Table of Contents for this page:

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

    JVI Current Issue

  • Editorial Board [Masthead]

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

  • Human Papillomavirus E2 Protein: Linking Replication, Transcription, and RNA Processing [Gems]

  • The human papillomavirus (HPV) life cycle is tightly linked to differentiation of the infected epithelium. This means that viral proteins must exert control over epithelial gene expression in order to optimize viral production. The HPV E2 protein controls replication, transcription, and viral genome partitioning during the viral infectious life cycle. It consists of a nucleic acid-binding domain and a protein-protein interaction domain separated by a flexible serine and arginine-rich hinge region. Over the last few years, mounting evidence has uncovered an important new role for E2 in viral and cellular RNA processing. This Gem discusses the role of E2 in controlling the epithelial cellular environment and how E2 might act to coordinate late events in the viral replication cycle.

  • To Conquer the Host, Influenza Virus Is Packing It In: Interferon-Antagonistic Strategies beyond NS1 [Gems]

  • The nonstructural protein NS1 is well established as a virulence factor of influenza A virus counteracting induction of the antiviral type I interferon system. Recent studies now show that viral structural proteins, their derivatives, and even the genome itself also contribute to keeping the host defense under control. Here, we summarize the current knowledge on these NS1-independent interferon escape strategies.

  • A Highly Conserved gp120 Inner Domain Residue Modulates Env Conformation and Trimer Stability [Pathogenesis and Immunity]

  • Previous studies have shown that highly conserved residues in the inner domain of gp120 are required for HIV-1 envelope glycoprotein (Env) transitions to the CD4-bound conformation (A. Finzi, S. H. Xiang, B. Pacheco, L. Wang, J. Haight, et al., Mol Cell 37:656nndash;667, 2010, http://dx.doi.org/10.1016/j.molcel.2010.02.012; A. Desormeaux, M. Coutu, H. Medjahed, B. Pacheco, A. Herschhorn, et al., J Virol 87:2549nndash;2562, 2013, http://dx.doi.org/10.1128/JVI.03104-12). Moreover, W69, a highly conserved residue located at the interface between layer 1 and layer 2 of the inner domain, was recently shown to be important for efficient Env recognition by CD4-induced (CD4i) antibodies capable of potent antibody-dependent cellular cytotoxicity (W. D. Tolbert, N. Gohain, M. Veillette, J. P. Chapleau, C. Orlandi, et al., 2016, Structure 24:697nndash;709, http://dx.doi.org/10.1016/j.str.2016.03.005; S. Ding, M. Veillette, M. Coutu, J. Prevost, L. Scharf, et al., 2016, J Virol 90:2127nndash;2134, http://dx.doi.org/10.1128/JVI.02779-15). We evaluated the contribution of the hydrophobicity of W69 to conformational changes of Env by replacing it with a series of residues with aliphatic or aromatic side chains of decreasing chain length. We have found that the hydrophobicity of residue 69 is important for Env processing, CD4 binding, and its transition to the CD4-bound conformation. The most deleterious effect was observed when W69 was replaced with alanine or glycine residues. However, the functions lost due to W69 mutations could be progressively restored with amino acids of increasing aliphatic chain length and fully recovered with residues bearing an aromatic ring. Interestingly, poor CD4 binding of W69A could be fully restored by introducing a compensatory mutation within layer 2 (S115W). Structural studies of HIV-1 gp120 coree W69A/S115W mutant bound to the CD4 peptide mimetic M48U1 and Fab of anti-cluster A antibody N60-i3 revealed no perturbations to the overall structure of the double mutant compared to the wild-type protein but identified higher mobility within the interface between layer 1 and layer 2, the bridging sheet region, and the CD4 binding site.

    IMPORTANCE HIV-1 Env transitions to the CD4-bound conformation are required for viral entry. Previous studies identified a highly conserved residue of the inner domain, W69, as being involved in these conformational transitions (A. Finzi, S. H. Xiang, B. Pacheco, L. Wang, J. Haight, et al., Mol Cell 37:656nndash;667, 2010, http://dx.doi.org/10.1016/j.molcel.2010.02.012). Here, we show that W69, located at the interface between gp120 and gp41 in the PGT151-bound trimer, plays a critical role in the interprotomer signaling induced by CD4 binding. This new information might be useful in immunogen design.

  • Poliovirus Polymerase Leu420 Facilitates RNA Recombination and Ribavirin Resistance [Genome Replication and Regulation of Viral Gene Expression]

  • RNA recombination is important in the formation of picornavirus species groups and the ongoing evolution of viruses within species groups. In this study, we examined the structure and function of poliovirus polymerase, 3Dpol, as it relates to RNA recombination. Recombination occurs when nascent RNA products exchange one viral RNA template for another during RNA replication. Because recombination is a natural aspect of picornavirus replication, we hypothesized that some features of 3Dpol may exist, in part, to facilitate RNA recombination. Furthermore, we reasoned that alanine substitution mutations that disrupt 3Dpol-RNA interactions within the polymerase elongation complex might increase and/or decrease the magnitudes of recombination. We found that an L420A mutation in 3Dpol decreased the frequency of RNA recombination, whereas alanine substitutions at other sites in 3Dpol increased the frequency of recombination. The 3Dpol Leu420 side chain interacts with a ribose in the nascent RNA product 3 nucleotides from the active site of the polymerase. Notably, the L420A mutation that reduced recombination also rendered the virus more susceptible to inhibition by ribavirin, coincident with the accumulation of ribavirin-induced G-ggt;A and C-ggt;U mutations in viral RNA. We conclude that 3Dpol Leu420 is critically important for RNA recombination and that RNA recombination contributes to ribavirin resistance.

    IMPORTANCE Recombination contributes to the formation of picornavirus species groups and the emergence of circulating vaccine-derived polioviruses (cVDPVs). The recombinant viruses that arise in nature are occasionally more fit than either parental strain, especially when the two partners in recombination are closely related, i.e., members of characteristic species groups, such as enterovirus species groups A to H or rhinovirus species groups A to C. Our study shows that RNA recombination requires conserved features of the viral polymerase. Furthermore, a polymerase mutation that disables recombination renders the virus more susceptible to the antiviral drug ribavirin, suggesting that recombination contributes to ribavirin resistance. Elucidating the molecular mechanisms of RNA replication and recombination may help mankind achieve and maintain poliovirus eradication.

  • Identification of a New Benzimidazole Derivative as an Antiviral against Hepatitis C Virus [Vaccines and Antiviral Agents]

  • Aminoquinolines and piperazines, linked or not, have been used successfully to treat malaria, and some molecules of this family also exhibit antiviral properties. Here we tested several derivatives of 4-aminoquinolines and piperazines for their activity against hepatitis C virus (HCV). We screened 11 molecules from three different families of compounds, and we identified anti-HCV activity in cell culture for six of them. Of these, we selected a compound (B5) that is currently ending clinical phase I evaluation for neurodegenerative diseases. In hepatoma cells, B5 inhibited HCV infection in a pangenotypic and dose-dependent manner, and its antiviral activity was confirmed in primary hepatocytes. B5 also inhibited infection by pseudoparticles expressing HCV envelope glycoproteins E1 and E2, and we demonstrated that it affects a postattachment stage of the entry step. Virus with resistance to B5 was selected by sequential passage in the presence of the drug, and reverse genetics experiments indicated that resistance was conferred mainly by a single mutation in the putative fusion peptide of E1 envelope glycoprotein (F291I). Furthermore, analyses of the effects of other closely related compounds on the B5-resistant mutant suggest that B5 shares a mode of action with other 4-aminoquinoline-based molecules. Finally, mice with humanized liver that were treated with B5 showed a delay in the kinetics of the viral infection. In conclusion, B5 is a novel interesting anti-HCV molecule that could be used to decipher the early steps of the HCV life cycle.

    IMPORTANCE In the last 4 years, HCV therapy has been profoundly improved with the approval of direct-acting antivirals in clinical practice. Nevertheless, the high costs of these drugs limit access to therapy in most countries. The present study reports the identification and characterization of a compound (B5) that inhibits HCV propagation in cell culture and is currently ending clinical phase I evaluation for neurodegenerative diseases. This molecule inhibits the HCV life cycle by blocking virus entry. Interestingly, after selection of drug-resistant virus, a resistance mutation in the putative fusion peptide of E1 envelope glycoprotein was identified, indicating that B5 could be used to further investigate the fusion mechanism. Furthermore, mice with humanized liver treated with B5 showed a delay in the kinetics of the viral infection. In conclusion, B5 is a novel interesting anti-HCV molecule that could be used to decipher the early steps of the HCV life cycle.

  • Derivation and Characterization of Pathogenic Transmitted/Founder Molecular Clones from Simian Immunodeficiency Virus SIVsmE660 and SIVmac251 following Mucosal Infection [Genetic Diversity and Evolution]

  • Currently available simian immunodeficiency virus (SIV) infectious molecular clones (IMCs) and isolates used in nonhuman primate (NHP) models of AIDS were originally derived from infected macaques during chronic infection or end stage disease and may not authentically recapitulate features of transmitted/founder (T/F) genomes that are of particular interest in transmission, pathogenesis, prevention, and treatment studies. We therefore generated and characterized T/F IMCs from genetically and biologically heterogeneous challenge stocks of SIVmac251 and SIVsmE660. Single-genome amplification (SGA) was used to identify full-length T/F genomes present in plasma during acute infection resulting from atraumatic rectal inoculation of Indian rhesus macaques with low doses of SIVmac251 or SIVsmE660. All 8 T/F clones yielded viruses that were infectious and replication competent in vitro, with replication kinetics similar to those of the widely used chronic-infection-derived IMCs SIVmac239 and SIVsmE543. Phenotypically, the new T/F virus strains exhibited a range of neutralization sensitivity profiles. Four T/F virus strains were inoculated into rhesus macaques, and each exhibited typical SIV replication kinetics. The SIVsm T/F viruses were sensitive to TRIM5aalpha; restriction. All T/F viruses were pathogenic in rhesus macaques, resulting in progressive CD4+ T cell loss in gastrointestinal tissues, peripheral blood, and lymphatic tissues. The animals developed pathological immune activation; lymphoid tissue damage, including fibrosis; and clinically significant immunodeficiency leading to AIDS-defining clinical endpoints. These T/F clones represent a new molecular platform for the analysis of virus transmission and immunopathogenesis and for the generation of novel "bar-coded" challenge viruses and next-generation simian-human immunodeficiency viruses that may advance the HIV/AIDS vaccine agenda.

    IMPORTANCE Nonhuman primate research has relied on only a few infectious molecular clones for a myriad of diverse research projects, including pathogenesis, preclinical vaccine evaluations, transmission, and host-versus-pathogen interactions. With new data suggesting a selected phenotype of the virus that causes infection (i.e., the transmitted/founder virus), we sought to generate and characterize infectious molecular clones from two widely used simian immunodeficiency virus lineages (SIVmac251 and SIVsmE660). Although the exact requirements necessary to be a T/F virus are not yet fully understood, we generated cloned viruses with all the necessary characteristic of a successful T/F virus. The cloned viruses revealed typical acute and set point viral-load dynamics with pathological immune activation, lymphoid tissue damage progressing to significant immunodeficiency, and AIDS-defining clinical endpoints in some animals. These T/F clones represent a new molecular platform for studies requiring authentic T/F viruses.

  • Reversion of Cold-Adapted Live Attenuated Influenza Vaccine into a Pathogenic Virus [Vaccines and Antiviral Agents]

  • The only licensed live attenuated influenza A virus vaccines (LAIVs) in the United States (FluMist) are created using internal protein-coding gene segments from the cold-adapted temperature-sensitive master donor virus A/Ann Arbor/6/1960 and HA/NA gene segments from circulating viruses. During serial passage of A/Ann Arbor/6/1960 at low temperatures to select the desired attenuating phenotypes, multiple cold-adaptive mutations and temperature-sensitive mutations arose. A substantial amount of scientific and clinical evidence has proven that FluMist is safe and effective. Nevertheless, no study has been conducted specifically to determine if the attenuating temperature-sensitive phenotype can revert and, if so, the types of substitutions that will emerge (i.e., compensatory substitutions versus reversion of existing attenuating mutations). Serial passage of the monovalent FluMist 2009 H1N1 pandemic vaccine at increasing temperatures in vitro generated a variant that replicated efficiently at higher temperatures. Sequencing of the variant identified seven nonsynonymous mutations, PB1-E51K, PB1-I171V, PA-N350K, PA-L366I, NP-N125Y, NP-V186I, and NS2-G63E. None occurred at positions previously reported to affect the temperature sensitivity of influenza A viruses. Synthetic genomics technology was used to synthesize the whole genome of the virus, and the roles of individual mutations were characterized by assessing their effects on RNA polymerase activity and virus replication kinetics at various temperatures. The revertant also regained virulence and caused significant disease in mice, with severity comparable to that caused by a wild-type 2009 H1N1 pandemic virus.

    IMPORTANCE The live attenuated influenza vaccine FluMist has been proven safe and effective and is widely used in the United States. The phenotype and genotype of the vaccine virus are believed to be very stable, and mutants that cause disease in animals or humans have never been reported. By propagating the virus under well-controlled laboratory conditions, we found that the FluMist vaccine backbone could regain virulence to cause severe disease in mice. The identification of the responsible substitutions and elucidation of the underlying mechanisms provide unique insights into the attenuation of influenza virus, which is important to basic research on vaccines, attenuation reversion, and replication. In addition, this study suggests that the safety of LAIVs should be closely monitored after mass vaccination and that novel strategies to continue to improve LAIV vaccine safety should be investigated.

  • Human Cathelicidin Compensates for the Role of Apolipoproteins in Hepatitis C Virus Infectious Particle Formation [Virus-Cell Interactions]

  • Exchangeable apolipoproteins (ApoA, -C, and -E) have been shown to redundantly participate in the formation of infectious hepatitis C virus (HCV) particles during the assembly process, although their precise role in the viral life cycle is not well understood. Recently, it was shown that the exogenous expression of only short sequences containing amphipathic aalpha;-helices from various apolipoproteins is sufficient to restore the formation of infectious HCV particles in ApoB and ApoE double-gene-knockout Huh7 (BE-KO) cells. In this study, through the expression of a small library of human secretory proteins containing amphipathic aalpha;-helix structures, we identified the human cathelicidin antimicrobial peptide (CAMP), the only known member of the cathelicidin family of antimicrobial peptides (AMPs) in humans and expressed mainly in bone marrow and leukocytes. We showed that CAMP is able to rescue HCV infectious particle formation in BE-KO cells. In addition, we revealed that the LL-37 domain in CAMP containing amphipathic aalpha;-helices is crucial for the compensation of infectivity in BE-KO cells, and the expression of CAMP in nonhepatic 293T cells expressing claudin 1 and microRNA miR-122 confers complete propagation of HCV. These results suggest the possibility of extrahepatic propagation of HCV in cells with low-level or no expression of apolipoproteins but expressing secretory proteins containing amphipathic aalpha;-helices such as CAMP.

    IMPORTANCE Various exchangeable apolipoproteins play a pivotal role in the formation of infectious HCV during the assembly of viral particles, and amphipathic aalpha;-helix motifs in the apolipoproteins have been shown to be a key factor. To the best of our knowledge, we have identified for the first time the human cathelicidin CAMP as a cellular protein that can compensate for the role of apolipoproteins in the life cycle of HCV. We have also identified the domain in CAMP that contains amphipathic aalpha;-helices crucial for compensation and show that the expression of CAMP in nonhepatic cells expressing claudin 1 and miR-122 confers complete propagation of HCV. We speculate that low levels of HCV propagation might be possible in extrahepatic tissues expressing secretory proteins containing amphipathic aalpha;-helices without the expression of apolipoproteins.

  • Viral Macro Domains Reverse Protein ADP-Ribosylation [Virus-Cell Interactions]

  • ADP-ribosylation is a posttranslational protein modification in which ADP-ribose is transferred from NAD+ to specific acceptors to regulate a wide variety of cellular processes. The macro domain is an ancient and highly evolutionarily conserved protein domain widely distributed throughout all kingdoms of life, including viruses. The human TARG1/C6orf130, MacroD1, and MacroD2 proteins can reverse ADP-ribosylation by acting on ADP-ribosylated substrates through the hydrolytic activity of their macro domains. Here, we report that the macro domain from hepatitis E virus (HEV) serves as an ADP-ribose-protein hydrolase for mono-ADP-ribose (MAR) and poly(ADP-ribose) (PAR) chain removal (de-MARylation and de-PARylation, respectively) from mono- and poly(ADP)-ribosylated proteins, respectively. The presence of the HEV helicase in cis dramatically increases the binding of the macro domain to poly(ADP-ribose) and stimulates the de-PARylation activity. Abrogation of the latter dramatically decreases replication of an HEV subgenomic replicon. The de-MARylation activity is present in all three pathogenic positive-sense, single-stranded RNA [(+)ssRNA] virus families which carry a macro domain: Coronaviridae (severe acute respiratory syndrome coronavirus and human coronavirus 229E), Togaviridae (Venezuelan equine encephalitis virus), and Hepeviridae (HEV), indicating that it might be a significant tropism and/or pathogenic determinant.

    IMPORTANCE Protein ADP-ribosylation is a covalent posttranslational modification regulating cellular protein activities in a dynamic fashion to modulate and coordinate a variety of cellular processes. Three viral families, Coronaviridae, Togaviridae, and Hepeviridae, possess macro domains embedded in their polyproteins. Here, we show that viral macro domains reverse cellular ADP-ribosylation, potentially cutting the signal of a viral infection in the cell. Various poly(ADP-ribose) polymerases which are notorious guardians of cellular integrity are demodified by macro domains from members of these virus families. In the case of hepatitis E virus, the adjacent viral helicase domain dramatically increases the binding of the macro domain to PAR and simulates the demodification activity.

  • Relationship between Vaccine-Induced Antibody Capture of Infectious Virus and Infection Outcomes following Repeated Low-Dose Rectal Challenges with Simian Immunodeficiency Virus SIVmac251 [Pathogenesis and Immunity]

  • Antibodies are known to enhance in vitro infection by human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). We measured the ability of antibodies induced by ALVAC-SIV/gp120 vaccination, given with alum or MF59 adjuvant, to capture infectious SIVmac251 and determined the association between capture and infection outcomes following low-dose, repeated rectal challenge of rhesus macaques. We found that capture correlated with the number of transmitted/founder (T/F) variants that established infection, such that animals whose plasma captured more virus were infected with a higher number of T/F strains. Capture also correlated with results of Env binding assays, indicating that greater immunogenicity resulted in greater capture. Although vaccination elicited negligible neutralizing activity against the challenge strain (50% inhibitory dilutions of ggt;1/80 in all cases), animals with low capture and whose plasma, at a fixed dilution, inhibited a higher fraction of virus were infected at a lower rate than animals with high capture and low neutralization (P = 0.039); only animals with the low capture/high neutralization response profile were protected compared with unvaccinated control animals (P = 0.026). In a sieve analysis, high capture and low capture were distinguishable on the basis of polymorphisms in the V1 loop of Env at amino acids 144 and 145. Our results indicate that vaccine-induced antibody that binds to and captures infectious virus but does not inhibit its infectivity may enhance the likelihood of infection following rectal challenge with SIVmac251. Higher immunogenicity resulting in better antibody capture but similar anti-infectivity may not improve vaccine efficacy.

    IMPORTANCE Vaccines generally prevent viral infections by eliciting antibodies that inhibit virus infectivity. However, antibodies, including those induced by vaccination, have the potential to enhance, rather than prevent infection. We measured the ability of vaccine-induced antibodies to capture infectious simian immunodeficiency virus (SIV) and explored the relationship between virus capture and infection outcomes. We found that capture correlated with the number of SIV variants that established infection, such that animals whose plasma captured more virus were infected with a higher number of unique strains. In addition, animals whose sera had high capture but weak anti-infectivity activity were infected at a higher rate than were animals with low capture and stronger anti-infectivity activity. These results suggest that vaccines that induce antibodies that bind to and capture infectious virus but do not inhibit virus infectivity will not be effective in preventing infection.

  • Unmasking Stem-Specific Neutralizing Epitopes by Abolishing N-Linked Glycosylation Sites of Influenza Virus Hemagglutinin Proteins for Vaccine Design [Vaccines and Antiviral Agents]

  • Influenza virus hemagglutinin (HA) protein consists of two components, i.e., a globular head region and a stem region that are folded within six disulfide bonds, plus several N-linked glycans that produce a homotrimeric complex structure. While N-linked glycosylation sites on the globular head are variable among different strains and different subtypes, N-linked glycosylation sites in the stem region are mostly well conserved among various influenza virus strains. Targeting highly conserved HA stem regions has been proposed as a useful strategy for designing universal influenza vaccines. Since the HA stem region is constituted by an HA1 N-terminal part and a full HA2 part, we expressed a series of recombinant HA mutant proteins with deleted N-linked glycosylation sites in the HA1 stem and HA2 stem regions of H5N1 and pH1N1 viruses. Unmasking N-glycans in the HA2 stem region (H5 N484A and H1 N503A) was found to elicit more potent neutralizing antibody titers against homologous, heterologous, and heterosubtypic viruses. Unmasking the HA2 stem N-glycans of H5HA but not H1HA resulted in more CR6261-like and FI6v3-like antibodies and also correlated with the increase of cell fusion inhibition activity in antisera. Only H5 N484A HA2 stem mutant protein immunization increased the numbers of antibody-secreting cells, germinal center B cells, and memory B cells targeting the stem helix A epitopes in splenocytes. Unmasking the HA2 stem N-glycans of H5HA mutant proteins showed a significantly improvement in the protection against homologous virus challenges but did so to a less degree for the protection against heterosubtypic pH1N1 virus challenges. These results may provide useful information for designing more effective influenza vaccines.

    IMPORTANCE N-linked glycosylation sites in the stem regions of influenza virus hemagglutinin (HA) proteins are mostly well conserved among various influenza virus strains. Targeting highly conserved HA stem regions has been proposed as a useful strategy for designing universal influenza vaccines. Our studies indicate that unmasking the HA2 stem N-glycans of recombinant HA proteins from H5N1 and pH1N1 viruses induced more potent neutralizing antibody titers against homologous and heterosubtypic viruses. However, only immunization with the H5N1 HA2 stem mutant protein can refocus B antibody responses to the helix A epitope for inducing more CR6261-like/FI6v3-like and fusion inhibition antibodies in antisera, resulting in a significant improvement for the protection against lethal H5N1 virus challenges. These results may provide useful information for designing more effective influenza vaccines.

  • Molecular Determinants Directing HIV-1 Gag Assembly to Virus-Containing Compartments in Primary Macrophages [Structure and Assembly]

  • The subcellular sites of HIV-1 assembly, determined by the localization of the structural protein Gag, vary in a cell-type-dependent manner. In T cells and transformed cell lines used as model systems, HIV-1 assembles at the plasma membrane (PM). The binding and localization of HIV-1 Gag to the PM are mediated by the interaction between the matrix (MA) domain, specifically the highly basic region, and a PM-specific acidic phospholipid, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. In primary macrophages, prominent accumulation of assembling or assembled particles is found in the virus-containing compartments (VCCs), which largely consist of convoluted invaginations of the PM. To elucidate the molecular mechanism of HIV-1 Gag targeting to the VCCs, we examined the impact of overexpression of polyphosphoinositide 5-phosphatase IV (5ptaseIV), which depletes cellular PI(4,5)P2, in primary macrophages. We found that the VCC localization and virus release of HIV-1 are severely impaired upon 5ptaseIV overexpression, suggesting an important role for the MA-PI(4,5)P2 interaction in HIV-1 assembly in primary macrophages. However, our analysis of HIV-1 Gag derivatives with MA changes showed that this interaction contributes to Gag membrane binding but is dispensable for specific targeting of Gag to the VCCs per se. We further determined that deletion of the NC domain abolishes VCC-specific localization of HIV-1 Gag. Notably, HIV-1 Gag localized efficiently to the VCCs when the NC domain was replaced with a leucine zipper dimerization motif that promotes Gag multimerization. Altogether, our data revealed that targeting of HIV-1 Gag to the VCCs requires NC-dependent multimerization.

    IMPORTANCE In T cells and model cell lines, HIV-1 Gag localizes to the PM in a manner dependent on the MA-PI(4,5)P2 interaction. On the other hand, in primary macrophages, HIV-1 Gag localizes to convoluted intracellular membrane structures termed virus-containing compartments (VCCs). Although these compartments have been known for decades, and despite the implication of viruses in VCCs being involved in virus reservoir maintenance and spread, the viral determinant(s) that promotes Gag targeting to VCCs is unknown. In this study, we found that the MA-PI(4,5)P2 interaction facilitates efficient Gag membrane binding in macrophages but is not essential for Gag targeting to VCCs. Rather, our results revealed that NC-dependent multimerization promotes VCC targeting. Our findings highlight the differential roles played by MA and NC in HIV-1 Gag membrane binding and targeting and suggest a multimerization-dependent mechanism for Gag trafficking in primary macrophages similar to that for Gag localization to uropods in polarized T cells.

  • Epstein-Barr Virus Oncoprotein LMP1 Mediates Epigenetic Changes in Host Gene Expression through PARP1 [Transformation and Oncogenesis]

  • The latent infection of Epstein-Barr virus (EBV) is associated with 1% of human cancer incidence. Poly(ADP-ribosyl)ation (PARylation) is a posttranslational modification catalyzed by poly(ADP-ribose) polymerases (PARPs) that mediate EBV replication during latency. In this study, we detail the mechanisms that drive cellular PARylation during latent EBV infection and the effects of PARylation on host gene expression and cellular function. EBV-infected B cells had higher PAR levels than EBV-negative B cells. Moreover, cellular PAR levels were up to 2-fold greater in type III than type I latently infected EBV B cells. We identified a positive association between expression of the EBV genome-encoded latency membrane protein 1 (LMP1) and PAR levels that was dependent upon PARP1. PARP1 regulates gene expression by numerous mechanisms, including modifying chromatin structure and altering the function of chromatin-modifying enzymes. Since LMP1 is essential in establishing EBV latency and promoting tumorigenesis, we explored the model that disruption in cellular PARylation, driven by LMP1 expression, subsequently promotes epigenetic alterations to elicit changes in host gene expression. PARP1 inhibition resulted in the accumulation of the repressive histone mark H3K27me3 at a subset of LMP1-regulated genes. Inhibition of PARP1, or abrogation of PARP1 expression, also suppressed the expression of LMP1-activated genes and LMP1-mediated cellular transformation, demonstrating an essential role for PARP1 activity in LMP1-induced gene expression and cellular transformation associated with LMP1. In summary, we identified a novel mechanism by which LMP1 drives expression of host tumor-promoting genes by blocking generation of the inhibitory histone modification H3K27me3 through PARP1 activation.

    IMPORTANCE EBV is causally linked to several malignancies and is responsible for 1% of cancer incidence worldwide. The EBV-encoded protein LMP1 is essential for promoting viral tumorigenesis by aberrant activation of several well-known intracellular signaling pathways. We have identified and defined an additional novel molecular mechanism by which LMP1 regulates the expression of tumor-promoting host genes. We found that LMP1 activates the cellular protein PARP1, leading to a decrease in a repressive histone modification, accompanied by induction in expression of multiple cancer-related genes. PARP1 inhibition or depletion led to a decrease in LMP1-induced cellular transformation. Therefore, targeting PARP1 activity may be an effective treatment for EBV-associated malignancies.

  • Assessing Host-Virus Codivergence for Close Relatives of Merkel Cell Polyomavirus Infecting African Great Apes [Genetic Diversity and Evolution]

  • It has long been hypothesized that polyomaviruses (PyV; family Polyomaviridae) codiverged with their animal hosts. In contrast, recent analyses suggested that codivergence may only marginally influence the evolution of PyV. We reassess this question by focusing on a single lineage of PyV infecting hominine hosts, the Merkel cell polyomavirus (MCPyV) lineage. By characterizing the genetic diversity of these viruses in seven African great ape taxa, we show that they exhibit very strong host specificity. Reconciliation analyses identify more codivergence than noncodivergence events. In addition, we find that a number of host and PyV divergence events are synchronous. Collectively, our results support codivergence as the dominant process at play during the evolution of the MCPyV lineage. More generally, our results add to the growing body of evidence suggesting an ancient and stable association of PyV and their animal hosts.

    IMPORTANCE The processes involved in viral evolution and the interaction of viruses with their hosts are of great scientific interest and public health relevance. It has long been thought that the genetic diversity of double-stranded DNA viruses was generated over long periods of time, similar to typical host evolutionary timescales. This was also hypothesized for polyomaviruses (family Polyomaviridae), a group comprising several human pathogens, but this remains a point of controversy. Here, we investigate this question by focusing on a single lineage of polyomaviruses that infect both humans and their closest relatives, the African great apes. We show that these viruses exhibit considerable host specificity and that their evolution largely mirrors that of their hosts, suggesting that codivergence with their hosts played a major role in their diversification. Our results provide statistical evidence in favor of an association of polyomaviruses and their hosts over millions of years.

  • Cryo-electron Microscopy Reconstruction and Stability Studies of the Wild Type and the R432A Variant of Adeno-associated Virus Type 2 Reveal that Capsid Structural Stability Is a Major Factor in Genome Packaging [Structure and Assembly]

  • The adeno-associated viruses (AAV) are promising therapeutic gene delivery vectors and better understanding of their capsid assembly and genome packaging mechanism is needed for improved vector production. Empty AAV capsids assemble in the nucleus prior to genome packaging by virally encoded Rep proteins. To elucidate the capsid determinants of this process, structural differences between wild-type (wt) AAV2 and a packaging deficient variant, AAV2-R432A, were examined using cryo-electron microscopy and three-dimensional image reconstruction both at an ~5.0-AAring; resolution (medium) and also at 3.8- and 3.7-AAring; resolutions (high), respectively. The high resolution structures showed that removal of the arginine side chain in AAV2-R432A eliminated hydrogen bonding interactions, resulting in altered intramolecular and intermolecular interactions propagated from under the 3-fold axis toward the 5-fold channel. Consistent with these observations, differential scanning calorimetry showed an ~10ddeg;C decrease in thermal stability for AAV2-R432A compared to wt-AAV2. In addition, the medium resolution structures revealed differences in the juxtaposition of the less ordered, N-terminal region of their capsid proteins, VP1/2/3. A structural rearrangement in AAV2-R432A repositioned the bbeta;A strand region under the icosahedral 2-fold axis rather than antiparallel to the bbeta;B strand, eliminating many intramolecular interactions. Thus, a single amino acid substitution can significantly alter the AAV capsid integrity to the extent of reducing its stability and possibly rendering it unable to tolerate the stress of genome packaging. Furthermore, the data show that the 2-, 3-, and 5-fold regions of the capsid contributed to producing the packaging defect and highlight a tight connection between the entire capsid in maintaining packaging efficiency.

    IMPORTANCE The mechanism of AAV genome packaging is still poorly understood, particularly with respect to the capsid determinants of the required capsid-Rep interaction. Understanding this mechanism may aid in the improvement of AAV packaging efficiency, which is currently ~1:10 (10%) genome packaged to empty capsid in vector preparations. This report identifies regions of the AAV capsid that play roles in genome packaging and that may be important for Rep recognition. It also demonstrates the need to maintain capsid stability for the success of this process. This information is important for efforts to improve AAV genome packaging and will also inform the engineering of AAV capsid variants for improved tropism, specific tissue targeting, and host antibody escape by defining amino acids that cannot be altered without detriment to infectious vector production.

  • Nonhuman TRIM5 Variants Enhance Recognition of HIV-1-Infected Cells by CD8+ T Cells [Pathogenesis and Immunity]

  • Tripartite motif-containing protein 5 (TRIM5) restricts human immunodeficiency virus type 1 (HIV-1) in a species-specific manner by uncoating viral particles while activating early innate responses. Although the contribution of TRIM5 proteins to cellular immunity has not yet been studied, their interactions with the incoming viral capsid and the cellular proteasome led us to hypothesize a role for them. Here, we investigate whether the expression of two nonhuman TRIM5 orthologs, rhesus TRIM5aalpha; (RhT5) and TRIM-cyclophilin A (TCyp), both of which are potent restrictors of HIV-1, could enhance immune recognition of infected cells by CD8+ T cells. We illustrate how TRIM5 restriction improves CD8+ T-cell-mediated HIV-1 inhibition. Moreover, when TRIM5 activity was blocked by the nonimmunosuppressive analog of cyclosporine (CsA), sarcosine-3(4-methylbenzoate)nndash;CsA (SmBz-CsA), we found a significant reduction in CD107a/MIP-1bbeta; expression in HIV-1-specific CD8+ T cells. This finding underscores the direct link between TRIM5 restriction and activation of CD8+ T-cell responses. Interestingly, cells expressing RhT5 induced stronger CD8+ T-cell responses through the specific recognition of the HIV-1 capsid by the immune system. The underlying mechanism of this process may involve TRIM5-specific capsid recruitment to cellular proteasomes and increase peptide availability for loading and presentation of HLA class I antigens. In summary, we identified a novel function for nonhuman TRIM5 variants in cellular immunity. We hypothesize that TRIM5 can couple innate viral sensing and CD8+ T-cell activation to increase species barriers against retrovirus infection.

    IMPORTANCE New therapeutics to tackle HIV-1 infection should aim to combine rapid innate viral sensing and cellular immune recognition. Such strategies could prevent seeding of the viral reservoir and the immune damage that occurs during acute infection. The nonhuman TRIM5 variants, rhesus TRIM5aalpha; (RhT5) and TRIM-cyclophilin A (TCyp), are attractive candidates owing to their potency in sensing HIV-1 and blocking its activity. Here, we show that expression of RhT5 and TCyp in HIV-1-infected cells improves CD8+ T-cell-mediated inhibition through the direct activation of HIV-1-specific CD8+ T-cell responses. We found that the potency in CD8+ activation was stronger for RhT5 variants and capsid-specific CD8+ T cells in a mechanism that relies on TRIM5-dependent particle recruitment to cellular proteasomes. This novel mechanism couples innate viral sensing with cellular immunity in a single protein and could be exploited to develop innovative therapeutics for control of HIV-1 infection.

  • Complementary Effects of Interleukin-15 and Alpha Interferon Induce Immunity in Hepatitis B Virus Transgenic Mice [Gene Delivery]

  • In chronic hepatitis B (CHB), failure to control hepatitis B virus (HBV) is associated with T cell dysfunction. HBV transgenic mice mirror many features of the human disease, including T cell unresponsiveness, and thus represent an appropriate model in which to test novel therapeutic strategies. To date, the tolerant state of CD8+ T cells in these animals could be altered only by strong immunogens or by immunization with HBV antigen-pulsed dendritic cells; however, the effectors induced were unable to suppress viral gene expression or replication. Because of the known stimulatory properties of alpha interferon (IFN-aalpha;) and interleukin-15 (IL-15), this study explored the therapeutic potential of liver-directed gene transfer of these cytokines in a murine model of CHB using adeno-associated virus (AAV) delivery. This combination not only resulted in a reduction in the viral load in the liver and the induction of an antibody response but also gave rise to functional and specific CD8+ immunity. Furthermore, when splenic and intrahepatic lymphocytes from IFN-aalpha;- and IL-15-treated animals were transferred to new HBV carriers, partial antiviral immunity was achieved. In contrast to previous observations made using either cytokine alone, markedly attenuated PD-L1 induction in hepatic tissue was observed upon coadministration. An initial study with CHB patient samples also gave promising results. Hence, we demonstrated synergy between two stimulating cytokines, IL-15 and IFN-aalpha;, which, given together, constitute a potent approach to significantly enhance the CD8+ T cell response in a state of immune hyporesponsiveness. Such an approach may be useful for treating chronic viral infections and neoplastic conditions.

    IMPORTANCE With 350 million people affected worldwide and 600,000 annual deaths due to HBV-induced liver cirrhosis and/or hepatocellular carcinoma, chronic hepatitis B (CHB) is a major health problem. However, current treatment options are costly and not very effective and/or need to be administered for life. The unprecedented efficacy of the strategy described in our paper may offer an alternative and is relevant for a broad spectrum of readers because of its clear translational importance to other chronic viral infections in which a hyporesponsive antigen-specific T cell repertoire prevents clearance of the pathogen.

  • An RNA Element That Facilitates Programmed Ribosomal Readthrough in Turnip Crinkle Virus Adopts Multiple Conformations [Genome Replication and Regulation of Viral Gene Expression]

  • Ribosome recoding is used by RNA viruses for translational readthrough or frameshifting past termination codons for the synthesis of extension products. Recoding sites, along with downstream recoding stimulatory elements (RSEs), have long been studied in reporter constructs, because these fragments alone mediate customary levels of recoding and are thus assumed to contain complete instructions for establishment of the proper ratio of termination to recoding. RSEs from the Tombusviridae and Luteoviridae are thought to be exceptions, since they contain a long-distance RNA-RNA connection with the 3' end. This interaction has been suggested to substitute for pseudoknots, thought to be missing in tombusvirid RSEs. We provide evidence that the phylogenetically conserved RSE of the carmovirus Turnip crinkle virus (TCV) adopts an alternative, smaller structure that extends an upstream conserved hairpin and that this alternative structure is the predominant form of the RSE within nascent viral RNA in plant cells and when RNA is synthesized in vitro. The TCV RSE also contains an internal pseudoknot along with the long-distance interaction, and the pseudoknot is not compatible with the phylogenetically conserved structure. Conserved residues just past the recoding site are important for recoding, and these residues are also conserved in the RSEs of gammaretroviruses. Our data demonstrate the dynamic nature of the TCV RSE and suggest that studies using reporter constructs may not be effectively recapitulating RSE-mediated recoding within viral genomes.

    IMPORTANCE Ribosome recoding is used by RNA viruses to enable ribosomes to extend translation past termination codons for the synthesis of longer products. Recoding sites and a downstream recoding stimulatory element (RSE) mediate expected levels of recoding when excised and placed in reporter constructs and thus are assumed to contain complete instructions for the establishment of the proper ratio of termination to recoding. We provide evidence that most of the TCV RSE adopts an alternative structure that extends an upstream conserved hairpin and that this alternative structure, and not the phylogenetically conserved structure, is the predominant form of the RSE in RNA synthesized in vitro and in plant cells. The TCV RSE also contains an internal pseudoknot that is not compatible with the phylogenetically conserved structure and an RNA bridge to the 3' end. These data suggest that the TCV RSE is structurally dynamic and that multiple conformations are likely required to regulate ribosomal readthrough.

  • A Single Mutation in the VP1 of Enterovirus 71 Is Responsible for Increased Virulence and Neurotropism in Adult Interferon-Deficient Mice [Pathogenesis and Immunity]

  • Hand, foot, and mouth disease (HFMD) has spread throughout the Asia-Pacific region, affecting millions of young children, who develop symptoms ranging from painful blisters around their mouths and hands to neurological complications. Many members of the genus Enterovirus (family Picornaviridae) cause HFMD. Enterovirus 71 (EV71) is one of the primary causative agents and has been linked to severe disease. Vaccine efficacy and pathogenesis studies for EV71 have been limited because there is a lack of suitable animal models. Previously, we generated a mouse-adapted EV71 (mEV71) capable of infecting 12-week-old interferon receptor-deficient AG129 mice and used the model to evaluate the efficacy of candidate HFMD vaccines. Here, we present data investigating the genetic correlates of EV71 adaptation and characterize the virus's tissue tropism in mice. Using reverse genetics, a VP1 mutation (K244E) was shown to be necessary for mEV71 virulence in adult mice. Another VP1 mutation (H37R) was required for mEV71 recovery on rhabdomyosarcoma (RD) cells. Viral loads determined by real-time reverse transcription (RT)-PCR confirmed the presence of mEV71 in the sera and multiple organs of mice. Histological analysis revealed signs of meningitis and encephalitis, characteristic of severe human disease. The further description of this model has provided insight into EV71 pathogenesis and demonstrates the importance of the VP1 region in facilitating mEV71 adaptation.

    IMPORTANCE EV71 is a reemerging pathogen, and little is known about the genetic determinants involved in its pathogenesis. The absence of animal models has contributed to this lack of knowledge. The data presented here improve upon the existing animal models by characterizing a mouse-adapted strain of EV71. We determined that a VP1 mutation (K244E) was needed for EV71 virulence in adult AG129 mice. While this mutation was found previously for EV71 adaptation in 5-day-old BALB/c mice, neurotropic disease did not develop. Using interferon-deficient mice, we raised the age of susceptibility beyond 6 weeks and provided clear evidence that our model mimics severe human infections. The model can be exploited to identify determinants of EV71 virulence and to reveal molecular mechanisms that control the virus-host interaction, especially those associated with neurotropic disease. Furthermore, these data provide useful information regarding the importance of VP1, specifically position 244, in host adaptation and tissue dissemination.

  • Analysis of Major Histocompatibility Complex-Bound HIV Peptides Identified from Various Cell Types Reveals Common Nested Peptides and Novel T Cell Responses [Pathogenesis and Immunity]

  • Despite the critical role of epitope presentation for immune recognition, we still lack a comprehensive definition of HIV peptides presented by HIV-infected cells. Here we identified 107 major histocompatibility complex (MHC)-bound HIV peptides directly from the surface of live HIV-transfected 293T cells, HIV-infected B cells, and primary CD4 T cells expressing a variety of HLAs. The majority of peptides were 8 to 12 amino acids (aa) long and mostly derived from Gag and Pol. The analysis of the total MHC-peptidome and of HLA-A02-bound peptides identified new noncanonical HIV peptides of up to 16 aa that could not be predicted by HLA anchor scanning and revealed an heterogeneous surface peptidome. Nested sets of surface HIV peptides included optimal and extended HIV epitopes and peptides partly overlapping or distinct from known epitopes, revealing new immune responses in HIV-infected persons. Surprisingly, in all three cell types, a majority of Gag peptides derived from p15 rather than from the most immunogenic p24. The cytosolic degradation of peptide precursors in corresponding cells confirmed the generation of identified surface-nested peptides. Cytosolic degradation revealed peptides commonly produced in all cell types and displayed by various HLAs, peptides commonly produced in all cell types and selectively displayed by specific HLAs, and peptides produced in only one cell type. Importantly, we identified areas of proteins leading to common presentations of noncanonical peptides by several cell types with distinct HLAs. These peptides may benefit the design of immunogens, focusing T cell responses on relevant markers of HIV infection in the context of HLA diversity.

    IMPORTANCE The recognition of HIV-infected cells by immune T cells relies on the presentation of HIV-derived peptides by diverse HLA molecules at the surface of cells. The landscape of HIV peptides displayed by HIV-infected cells is not well defined. Considering the diversity of HLA molecules in the human population, it is critical for vaccine design to identify HIV peptides that may be displayed despite the HLA diversity. We identified 107 HIV peptides directly from the surface of three cell types infected with HIV. They corresponded to nested sets of HIV peptides of canonical and novel noncanonical lengths not predictable by the presence of HLA anchors. Importantly, we identified areas of HIV proteins leading to presentation of noncanonical peptides by several cell types with distinct HLAs. Including such peptides in vaccine immunogen may help to focus immune responses on common markers of HIV infection in the context of HLA diversity.

  • MORC3, a Component of PML Nuclear Bodies, Has a Role in Restricting Herpes Simplex Virus 1 and Human Cytomegalovirus [Virus-Cell Interactions]

  • We previously reported that MORC3, a protein associated with promyelocytic leukemia nuclear bodies (PML NBs), is a target of herpes simplex virus 1 (HSV-1) ICP0-mediated degradation (E. Sloan, et al., PLoS Pathog 11:e1005059, 2015, http://dx.doi.org/10.1371/journal.ppat.1005059). Since it is well known that certain other components of the PML NB complex play an important role during an intrinsic immune response to HSV-1 and are also degraded or inactivated by ICP0, here we further investigate the role of MORC3 during HSV-1 infection. We demonstrate that MORC3 has antiviral activity during HSV-1 infection and that this antiviral role is counteracted by ICP0. In addition, MORC3's antiviral role extends to wild-type (wt) human cytomegalovirus (HCMV) infection, as its plaque-forming efficiency increased in MORC3-depleted cells. We found that MORC3 is recruited to sites associated with HSV-1 genomes after their entry into the nucleus of an infected cell, and in wt infections this is followed by its association with ICP0 foci prior to its degradation. The RING finger domain of ICP0 was required for degradation of MORC3, and we confirmed that no other HSV-1 protein is required for the loss of MORC3. We also found that MORC3 is required for fully efficient recruitment of PML, Sp100, hDaxx, and H2AX to sites associated with HSV-1 genomes entering the host cell nucleus. This study further unravels the intricate ways in which HSV-1 has evolved to counteract the host immune response and reveals a novel function for MORC3 during the host intrinsic immune response.

    IMPORTANCE Herpesviruses have devised ways to manipulate the host intrinsic immune response to promote their own survival and persistence within the human population. One way in which this is achieved is through degradation or functional inactivation of PML NB proteins, which are recruited to viral genomes in order to repress viral transcription. Because MORC3 associates with PML NBs in uninfected cells and is a target for HSV-1-mediated degradation, we investigated the role of MORC3 during HSV-1 infection. We found that MORC3 is also recruited to viral HSV-1 genomes, and importantly it contributes to the fully efficient recruitment of PML, hDaxx, Sp100, and H2AX to these sites. Depletion of MORC3 resulted in an increase in ICP0-null HSV-1 and wt HCMV replication and plaque formation; therefore, this study reveals that MORC3 is an antiviral factor which plays an important role during HSV-1 and HCMV infection.

  • Human Cytomegalovirus Can Procure Deoxyribonucleotides for Viral DNA Replication in the Absence of Retinoblastoma Protein Phosphorylation [Virus-Cell Interactions]

  • Viral DNA replication requires deoxyribonucleotide triphosphates (dNTPs). These molecules, which are found at low levels in noncycling cells, are generated either by salvage pathways or through de novo synthesis. Nucleotide synthesis utilizes the activity of a series of nucleotide-biosynthetic enzymes (NBEs) whose expression is repressed in noncycling cells by complexes between the E2F transcription factors and the retinoblastoma (Rb) tumor suppressor. Rb-E2F complexes are dissociated and NBE expression is activated during cell cycle transit by cyclin-dependent kinase (Cdk)-mediated Rb phosphorylation. The DNA virus human cytomegalovirus (HCMV) encodes a viral Cdk (v-Cdk) (the UL97 protein) that phosphorylates Rb, induces the expression of cellular NBEs, and is required for efficient viral DNA synthesis. A long-held hypothesis proposed that viral proteins with Rb-inactivating activities functionally similar to those of UL97 facilitated viral DNA replication in part by inducing the de novo production of dNTPs. However, we found that dNTPs were limiting even in cells infected with wild-type HCMV in which UL97 is expressed and Rb is phosphorylated. Furthermore, we revealed that both de novo and salvage pathway enzymes contribute to viral DNA replication during HCMV infection and that Rb phosphorylation by cellular Cdks does not correct the viral DNA replication defect observed in cells infected with a UL97-deficient virus. We conclude that HCMV can obtain dNTPs in the absence of Rb phosphorylation and that UL97 can contribute to the efficiency of DNA replication in an Rb phosphorylation-independent manner.

    IMPORTANCE Transforming viral oncoproteins, such as adenovirus E1A and papillomavirus E7, inactivate Rb. The standard hypothesis for how Rb inactivation facilitates infection with these viruses is that it is through an increase in the enzymes required for DNA synthesis, which include nucleotide-biosynthetic enzymes. However, HCMV UL97, which functionally mimics these viral oncoproteins through phosphorylation of Rb, fails to induce the production of nonlimiting amounts of dNTPs. This finding challenges the paradigm of the role of Rb inactivation during DNA virus infection and uncovers the existence of an alternative mechanism by which UL97 contributes to HCMV DNA synthesis. The ineffectiveness of the UL97 inhibitor maribavir in clinical trials might be better explained with a fuller understanding of the role of UL97 during infection. Furthermore, as the nucleoside analog ganciclovir is the current drug of choice for treating HCMV, knowing the provenance of the dNTPs incorporated into viral DNA may help inform antiviral therapeutic regimens.

  • Induction of Heterologous Tier 2 HIV-1-Neutralizing and Cross-Reactive V1/V2-Specific Antibodies in Rabbits by Prime-Boost Immunization [Vaccines and Antiviral Agents]

  • Poxvirus prime-protein boost used in the RV144 trial remains the only immunization strategy shown to elicit a modest level of protection against HIV-1 acquisition in humans. Although neutralizing antibodies (NAb) were generated, they were against sensitive viruses, not the more resistant "tier 2" isolates that dominate circulating strains. Instead, risk reduction correlated with antibodies recognizing epitopes in the V1/V2 region of HIV-1 envelope glycoprotein (Env). Here, we examined whether tier 2 virus NAb and V1/V2-specific non-NAb could be elicited by a poxvirus prime-gp120 boost strategy in a rabbit model. We studied two clade B Envs that differ in multiple parameters, including tissue origin, neutralization sensitivity, and presence of the N197 (N7) glycan that was previously shown to modulate the exposure of conserved epitopes on Env. We demonstrate that immunized rabbits generated cross-reactive neutralizing activities against ggt;50% of the tier 2 global HIV-1 isolates tested. Some of these activities were directed against the CD4 binding site (CD4bs). These rabbits also generated antibodies that recognized protein scaffolds bearing V1/V2 sequences from diverse HIV-1 isolates and mediated antibody-dependent cellular cytotoxicity. However, there are subtle differences in the specificities and the response rates of V1/V2-specific antibodies between animals immunized with different Envs, with or without the N7 glycan. These findings demonstrate that antibody responses that have been correlated with protection against HIV-1 acquisition in humans can be elicited in a preclinical model by a poxvirus prime-gp120 boost strategy and that improvements may be achievable by optimizing the nature of the priming and boosting immunogens.

    IMPORTANCE The only vaccine approach shown to elicit any protective efficacy against HIV-1 acquisition is based on a poxvirus prime-protein boost regimen (RV144 Thai trial). Reduction of risk was associated with nonneutralizing antibodies targeting the V1/V2 loops of the envelope protein gp120. However, the modest efficacy (31.2%) achieved in this trial highlights the need to examine approaches and factors that may improve vaccine-induced responses, including cross-reactive neutralizing activities. We show here that rabbits immunized with a novel recombinant vaccinia virus prime-gp120 protein boost regimen generated antibodies that recognize protein scaffolds bearing V1/V2 sequences from diverse HIV-1 isolates and mediated antibody-dependent cellular cytotoxicity. Importantly, immunized rabbits also showed neutralizing activities against heterologous tier 2 HIV-1 isolates. These findings may inform the design of prime-boost immunization approaches and help improve the protective efficacy of candidate HIV-1 vaccines.

  • Herpes Simplex Virus 1 Interaction with Myeloid Cells In Vivo [Pathogenesis and Immunity]

  • Herpes simplex virus 1 (HSV-1) enters mice via olfactory epithelial cells and then colonizes the trigeminal ganglia (TG). Most TG nerve endings are subepithelial, so this colonization implies subepithelial viral spread, where myeloid cells provide an important line of defense. The outcome of infection of myeloid cells by HSV-1 in vitro depends on their differentiation state; the outcome in vivo is unknown. Epithelial HSV-1 commonly infected myeloid cells, and Cre-Lox virus marking showed nose and lung infections passing through LysM-positive (LysM+) and CD11c+ cells. In contrast, subcapsular sinus macrophages (SSMs) exposed to lymph-borne HSV-1 were permissive only when type I interferon (IFN-I) signaling was blocked; normally, their infection was suppressed. Thus, the outcome of myeloid cell infection helped to determine the HSV-1 distribution: subepithelial myeloid cells provided a route of spread from the olfactory epithelium to TG neurons, while SSMs blocked systemic spread.

    IMPORTANCE Herpes simplex virus 1 (HSV-1) infects most people and can cause severe disease. This reflects its persistence in nerve cells that connect to the mouth, nose, eye, and face. Established infection seems impossible to clear. Therefore, we must understand how it starts. This is difficult in humans, but mice show HSV-1 entry via the nose and then spread to its preferred nerve cells. We show that this spread proceeds in part via myeloid cells, which normally function in host defense. Myeloid infection was productive in some settings but was efficiently suppressed by interferon in others. Therefore, interferon acting on myeloid cells can stop HSV-1 spread, and enhancing this defense offers a way to improve infection control.

  • Exocytosis of Varicella-Zoster Virus Virions Involves a Convergence of Endosomal and Autophagy Pathways [Virus-Cell Interactions]

  • Varicella-zoster virus (VZV) is an extremely cell-associated herpesvirus with limited egress of viral particles. The induction of autophagy in VZV-infected monolayers is easily detectable; inhibition of autophagy leads to decreased VZV glycoprotein biosynthesis and diminished viral titers. To explain how autophagic flux could exert a proviral effect on the VZV infectious cycle, we postulated that the VZV exocytosis pathway following secondary envelopment may converge with the autophagy pathway. This hypothesis depended on known similarities between VZV gE and autophagy-related (Atg) Atg9/Atg16L1 trafficking pathways. Investigations were carried out with highly purified fractions of VZV virions. When the virion fraction was tested for the presence of autophagy and endosomal proteins, microtubule-associated protein 1 light chain (MAP1LC3B) and Ras-like GTPase 11 (Rab11) were detected. By two-dimensional (2D) and 3D imaging after immunolabeling, both proteins also colocalized with VZV gE in a proportion of cytoplasmic vesicles. When purified VZV virions were enumerated after immunoelectron microscopy, gold beads were detected on viruses following incubation with antibodies to VZV gE (~100%), Rab11 (50%), and LC3B (30%). Examination of numerous electron micrographs demonstrated that enveloped virions were housed in single-membraned vesicles; viral particles were not observed in autophagosomes. Taken together, our data suggested that some viral particles after secondary envelopment accumulated in a heterogeneous population of single-membraned vesicular compartments, which were decorated with components from both the endocytic pathway (Rab11) and the autophagy pathway (LC3B). The latter cytoplasmic viral vesicles resembled an amphisome.

    IMPORTANCE VZV infection leads to increased autophagic flux, while inhibition of autophagy leads to a marked reduction in virus spread. In this investigation of the proviral role of autophagy, we found evidence for an intersection of viral exocytosis and autophagy pathways. Specifically, both LC3-II and Rab11 proteins copurified with some infectious VZV particles. The results suggested that a subpopulation of VZV particles were carried to the cell surface in single-walled vesicles with attributes of an amphisome, an organelle formed from the fusion of an endosome and an autophagosome. Our results also addressed the interpretation of autophagy/xenophagy results with mutated herpes simplex virus lacking its ICP34.5 neurovirulence gene (HSV34.5). The VZV genome lacks an ICP34.5 ortholog, yet we found no evidence of VZV particles housed in a double-membraned autophagosome. In other words, xenophagy, a degradative process documented after infection with HSV34.5, was not observed in VZV-infected cells.

  • Varicella-Zoster Virus Activates CREB, and Inhibition of the pCREB-p300/CBP Interaction Inhibits Viral Replication In Vitro and Skin Pathogenesis In Vivo [Virus-Cell Interactions]

  • Varicella-zoster virus (VZV) is an alphaherpesvirus that causes varicella upon primary infection and zoster upon reactivation from latency in sensory ganglion neurons. The replication of herpesviruses requires manipulation of cell signaling pathways. Notably, CREB, a factor involved in the regulation of several cellular processes, is activated upon infection of T cells with VZV. Here, we report that VZV infection also induced CREB phosphorylation in fibroblasts and that XX-650-23, a newly identified inhibitor of the phosphorylated-CREB (pCREB) interaction with p300/CBP, restricted cell-cell spread of VZV in vitro. CREB phosphorylation did not require the viral open reading frame 47 (ORF47) and ORF66 kinases encoded by VZV. Evaluating the biological relevance of these observations during VZV infection of human skin xenografts in the SCID mouse model of VZV pathogenesis showed both that pCREB was upregulated in infected skin and that treatment with XX-650-23 reduced infectious-virus production and limited lesion formation compared to treatment with a vehicle control. Thus, processes of CREB activation and p300/CBP binding are important for VZV skin infection and may be targeted for antiviral drug development.

    IMPORTANCE Varicella-zoster virus (VZV) is a common pathogen that causes chicken pox and shingles. As with all herpesviruses, the infection is acquired for life, and the virus can periodically reactivate from latency. Although VZV infection is usually benign with few or no deleterious consequences, infection can be life threatening in immunocompromised patients. Otherwise healthy elderly individuals who develop zoster as a consequence of viral reactivation are at risk for postherpetic neuralgia (PHN), a painful and long-lasting complication. Current vaccines use a live attenuated virus that is usually safe but cannot be given to many immunodeficient patients and retains the capacity to establish latency and reactivate, causing zoster. Antiviral drugs are effective against severe VZV infections but have little impact on PHN. A better understanding of virus-host cell interactions is relevant for developing improved therapies to safely interfere with cellular processes that are crucial for VZV pathogenesis.

  • Deep Sequencing of Distinct Preparations of the Live Attenuated Varicella-Zoster Virus Vaccine Reveals a Conserved Core of Attenuating Single-Nucleotide Polymorphisms [Vaccines and Antiviral Agents]

  • The continued success of the live attenuated varicella-zoster virus vaccine in preventing varicella-zoster and herpes zoster is well documented, as are many of the mutations that contribute to the attenuation of the vOka virus for replication in skin. At least three different preparations of vOka are marketed. Here, we show using deep sequencing of seven batches of vOka vaccine (including ZostaVax, VariVax, VarilRix, and the Oka/Biken working seed) from three different manufacturers (VariVax, GSK, and Biken) that 137 single-nucleotide polymorphism (SNP) mutations are present in all vaccine batches. This includes six sites at which the vaccine allele is fixed or near fixation, which we speculate are likely to be important for attenuation. We also show that despite differences in the vaccine populations between preparations, batch-to-batch variation is minimal, as is the number and frequency of mutations unique to individual batches. This suggests that the vaccine manufacturing processes are not introducing new mutations and that, notwithstanding the mixture of variants present, VZV live vaccines are extremely stable.

    IMPORTANCE The continued success of vaccinations to prevent chickenpox and shingles, combined with the extremely low incidence of adverse reactions, indicates the quality of these vaccines. The vaccine itself is comprised of a heterogeneous live attenuated virus population and thus requires deep-sequencing technologies to explore the differences and similarities in the virus populations between different preparations and batches of the vaccines. Our data demonstrate minimal variation between batches, an important safety feature, and provide new insights into the extent of the mutations present in this attenuated virus.

  • Hepatitis B Virus Polymerase Localizes to the Mitochondria, and Its Terminal Protein Domain Contains the Mitochondrial Targeting Signal [Virus-Cell Interactions]

  • To understand subcellular sites of hepatitis B virus (HBV) replication, we visualized core (Cp), polymerase (Pol), and pregenomic RNA (pgRNA) in infected cells. Interestingly, we found that the majority of Pol localized to the mitochondria in cells undergoing viral replication. The mitochondrial localization of Pol was independent of both the cell type and other viral components, indicating that Pol contains an intrinsic mitochondrial targeting signal (MTS). Neither Cp nor pgRNA localized to the mitochondria during active replication, suggesting a role other than DNA synthesis for Pol at the mitochondria. The Pol of duck hepatitis B virus (DHBV) also localized to the mitochondria. This result indicates that localization of Pol to mitochondria is likely a feature of all hepadnaviruses. To map the MTS within HBV Pol, we generated a series of Pol-green fluorescent protein (Pol-GFP) fusions and found that a stretch spanning amino acids (aa) 141 to 160 of Pol was sufficient to target GFP to the mitochondria. Surprisingly, deleting aa 141 to 160 in full-length Pol did not fully ablate Pol's mitochondrial localization, suggesting that additional sequences are involved in mitochondrial targeting. Only by deleting the N-terminal 160 amino acids in full-length Pol was mitochondrial localization ablated. Crucial residues for pgRNA packaging are contained within aa 141 to 160, indicating a multifunctional role of this region of Pol in the viral life cycle. Our studies show an unexpected Pol trafficking behavior that is uncoupled from its role in viral DNA synthesis.

    IMPORTANCE Chronic infection by HBV is a serious health concern. Existing therapies for chronically infected individuals are not curative, underscoring the need for a better understanding of the viral life cycle to develop better antiviral therapies. To date, the most thoroughly studied function of Pol is to package the pgRNA and reverse transcribe it to double-stranded DNA within capsids. This study provides evidence for mitochondrial localization of Pol and defines the MTS. Recent findings have implicated a non-reverse transcription role for Pol in evading host innate immune responses. Mitochondria play an important role in controlling cellular metabolism, apoptosis, and innate immunity. Pol may alter one or more of these host mitochondrial functions to gain a replicative advantage and persist in chronically infected individuals.

  • An Ebola Virus-Like Particle-Based Reporter System Enables Evaluation of Antiviral Drugs In Vivo under Non-Biosafety Level 4 Conditions [Vaccines and Antiviral Agents]

  • Ebola virus (EBOV) is a highly contagious lethal pathogen. As a biosafety level 4 (BSL-4) agent, however, EBOV is restricted to costly BSL-4 laboratories for experimentation, thus significantly impeding the evaluation of EBOV vaccines and drugs. Here, we report an EBOV-like particle (EBOVLP)-based luciferase reporter system that enables the evaluation of anti-EBOV agents in vitro and in vivo outside BSL-4 facilities. Cotransfection of HEK293T cells with four plasmids encoding the proteins VP40, NP, and GP of EBOV and firefly luciferase (Fluc) resulted in the production of Fluc-containing filamentous particles that morphologically resemble authentic EBOV. The reporter EBOVLP was capable of delivering Fluc into various cultured cells in a GP-dependent manner and was recognized by a conformation-dependent anti-EBOV monoclonal antibody (MAb). Significantly, inoculation of mice with the reporter EBOVLP led to the delivery of Fluc protein into target cells and rapid generation of intense bioluminescence signals that could be blocked by the administration of EBOV neutralizing MAbs. This BSL-4-free reporter system should facilitate high-throughput screening for anti-EBOV drugs targeting viral entry and efficacy testing of candidate vaccines.

    IMPORTANCE Ebola virus (EBOV) researches have been limited to costly biosafety level 4 (BSL-4) facilities due to the lack of animal models independent of BSL-4 laboratories. In this study, we reveal that a firefly luciferase-bearing EBOV-like particle (EBOVLP) with typical filamentous EBOV morphology is capable of delivering the reporter protein into murine target cells both in vitro and in vivo. Moreover, we demonstrate that the reporter delivery can be inhibited both in vitro and in vivo by a known anti-EBOV protective monoclonal antibody, 13C6. Our work provides a BSL-4-free system that can facilitate the in vivo evaluation of anti-EBOV antibodies, drugs, and vaccines. The system may also be useful for mechanistic study of the viral entry process.

  • The PB2 Subunit of the Influenza A Virus RNA Polymerase Is Imported into the Mitochondrial Matrix [Cellular Response to Infection]

  • The polymerase basic 2 (PB2) subunit of the RNA polymerase complex of seasonal human influenza A viruses has been shown to localize to the mitochondria. Various roles, including the regulation of apoptosis and innate immune responses to viral infection, have been proposed for mitochondrial PB2. In particular, PB2 has been shown to inhibit interferon expression by associating with the mitochondrial antiviral signaling (MAVS) protein, which acts downstream of RIG-I and MDA-5 in the interferon induction pathway. However, in spite of a growing body of literature on the potential roles of mitochondrial PB2, the exact location of PB2 in mitochondria has not been determined. Here, we used enhanced ascorbate peroxidase (APEX)-tagged PB2 proteins and electron microscopy to study the localization of PB2 in mitochondria. We found that PB2 is imported into mitochondria, where it localizes to the mitochondrial matrix. We also demonstrated that MAVS is not required for the import of PB2 into mitochondria by showing that PB2 associates with mitochondria in MAVS knockout mouse embryo fibroblasts. Instead, we found that amino acid residue 9 in the N-terminal mitochondrial targeting sequence is a determinant of the mitochondrial import of PB2, differentiating the localization of PB2 of human from that of avian influenza A virus strains. We also showed that a virus encoding nonmitochondrial PB2 is attenuated in mouse embryonic fibroblasts (MEFs) compared with an isogenic virus encoding mitochondrial PB2, in a MAVS-independent manner, suggesting a role for PB2 within the mitochondrial matrix. This work extends our understanding of the interplay between influenza virus and mitochondria.

    IMPORTANCE The PB2 subunit of the influenza virus RNA polymerase is a major determinant of viral pathogenicity. However, the molecular mechanisms of how PB2 determines pathogenicity remain poorly understood. PB2 associates with mitochondria and inhibits the function of the mitochondrial antiviral signaling protein MAVS, implicating PB2 in the regulation of innate immune responses. We found that PB2 is imported into the mitochondrial matrix and showed that amino acid residue 9 is a determinant of mitochondrial import. The presence of asparagine or threonine in over 99% of all human seasonal influenza virus pre-2009 H1N1, H2N2, and H3N2 strains is compatible with mitochondrial import, whereas the presence of an aspartic acid in over 95% of all avian influenza viruses is not, resulting in a clear distinction between human-adapted and avian influenza viruses. These findings provide insights into the interplay between influenza virus and mitochondria and suggest mechanisms by which PB2 could affect pathogenicity.

  • HIV-1 Vpr Inhibits Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication by Inducing MicroRNA miR-942-5p and Activating NF-{kappa}B Signaling [Genome Replication and Regulation of Viral Gene Expression]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) infection is required for the development of several AIDS-related malignancies, including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). The high incidence of AIDS-KS has been ascribed to the interaction of KSHV and HIV-1. We have previously shown that HIV-1-secreted proteins Tat and Nef regulate the KSHV life cycle and synergize with KSHV oncogenes to promote angiogenesis and tumorigenesis. Here, we examined the regulation of KSHV latency by HIV-1 viral protein R (Vpr). We found that soluble Vpr inhibits the expression of KSHV lytic transcripts and proteins, as well as viral particle production by activating NF-B signaling following internalization into PEL cells. By analyzing the expression profiles of microRNAs combined with target search by bioinformatics and luciferase reporter analyses, we identified a Vpr-upregulated cellular microRNA (miRNA), miR-942-5p, that directly targeted IBaalpha;. Suppression of miR-942-5p relieved the expression of IBaalpha; and reduced Vpr inhibition of KSHV lytic replication, while overexpression of miR-942-5p enhanced Vpr inhibition of KSHV lytic replication. Our findings collectively illustrate that, by activating NF-B signaling through upregulating a cellular miRNA to target IBaalpha;, internalized HIV-1 Vpr inhibits KSHV lytic replication. These results have demonstrated an essential role of Vpr in the life cycle of KSHV.

    IMPORTANCE Coinfection by HIV-1 promotes the aggressive growth of Kaposi's sarcoma-associated herpesvirus (KSHV)-related malignancies, including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). In this study, we have shown that soluble HIV-1 Vpr inhibits KSHV lytic replication by activating NF-B signaling following internalization into PEL cells. Mechanistic studies revealed that a cellular microRNA upregulated by Vpr, miR-942-5p, directly targeted IBaalpha;. Suppression of miR-942-5p relieved IBaalpha; expression and reduced Vpr inhibition of KSHV replication, while overexpression of miR-942-5p enhanced Vpr inhibition of KSHV replication. These results indicate that by activating NF-B signaling through upregulating a cellular miRNA to target IBaalpha;, internalized Vpr inhibits KSHV lytic replication. This work illustrates a molecular mechanism by which HIV-1-secreted regulatory protein Vpr regulates KSHV latency and the pathogenesis of AIDS-related malignancies.

  • The Interaction between Herpes Simplex Virus 1 Tegument Proteins UL51 and UL14 and Its Role in Virion Morphogenesis [Structure and Assembly]

  • To investigate the molecular mechanism(s) by which herpes simplex virus 1 (HSV-1) tegument protein UL51 promotes viral replication, we screened for viral proteins that interact with UL51 in infected cells. Affinity purification of tagged UL51 in HSV-1-infected Vero cells was coupled with immunoblotting of the purified UL51 complexes with various antibodies to HSV-1 virion proteins. Subsequent analyses revealed that UL51 interacted with another tegument protein, UL14, in infected cells. Mutational analyses of UL51 showed that UL51 amino acid residues Leu-111, Ile-119, and Tyr-123 were required for interaction with UL14 in HSV-1-infected cells. Alanine substitutions of these UL51 amino acid residues reduced viral replication and produced an accumulation of unenveloped and partially enveloped nucleocapsids in the cytoplasm at levels comparable to those of UL51-null, UL14-null, and UL51/UL14 double-null mutations. In addition, although UL51 and UL14 colocalized at juxtanuclear domains in HSV-1-infected cells, the amino acid substitutions in UL51 produced aberrant localization of UL51 and UL14. The effects of these substitutions on localization of UL51 and UL14 were similar to those of the UL51-null and UL14-null mutations on localization of UL14 and UL51, respectively. These results suggested that the interaction between UL51 and UL14 was required for proper localization of these viral proteins in infected cells and that the UL51-UL14 complex regulated final viral envelopment for efficient viral replication.

    IMPORTANCE Herpesviruses contain a unique virion structure designated the tegument, which is a protein layer between the nucleocapsid and the envelope. HSV-1 has dozens of viral proteins in the tegument, which are thought to facilitate viral envelopment by interacting with other virion components. However, although numerous interactions among virion proteins have been reported, data on how these interactions facilitate viral envelopment is limited. In this study, we have presented data showing that the interaction of HSV-1 tegument proteins UL51 and UL14 promoted viral final envelopment for efficient viral replication. In particular, prevention of this interaction induced aberrant accumulation of partially enveloped capsids in the cytoplasm, suggesting that the UL51-UL14 complex acted in the envelopment process but not in an upstream event, such as transport of capsids to the site for envelopment. This is the first report showing that an interaction between HSV-1 tegument proteins directly regulated final virion envelopment.

  • Concurrence of Iridovirus, Polyomavirus, and a Unique Member of a New Group of Fish Papillomaviruses in Lymphocystis Disease-Affected Gilthead Sea Bream [Genetic Diversity and Evolution]

  • Lymphocystis disease is a geographically widespread disease affecting more than 150 different species of marine and freshwater fish. The disease, provoked by the iridovirus lymphocystis disease virus (LCDV), is characterized by the appearance of papillomalike lesions on the skin of affected animals that usually self-resolve over time. Development of the disease is usually associated with several environmental factors and, more frequently, with stress conditions provoked by the intensive culture conditions present in fish farms. In gilthead sea bream (Sparus aurata), an economically important cultured fish species in the Mediterranean area, a distinct LCDV has been identified but not yet completely characterized. We have used direct sequencing of the virome of lymphocystis lesions from affected S. aurata fish to obtain the complete genome of a new LCDV-Sa species that is the largest vertebrate iridovirus sequenced to date. Importantly, this approach allowed us to assemble the full-length circular genome sequence of two previously unknown viruses belonging to the papillomaviruses and polyomaviruses, termed Sparus aurata papillomavirus 1 (SaPV1) and Sparus aurata polyomavirus 1 (SaPyV1), respectively. Epidemiological surveys showed that lymphocystis disease was frequently associated with the concurrent appearance of one or both of the new viruses. SaPV1 has unique characteristics, such as an intron within the L1 gene, and as the first member of the Papillomaviridae family described in fish, provides evidence for a more ancient origin of this family than previously thought.

    IMPORTANCE Lymphocystis disease affects marine and freshwater fish species worldwide. It is characterized by the appearance of papillomalike lesions on the skin that contain heavily enlarged cells (lymphocysts). The causative agent is the lymphocystis disease virus (LCDV), a large icosahedral virus of the family Iridoviridae. In the Mediterranean area, the gilthead sea bream (Sparus aurata), an important farmed fish, is frequently affected. Using next-generation sequencing, we have identified within S. aurata lymphocystis lesions the concurrent presence of an additional LCDV species (LCDV-Sa) as well as two novel viruses. These are members of polyomavirus and papillomavirus families, and here we report them to be frequently associated with the presence of lymphocysts in affected fish. Because papillomaviruses have not been described in fish before, these findings support a more ancient origin of this virus family than previously thought and evolutionary implications are discussed.

  • The Interferon-Stimulated Gene IFITM3 Restricts Infection and Pathogenesis of Arthritogenic and Encephalitic Alphaviruses [Pathogenesis and Immunity]

  • Host cells respond to viral infections by producing type I interferon (IFN), which induces the expression of hundreds of interferon-stimulated genes (ISGs). Although ISGs mediate a protective state against many pathogens, the antiviral functions of the majority of these genes have not been identified. IFITM3 is a small transmembrane ISG that restricts a broad range of viruses, including orthomyxoviruses, flaviviruses, filoviruses, and coronaviruses. Here, we show that alphavirus infection is increased in Ifitm3nndash;/nndash; and Ifitm locus deletion (Ifitm-del) fibroblasts and, reciprocally, reduced in fibroblasts transcomplemented with Ifitm3. Mechanistic studies showed that Ifitm3 did not affect viral binding or entry but inhibited pH-dependent fusion. In a murine model of chikungunya virus arthritis, Ifitm3nndash;/nndash; mice sustained greater joint swelling in the ipsilateral ankle at days 3 and 7 postinfection, and this correlated with higher levels of proinflammatory cytokines and viral burden. Flow cytometric analysis suggested that Ifitm3nndash;/nndash; macrophages from the spleen were infected at greater levels than observed in wild-type (WT) mice, results that were supported by experiments with Ifitm3nndash;/nndash; bone marrow-derived macrophages. Ifitm3nndash;/nndash; mice also were more susceptible than WT mice to lethal alphavirus infection with Venezuelan equine encephalitis virus, and this was associated with greater viral burden in multiple organs. Collectively, our data define an antiviral role for Ifitm3 in restricting infection of multiple alphaviruses.

    IMPORTANCE The interferon-induced transmembrane protein 3 (IFITM3) inhibits infection of multiple families of viruses in cell culture. Compared to other viruses, much less is known about the antiviral effect of IFITM3 on alphaviruses. In this study, we characterized the antiviral activity of mouse Ifitm3 against arthritogenic and encephalitic alphaviruses using cells and animals with a targeted gene deletion of Ifitm3 as well as deficient cells transcomplemented with Ifitm3. Based on extensive virological analysis, we demonstrate greater levels of alphavirus infection and disease pathogenesis when Ifitm3 expression is absent. Our data establish an inhibitory role for Ifitm3 in controlling infection of alphaviruses.

  • Characterization of Simian Immunodeficiency Virus Variants Anatomically Compartmentalized in Plasma and Milk in Chronically Infected African Green Monkeys [Genetic Diversity and Evolution]

  • Unlike human immunodeficiency virus type 1 (HIV-1)-infected humans, African-origin, natural simian immunodeficiency virus (SIV) hosts, such as African green monkeys (AGMs), sustain nonpathogenic SIV infections and rarely vertically transmit SIV to their infants. Interestingly, chronically SIV-infected AGMs have anatomically compartmentalized SIV variants in plasma and milk, whereas humans and SIV-infected rhesus monkeys (RMs), Asian-origin nonnatural SIV hosts, do not exhibit this compartmentalization. Thus, it is possible that AGM SIV populations in milk have unique phenotypic features that contribute to the low postnatal transmission rates observed in this natural host species. In this study, we explored this possibility by characterizing the infectivity, tropism, and neutralization susceptibility of plasma and milk SIVsab env variants isolated from chronically SIVsab92018ivTF-infected AGMs. AGM plasma and milk SIVsab env pseudovirus variants exhibited similar infectivities, neutralization susceptibilities to autologous and heterologous plasma, and chemokine coreceptor usages for cell entry, suggesting similar abilities to initiate infection in a new host. We also assessed the cytokine milieu in SIV-infected AGM milk and compared it to that of SIV-infected RMs. MIP-1bbeta;, granulocyte colony-stimulating factor (G-CSF), interleukin-12/23 (IL-12/23), and IL-13 trended significantly higher in SIV-infected AGM milk than in that of RMs, while IL-18 and IL-6 trended significantly higher in SIV-infected RM milk than in that of AGMs. Taken together, our findings imply that nonviral maternal factors, such as the cytokine milieu, rather than unique characteristics of SIV populations in the milk contribute to the low postnatal transmission rates observed in AGMs.

    IMPORTANCE Due to the ongoing global incidence of pediatric HIV-1 infections, including many that occur via breastfeeding, development of effective vaccine strategies capable of preventing vertical HIV transmission through breastfeeding remains an important goal. Unlike HIV-1-infected humans, African green monkeys (AGMs), the natural SIV host species, sustain nonpathogenic SIV infections, rarely transmit the virus postnatally to their infants, and exhibit anatomically compartmentalized SIV populations in milk and plasma. Identifying unique features of the anatomically compartmentalized milk SIV populations could enhance our understanding of how AGMs may have evolved to avoid transmission through breastfeeding. While this study identified limited phenotypic distinctions between AGM plasma and milk SIV populations, potential differences in milk cytokine profiles of natural and nonnatural SIV hosts were observed. These findings imply the potential importance of nonviral factors in natural SIV host species, such as innate SIV/HIV immune factors in milk, as a means of naturally preventing vertical transmission.

  • Constitutively Active IRF7/IRF3 Fusion Protein Completely Protects Swine against Foot-and-Mouth Disease [Vaccines and Antiviral Agents]

  • Foot-and-mouth disease (FMD) remains one of the most devastating livestock diseases around the world. Several serotype-specific vaccine formulations exist, but they require about 5 to 7 days to induce protective immunity. Our previous studies have shown that a constitutively active fusion protein of porcine interferon (IFN) regulatory factors (IRF) 7 and 3 [IRF7/3(5D)] strongly induced type I IFN and antiviral genes in vitro and prevented mortality in an FMD mouse model when delivered with a replication-defective adenoviral vector [Ad5-poIRF7/3(5D)]. Here, we demonstrate that pigs treated with 108, 109, or 1010 PFU of Ad5-poIRF7/3(5D) 24 h before FMDV challenge were fully protected from FMD clinical signs and did not develop viremia, virus shedding or antibodies against FMDV nonstructural proteins. Pigs treated with Ad5-poIRF7/3(5D) had higher levels of IFN and antiviral activity in serum, and upregulated expression of several IFN-stimulated genes in peripheral blood mononuclear cells, compared to pigs treated with Ad5-Blue vector control. Importantly, treatment of porcine cultured cells with Ad5-poIRF7/3(5D) inhibited the replication of all 7 FMDV serotypes. In vitro experiments using cultured embryonic fibroblasts derived from IFN receptor knockout mice suggested that the antiviral response induced by Ad5-poIRF7/3(5D) was dependent on type I and III IFN pathways; however, experiments with mice demonstrated that a functional type I IFN pathway mediates Ad5-poIRF7/3(5D) protection conferred in vivo. Our studies demonstrate that inoculation with Ad5-poIRF7/3(5D) completely protects swine against FMD by inducing a strong type I IFN response and highlights its potential application to rapidly and effectively prevent FMDV replication and dissemination.

    IMPORTANCE Foot-and-mouth disease virus (FMDV) causes a fast-spreading disease that affects farm animals, with economically and socially devastating consequences. Our study shows that inoculation with a constitutively active transcription factor, namely, a fusion protein of porcine interferon (IFN) regulatory factors (IRF) 7 and 3 delivered by an adenovirus vector [Ad5-poIRF7/3(5D)], is a new effective treatment to prevent FMD in swine. Animals pretreated with Ad5-poIRF7/3(5D) 1 day before being exposed to FMDV were completely protected from viral replication and clinical disease. It is noteworthy that the doses of Ad5-poIRF7/3(5D) required for protection are lower than those previously reported for similar approaches using Ad5 vectors delivering type I, II, or III IFN, suggesting that this novel strategy would be economically appealing to counteract FMD. Our results also indicate that a dynamic interplay among different components of pigs' innate immune defenses allows potent antiviral effects after Ad5-poIF7/3(5D) administration.

  • Nuclear Innate Immune DNA Sensor IFI16 Is Degraded during Lytic Reactivation of Kaposi's Sarcoma-Associated Herpesvirus (KSHV): Role of IFI16 in Maintenance of KSHV Latency [Virus-Cell Interactions]

  • IFI16 (interferon gamma-inducible protein 16) recognizes nuclear episomal herpesvirus (Kaposi's sarcoma-associated herpesvirus [KSHV], Epstein-Barr virus [EBV], and herpes simplex virus 1 [HSV-1]) genomes and induces the inflammasome and interferon beta responses. It also acts as a lytic replication restriction factor and inhibits viral DNA replication (human cytomegalovirus [HCMV] and human papillomavirus [HPV]) and transcription (HSV-1, HCMV, and HPV) through epigenetic modifications of the viral genomes. To date, the role of IFI16 in the biology of latent viruses is not known. Here, we demonstrate that knockdown of IFI16 in the latently KSHV-infected B-lymphoma BCBL-1 and BC-3 cell lines results in lytic reactivation and increases in levels of KSHV lytic transcripts, proteins, and viral genome replication. Similar results were also observed during KSHV lytic cycle induction in TREX-BCBL-1 cells with the doxycycline-inducible lytic cycle switch replication and transcription activator (RTA) gene. Overexpression of IFI16 reduced lytic gene induction by the chemical agent 12-O-tetradecoylphorbol-13-acetate (TPA). IFI16 protein levels were significantly reduced or absent in TPA- or doxycycline-induced cells expressing lytic KSHV proteins. IFI16 is polyubiquitinated and degraded via the proteasomal pathway. The degradation of IFI16 was absent in phosphonoacetic acid-treated cells, which blocks KSHV DNA replication and, consequently, late lytic gene expression. Chromatin immunoprecipitation assays of BCBL-1 and BC-3 cells demonstrated that IFI16 binds to KSHV gene promoters. Uninfected epithelial SLK and osteosarcoma U2OS cells transfected with KSHV luciferase promoter constructs confirmed that IFI16 functions as a transcriptional repressor. These results reveal that KSHV utilizes the innate immune nuclear DNA sensor IFI16 to maintain its latency and repression of lytic transcripts, and a late lytic KSHV gene product(s) targets IFI16 for degradation during lytic reactivation.

    IMPORTANCE Like all herpesviruses, latency is an integral part of the life cycle of Kaposi's sarcoma-associated herpesvirus (KSHV), an etiological agent for many human cancers. Herpesviruses utilize viral and host factors to successfully evade the host immune system to maintain latency. Reactivation is a complex event where the latent episomal viral genome springs back to active transcription of lytic cycle genes. Our studies reveal that KSHV has evolved to utilize the innate immune sensor IFI16 to keep lytic cycle transcription in dormancy. We demonstrate that IFI16 binds to the lytic gene promoter, acts as a transcriptional repressor, and thereby helps to maintain latency. We also discovered that during the late stage of lytic replication, KSHV selectively degrades IFI16, thus relieving transcriptional repression. This is the first report to demonstrate the role of IFI16 in latency maintenance of a herpesvirus, and further understanding will lead to the development of strategies to eliminate latent infection.

  • Virus-Like Particles Displaying Trimeric Simian Immunodeficiency Virus (SIV) Envelope gp160 Enhance the Breadth of DNA/Modified Vaccinia Virus Ankara SIV Vaccine-Induced Antibody Responses in Rhesus Macaques [Vaccines and Antiviral Agents]

  • The encouraging results of the RV144 vaccine trial have spurred interest in poxvirus prime-protein boost human immunodeficiency virus (HIV) vaccine modalities as a strategy to induce protective immunity. Because vaccine-induced protective immunity is critically determined by HIV envelope (Env) conformation, significant efforts are directed toward generating soluble trimeric Env immunogens that assume native structures. Using the simian immunodeficiency virus (SIV)-macaque model, we tested the immunogenicity and efficacy of sequential immunizations with DNA (D), modified vaccinia virus Ankara (MVA) (M), and protein immunogens, all expressing virus-like particles (VLPs) displaying membrane-anchored trimeric Env. A single VLP protein boost displaying trimeric gp160 adjuvanted with nanoparticle-encapsulated Toll-like receptor 4/7/8 (TLR4/7/8) agonists, administered 44 weeks after the second MVA immunization, induced up to a 3-fold increase in Env-specific IgG binding titers in serum and mucosa. Importantly, the VLP protein boost increased binding antibody against scaffolded V1V2, antibody-dependent phagocytic activity against VLP-coated beads, and antibody breadth and neutralizing antibody titers against homologous and heterologous tier 1 SIVs. Following 5 weekly intrarectal SIVmac251 challenges, two of seven DNA/MVA and VLP (DM+VLP)-vaccinated animals were completely protected compared to productive infection in all seven DM-vaccinated animals. Vaccinated animals demonstrated stronger acute viral pulldown than controls, but a trend for higher acute viremia was observed in the DM+VLP group, likely due to a slower recall of Gag-specific CD8 T cells. Our findings support immunization with VLPs containing trimeric Env as a strategy to augment protective antibody but underscore the need for optimal engagement of CD8 T cells to achieve robust early viral control.

    IMPORTANCE The development of an effective HIV vaccine remains a global necessity for preventing HIV infection and reducing the burden of AIDS. While this goal represents a formidable challenge, the modest efficacy of the RV144 trial indicates that multicomponent vaccination regimens that elicit both cellular and humoral immune responses can prevent HIV infection in humans. However, whether protein immunizations synergize with DNA prime-viral vector boosts to enhance cellular and humoral immune responses remains poorly understood. We addressed this question in a nonhuman primate model, and our findings show benefit for sequential protein immunization combined with a potent adjuvant in boosting antibody titers induced by a preceding DNA/MVA immunization. This promising strategy can be further developed to enhance neutralizing antibody responses and boost CD8 T cells to provide robust protection and viral control.

  • Multiple Transcripts Encode Full-Length Human Cytomegalovirus IE1 and IE2 Proteins during Lytic Infection [Genome Replication and Regulation of Viral Gene Expression]

  • Expression of the human cytomegalovirus (HCMV) IE1 and IE2 proteins is critical for the establishment of lytic infection and reactivation from viral latency. Defining the mechanisms controlling IE1 and IE2 expression is therefore important for understanding how HCMV regulates its replicative cycle. Here we identify several novel transcripts encoding full-length IE1 and IE2 proteins during HCMV lytic replication. Two of the alternative major immediate early (MIE) transcripts initiate in the first intron, intron A, of the previously defined MIE transcript, while others extend the 5' untranslated region. Each of the MIE transcripts associates with polyribosomes in infected cells and therefore contributes to IE1 and IE2 protein levels. Surprisingly, deletion of the core promoter region of the major immediate early promoter (MIEP) from a plasmid containing the MIE genomic locus did not completely abrogate IE1 and IE2 expression. Instead, deletion of the MIEP core promoter resulted in increased expression of alternative MIE transcripts, suggesting that the MIEP suppresses the activity of the alternative MIE promoters. While the canonical MIE mRNA was the most abundant transcript at immediate early times, the novel MIE transcripts accumulated to levels equivalent to that of the known MIE transcript later in infection. Using two HCMV recombinants, we found that sequences in intron A of the previously defined MIE transcript are required for efficient IE1 and IE2 expression and viral replication. Together, our results identify new regulatory sequences controlling IE1 and IE2 expression and suggest that multiple transcription units act in concert to regulate IE1 and IE2 expression during lytic infection.

    IMPORTANCE The HCMV IE1 and IE2 proteins are critical regulators of HCMV replication, both during primary infection and reactivation from viral latency. This study expands our understanding of the sequences controlling IE1 and IE2 expression by defining novel transcriptional units controlling the expression of full-length IE1 and IE2 proteins. Our results suggest that alternative promoters may allow for IE1 and IE2 expression when MIEP activity is limiting, as occurs in latently infected cells.

  • Modification of Three Amino Acids in Sodium Taurocholate Cotransporting Polypeptide Renders Mice Susceptible to Infection with Hepatitis D Virus In Vivo [Virus-Cell Interactions]

  • Sodium taurocholate cotransporting polypeptide (NTCP) was identified as a functional receptor for hepatitis D virus (HDV) and its helper hepatitis B virus (HBV). In cultured cell lines, HDV infection through mouse NTCP is restricted by residues 84 to 87 of the receptor. This study shows that mice with these three amino acids altered their corresponding human residues (H84R, T86K, and S87N) in endogenous mouse NTCP support de novo HDV infection in vivo. HDV infection was documented by the presence of replicative forms of HDV RNA and HDV proteins in liver cells at day 6 after viral inoculation. Monoclonal antibody specifically binding to the motif centered on K86 in NTCP partially inhibited HDV infection. These studies demonstrated specific interaction between the receptor and the viral envelopes in vivo and established a novel mouse model with minimal genetic manipulation for studying HDV infection. The model will also be useful for evaluating entry inhibitors against HDV and its helper HBV.

    IMPORTANCE NTCP was identified as a functional receptor for both HDV and HBV in cell cultures. We recently showed that neonatal C57BL/6 transgenic (Tg) mice exogenously expressing human NTCP (hNTCP-Tg) in liver support transient HDV infection. In this study, we introduced alterations of three amino acids in the endogenous NTCP of FVB mice through genome editing. The mice with the humanized NTCP residues (H84R, T86K, and S87N) are susceptible to HDV infection, and the infection can be established in both neonatal and adult mice with this editing. We also developed a monoclonal antibody specifically targeting the region of NTCP centered on lysine residue 86, and it can differentiate the modified mouse NTCP from that of the wild type and partially inhibited HDV infection. These studies shed new light on NTCP-mediated HDV infection in vivo, and the NTCP-modified mice provide a useful animal model for studying HDV infection and evaluating antivirals against the infection.

  • Generation of Long-Lived Bone Marrow Plasma Cells Secreting Antibodies Specific for the HIV-1 gp41 Membrane-Proximal External Region in the Absence of Polyreactivity [Vaccines and Antiviral Agents]

  • An effective preventive vaccine is highly sought after in order to stem the current HIV-1 pandemic. Both conservation of contiguous gp41 membrane-proximal external region (MPER) amino acid sequences across HIV-1 clades and the ability of anti-MPER broadly neutralizing antibodies (BNAbs) to block viral hemifusion/fusion establish the MPER as a prime vaccination target. In earlier studies, we described the development of an MPER vaccine formulation that takes advantage of liposomes to array the MPER on a lipid bilayer surface, paralleling its native configuration on the virus membrane while also incorporating molecular adjuvant and CD4 T cell epitope cargo. Here we demonstrate that several immunizations with MPER/liposomes induce high levels of bone marrow long-lived plasma cell (LLPC) antibody production. Single-cell immunoglobulin gene retrieval analysis shows that these plasma cells are derived from a germ line repertoire of B cells with a diverse representation of immunoglobulin genes, exhibiting antigen-driven positive selection. Characterization of LLPC recombinant monoclonal antibodies (rMAbs) indicates that antigen recognition is achieved through convergence on a common epitopic focus by utilizing various complementarity-determining region H3 (CDRH3) lengths. Importantly, the vast majority of rMAbs produced from these cells lack polyreactivity yet manifest antigen specificity in the context of lipids, shaping MPER-specific paratopes through selective pressure. Taken together, these findings demonstrate that the MPER is a vaccine target with minimal risk of generating off-target autoimmunity.

    IMPORTANCE A useful vaccine must generate desired long-term, antigen-specific antibody responses devoid of polyreactivity or autoreactivity. The common polyreactive features of some HIV-1 BNAbs have raised concern about elicitation of anti-MPER antibodies. Utilizing single-LLPC repertoire analysis and biophysical characterization of anti-MPER rMAbs, we show that their fine specificities require a structural fitness of the antibody combining site involving heavy and light chain variable domains shaped by somatic hypermutation and affinity maturation of B cells in the germinal center. Perhaps more importantly, our results demonstrate that the majority of MPER-specific antibodies are not inherently polyspecific and/or autoreactive, suggesting that polyreactivity of MPER-specific antibodies is separable from their antigen specificity.

  • Retrograde Transport from Early Endosomes to the trans-Golgi Network Enables Membrane Wrapping and Egress of Vaccinia Virus Virions [Virus-Cell Interactions]

  • The anterograde pathway, from the endoplasmic reticulum through the trans-Golgi network to the cell surface, is utilized by trans-membrane and secretory proteins. The retrograde pathway, which directs traffic in the opposite direction, is used following endocytosis of exogenous molecules and recycling of membrane proteins. Microbes exploit both routes: viruses typically use the anterograde pathway for envelope formation prior to exiting the cell, whereas ricin and Shiga-like toxins and some nonenveloped viruses use the retrograde pathway for cell entry. Mining a human genome-wide RNA interference (RNAi) screen revealed a need for multiple retrograde pathway components for cell-to-cell spread of vaccinia virus. We confirmed and extended these results while discovering that retrograde trafficking was required for virus egress rather than entry. Retro-2, a specific retrograde trafficking inhibitor of protein toxins, potently prevented spread of vaccinia virus as well as monkeypox virus, a human pathogen. Electron and confocal microscopy studies revealed that Retro-2 prevented wrapping of virions with an additional double-membrane envelope that enables microtubular transport, exocytosis, and actin polymerization. The viral B5 and F13 protein components of this membrane, which are required for wrapping, normally colocalize in the trans-Golgi network. However, only B5 traffics through the secretory pathway, suggesting that F13 uses another route to the trans-Golgi network. The retrograde route was demonstrated by finding that F13 was largely confined to early endosomes and failed to colocalize with B5 in the presence of Retro-2. Thus, vaccinia virus makes novel use of the retrograde transport system for formation of the viral wrapping membrane.

    IMPORTANCE Efficient cell-to-cell spread of vaccinia virus and other orthopoxviruses depends on the wrapping of infectious particles with a double membrane that enables microtubular transport, exocytosis, and actin polymerization. Interference with wrapping or subsequent steps results in severe attenuation of the virus. Some previous studies had suggested that the wrapping membrane arises from the trans-Golgi network, whereas others suggested an origin from early endosomes. Some nonenveloped viruses use retrograde trafficking for entry into the cell. In contrast, we provided evidence that retrograde transport from early endosomes to the trans-Golgi network is required for the membrane-wrapping step in morphogenesis of vaccinia virus and egress from the cell. The potent in vitro inhibition of this step by the drug Retro-2 suggests that derivatives with enhanced pharmacological properties might serve as useful antipoxviral agents.

  • Replication of Human Norovirus RNA in Mammalian Cells Reveals Lack of Interferon Response [Pathogenesis and Immunity]

  • Human noroviruses (HuNoVs), named after the prototype strain Norwalk virus (NV), are a leading cause of acute gastroenteritis outbreaks worldwide. Studies on the related murine norovirus (MNV) have demonstrated the importance of an interferon (IFN) response in host control of virus replication, but this remains unclear for HuNoVs. Despite the lack of an efficient cell culture infection system, transfection of stool-isolated NV RNA into mammalian cells leads to viral RNA replication and virus production. Using this system, we show here that NV RNA replication is sensitive to type I (aalpha;/bbeta;) and III (interleukin-29 [IL-29]) IFN treatment. However, in cells capable of a strong IFN response to Sendai virus (SeV) and poly(Immiddot;C), NV RNA replicates efficiently and generates double-stranded RNA without inducing a detectable IFN response. Replication of HuNoV genogroup GII.3 strain U201 RNA, generated from a reverse genetics system, also does not induce an IFN response. Consistent with a lack of IFN induction, NV RNA replication is enhanced neither by neutralization of type I/III IFNs through neutralizing antibodies or the soluble IFN decoy receptor B18R nor by short hairpin RNA (shRNA) knockdown of mitochondrial antiviral signaling protein (MAVS) or interferon regulatory factor 3 (IRF3) in the IFN induction pathways. In contrast to other positive-strand RNA viruses that block IFN induction by targeting MAVS for degradation, MAVS is not degraded in NV RNA-replicating cells, and an SeV-induced IFN response is not blocked. Together, these results indicate that HuNoV RNA replication in mammalian cells does not induce an IFN response, suggesting that the epithelial IFN response may play a limited role in host restriction of HuNoV replication.

    IMPORTANCE Human noroviruses (HuNoVs) are a leading cause of epidemic gastroenteritis worldwide. Due to lack of an efficient cell culture system and robust small-animal model, little is known about the innate host defense to these viruses. Studies on murine norovirus (MNV) have shown the importance of an interferon (IFN) response in host control of MNV replication, but this remains unclear for HuNoVs. Here, we investigated the IFN response to HuNoV RNA replication in mammalian cells using Norwalk virus stool RNA transfection, a reverse genetics system, IFN neutralization reagents, and shRNA knockdown methods. Our results show that HuNoV RNA replication in mammalian epithelial cells does not induce an IFN response, nor can it be enhanced by blocking the IFN response. These results suggest a limited role of the epithelial IFN response in host control of HuNoV RNA replication, providing important insights into our understanding of the host defense to HuNoVs that differs from that to MNV.

  • Abelson Kinase Inhibitors Are Potent Inhibitors of Severe Acute Respiratory Syndrome Coronavirus and Middle East Respiratory Syndrome Coronavirus Fusion [Vaccines and Antiviral Agents]

  • The highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) cause significant morbidity and morality. There is currently no approved therapeutic for highly pathogenic coronaviruses, even as MERS-CoV is spreading throughout the Middle East. We previously screened a library of FDA-approved drugs for inhibitors of coronavirus replication in which we identified Abelson (Abl) kinase inhibitors, including the anticancer drug imatinib, as inhibitors of both SARS-CoV and MERS-CoV in vitro. Here we show that the anti-CoV activity of imatinib occurs at the early stages of infection, after internalization and endosomal trafficking, by inhibiting fusion of the virions at the endosomal membrane. We specifically identified the imatinib target, Abelson tyrosine-protein kinase 2 (Abl2), as required for efficient SARS-CoV and MERS-CoV replication in vitro. These data demonstrate that specific approved drugs can be characterized in vitro for their anticoronavirus activity and used to identify host proteins required for coronavirus replication. This type of study is an important step in the repurposing of approved drugs for treatment of emerging coronaviruses.

    IMPORTANCE Both SARS-CoV and MERS-CoV are zoonotic infections, with bats as the primary source. The 2003 SARS-CoV outbreak began in Guangdong Province in China and spread to humans via civet cats and raccoon dogs in the wet markets before spreading to 37 countries. The virus caused 8,096 confirmed cases of SARS and 774 deaths (a case fatality rate of ~10%). The MERS-CoV outbreak began in Saudi Arabia and has spread to 27 countries. MERS-CoV is believed to have emerged from bats and passed into humans via camels. The ongoing outbreak of MERS-CoV has resulted in 1,791 cases of MERS and 640 deaths (a case fatality rate of 36%). The emergence of SARS-CoV and MERS-CoV provides evidence that coronaviruses are currently spreading from zoonotic sources and can be highly pathogenic, causing serious morbidity and mortality in humans. Treatment of SARS-CoV and MERS-CoV infection is limited to providing supportive therapy consistent with any serious lung disease, as no specific drugs have been approved as therapeutics. Highly pathogenic coronaviruses are rare and appear to emerge and disappear within just a few years. Currently, MERS-CoV is still spreading, as new infections continue to be reported. The outbreaks of SARS-CoV and MERS-CoV and the continuing diagnosis of new MERS cases highlight the need for finding therapeutics for these diseases and potential future coronavirus outbreaks. Screening FDA-approved drugs streamlines the pipeline for this process, as these drugs have already been tested for safety in humans.

  • Demethylation of the PD-1 Promoter Is Imprinted during the Effector Phase of CD8 T Cell Exhaustion [Pathogenesis and Immunity]

  • PD-1 is an inhibitory receptor that has a major role in T cell dysfunction during chronic infections and cancer. While demethylation of the PD-1 promoter DNA is observed in exhausted T cells isolated from chronically infected individuals, little is known about when this stable demethylation of PD-1 promoter DNA is programmed during the course of a chronic infection. To assess if PD-1 promoter DNA demethylation is impacted by prolonged stimulation during effector phase of chronic infection, we adoptively transferred virus-specific day 8 effector CD8 T cells from mice infected with lymphocytic choriomeningitis virus (LCMV) clone 13 into recipient mice that had cleared an acute infection. We observed that LCMV-specific CD8 T cells from chronically infected mice maintained their surface expression of PD-1 even after transfer into acute immune mice until day 45 posttransfer. Interestingly, the PD-1 transcriptional regulatory region continued to remain unmethylated in these donor CD8 T cells generated from a chronic infection. The observed maintenance of PD-1 surface expression and the demethylated PD-1 promoter were not a result of residual antigen in the recipient mice, because similar results were seen when chronic infection-induced effector cells were transferred into mice infected with a variant strain of LCMV (LCMV V35A) bearing a mutation in the cognate major histocompatibility complex class I (MHC-I) epitope that is recognized by the donor CD8 T cells. Importantly, the maintenance of PD-1 promoter demethylation in memory CD8 T cells was coupled with impaired clonal expansion and higher PD-1 re-expression upon secondary challenge. These data show that the imprinting of the epigenetic program of the inhibitory receptor PD-1 occurs during the effector phase of chronic viral infection.

    IMPORTANCE Since PD-1 is a major inhibitory receptor regulating T cell dysfunction during chronic viral infection and cancers, a better understanding of the mechanisms that regulate PD-1 expression is important. In this work, we demonstrate that the PD-1 epigenetic program in antigen-specific CD8 T cells is fixed during the priming phase of chronic infection.

  • Correction for Rife et al., Evolution of Neuroadaptation in the Periphery and Purifying Selection in the Brain Contribute to Compartmentalization of Simian Immunodeficiency Virus (SIV) in the Brains of Rhesus Macaques with SIV-Associated Encephalitis [Author Correction]

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    JVI Accepts: Articles Published Ahead of Print

  • Resistance to mutant group-2 influenza neuraminidases of an oseltamivir-zanamivir hybrid inhibitor [PublishAheadOfPrint]

  • Influenza neuraminidase (NA) drug-resistance is one of the challenges to preparedness against epidemic and pandemic influenza infections. NA N1- and N2-containing influenza viruses are the primary cause of seasonal epidemics and past pandemics. The structural and functional basis underlying drug-resistance of the influenza N1 NA is well characterized. Yet, drug-resistance of the N2 strain is not well understood. Here, we confirm that substitution of N2 E119 or I222 results in multi-drug resistance, and when occurring together the sensitivity to NA inhibitors (NAI) is reduced severely. Using crystallographic studies, we show that E119 substitution results in a loss of hydrogen bonding to oseltamivir and zanamivir whereas I222 substitution results in a change in the hydrophobic environment that is critical for oseltamivir binding. Moreover, we find that MS-257, a zanamivir/oseltamivir hybrid inhibitor, is less susceptible to drug-resistance. The binding mode of MS-257 shows that increased hydrogen bonding interactions between the inhibitor and NA active site anchor the inhibitor within the active site and allow adjustments in response to active-site modifications. Such stability is likely responsible for the observed reduced susceptibility to drug-resistant .MS-257 serves as a next-generation anti-influenza drug candidate and serves also as a scaffold for further design of NAIs.

    Importance Oseltamivir and zanamivir are the two major antiviral drugs available for the treatment of influenza virus infections. However, multi-drug resistant viruses have emerged in clinical cases, which pose a challenge for the development of new drugs. N1 and N2 subtypes exist in the viruses which cause seasonal epidemics and past pandemics. Although N1 drug resistance is well characterized, the molecular mechanisms underlying N2 drug resistance are unknown. A previous report showed that an N2 E119V/I222L dual mutant conferred drug resistance to seasonal influenza virus. Here, we confirm that these substitutions result in multi-drug resistance and dramatically reduced sensitivity to NAI. We further elucidate the molecular mechanism underlying N2 drug resistance by solving crystal structures of N2 E119V, I222L, and the dual mutant. Most importantly, we found that a novel oseltamivir/zanamivir hybrid inhibitor, MS-257, remains more effective against drug-resistant N2 and is a promising candidate as a next generation anti-influenza drug.

  • Hepaciviral NS3/4A proteases interfere with MAVS signalling of their cognate animal hosts and also with human MAVS: implications for zoonotic transmission [PublishAheadOfPrint]

  • Multiple novel members of the genus Hepacivirus have recently been discovered in diverse mammalian species. However, to date, their replication mechanisms and zoonotic potential have not been explored in detail. The NS3/4A serine protease of HCV is critical for cleavage of the viral polyprotein. It also cleaves the cellular innate immune adaptor MAVS, thus decreasing IFN production and contributing to HCV persistence in the human host.

    To investigate conservation of fundamental aspects of the hepaciviral life-cycle, we explored if MAVS cleavage and suppression of innate immune signaling represents a common mechanism employed across different clades of the genus Hepacivirus to enhance viral replication. To estimate the zoonotic potential of these non-human hepaciviruses, we assessed if their NS3/4A proteases were capable of cleaving human MAVS.

    NS3/4A proteases of viruses infecting Colobus monkeys, rodents, horses, and cows cleaved the MAVS protein of their cognate hosts and interfered with its ability to induce the IFN-bbeta; promoter. All NS3/4A proteases from non-human viruses readily cleaved human MAVS. Thus, NS3/4A-dependent cleavage of MAVS is a conserved replication strategy across multiple clades within the genus Hepacivirus. Human MAVS is susceptible to cleavage by these non-human viral proteases indicating that it does not pose a barrier for zoonotic transmission of these viruses to humans.

    IMPORTANCE Virus infection is recognized by cellular sensor proteins triggering innate immune signaling and antiviral defenses. While viruses have evolved strategies to thwart these antiviral programs in their cognate host species, these evasion mechanisms are often ineffective in a novel host, thus limiting viral transmission across species. HCV, the best characterized member of the genus Hepacivirus within the family Flaviviridae, uses its NS3/4A protease to disrupt innate immune signaling by cleaving the cellular adaptor protein MAVS. Recently, a large number of HCV-related viruses were discovered in various animal species including wild, live-stock and companion animals. We show that the NS3/4A proteases of these hepaciviruses from different animals and representing various clades of the genus cleave their cognate host MAVS proteins, in addition to human MAVS. Therefore, cleavage of MAVS is a common strategy of hepaciviruses and human MAVS is likely unable to limit replication of these non-human viruses upon zoonotic exposure.

  • HIV-1 escape from a peptidic anchor inhibitor by envelope glycoprotein spike stabilization [PublishAheadOfPrint]

  • The trimeric HIV-1 envelope glycoprotein spike (Env) mediates viral entry into cells using a spring-loaded mechanism that allows for the controlled insertion of the Env fusion peptide into the target membrane, followed by membrane fusion. Env is the focus of vaccine research aimed at inducing protective immunity by antibodies as well as efforts to develop drugs that inhibit the viral entry process. The molecular factors contributing to Env stability and decay need to be better understood in order to optimally design vaccines and therapeutics. We generated escape viruses against VIR165, a peptidic inhibitor that binds the fusion peptide of the gp41 subunit and prevents its insertion into the target membrane. Interestingly, a number of escape viruses acquired substitutions in the C1 domain of the gp120 subunit (A60E, E64K and H66R) that rendered these viruses dependent on the inhibitor. These viruses could only infect target cells when VIR165 was present after CD4 binding. Furthermore, the VIR165-dependent viruses were resistant to soluble CD4-induced Env destabilization and decay. These data suggest that VIR165-dependent Env proteins are kinetically trapped in the unliganded state and require the drug to negotiate CD4-induced conformational changes. These studies provide mechanistic insight into the action of the gp41 fusion peptide and its inhibitors, and provide new ways to stabilize Env trimer vaccines.

    IMPORTANCE Because of the rapid development of HIV-1 drug resistance new drug targets need to be continuously explored. The fusion peptide of the envelope glycoprotein can be targeted by anchor inhibitors. Here we describe virus escape from the anchor inhibitor VIR165. Interestingly, some escape viruses became dependent on the inhibitor for cell entry. We show that the identified escape mutations stabilize the ground state of the envelope glycoprotein and should thus be useful in the design of stabilized envelope-based HIV vaccines.

  • PrPC governs prion strain susceptibility in bank vole while others host factors modulate strain features [PublishAheadOfPrint]

  • Bank vole is a rodent species that shows a differential susceptibility to the experimental transmission of different prion strains. In this work, the transmission features of a panel of diverse prions with distinct origin have been assayed in both bank vole expressing methionine at codon 109 (Bv109M) or in transgenic mice expressing physiological levels of bank vole PrPC (BvPrP-Tg407 mouse line). This work compiles the first systematic comparison of the transmission features of a collection of prion isolates, representing a panel of diverse prion strains, in a transgenic mouse model and in its natural counterpart. The results showed very similar transmission properties in both natural species and transgenic mouse model demonstrating the key role of PrP amino acid sequence in prion transmission susceptibility. However, differences in the PrpSc type propagated by Bv109M and BvPrP-Tg407 suggest that host factors other than PrPC would modulate prion strain features.

    IMPORTANCE The differential susceptibility of bank vole to prion strains can be modeled in transgenic mice, suggesting that this selective susceptibility is only controlled by the vole PrP sequence rather than by other species specific factors. Differences in the phenotypes observed after prion transmissions in bank voles and in the transgenic mice suggest that other host factors than PrPC sequence may affect the selection of the substrain replicating in the animal model.

  • Glycosyl Phosphatidylinositol-Anchored Variable Region of Llama Heavy Chain Only Antibody JM4 Efficiently Blocks Both Cell-Free and T Cell-T Cell Transmission of Human Immunodeficiency Virus Type 1 [PublishAheadOfPrint]

  • JM2 and JM4 are two recently isolated variable regions (VHH) of heavy chain only antibodies from llamas that have been immunized with a trimeric gp140 bound to a CD4 mimic. JM2 binds the CD4-binding site of gp120 and neutralizes HIV-1 strains from subtypes B, C and G. JM4 binds gp120 and neutralizes HIV-1 strains from subtypes A, B, C, A/E and G in a CD4-dependent manner. In the present study, we constructed glycosyl-phosphatidylinositol (GPI)-anchored VHH JM2 and JM4 along with a E4 control and transduced them into human CD4+ cell lines and primary CD4 T cells. We report that by genetically linking the VHHs with a GPI attachment signal, VHHs are targeted to the lipid rafts of the plasma membranes. Expression of GPI-VHH JM4, but not GPI-VHH E4 and JM2, on the surface of transduced TZM.bl cells potently neutralizes multiple subtypes of HIV-1 isolates including tier 2 or 3 strains, transmitted/founders, quasispecies and soluble single domain antibody (sdAb) JM4-resistant viruses. Moreover, transduction of CEMss-CCR5 cells with GPI-VHH JM4, but not with GPI-VHH E4, confers resistance to both cell-free and T cell-T cell transmission of HIV-1 and HIV-1 envelope-mediated fusion. Finally, GPI-VHH JM4-transduced human primary CD4 T cells efficiently resist both cell-free and T cell-T cell transmission of HIV-1. Thus, we conclude that the VHH JM4, when targeted to the lipid rafts of the plasma membrane, efficiently neutralizes HIV-1 infection via both cell-free and T cell-T cell transmission. Our findings should have important implications for GPI-anchored antibody-based therapy against HIV-1.

    Importance Lipid rafts are specialized dynamic microdomains of the plasma membrane and have been shown to be gateways for HIV-1 budding as well as entry into T cells and macrophages. In nature, many glycosyl-phosphatidylinositol (GPI)-anchored proteins localize in the lipid rafts. In the present study, we developed GPI-anchored variable region (VHH) of two heavy chain only antibodies JM2 and JM4 from immunized llamas. We show that by genetically linking the VHHs with a GPI attachment signal, VHHs are targeted to the lipid rafts of the plasma membranes. GPI-VHH JM4, but not GPI-VHH JM2, in transduced CD4+ cell lines and human primary CD4 T cells not only efficiently blocks diverse HIV-1 strains including tier 2 or 3 strains, transmitted founders, quasispecies and soluble sdAb JM4-resistant strains, but also efficiently interferes T cell-T cell transmissions of HIV-1 and HIV-1 envelope-mediated fusion. Our findings should have important implications in GPI-anchored antibody-based therapy against HIV-1.

  • Extragenic Suppression of a Mutation in Herpes Simplex Virus Type 1 (HSV-1) UL34 That Affects Lamina Disruption and Nuclear Egress [PublishAheadOfPrint]

  • Nuclear egress of herpesviruses is accompanied by changes in the architecture of the nuclear membrane and nuclear lamina that are thought to facilitate capsid access to the INM and curvature of patches of the INM around the capsid during budding. Here we report the properties of a point mutant of pUL34 (Q163A) that fails to induce gross changes in nuclear architecture or redistribution of lamin A/C. UL34(Q163A) shows a roughly 100-fold defect in single-step growth, and forms small plaques. This mutant has a defect in nuclear egress and furthermore fails to disrupt nuclear shape or cause observable displacement of lamin A/C despite retaining the ability to recruit pUS3 and PKC protein kinases, and to mediate phosphorylation of emerin. Extragenic suppressors of the UL34(Q163A) phenotype were isolated, all of which carry a single mutation of arginine 229 to leucine in UL31. Surprisingly, although this UL31 mutation largely restores virus replication, it does not correct the lamina disruption defect suggesting that, in Vero cells, changes in nuclear shape and gross displacements of lamin A/C may facilitate, but are unnecessary for nuclear egress.

    Importance Herpesvirus nuclear egress is an essential and conserved process that requires close association of the virus capsid with the inner nuclear membrane and budding of the capsid into that membrane. Access to the nuclear membrane and tight curvature of that membrane is thought to require disruption of the nuclear lamina that underlies the inner nuclear membrane and, consistent with this idea, herpesvirus infection induces biochemical and architectural changes at the nuclear membrane. The significance of the nuclear membrane architectural changes is poorly characterized. The results presented here address that deficiency in our understanding and show that a combination of mutations in two of the viral nuclear egress factors results in failure to accomplish at least two components of lamina disruption while still allowing relatively efficient viral replication, suggesting that changes in nuclear shape and displacement of lamins are not necessary for HSV-1 nuclear egress.

  • Multiple Strategies Reveal a Bi-dentate Interaction Between the Nipah Virus Attachment and Fusion Glycoproteins [PublishAheadOfPrint]

  • The paramyxoviral family contains many medically important viruses including measles virus, mumps virus, parainfluenza viruses, respiratory syncytial virus, human metapneumovirus, and the deadly zoonotic henipaviruses Hendra and Nipah virus (NiV). To both enter host cells and spread from cell to cell within infected hosts, the vast majority of paramyxoviruses utilize two viral envelope glycoproteins: the attachment glycoprotein (G, H, or HN) and the fusion glycoprotein (F). Binding of G/H/HN to a host cell receptor triggers structural changes in G/H/HN that in turn trigger F to undergo a series of conformational changes that result in viral-cell (viral entry) or cell-cell (syncytia) membrane fusion. The actual regions of G/H/HN and F that interact during the membrane fusion process remain relatively unknown, though it is generally thought that the paramyxoviral G/H/HN stalk region interacts with the F head region. Studies to determine such interactive regions have relied heavily on co-immunoprecipitation approaches, whose limitations include the use of detergents and the micelle-mediated association of proteins. Here we developed a flow cytometric strategy capable of detecting membrane protein-protein interactions by interchangeably using the full-length form of G and a soluble form of F, or vice versa. Using both co-immunoprecipitation and flow cytometric strategies, we found a bi-dentate interaction between NiV G and F, where both the stalk and head regions of NiV G interact with F. This is a new structural-biological finding for the paramyxoviruses. Additionally, our studies disclosed regions of the NiV G and F glycoproteins dispensable for the G and F interactions.

    IMPORTANCE Nipah virus (NiV) is a zoonotic paramyxovirus that causes high mortality rates in humans, with no approved treatment or vaccine available for human use. Viral entry into host cells relies on two viral envelope glycoproteins: the attachment (G) and fusion (F) glycoproteins. Binding of G to the ephrinB2 or ephrinB3 cell receptors triggers conformational changes in G that in turn cause F to undergo conformational changes that result in viral/cell membrane fusion and viral entry. It is currently unknown, however, which specific regions of G and F interact during membrane fusion. Past efforts to determine the interacting regions have relied mainly on co-immunoprecipitation, a technique with some pitfalls. We developed a flow cytometric assay to study membrane protein-protein interactions, and using this assay we report a bi-dentate interaction whereby both the head and stalk regions of NiV G interact with NiV F, a new finding for the paramyxovirus family.

  • Tetherin Antagonism by HIV-1 Group M Nef Proteins [PublishAheadOfPrint]

  • Although Nef is the viral gene product used by most simian immunodeficiency viruses to overcome restriction by tetherin, this activity was acquired by the Vpu protein of HIV-1 group M viruses due to the absence of sequences in human tetherin that confer susceptibility to Nef. Thus, it is widely accepted that HIV-1 group M uses Vpu instead of Nef to counteract tetherin. Challenging this paradigm, we identified Nef alleles of HIV-1 group M isolates with significant activity against human tetherin. These Nef proteins promoted virus release and tetherin downmodulation from the cell surface, and in the context of vpu-deleted HIV-1 recombinants, enhanced virus replication and resistance to antibody-dependent cell-mediated cytotoxicity (ADCC). Further analysis revealed that the Vpu proteins from several of these viruses lack anti-tetherin activity, suggesting that under certain circumstances, HIV-1 group M Nef may acquire the ability to counteract tetherin to compensate for the loss of this function by Vpu. These observations illustrate the remarkable plasticity of HIV-1 in overcoming restriction by tetherin and challenge the prevailing view that all HIV-1 group M isolates use Vpu to counteract tetherin.

    IMPORTANCE Most HIV-1 group M viruses, the main group of HIV-1 responsible for the global AIDS pandemic, use their Vpu proteins to overcome restriction by tetherin (BST-2 or CD317), which is a transmembrane protein that inhibits virus release from infected cells. Here we show that the Nef proteins of certain HIV-1 group M isolates can acquire the ability to counteract tetherin. These results challenge the current paradigm that HIV-1 group M exclusively uses Vpu to counteract tetherin and underscore the importance of tetherin antagonism for efficient viral replication.

  • The P Protein of Spring Viremia of Carp Virus Negatively Regulates Fish IFN Response by Inhibiting the Kinase Activity of TANK-binding kinase 1 [PublishAheadOfPrint]

  • Spring viremia of carp virus (SVCV) is an efficient pathogen causing high mortality in the common carp. Fish interferon (IFN) is a powerful cytokine enabling host cells to establish an antiviral response; therefore, the strategies that SVCV uses to avoid the cellular IFN response were investigated. Here, we report that the SVCV P protein is phosphorylated by cellular TANK-binding kinase 1 (TBK1), which decreases IFN regulatory factor 3 (IRF3) phosphorylation and suppresses IFN production. First, overexpression of P protein inhibited the IFN promoter activation induced by SVCV and the IFN activity activated by mitochondrial antiviral signaling protein (MAVS), although TBK1 activity was not blocked by P protein. Second, P protein co-localized and interacted with TBK1. Dominant-negative experiments suggested that the TBK1 N terminal kinase domain interacted with P protein and was essential for P protein and IRF3 phosphorylation. Finally, P protein overexpression reduced the IRF3 phosphorylation activated by TBK1 and reduced host cellular ifn transcription. Collectively, our data demonstrated that the SVCV P protein is a decoy substrate for the host phosphokinase TBK1, preventing IFN production and facilitating SVCV replication.

    IMPORTANCE TBK1 is a pivotal phosphokinase that activates host IFN production to defend against viral infection, thus, it is a potential target for viruses to negatively regulate IFN response and facilitate viral evasion. We report that the SVCV P protein functions as a decoy substrate for cellular TBK1, leading to the reduction of IRF3 phosphorylation and suppression of IFN expression. These findings reveal a novel immune evasion mechanism of SVCV.

  • A Novel Benzodiazepine Compound Inhibits Yellow Fever Virus Infection by Specifically Targeting NS4B Protein [PublishAheadOfPrint]

  • Although a highly effective vaccine is available, the number of yellow fever cases has increased over the past two decades, which highlights the pressing need for antiviral therapeutics. In a high throughput screening campaign, we identified an acetic acid benzodiazepine (BDAA) compound, which potently inhibits yellow fever virus (YFV). Interestingly, while treatment of YFV infected cultures with 2 mmu;M of BDAA reduced the virion production by greater than 2 logs, the compound is not active against 21 other viruses from 14 different viral families. Selection and genetic analysis of drug resistant viruses revealed that substitution of proline at amino acid 219 (P219) of the nonstructural protein 4B (NS4B) with serine, threonine or alanine confers YFV resistance to BDAA without apparent loss of replication fitness in cultured mammalian cells. However, substitution of P219 with glycine confers BDAA resistance with significant loss of replication ability. Bioinformatics analysis predicts that the P219 localizes at the endoplasmic reticulum lumen side of the fifth putative trans-membrane domain of NS4B and the mutation may render the viral protein incapable of interacting with BDAA. Our studies thus revealed important role and structural basis for NS4B protein in supporting YFV replication. Moreover, in YFV-infected hamsters, oral administration of BDAA protected 90% of the animals from death, significantly reduced viral load by greater than 2 logs and attenuated viral infection-induced liver injury and body weight loss. The encouraging preclinical results thus warrant further development of BDAA or its derivatives as antiviral agents to treat yellow fever.

    Importance Yellow fever is an acute viral hemorrhagic disease which threatens approximately one billion people living in tropical areas of Africa and Latin America. Although a highly effective yellow fever vaccine has been available for more than seven decades, the low vaccination rate fails to prevent outbreaks in at-risk regions. It has been estimated that up to 1.7 million YFV infections occur in Africa each year, resulting in 29,000 to 60,000 death. Thus far, there is no specific antiviral treatment for yellow fever. To cope with this medical challenge, we identified a benzodiazepine compound that selectively inhibits YFV by targeting the viral NS4B protein. To our knowledge, this is the first report demonstrating in vivo safety and antiviral efficacy of an YFV NS4B inhibitor in an animal model. We have thus reached a critical milestone toward the development of specific antiviral therapeutics for clinical management of yellow fever.

  • The cytoskeletal adaptor obscurin-like 1 interacts with the HPV16 capsid protein L2 and is required for HPV16 endocytosis [PublishAheadOfPrint]

  • The human papillomavirus (HPV) capsid protein L2 is essential for viral entry. To gain a deeper understanding of the role of L2, we searched for novel cellular L2-interacting proteins. A yeast two-hybrid analysis uncovered the actin depolymerizing factor gelsolin, the membrane glycoprotein dysadherin, the centrosomal protein 68 (Cep68), and the cytoskeletal adaptor protein obscurin-like 1 protein (OBSL1) as putative L2 binding molecules. Pseudovirus (PsV) infection assays identified OBSL1 as a host factor required for gene transduction by three oncogenic human papillomavirus types, HPV16, HPV18, and HPV31. In addition, we detected OBSL1 expression in cervical tissue sections and noted the involvement of OBSL1 during gene transduction of primary keratinocytes by HPV16 PsV. Complex formation of HPV16 L2 with OBSL1 was demonstrated in co-immunofluorescence and co-immunoprecipitation studies after overexpression of L2 or after PsV exposure. We observed a strong co-localization of OBSL1 with HPV16 PsV and CD151 at the plasma membrane suggesting a role of OBSL1 in viral endocytosis. Indeed, viral entry assays exhibited a reduction of viral endocytosis in OBSL1 depleted cells. Our results suggest OBSL1 as a novel L2-interacting protein and endocytosis factor in HPV infection.

    Importance Human papillomaviruses infect mucosal and cutaneous epithelium and the high risk HPV types account for 5% of cancer cases worldwide. As recently discovered, HPV entry occurs by a clathrin-, caveolin- and dynamin-independent endocytosis via tetraspanin-enriched microdomains. At present, the cellular proteins involved in the underlying mechanism of this type of endocytosis are under investigation. In this study, the cytoskeletal adaptor OBSL1 was discovered as a previously unrecognized interaction partner of the minor capsid protein L2 and was identified as a proviral host factor required for HPV16 endocytosis into target cells. The findings of this study advance the understanding of a so far less well characterized endocytic pathway that is used by oncogenic HPV subtypes.

  • Influenza virus susceptibility of wild-derived CAST/EiJ mice results from two amino acid changes in the MX1 restriction factor [PublishAheadOfPrint]

  • The interferon-regulated Mx1 gene of the A2G mouse strain confers a high degree of resistance against influenza A and Thogoto viruses. Most other laboratory inbred mouse strains carry truncated non-functional Mx1 alleles and, consequently, exhibit high virus susceptibility. Interestingly, CAST/EiJ mice derived from wild Mus musculus castaneus possess a seemingly intact Mx1 gene, but are highly susceptible to influenza A virus challenge. To determine whether enhanced influenza virus susceptibility is due to intrinsically reduced antiviral activity of the CAST-derived Mx1 allele, we generated a congenic C57BL/6J mouse line that carries the Mx locus of CAST/EiJ. Adults animals of this line were almost as susceptible to influenza virus challenge as standard C57BL/6J mice lacking functional Mx1 alleles but exhibited far more pronounced resistance to Thogoto virus. Sequencing revealed that CAST-derived MX1 differs from A2G-derived MX1 by two amino acids (G83R and A222V) in the GTPase domain. Especially the A222V mutation reduced GTPase activity of purified MX1 and diminished the inhibitory effect of MX1 in influenza A virus polymerase activity assays. Further, MX1 protein was substantially less abundant in organs of interferon-treated mice carrying the CAST Mx1 allele in comparison to mice carrying the A2G Mx1 allele. We found that the CAST-specific mutations reduced the metabolic stability of the MX1 protein although Mx1 mRNA levels were unchanged. Thus, enhanced influenza virus susceptibility of CAST/EiJ mice can be explained by minor alterations in the MX1 restriction factor that negatively affect its enzymatic activity and reduce its half-life.

    IMPORTANCE Although the crystal structures of the prototypic human MXA protein is known, the importance of specific protein domains for antiviral activity is still incompletely understood. Novel insights might come from studying naturally occuring MX protein variants with altered antiviral activity. Here we identified two seemingly minor amino acid changes in the GTPase domain that negatively affect enzymatic activity and metabolic stability of murine MX1 and thus dramatically reduce influenza virus resistance of the respective mouse inbred strain. These observations highlight our current inability to predict the biological consequences of previously uncharacterized MX mutations in mice. Since this is probably also true for naturally occurring mutations in Mx genes of humans, careful experimental analysis of any natural MXA variants for altered activity is necessary in order to assess possible consequences of such mutations on innate antiviral immunity.

  • The Respiratory Syncytial Virus Phosphoprotein, Matrix Protein, and Fusion Protein Carboxy-Terminal Domain Drive Efficient Filamentous Virus-Like Particle Formation. [PublishAheadOfPrint]

  • Virus-like particles (VLP) are attractive as a vaccine concept. For human respiratory syncytial virus (hRSV), VLP assembly is poorly understood and appears inefficient. Hence hRSV antigens are often incorporated into foreign VLP systems to generate anti-RSV vaccine candidates. To better understand assembly and utimately enable efficient production of authentic hRSV VLPs, we examined the associated requirements and mechanisms. In a previous analysis in HEp-2 cells, the nucleoprotein (N), phosphoprotein (P), matrix protein (M) and fusion protein (F) were required for formation of filamentous VLPs, which, similar to wildtype virus, were associated with the cell surface. Using fluorescence and electron microscopy combined with immuno-gold labeling we examined the surface of transfected HEp-2 cells and further dissected the process of filamentous VLP formation. Our results show that N was not required. Co-expression of P+M+F, but not P+M, M+F, or P+F, induced both viral protein coalescence and formation of filamentous VLPs which resembled wildtype virions. Despite sub-optimal coalescence in absence of P, the M and F proteins when co-expressed formed cell surface -associated filaments of abnormal morphology, appearing longer and thinner than wildtype virions. For F, only the carboxy-terminus (Fstem) was required, and addition of foreign protein sequences to Fstem allowed incorporation into VLPs. Together, the data show that P, M, and the F carboxy-terminus are sufficient for robust viral protein coalescence and filamentous VLP formation, and suggest that M-F interaction drives viral filament formation with P acting as a type of co-factor facilitating the process and exerting control over particle morphology.

    IMPORTANCE hRSV is responsible for ggt;100,000 deaths in children worldwide, and a vaccine is not available. Among the potential anti-hRSV approaches are virus-like particle (VLP) vaccines, which, based on resemblance to virus or viral components, can induce protective immunity. For hRSV, few reports are available concerning authentic VLP production or testing, in large part because VLP production is inefficient and the mechanisms underlying particle assembly are poorly understood. Here, we took advantage of the cell-associated nature of RSV particles, and used high-resolution microscopy analyses to examine the viral proteins required for formation of wildtype virus-resembling VLPs, the contribution of these proteins to morphology, and the domains involved in incorporation of the antigenically important viral F protein. The results provide new insights that will facilitate future production of hRSV VLPs of defined shape and composition and may translate into improved manufacture of live-attenuated hRSV vaccines.

  • Comprehensive Genome-Scale Phylogenetic Study Provides New Insights on the Global Expansion of Chikungunya Virus [PublishAheadOfPrint]

  • Since the India and Indian Ocean outbreaks of 2005-2006, the global distribution of chikungunya virus (CHIKV) and the locations of epidemics have dramatically shifted. First, the Indian Ocean lineage (IOL) caused sustained epidemics in India and has radiated to many other countries. Second, the Asian lineage has caused frequent outbreaks in the Pacific islands and in 2013 was introduced to the Caribbean, followed by rapid spread to nearly all of the neotropics. Further, CHIKV epidemics as well as exported cases have been reported in central Africa after a long period of perceived silence. To understand these changes and anticipate the future of this virus, the exact distribution, genetic diversity, transmission routes, and future epidemic potential of CHIKV require further assessment. To do so, we conducted the most comprehensive phylogenetic analysis to date, examined CHIKV evolution and transmission, and explored distinct genetic factors associated with the emergence of the East/Central/South African (ECSA), IOL, and Asian lineages. Our results reveal contrasting evolutionary patterns among lineages with growing genetic diversities observed in each, and suggest that CHIKV will continue to be a major public health threat with the potential for further emergence and spread.

    IMPORTANCE Chikungunya fever is a re-emerging infectious disease that is transmitted by Aedes mosquitoes and causes severe health and economic burden in affected populations. Since the unprecedented Indian Ocean and Indian Subcontinent outbreaks of 2005-2006, CHIKV has further expanded its geographic range including to the Americas in 2013. Its evolution and transmission during and following these epidemics, as well as the recent evolution and spread of other lineages require optimal assessment. Using newly obtained genome sequences, we provide a comprehensive update of the global distribution of CHIKV genetic diversity and analyze factors associated with recent outbreaks. These results provide a solid foundation for future evolutionary studies of CHIKV that not can only elucidate emergence mechanisms, and also may help to predict future epidemics.

  • Assessment of the PrPc amino-terminal domain in prion species barriers [PublishAheadOfPrint]

  • Chronic wasting disease (CWD) in cervids and bovine spongiform encephalopathy (BSE) in cattle are prion diseases that are caused by the same protein-misfolding mechanism, but appear to pose different risks to humans. We are interested in understanding the differences between the species barriers of CWD and BSE. We used real-time, quaking-induced conversion (RT-QuIC) to model the central molecular event in prion disease, the templated misfolding of the normal prion protein, PrPc, to a pathogenic, amyloid isoform, PrPSc. We examined the role of the PrPc amino-terminal domain (NTD, aa23-90) in cross-species conversion by comparing the conversion efficiency of various prion seeds in either full-length (aa23-231) or truncated (aa90-231) PrPc. We demonstrate that the presence of white-tailed deer and bovine NTDs hindered seeded conversion of PrPc, but human and bank vole NTDs did the opposite. Additionally, full-length human and bank vole PrPc were more likely to be converted to amyloid by CWD prions than were their truncated forms. A chimera with replacement of the human NTD by the bovine NTD resembled human PrPc. The requirement for an NTD, but not for the specific human sequence, suggests that the NTD interacts with other regions of the human PrPc to increase promiscuity. These data contribute to the evidence that, in addition to primary sequence, prion species barriers are controlled by interactions of the substrate NTD with the rest of the substrate PrPc molecule.

    Importance We demonstrate that the amino-terminal domain of the normal prion protein, PrPc, hinders seeded conversion of bovine and white-tailed deer PrPc to the prion form, but it facilitates conversion of the human and bank vole PrPc to the prion form. Additionally, we demonstrate that the amino-terminal domain of human and bank vole PrPc requires interaction with the rest of the molecule to facilitate conversion by CWD prions. These data suggest that interactions of the amino-terminal domain with the rest of the PrPc molecule play an important role in the susceptibility of humans to CWD prions.

  • Convallatoxin-induced reduction of methionine import effectively inhibits human cytomegalovirus infection and replication [PublishAheadOfPrint]

  • Cytomegalovirus (CMV) is a ubiquitous human pathogen that increases morbidity and mortality of immunocompromised individuals. The current FDA approved treatments for CMV infection are intended to be virus specific, yet they have significant adverse side effects including nephrotoxicity and hematological toxicity. Thus, there is a medical need for safer and more effective CMV therapeutics. Using a high-content screen, we have identified the cardiac glycoside convallatoxin as an effective compound that inhibits CMV infection. Using a panel of cardiac glycoside variants, we assessed the structural elements critical for anti-CMV activity by both experimental and in silico methods. Analysis of the antiviral effect, toxicity, and pharmacodynamics of different variants of cardiac glycosides identified the mechanism of inhibition as reduction of methionine import leading to decreased immediate early gene translation without significant toxicity. Also, convallatoxin was found to dramatically reduce viral proliferation of clinical CMV strains implying its mechanism of action as an effective strategy to block CMV dissemination. Our study has uncovered the mechanism and structural elements of convallatoxin that are important for effectively inhibiting CMV infection by targeting the expression of immediate early genes.

    Importance: Cytomegalovirus is a highly prevalent virus capable of causing severe disease in certain populations. The current FDA approved therapeutics all target the same stage of the viral life cycle and induce toxicity and viral resistance. We identified convallatoxin, a novel, cell-targeting antiviral that inhibits CMV infection by decreasing the synthesis of viral proteins. At doses low enough for cells to tolerate, convallatoxin was able to inhibit primary isolates of CMV, including those resistant to the anti-CMV drug ganciclovir. In addition to identifying convallatoxin as a novel antiviral, limiting mRNA translation has a dramatic impact on CMV infection and proliferation.

  • Multiple antibody lineages in one donor target the glycan-V3 supersite of the HIV-1 envelope glycoprotein and display a preference for quaternary binding [PublishAheadOfPrint]

  • One of the goals of HIV-1 vaccine development is the elicitation of neutralizing antibodies against vulnerable regions on the envelope glycoproteins (Env) viral spike. Broadly neutralizing antibodies targeting the Env glycan-V3 region (also called the N332-glycan supersite) have previously been described, with several single lineages each derived from individual donors. We used a high-throughput B-cell culture method to isolate neutralizing antibodies from an HIV-1 infected donor with high serum neutralization breadth. Clonal relatives from three distinct antibody lineages were isolated. Each of these antibody lineages displayed modest breadth and potency, but shared several characteristics with the well-characterized glycan-V3 antibodies including dependence on glycans N332 and N301, VH4 family gene utilization, a CDRH2 insertion, and a longer than average CDRH3. In contrast to previously described glycan-V3 antibodies, the most potent of these antibodies preferentially recognized the native Env trimer compared to monomeric gp120. These data indicate the diversity of antibody specificities that target the glycan-V3 site. The quaternary binding preference of these antibodies suggests that that their elicitation likely requires presentation of a native-like trimeric Env immunogen.

    Importance Broadly neutralizing antibodies targeting the HIV-1 glycan-V3 region have previously been described with single lineages from individual donors. Here we describe three lineages from a single donor that each target glycan-V3. Unlike the previously described glycan-V3 antibodies, these mature antibodies bind preferentially to the native Env trimer and weakly to gp120 monomer. These data extend our knowledge of immune response recognition of the N332 supersite region and suggest that the mode of epitope recognition is more complex than previously anticipated.

  • SUMO Modification Stabilizes Enterovirus 71 Polymerase 3D to Facilitate Viral Replication [PublishAheadOfPrint]

  • Accumulating evidences suggest that viruses hijack cellular proteins to circumvent the host immune system. Ubiquitination and SUMOylation are extensively studied post-translational modifications (PTMs) that play critical roles in diverse biological processes. Crosstalk between ubiquitination and SUMOylation of both host and viral proteins has been reported to result in distinct functional conses1quences. Enterovirus 71 (EV71), an RNA virus belonging to Picornaviridae family, is a common cause of hand, foot and mouth disease. Little is known concerning how host PTM systems interact with enteroviruses. Here, we demonstrated that the 3D protein, an RNA-dependent RNA polymerase (RdRp) of EV71, is modified by small ubiquitin-like modifier-1 both during infection and in vitro. Residues K159 and L150/D151/L152 were responsible for 3D SUMOylation determined by bioinformatic prediction combined with site-directed mutagenesis. And primer-dependent polymerase assays indicated that mutation of SUMOylation sites impaired 3D polymerase activity and virus replication. Moreover, 3D is ubiquitinated in a SUMO-dependent manner, and SUMOylation is crucial for 3D stability which may be due to the interplay between the two PTMs. Of importance, increasing the level of SUMO-1 in EV71-infected cells augmented the SUMOylation and ubiquitination level of 3D, leading to enhanced replication of EV71. These results together suggested that SUMO and ubiquitin cooperatively regulated EV71 infection either by SUMO-ubiquitin hybrid chains or by ubiquitin conjugating to the exposed lysine residue through SUMOylation. Our study provides a new insight into how a virus utilizes cellular pathways to facilitate its replication.

    Importance Infection with Enterovirus 71(EV71) often causes neurological diseases in children and EV71 is responsible for the majority of fatalities. Based on a better understanding of interplay between virus and host cell, antiviral drugs against enteroviruses may be developed. As a dynamic cellular process of post-translational modification, SUMOylation regulates global cellular protein localization, interaction, stability, and enzymatic activity. However, little is known concerning the SUMOylation directly influence virus replication by targeting viral polymerase. Herein, we found that EV71 polymerase 3D was SUMOylated during EV71 infection and in vitro. Moreover, the SUMOylation sites were determined. And in vitro polymerase assays indicated that mutations at SUMOylation sites could impair polymerase synthesis. Importantly, 3D is ubiquitinated in a SUMOylation-dependent manner which enhances the stability of the viral polymerase. Our findings indicate that the two modifications likely cooperatively enhance virus replication. Our study may offer a new therapeutic strategy against virus replication.

  • Immunodominant T-cell epitopes in the VP1 capsid protein of rhinovirus species A and C. [PublishAheadOfPrint]

  • Rhinovirus (RV) species A and C are the most frequent cause of respiratory viral illness worldwide and RV-C has been linked to more severe exacerbations of asthma in young children. Little is known about the immune response against the different RV species, although studies comparing IgG1 antibody titers found impaired antibody responses to RV-C. Therefore, the aim of this study was to assess whether T-cell immunity to RV-C is similarly impaired. We measured T-cell proliferation to overlapping synthetic peptides covering the entire VP1 capsid protein of an RV-A and RV-C genotype for 20 healthy adult donors. Human leukocyte antigen (HLA) was typed in all donors in order to investigate possible associations between HLA type and RV peptide recognition. Total and specific IgG1 antibody titers to VP1 of both RV-A and RV-C were also measured to examine associations between the antibody and T-cell responses. We identified T-cell epitopes that are specific of, and representative for each RV-A and RV-C species. These epitopes stimulated CD4+ specific T-cell proliferation with a similar magnitude of response for both RV species. All donors, independent of their HLA-DR or DQ type, were able to recognize the immunodominant RV-A and C regions of VP1. Furthermore, the presence or absence of specific antibody titers was not related with changes in T-cell recognition. Our results indicate a dissociation between the antibody and T-cell response against rhinoviruses. The species-representative T-cell epitopes identified in this study are valuable tools for future studies investigating T-cell responses to the different RV species.

    Importance Rhinoviruses (RV) are mostly associated with the common cold and asthma exacerbations, although their contribution in most upper and lower respiratory tract diseases have increasingly been reported. Species C (RV-C) has been associated with more frequent and severe asthma exacerbations in young children, and with RV-A are the most clinically relevant species. Little is known about how our immune system responds to rhinoviruses and there are limited tools to study specific adaptive immunity against each rhinovirus species.

    In this study, we identified immunodominant T-cell epitopes of the VP1 protein of RV-A and RV-C, which are representative of each species. The study found T-cell responses to RV-A and RV-C were of a similar magnitude, in contrast with previous findings showing RV-C specific antibody responses were low. These findings will provide the basis for future studies on immune response against rhinoviruses and can help elucidate the mechanisms of severity of rhinovirus-induced infections.

  • Hepatitis C virus is released via a non-canonical secretory route. [PublishAheadOfPrint]

  • We analyzed HCV morphogenesis using viral genomes encoding for a mCherry-tagged E1 glycoprotein. HCV-E1-mCherry polyprotein expression, intracellular localization and replication kinetics were comparable to untagged HCV and E1-mCherry tagged viral particles were assembled and released into cell culture supernatants. Expression and localization of structural E1 and non-structural NS5A followed a tempo-spatial pattern with succinct decrease in replication complexes and the appearance of E1-mCherry punctae. Interaction of the structural proteins E1, Core and E2 increased at E1-mCherry punctae in a time-dependent manner, indicating that E1-mCherry punctae represent assembled or assembling virions. E1-mCherry did not colocalize with Golgi markers. Furthermore, the bulk of viral glycoproteins within released particles revealed an EndoH-sensitive glycosylation pattern, indicating absence of viral glycoprotein processing by the Golgi. In contrast, HCV-E1-mCherry trafficked with Rab9-positive compartments and inhibition of endosomes specifically suppressed HCV release. Our data suggests that assembled HCV particles are released via a non-canonical secretory route involving the endosomal compartment.

    IMPORTANCE STATEMENT The goal of this study was to shed light on the poorly understood trafficking and release routes of hepatitis C virus (HCV). For this, we generated novel HCV genomes which result in the production of fluorescently labeled viral particles. We used live cell microscopy and other imaging techniques to follow up on the temporal dynamics of virus particle formation and trafficking in HCV-expressing liver cells. While viral particles and viral structural protein were found in endosomal compartments, no overlap with Golgi structures could be observed. Furthermore, biochemical and inhibitor-based experiments support a HCV release route which is distinguishable from canonical Golgi-mediated secretion. Since viruses hijack cellular pathways to generate viral progeny, our results point towards the possible existence of a not yet described cellular secretion route.

  • FIV Vif N-Terminal Residues Selectively Counteract Feline APOBEC3s [PublishAheadOfPrint]

  • Feline immunodeficiency virus (FIV) Vif protein counteracts feline APOBEC3s (FcaA3s) restriction factors by inducing their proteasomal degradation. The functional domains in FIV Vif for interaction with FcaA3s are poorly understood. Here, we have identified several motifs in FIV Vif that are important for selective degradation of different FcaA3s. Cats (Felis catus) express three types of A3s: single-domain A3Z2, A3Z3 and double-domain A3Z2Z3. We proposed that FIV Vif would selectively interact with the Z2 and the Z3 A3s. Indeed, we identified two N-terminal Vif motifs (12LF13 and 18GG19) that specifically interacted with the FcaA3Z2 protein but not with A3Z3. In contrast, the exclusive degradation of FcaA3Z3 was regulated by a region of three residues (M24, L25 and I27). Only a FIV Vif carrying a combination of mutations from both interaction sites lost the capacity to degrade and counteract FcaA3Z2Z3. However, alterations in the specific A3s interaction sites did not affect the cellular localization of the FIV Vif protein and binding to feline A3s. Pull-down experiments demonstrated that the A3 binding region localized to FIV Vif residues 50 to 80, outside the specific A3 interaction domain. Finally, we found that the Vif sites specific to individual A3s are conserved in several FIV lineages of domestic cat and non-domestic cats, while being absent in the FIV Vif of pumas. Our data support a complex model of multiple Vif-A3 interactions in which the specific region for selective A3 counteraction is discrete from a general A3 binding domain.

    IMPORTANCE Both human immunodeficiency virus (HIV) and feline immunodeficiency virus (FIV) Vif proteins counteract their host's APOBEC3 restriction factors. However, these two Vif proteins have limited sequence homology. The molecular interaction between FIV Vif and feline APOBEC3s are not well understood. Here we have identified N-terminal FIV Vif sites that regulate the selective interaction of Vif with either feline APOBEC3Z2 or APOBEC3Z3. These specific Vif sites are conserved in several FIV lineages of domestic cat and non-domestic cats, while being absent in FIV Vif from puma. Our findings provide important insights for future experiments describing the FIV Vif interaction with feline APOBEC3s and also indicate that the conserved feline APOBEC3s interaction sites of FIV Vif allow FIV transmissions in Felidae.

  • HSV1 Enters Human Keratinocytes by a Nectin-1-Dependent, Rapid Plasma Membrane Fusion Pathway that Functions at Low Temperature [PublishAheadOfPrint]

  • Herpes simplex virus 1 (HSV1) infects humans through stratified epithelia that are composed primarily of keratinocytes. The route of HSV1 entry into keratinocytes has been the subject of limited investigation, but is proposed to involve pH-dependent endocytosis, requiring the gD-binding receptor, nectin-1. Here, we have utilized the nTERT human keratinocyte cell line as a new model for dissecting the mechanism of HSV1 entry in to the host. Although immortalised, these cells nonetheless retain normal growth and differentiation properties of primary cells. Using siRNA depletion studies, we confirm that, despite nTERT cells expressing high levels of the alternative gD receptor HVEM, HSV1 requires nectin-1, not HVEM, to enter these cells. Strikingly, virus entry into nTERT cells occured with unusual rapidity, such that maximum penetration was achieved within 5 minutes. Moreover, HSV1 was able to enter keratinocytes but not other cell types at temperatures as low as 7ddeg;C, conditions where endocytosis was shown to be completely inhibited. Transmission electron microscopy of early entry events at both 37ddeg;C and 7ddeg;C identified numerous examples of naked virus capsids located immediately beneath the plasma membrane, with no evidence of virions in cytoplasmic vesicles. Taken together, these results imply that HSV1 uses the nectin-1 receptor to enter human keratinocyte cells via a previously uncharacterised rapid plasma membrane fusion pathway that functions at low temperature. These studies have important implications for current understanding of the relationship between HSV1 and its relevant in vivo target cell.

    IMPORTANCE The gold standard of antiviral treatment for any human virus infection is the prevention of virus entry into the host cell. In the case of HSV1, primary infection in the human begins in the epidermis of the skin or the oral mucosa, where the virus infects keratinocytes, and it is therefore important to understand the molecular events involved in HSV1 entry into this cell type. Nonetheless, few studies have looked specifically at entry into these relevant human cells. Our results reveal a new route for virus entry that is specific to keratinocytes, involves rapid entry, and functions at low temperatures. This may reflect the environmental conditions encountered by HSV1 when entering its host through the skin and emphasizes the importance of studying virus-host interactions in physiologically relevant cells.

  • Varicella Zoster Virus Downregulates Programmed Death Ligand 1 and Major Histocompatibility Complex 1 in Human Brain Vascular Adventitial Fibroblasts, Perineurial Cells and Lung Fibroblasts [PublishAheadOfPrint]

  • Varicella zoster virus (VZV) vasculopathy produces stroke, giant cell arteritis and granulomatous aortitis and develops after virus reactivates from ganglia and spreads transaxonally to arterial adventitia, resulting in persistent inflammation and pathological vascular remodeling. The mechanism(s) by which inflammatory cells persist in VZV-infected arteries is unknown; however, virus-induced dysregulation of programmed death ligand 1 (PD-L1) may play a role. Specifically, PD-L1 is expressed on virtually all nucleated cells and suppresses the immune system by interacting with the programmed cell death protein receptor 1 found exclusively on immune cells; thus, downregulation of PD-L1 may promote inflammation as seen in some autoimmune diseases. Both flow cytometry and immunofluorescence analyses to test whether VZV infection of adventitial cells downregulates PD-L1 showed decreased PD-L1 expression in VZV-infected compared to mock-infected human brain vascular adventitial fibroblasts (HBVAFs), perineurial cells (HPNCs) and fetal lung fibroblasts (HFLs) at 72 hours post-infection. Quantitative RT-PCR analyses showed no change in PD-L1 transcript levels between mock- and VZV-infected cells, indicating a post-transcriptional mechanism for VZV-mediated downregulation of PD-L1. Flow cytometry analyses showed decreased major histocompatibility complex 1 (MHC-1) expression in VZV-infected cells and adjacent uninfected cells compared to mock-infected cells. These data suggest that reduced PD-L1 expression in VZV-infected adventitial cells may potentially contribute to persistent vascular inflammation observed in virus-infected arteries from patients with VZV vasculopathy, while downregulation of MHC-1 prevents viral clearance.

    IMPORTANCE Here, we provide the first demonstration that VZV downregulates PD-L1 expression in infected HBVAFs, HPNCs and HFLs, which together with the noted VZV-mediated downregulation of MHC-1, might foster persistent inflammation in vessels leading to pathological vascular remodeling during VZV vasculopathy, as well as persistent inflammation in infected lungs to promote subsequent infection of T cells and hematogenous virus spread. Identification of a potential mechanism by which persistent inflammation in the absence of effective viral clearance occurs in VZV vasculopathy and VZV infection of the lung is a step toward targeted therapy of VZV-induced disease.

  • HIV envelope gp120 alters T-cell receptor mobilization in the immunological synapse of uninfected CD4 T cells and augments the T cell activation [PublishAheadOfPrint]

  • HIV is transmitted most efficiently from cell to cell and productive infection occurs mainly in activated CD4 T cells. It is postulated that HIV exploits immunological synapses formed between CD4 T cells and antigen-presenting cells to facilitate the targeting and infection of activated CD4 T cells. This study sought to evaluate how the presence of the HIV envelope (Env) in the CD4 T cell immunological synapse affects synapse formation and intracellular signaling to impact the downstream T cell activation events. CD4 T cells were applied onto supported lipid bilayers (SLBs) that were reconstituted with HIV Env gp120, anti-TCR antibody OKT3 and ICAM-1 to represent the surface of HIV Env-bearing antigen-presenting cells. The results showed that the HIV Env did not disrupt immunological synapse formation. Instead, the HIV Env accumulated with TCR at the center of the synapse, altered the kinetics of TCR recruitment to the synapse, and affected the synapse morphology over time. The HIV Env also prolonged Lck phosphorylation at the synapse and enhanced TCR-induced CD69 upregulation, IL-2 secretion, and proliferation to promote virus infection. These results suggest that HIV uses the immunological synapse as a conduit not only for selective virus transmission to activated CD4 T cells, but also for boosting the T cell activation state, thereby increasing its likelihood for undergoing productive replication in the targeted CD4 T cells.

    IMPORTANCE There are about two million new HIV infections every year. A better understanding on how HIV is transmitted to the susceptible cells is critical to devise effective strategies to prevent HIV infection. Activated CD4 T cells are preferentially infected by HIV, although how this is accomplished is not fully understood. This study examined whether HIV co-opts the normal T-cell activation process through the so-called immunological synapse. We found that the HIV envelope is recruited to the center of the immunological synapse together with the T-cell receptor and enhances the T cell receptor-induced activation of the CD4 T cells. The heightened cellular activation promotes the capacity of the CD4 T cells to support productive HIV replication. This study provides evidence for the exploitation of the normal immunological synapse and T-cell activation process by HIV to boost the activation state of the targeted CD4 T cells and promote infection of these cells.

  • Regulation of HSV glycoprotein induced cascade of events governing cell-cell fusion [PublishAheadOfPrint]

  • Receptor dependent HSV-induced fusion requires glycoproteins gD, gH/gL, and gB. Our current model posits that during fusion receptor-activated conformational changes in gD activate gH/gL, which subsequently triggers transformation of the pre-fusion form of gB into a fusogenic state. To examine the role of each glycoprotein in receptor dependent cell-cell fusion we took advantage of our discovery that fusion by wild type HSV-2 glycoproteins occurs twice as fast as that achieved by HSV-1 glycoproteins. By sequentially swapping each glycoprotein between the two serotypes, we established that fusion speed was governed by gH/gL, with gH being the main contributor. While the mutant forms of gB fuse at distinct rates that are dictated by their molecular structure, these restrictions can be overcome by gH2/gL2, thereby enhancing their activity. We also found that deregulated forms of gD1 and gH2/gL2 can alter the fusogenic potential of gB, promoting cell fusion in the absence of a cellular receptor and that deregulated forms of gB can drive the fusion machinery to even higher levels. Low pH enhanced fusion by affecting the structure of both gB and gH/gL mutants. Together, our data highlight the complexity of the fusion machinery, the impact of the activation state of each glycoprotein on the fusion process and the critical role of gH/gL in regulating HSV induced fusion.

    IMPORTANCE Cell-cell fusion mediated by HSV glycoproteins requires gD, gH/gL, gB and a gD receptor. Here, we show that fusion by wild type HSV-2 glycoproteins occurs twice as fast as that achieved by HSV-1 glycoproteins. By sequentially swapping each glycoprotein between the two serotypes, we found that the fusion process was controlled by gH/gL. Restrictions imposed on the gB structure by mutations could be overcome by gH2/gL2, enhancing their activity. Using low pH or deregulated forms of gD1 and gH2/gL2 the fusogenic potential of gB could only be increased in the absence of receptor, underlining the exquisite regulation that occurs in the presence of receptor. Our data highlight the complexity of the fusion machinery, the impact of the activation state of each glycoprotein on the fusion process and the critical role of gH/gL in regulating HSV induced fusion.

  • Glycan specificity of P[19] rotavirus and comparison with those of other related P genotypes [PublishAheadOfPrint]

  • The P[19] genotype belongs to the P[II] genogroup of group A rotaviruses (RVs). However, unlike the other P[II] RVs that mainly infects humans, P[19] RVs commonly infect animals (porcine), making P[19] unique to study RV diversity and host ranges. Through in vitro binding assays and saturation transfer difference (STD) NMR, we found that P[19] could bind mucin cores 2, 4, and 6, as well as type 1 histo-blood group antigens (HBGAs). The common sequences of these glycans serve as minimal binding units, while additional residues, such as the A, B, H, and Lewis epitopes of the type 1 HBGAs, can further define the binding outcomes and therefore, likely the host ranges for P[19] RVs. This complex binding property of P[19] is shared with those of the other three P[II] RVs (P[4], P[6] and P[8]) in that all of them recognized the type 1 HBGA precursor, although P[4] and P[8], but not P[6], also bind to mucin cores. Moreover, while essential for P[4] and P[8] binding, the addition of the Lewis epitope blocked P[6] and P[19] binding to type 1 HBGAs. Chemical shift NMR of P[19] VP8* identified a ligand binding interface that has shifted away from the known RV P-genotype binding sites but is conserved among all P[II] RVs and two P[I] RVs (P[10] and P[12]), suggesting an evolutionary connection among these human and animal RVs. Taken together, these data are important for hypotheses on potential mechanisms for RV diversity, host ranges, and cross-species transmission.

    Importance In this study, we found that this P[19] strain and other P[II] RVs recognize mucin cores and the type 1 HBGA precursors as the minimal functional units and that additional saccharides adjacent to these units can alter binding outcomes and thereby possibly host ranges. These data may help to explain why some P[II] RVs, such as P[6] and P[19], commonly infect animals but rarely humans, while others, such as the P[4] and P[8] RVs, mainly infect humans and are predominant over other P genotypes. Elucidation of the molecular bases for strain-specific host ranges and cross-species transmission of these human and animal RVs is important to understand RV epidemiology and disease burden, which may impact development of control and prevention strategies against RV gastroenteritis.

  • Dynamics and Correlates of CD8 T-cell Counts in Africans with Primary Human Immunodeficient Virus Type I (HIV-1) Infection [PublishAheadOfPrint]

  • In individuals with HIV-1 infection, depletion of CD4+ T-cells is often accompanied by a malfunction of CD8+ T-cells that are persistently activated and/or exhausted. While the dynamics and correlates of CD4 counts have been well-documented, the same does not apply to CD8 counts. Here, we examined CD8 counts in a cohort of 497 Africans with primary HIV-1 infection and monthly to quarterly follow-up visits for up to three years in the absence of antiretroviral therapy. Statistical models revealed that i) CD8 counts were relatively steady in the 3-36 months period of infection and similar between men and women; ii) neither geography nor heterogeneity in HIV-1 set-point viral load could account for the roughly 10-fold range of CD8 counts in the cohort (P ggt;0.25 in all tests); and iii) factors independently associated with relatively high CD8 counts included demographics (age lle;40 years, adjusted P = 0.010) and several human leukocyte antigen class I (HLA-I) alleles, including HLA-A*03:01 (P = 0.013), B*15:10 (P = 0.007), and B*58:02 (P llt;0.001). Several sensitivity analyses provided supporting evidence for these novel relationships. Overall, these findings suggest that factors associated with CD8 count have little overlap with those previously reported for other HIV-1-related outcome measures, including viral load, CD4 count, and CD4:CD8 ratio.

    Importance Longitudinal data from 497 HIV-1 seroconverters have allowed us to systematically evaluate the dynamics and correlates of CD8+ T-cell counts during untreated primary HIV-1 infection in eastern and southern Africans. Our findings suggest that individuals with certain HLA-I alleles, including A*03 (exclusively A*03:01), persistently maintain a relatively high CD8 counts following HIV-1 infection, which may offer an intriguing explanation for the recently reported, negative association of A*03 with HIV-1-specific, broadly neutralizing antibody responses. In future studies, attention to HLA-I genotyping data may benefit in-depth understanding of both cellular and humoral immunity, as well as their intrinsic balances, especially in settings where there is emerging evidence of antagonism between the two arms of adaptive immunity.

  • Altered glycosylation patterns increase immunogenicity of a subunit HCV vaccine inducing neutralizing antibodies which confer protection in mice [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) infection is a global health problem for which no vaccine is available. HCV has a highly heterogeneous RNA genome and can be classified into seven genotypes. Due to the high genetic and resultant antigenic variation among genotypes, inducing antibodies capable of neutralizing most of the HCV genotypes by experimental vaccination has been challenging. Previous efforts focused on priming humoral immune responses with recombinant HCV envelope E2 protein produced in mammalian cells. Here, we report that a soluble form of HCV E2 (sE2) produced in insect cells possess different glycosylation patterns and is more immunogenic as evidenced by the induction of higher titers of broadly neutralizing antibodies (bNAbs) against cell culture-derived HCV (HCVcc) harboring structural proteins from a diverse array of HCV genotypes. We affirm that continuous and discontinuous epitopes of well-characterized bNAbs are conserved, thus suggesting that sE2 produced in insect cells is properly folded. In a genetically humanized mouse model, active immunization with sE2 efficiently protected against challenge with a heterologous HCV genotype. These data not only demonstrate that sE2 is a promising HCV vaccine candidate but also highlight the importance of glycosylation patterns in developing subunit viral vaccines.

    Importance: A prophylactic vaccine with high efficacy and low cost is greatly needed for global control of HCV infection. Induction of broadly neutralizing antibodies against most HCV genotypes has been challenging due to the antigenic diversity of the HCV genome. Herein, we refined a high-yield subunit HCV vaccine that elicited broadly neutralizing antibody responses in preclinical trials. We found that soluble HCV E2 protein (sE2) produced in insect cells is distinctly glycosylated and is more immunogenic than sE2 produced in mammalian cells, suggesting that glycosylation patterns should be taken into consideration in efforts to generate antibody-based, recombinant vaccines against HCV. We further showed that sE2 vaccination confers protection against HCV infection in a genetically humanized mouse model. Thus our work identified a promising broadly protective HCV vaccine candidate, which should be considered of further pre-clinical and clinical development.

  • Fc receptor mediated activities by Env-specific human mAbs generated from volunteers receiving the DNA prime-protein boost HIV vaccine DP6-001 [PublishAheadOfPrint]

  • HIV-1 is able to elicit broadly potent neutralizing antibodies in a very small subset of individuals only after several years' infection and, therefore, vaccines that elicit these types of antibodies have been difficult to design. The RV144 trial showed that a moderate protection is possible, which may correlate with ADCC activity. Our previous studies demonstrated that in an HIV vaccine phase I trial, DP6-001, a polyvalent Env DNA prime-protein boost formulation, could elicit potent and broadly reactive, gp120-specific antibodies with positive neutralization activities. Here we report the production and analysis of HIV-1 Env-specific human monoclonal antibodies (mAbs) isolated from DP6-001 vaccinees. For this initial report, 13 mAbs from four DP6-001 vaccinees showed broad binding activities to gp120 proteins of diverse subtypes, both autologous and heterologous to vaccine immunogens. Equally cross-reactive Fc-mediated functional activities, including ADCC and ADCP, were present with both immune sera and isolated mAbs, confirming the induction of non-neutralizing functional mAbs by the DNA prime-protein boost vaccination. Elicitation of broadly reactive mAbs by vaccination in healthy human volunteers confirms the value of the polyvalent formulation in this HIV vaccine design.

    Importance The roles of FcR mediated protective antibody responses are gaining more attention due their potential contribution to the low level protection against HIV-1 infection in RV144 trail. At the same time, the information from other HIV vaccine studies from humans is very limited. In the current report, both immune sera and monoclonal antibodies from vaccinated humans showed not only high level but also cross-subtype ADCC and ADCP activities using a polyvalent DNA prime-protein boost vaccine formulation.

  • RATIONALLY-DESIGNED VACCINES TARGETING THE V2 REGION OF HIV-1 gp120 INDUCE A FOCUSED, CROSS CLADE-REACTIVE, BIOLOGICALLY FUNCTIONAL ANTIBODY RESPONSE [PublishAheadOfPrint]

  • Strong antibody (Ab) responses against V1V2 epitopes of the HIV-1 gp120 envelope (Env) correlated with reduced infection rates in studies of HIV, SHIV, and SIV. In order to focus the Ab response on V1V2, we used six V1V2 sequences and nine scaffold proteins to construct immunogens which were tested using various immunization regimens for their ability to induce cross-reactive and biologically active V2 Abs in rabbits. A prime/boost immunization strategy was employed using gp120 DNA and various V1V2-scaffold proteins. The rabbit polyclonal Ab responses (a) were successfully focused on the V1V2 region, with weak or only transient responses to other Env epitopes, (b) displayed broad cross-reactive binding activity with gp120s and the V1V2 regions of diverse strains from clades B, C, and E, (c) included V2 Abs with specificities similar to those found in HIV-infected individuals, and (d) were detectable gge;1 year after the last boosting dose. Importantly, sera from rabbits receiving V1V2-scaffold immunogens displayed Ab-dependent cellular phagocytosis whereas sera from rabbits receiving only gp120 did not. The results represent the first fully successful example of reverse vaccinology in the HIV vaccine field with rationally-designed epitope-scaffold immunogens inducing Abs that recapitulate the epitope specificity and biologic activity of the human monoclonal Abs from which the immunogens were designed. Moreover, this is the first immunogenicity study using epitope-targeting rationally designed vaccine constructs that induced an Fc-mediated activity associated with protection from infection with HIV, SIV and SHIV.

    IMPORTANCE Novel immunogens were designed to focus the antibody response of rabbits on the V1V2 epitopes of HIV-1 gp120 since such antibodies were associated with reduced infection rates of HIV, SIV, and SHIV. The vaccine-induced antibodies were broadly cross-reactive with the V1V2 regions of HIV subtypes B, C and E, and, importantly, facilitated Fc-mediated phagocytosis, an activity not induced upon immunization of rabbits with gp120. This is the first immunogenicity study of vaccine constructs that focuses the antibody response on V1V2 and induces V2-specific antibodies with the ability to mediate phagocytosis, an activity that has been associated with protection from infection with HIV, SIV and SHIV.

  • Herpes Simplex Virus 1 Induces Phosphorylation and Reorganization of lamin A/C Through the {gamma}134.5 Protein That Facilitates Nuclear Egress [PublishAheadOfPrint]

  • Herpes simplex virus 1 (HSV-1) remodels nuclear membranes during virus egress. Although the UL31 and UL34 proteins control nucleocapsid transit in infected cells, the molecular interactions required for their function are unclear. Here we report that the 134.5 gene product of HSV-1 facilitates nucleocapsid release to the cytoplasm through bridging the UL31/UL34 complex, cellular p32 and protein kinase C. Unlike wild type virus, an HSV mutant devoid of 134.5 or its amino-terminus is crippled for viral growth and release. This is attributable to a defect in virus nuclear egress. In infected cells, wild type virus recruits protein kinase C to the nuclear membrane and triggers its activation whereas the 134.5 mutants fail to exert such effect. Accordingly, the 134.5 mutants are unable to induce phosphorylation and reorganization of lamin A/C. When expressed in host cells 134.5 targets p32 and protein kinase C. Meanwhile, it communicates with the UL31/UL34 complex through UL31. Deletion of the amino-terminus from 134.5 disrupts its activity. These results suggest that disintegration of the nuclear lamina mediated by 134.5 promotes HSV replication.

    IMPORTANCE HSV nuclear egress is a key step that determines the outcome of viral infection. While the nuclear egress complex mediates capsid transit across the nuclear membrane, the regulatory components are not clearly defined in virus infected cells. We report that the 134.5 gene product, a virulence factor of HSV-1, facilitates nuclear egress cooperatively with cellular p32, protein kinase C and the nuclear egress complex. This work highlights a viral mechanism that may contribute to the pathogenesis of HSV infection.

  • The presumed polyomavirus viroporin VP4 of SV40 or BKPyV is not required for viral progeny release [PublishAheadOfPrint]

  • The minor capsid protein of human BK polyomavirus (BKPyV), VP2, and its N-terminally truncated form VP3, are both important for viral entry. The closely related Simian Virus 40 (SV40) reportedly produces an additional truncated form of VP2/3, denoted VP4, apparently functioning as a viroporin promoting progeny release. The VP4 open reading frame is conserved in some polyomaviruses including BKPyV. In this study, we investigated the role of VP4 in BKPyV replication. By transfecting viral genomes into primary human renal proximal tubule epithelial cells we demonstrated that unaltered BKPyV and mutants with start codon substitutions in VP4 (VP2M229I and VP2M229A) abolishing putative VP4 production, were released at the same level to supernatants. However, during infection studies, VP2M229I and VP2M229A exhibited 90% and 65% reduced infectivity, respectively, indicating that isoleucine substitution inadvertently disrupted VP2/3 function to the detriment of viral entry, while inhibition of VP4 production during late infection was well tolerated. Unexpectedly, and similarly to BKPyV, SV40 WT and the corresponding VP4 start codon mutants (VP2M228I and VP2M228A) transfected into monkey kidney cell lines, were also released at equal levels. Upon infection, only the VP2M228I mutant exhibited reduced infectivity, a 43% reduction, which also subsequently lead to delayed host cell lysis. Mass spectrometry analysis of nuclear extracts from SV40 infected cells failed to identify VP4. Our results suggest that neither BKPyV nor SV40 require VP4 for progeny release. Moreover, our results reveal an important role in viral entry for the amino acid in VP2/VP3 unavoidably changed by VP4 start codon mutagenesis.

    IMPORTANCE Almost a decade ago, SV40 was reported to produce a late non-structural protein, VP4, which forms pores in the nuclear membrane, facilitating progeny release. By performing transfection studies with unaltered BKPyV and SV40 and their respective VP4-deficient mutants, we found that VP4 is dispensable for progeny release contrary to the original findings. However, infection studies demonstrated a counterintuitive reduction of infectivity of certain VP4-deficient mutants. In addition to the isoleucine-substituted SV40 mutant of the original study, we included alanine-substituted VP4-deficient mutants of BKPyV (VP2M229A) and SV40 (VP2M228A). These revealed that the reduction in infectivity was not caused by a lack of VP4 but rather depended on the identity of the single amino acid substituted within VP2/3 for VP4 start codon mutagenesis. Hopefully, our results will correct the longstanding misconception of VP4's role during infection, and stimulate continued work on unravelling the mechanism for release of polyomavirus progeny.

  • Human monoclonal antibody 81.39a effectively neutralizes emerging influenza A viruses of group 1 and 2 hemagglutinins [PublishAheadOfPrint]

  • The pandemic threat posed by emerging zoonotic influenza A viruses necessitate development of antiviral agents effective against various antigenic subtypes. Human monoclonal antibody (hmAb) targeting the hemagglutinin (HA) stalk offers a promising approach to control influenza virus infections. Here we investigated the ability of hmAb 81.39a to inhibit in vitro replication of human and zoonotic viruses, representing 16 HA subtypes. The majority of viruses were effectively neutralized by 81.39a, EC50 llt;0.01nndash;4.9mmu;g/ml. Among group 2 HA viruses tested, a single A(H7N9) virus was not neutralized at 50mmu;g/ml; it contained HA2-Asp19Gly, an amino acid position previously associated with resistance to neutralization by the group 2 HA-neutralizing mAb CR8020. Notably, among group 1 HA viruses, H11nndash;H13, and H16 subtypes were not neutralized at 50mmu;g/ml; they shared a substitution HA2-Asp19Asn/Ala. Conversely, H9 viruses harboring HA2-Asp19Ala were fully susceptible to neutralization. Therefore, amino acid variance at HA2-Asp19 has subtype-specific adverse effects on in vitro neutralization. Mice given a single injection (15 or 45 mg/kg) at 24 or 48 hours after infection with recently emerged A(H5N2), A(H5N8), A(H6N1) or A(H7N9) viruses were protected from mortality and showed drastically reduced lung viral titers. Furthermore, 81.39a protected mice infected with A(H7N9) harboring HA2-Asp19Gly, although the antiviral effect was lessened. A(H1N1)pdm09-infected ferrets receiving a single dose (25 mg/kg) had reduced viral titers and showed less lung tissue injury, despite 24nndash;72 hours delayed treatment. Taken together, this study provides experimental evidence for the therapeutic potential of 81.39a against diverse influenza A viruses.

    Importance Zoonotic influenza viruses, such as A(H5N1) and A(H7N9) subtypes, have caused severe disease and deaths in humans raising public health concerns. Development of novel anti-influenza therapeutics with a broad spectrum of activity against various subtypes is necessary to mitigate disease severity. Here we demonstrate that the hemagglutinin (HA) stalk-targeting human monoclonal antibody 81.39a effectively neutralized the majority of influenza A viruses tested, representing 16 HA subtypes. Furthermore, 81.39a delayed treatment significantly suppressed virus replication in the lungs, prevented dramatic body weight loss and increased survival rates of mice infected with A(H5Nx), A(H6N1) or A(H7N9) viruses. When tested in ferrets, 81.39a delayed treatment reduced viral titers, particularly in the lower respiratory tract, and substantially alleviated disease symptoms associated with severe A(H1N1)pdm09 influenza. Collectively, our data demonstrated the effectiveness of 81.39a against both seasonal and emerging influenza A viruses.

  • Herpes Simplex Virus Type 1 UL37 Protein Tyrosine Residues Conserved among all Alphaherpesviruses are Required for Interactions with Glycoprotein K (gK), Cytoplasmic Virion Envelopment, and Infectious Virus Production [PublishAheadOfPrint]

  • The HSV-1 UL37 protein functions in virion envelopment at trans-Golgi membranes, as well as in retrograde and anterograde transport of virion capsids. Recently, we reported that UL37 interacts with glycoprotein K(gK) and its interacting partner protein UL20 (Jambunathan, et. al, J. Virol. 88:5927-5935) facilitating cytoplasmic virion envelopment. Alignment of UL37 homologues encoded by alphaherpesviruses revealed the presence of highly conserved residues in the central portion of the UL37 protein. A cadre of nine UL37 site-specific mutations were produced and tested for their ability to inhibit virion envelopment and infectious virus production. Complementation analysis revealed that replacement of tyrosines 474 and 480 with alanine failed to complement the UL37-null virus, while all other mutated UL37 genes complemented efficiently. The recombinant virus DC474-480 constructed with tyrosines 474, 476, 477 and 480 mutated to alanine residues produced a gK-null-like phenotype characterized by the production of very small plaques and accumulation of capsids in the cytoplasm of infected cells. Recombinant viruses having either tyrosine 476 or 477 replaced with alanine residues produced a wild-type phenotype. Immunoprecipitation assays revealed that replacement of all four tyrosines to alanines substantially reduced the ability of gK to interact with UL37. Alignment of herpes simplex virus (HSV) UL37 to the human cytomegalovirus and Epstein Barr Virus UL37 homologues revealed that Y480 was conserved only for alphaherpesviruses. Collectively, these results suggest that UL37 conserved tyrosine residue Y480 plays crucial role in interactions with gK to facilitate cytoplasmic virion envelopment and infectious virus production.

    Importance: The HSV-1 UL37 protein is conserved among all herpesviruses, functions in both retrograde and anterograde transport of virion capsids, and plays critical roles in cytoplasmic virion envelopment by interacting with glycoprotein K (gK). We show here that UL37 tyrosine residues conserved among all alphaherpesviruses serve critical roles in cytoplasmic virion envelopment and interactions with gK.

  • Packaging and prefusion stabilization separately and additively increase the quantity and quality of RSV-neutralizing antibodies induced by respiratory syncytial virus (RSV) fusion protein expressed by a parainfluenza vector. [PublishAheadOfPrint]

  • Human respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are major pediatric respiratory pathogens that lack vaccines. A chimeric bovine/human PIV3 (rB/HPIV3) virus expressing unmodified, wild-type (wt) RSV fusion (F) protein from an added gene was previously evaluated in seronegative children as a bivalent intranasal RSV/HPIV3 vaccine, and was well tolerated but insufficiently immunogenic for RSV F. We recently showed that rB/HPIV3 expressing a partially stabilized prefusion form (pre-F) of RSV F efficiently induced "high quality" RSV neutralizing antibodies, defined as neutralizing RSV in vitro without added complement (Liang et al., J. Virol. 89: 89:9499 nndash;9510, 2015). In the present study, we modified RSV F by replacing its cytoplasmic tail (CT) domain, or its CT plus transmembrane (TM) domains, with counterparts from BPIV3 F, with or without pre-F stabilization. This resulted in RSV F being packaged in the B/HPIV3 particle with an efficiency similar to that of RSV particles. Enhanced packaging was substantially attenuating in hamsters (10- to 100-fold) and rhesus monkeys (100- to 1000-fold). Nonetheless, TMCT-directed packaging substantially increased the titers of "high quality" RSV serum neutralizing antibodies in hamsters. In rhesus monkeys, a strongly additive immunogenic effect of packaging and pre-F stabilization was observed, demonstrated by 8- and 30-fold increases of RSV serum neutralizing titers in the presence and absence of added complement, respectively, compared to pre-F stabilization alone. Analysis of vaccine-induced F-specific antibodies by binding assays indicated that packaging conferred substantial stabilization of RSV F in the pre-F conformation. This provides an improved version of this well-tolerated RSV/HPIV3 vaccine candidate with potently improved immunogenicity, which can be returned to clinical trials.

    Importance Human respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are major viral agents of acute pediatric bronchiolitis and pneumonia wordwide that lack vaccines. A bivalent intranasal RSV/HPIV3 vaccine candidate consisting of a chimeric bovine/human PIV3 (rB/HPIV3) expressing RSV fusion (F) protein previously was shown to be well tolerated by seronegative children, but was insufficiently immunogenic for RSV F. Here, RSV F protein was engineered to be efficiently packaged into the vaccine virus particles. This resulted in significantly enhanced quantity and quality of RSV-neutralizing antibodies in hamsters and non-human primates. In non-human primates, this effect was strongly additive to the previously described stabilization of the prefusion conformation of the F protein. The improved immunogenicity of RSV F by packaging appeared to involve prefusion stabilization. These findings provide a potently more immunogenic version of this well-tolerated vaccine candidate, and should be applicable to other vectored vaccines.

  • {gamma}134.5 neurovirulence gene of herpes simplex virus type 1 modifies the exosome secretion profile in epithelial cells [PublishAheadOfPrint]

  • Recently, it has been demonstrated that herpes simplex virus type 1 (HSV-1) -infected cells secrete exosomes that deliver to uninfected cells the innate immune sensor, STING, and viral RNAs (1, 2)....

  • Characterization of the glycoprotein stable signal peptide in mediating Pichinde viral replication and virulence [PublishAheadOfPrint]

  • Arenaviruses can cause lethal hemorrhagic fevers in humans with limited preventative and therapeutic measures. The arenaviral glycoprotein stable signal peptide (SSP) is unique among signal peptides in that it is an integral component of the mature glycoprotein complex (GPC) and plays important roles not only in the GPC expression and processing but also in the membrane fusion process during viral entry. Using the Pichinde virus (PICV) reverse genetics system, we analyzed the effects of alanine substitutions at many conserved residues within SSP on viral replication in cell culture and in a guinea-pig infection model. Our data showed that the K33A, F49A, and C57A mutations abolished GPC-mediated cell entry and, therefore, could not allow for the generation of viable recombinant viruses, demonstrating that these residues are essential for the PICV life cycle. The G2A mutation caused a marked reduction of cell entry at the membrane fusion step, and while this mutant virus was viable, it was significantly attenuated in vitro and in vivo. The N20A mutation also reduced membrane fusion activity and viral virulence in guinea pigs, but did not significantly affect cell entry or viral growth in cell culture. Two other mutations (N37A and R55A) did not affect membrane fusion or viral growth in vitro but significantly reduced viral virulence in vivo. Taken together, our data suggest that GPC SSP plays an essential role in mediating viral entry and also contributes to viral virulence in vivo.

    Authors' summary Several arenaviruses, such as Lassa fever virus, can cause severe and lethal hemorrhagic fever diseases with high mortality and morbidity, for which no FDA-approved vaccines or therapies are currently available. Viral entry into cells is mediated by arenavirus glycoprotein complex (GPC) that consists of a stable signal peptide (SSP), the receptor-binding GP1, and transmembrane GP2 protein subunits. Using a reverse genetics system of a prototypic arenavirus, Pichinde virus (PICV), we have shown for the first time in the context of infectious virus infections of cell culture and of guinea pigs that the SSP plays an essential role in mediating the membrane fusion step as well as at other yet-to-be-determined processes during viral infection. Our study provides important insights into the biological roles of GPC SSP and implicates it as a potentially good target for the development of antivirals against deadly human arenavirus pathogens.

  • Magnitude and quality of cytokine and chemokine storm during acute infection distinguish non-progressive and progressive simian immunodeficiency virus infections of nonhuman primates [PublishAheadOfPrint]

  • Acute HIV infection represents a period of intense immune perturbation and activation of the host immune system. Study of the eclipse and viral expansion phases of infection is difficult in humans, but studies in non-progressive and progressive nonhuman primate infection models can provide significant insight into critical events occurring during this time. Cytokines, chemokines and other soluble immune factors were measured in longitudinal samples from rhesus macaques infected with either SIVmac251 (progressive infection) or SIVmac239nef (attenuated/non-progressive infection), and from African green monkeys infected with SIVsab9315BR (non-pathogenic infection). Levels of acute-phase peak viral replication were highest in SIVmac251 infection, but correlated positively with viremia at three months post-infection in all three infection models. SIVmac251 infection was associated with stronger corresponding acute-phase cytokine/chemokine responses than the non-progressive infections. Production of IL-15, IL-18, IFN-, G-CSF, MCP-1, MIP-1bbeta; and SAA during acute SIVmac251 infection, but not SIVmac239nef or SIVsab9315BR infection, correlated positively with chronic viremia at three months post infection. Acute-phase production of MCP-1 correlated with viremia at three months post infection in both non-progressive infections. Finally, a positive correlation between the acute-phase area under the curve (AUC) IL-6 and sCD40L and chronic viremia was only observed in the non-progressive infections models. While we observed dynamic acute inflammatory immune responses in both progressive and non-progressive SIV infections, the responses in the non-progressive infections were not only lower in magnitude but also qualitatively different biomarkers of disease progression.

    IMPORTANCE NHP models of HIV infection constitute a powerful tool to study viral pathogenesis to gain critical information for a better understanding of HIV infection in humans. Here, we studied progressive and non-progressive SIV infection models in both natural and non-natural host NHP species. Regardless of the pathogenicity of the virus infection or NHP species studied, the magnitude of viremia, as measured by area under the curve during the first 4 weeks of infection, positively correlated with viremia in chronic infection. The magnitude of cytokine and chemokine responses during primary infection also correlated positively with both acute-phase and chronic viremia. However, the pattern and levels of specific cytokines and chemokines produced differed between non-progressive and progressive SIV infection models. The qualitative differences in the early immune response in progressive and non-progressive infections identified here correlate with and may provide insights into the basis of differences in the subsequent course of disease.

  • An E2 gene infectious HCV library reveals growth adaptive mutations that modulate interactions with the scavenger receptor class B type I [PublishAheadOfPrint]

  • While natural hepatitis C virus (HCV) infection results in highly diverse quasispecies of related viruses over time, mutations accumulate more slowly in tissue culture, in part because of the inefficiency of replication in cells. To create a highly diverse population of HCV particles in cell culture and identify novel growth-enhancing mutations, we engineered a library of infectious HCV with all codons represented at most positions in the ectodomain of the E2 gene. We identified many putative growth adaptive mutations and selected nine highly represented E2 mutants for further study: Q412R, T416R, S449P, T563V, A579R, L619T, V626S, K632T, and L644I. We evaluated these mutants for changes in particle to infectious unit ratio, sensitivity to neutralizing antibody or CD81 large extracellular loop (CD81-LEL) inhibition, entry factor usage, and buoyant density profiles. Q412R, T416R, S449P, T563V and L619T were neutralized more efficiently by anti-E2 antibodies and T416R, T563V and L619T by CD81-LEL. Remarkably, all nine variants showed reduced dependence on scavenger receptor class B type I (SR-BI) for infection. This shift from SR-BI usage did not correlate with a change in the buoyant density profiles of the variants, suggesting an altered E2-SR-BI interaction rather than changes in the virus-associated lipoprotein-E2 interaction. Our results demonstrate that residues influencing SR-BI usage are distributed across E2 and support the development of large scale mutagenesis studies to identify viral variants with unique functional properties.

    IMPORTANCE Characterizing variant viruses can reveal new information about the lifecycle of Hepatitis C virus (HCV) and the roles played by different viral genes. However, it is difficult to recapitulate high levels of diversity in the laboratory because of limitations in the HCV culture system. To overcome this limitation, we engineered a library of mutations into the E2 gene in the context of an infectious clone of the virus. We used this library of viruses to identify nine mutations that enhance the growth rate of HCV. These growth enhancing mutations reduced the dependence on a key entry receptor, scavenger receptor class B type I (SR-BI). By generating a highly diverse library of infectious HCV, we mapped regions of the E2 protein that influence a key virus-host interaction and provide proof-of-principle for the generation of large-scale mutant libraries for the study of pathogens with great sequence variability.

  • Monitoring integration over time supports a role for CTL and ongoing replication as determinants of reservoir size [PublishAheadOfPrint]

  • The dynamics of HIV reservoir accumulation off antiretroviral therapy (ART) is underexplored. Levels of integrated HIV DNA in peripheral blood mononuclear cells (PBMCs) were longitudinally monitored before and after antiviral therapy. HIV integration increased over time in both Elite Controllers (ECs, n=8) and Non-Controllers (NCs, n=6) before ART, whereas integration remained stable in patients on ART (n=4). The median annual fold-change was higher in NCs compared to ECs and negatively correlated with CD4/CD8 T-cell ratio. CTL function as assessed by infected CD4 T-cell elimination (ICE) and granzyme B activity did not significantly change over time in ECs, suggesting the gradual increase in integrated HIV DNA observed in ECs was not a result of progressive loss of immune-mediated control. Also, acutely infected (n=7) but not chronically infected (n=6) patients exhibited a significant drop in integrated HIV DNA 12 months after ART initiation. In conclusion, in the absence of ART, integrated HIV accumulates over time both in NCs and in ECs, at variable individual rates. Starting ART early in infection leads to a greater drop in integrated HIV DNA compared to initiating treatment after years of infection. The increase in integrated HIV DNA over time suggests early treatment may be of benefit in limiting HIV reservoirs.

    Importance The establishment of a latent reservoir represents a barrier to cure among HIV-infected individuals. The dynamics of HIV reservoir accumulation over time in patients before antiviral therapy is underexplored, in large part because it is difficult to accurately and reproducibly measure the size of HIV reservoir in this setting. In our study, we compared the dynamics of integrated HIV DNA over time in ECs and NCs before and after ART is initiated. We found that integrated HIV DNA levels progressively increase over time in the absence of ART, but with a faster, albeit variable, rate in NCs compared to ECs. In addition, integrated HIV DNA declines more dramatically when ART is initiated in acute rather than chronic HIV infection, suggesting important differences between acute and chronic infection. Our study highlights the role of HIV replication and CTL control in reservoir accumulation in sanctuary sites and why ART appears to be more effective in acute infection.

  • Characterization of VSV pseudotypes bearing essential entry glycoproteins gB, gD, gH, and gL of Herpes Simplex virus Type 1 [PublishAheadOfPrint]

  • Herpes Simplex viruses (HSV) are unusual in that unlike most enveloped viruses, they require at least four entry glycoproteins, gB, gD, gH, and gL, for entry into target cells in addition to a cellular receptor for gD. The dissection of the HSV-1 entry mechanism is complicated by the presence of over a dozen proteins on the viral envelope. To investigate HSV-1 entry requirements in a simplified system, we generated VSV virions pseudotyped with HSV-1 essential entry glycoproteins gB, gD, gH, and gL but lacking the native VSV fusogen G. These virions, referred to here as VSVG-BHLD virions, infected a cell line expressing a gD receptor, demonstrating for the first time that the four essential entry glycoproteins of HSV-1 are not only required but also sufficient for cell entry. To our knowledge, this is the first time the VSV pseudotyping system has been successfully extended beyond two proteins. Entry of pseudotyped virions required a gD receptor and was inhibited by HSV-1 specific anti-gB or anti-gH/gL neutralizing antibodies, which suggests that membrane fusion during the entry of the pseudotyped virions shares common requirements with the membrane fusion involved in HSV-1 entry and HSV-1-mediated syncytia formation. The HSV pseudotyping system established here presents a novel tool for systematic exploration of the HSV entry and membrane fusion mechanisms.

    IMPORTANCE Herpes simplex viruses (HSVs) are human pathogens that can cause cold sores, genital herpes, and blindness. No vaccines or preventatives are available. HSV entry into cells nndash; a prerequisite for a successful infection nndash; is a complex process that involves multiple viral and host proteins and occurs by different routes. The detailed mechanistic knowledge of the HSV entry is important for understanding its pathogenesis and would benefit antiviral and vaccine development; yet, the presence of over a dozen proteins on the viral envelope complicates the dissection of the HSV entry mechanisms. Here, we generated heterologous virions displaying the four essential entry proteins of HSV-1 and showed that they are capable of cell entry and, like HSV-1, require all four entry glycoproteins along with a gD receptor. This HSV pseudotyping system pioneered here opens doors for future systematic exploration of the herpesvirus entry mechanisms.

  • Cell-cell sensing of viral infection by pDCs [PublishAheadOfPrint]

  • All cells possess signaling pathways designed to trigger antiviral responses, notably characterized by type I interferon (IFN) production, upon recognition of invading viruses. Especially, host sensors recognize viral nucleic acids. Nonetheless, virtually all viruses have evolved potent strategies that preclude host responses within the infected cells. The plasmacytoid dendritic cell (pDC) is an immune cell type known as a robust type I IFN producer in response to viral infection. Evidence suggests that such functionality of the pDCs participates to viral clearance. Nonetheless, their contribution, which is likely complex and varies depending on the pathogen, is still enigmatic for many viruses. pDCs are not permissive to most viral infections, and consistently recent examples suggest that pDCs respond to immuno-stimulatory viral RNA transferred via non-infectious and/or non-canonical viral/cellular carriers. Therefore, the pDC response likely bypasses innate signaling blockage induced by virus within infected cells. Importantly, the requirement for cell-cell contact is increasingly recognized as a hallmark of the pDC-mediated antiviral state, triggered by evolutionarily divergent RNA viruses.

  • The role of LAMP1 binding and pH sensing by the spike complex of Lassa virus [PublishAheadOfPrint]

  • To effectively infect cells, Lassa virus needs to switch in endosomal compartment from its primary receptor aalpha;-dystroglycan to a protein termed LAMP1. A unique histidine triad on the surface of the receptor-binding domain from the glycoprotein spike complex of Lassa virus is important for LAMP1 binding. Here we investigate mutated spikes that have impaired ability to interact with LAMP1 and show that although LAMP1 is important for efficient infectivity it is not required for spike-mediated membrane fusion per se. Our studies reveal an important regulatory role for histidines from the triad in sensing acidic pH and preventing premature spike triggering. We further show that LAMP1 requires a positively charged His230 to engage with the spike complex and that LAMP1 binding promotes membrane fusion. These results elucidate the molecular role for LAMP1 binding during Lassa virus cell-entry and provide new insights for how pH is sensed by the spike.

    IMPORTANCE Lassa virus is a devastating disease-causing agent in West Africa with a significant yearly death toll and severe long-term complications associated with its infection in survivors. In recent years we learned that Lassa virus needs to switch receptors in a pH dependent manner to efficiently infect cells, but neither the molecular mechanisms that allow switching nor the actual effect of switching were known. Here we investigate the activity of the viral spike complex after abrogating its ability to switch receptors. These studies informed us about the role of switching receptors and provided new insights for how the spike senses acidic pH.

  • Alteration of a second putative fusion peptide of structural glycoprotein E2 of classical swine fever virus alters virus replication and virulence in swine [PublishAheadOfPrint]

  • E2, the major envelope glycoprotein of classical swine fever virus (CSFV), is involved in several critical virus functions including cell attachment, host range susceptibility, and virulence in natural hosts. Functional structural analysis of E2 based on Wimley-White interfacial hydrophobicity distribution predicted the involvement of a loop (residues 864-881) stabilized by a disulfide bond (869CKWGGNWTCV878, named FPII) in establishing interactions with the host cell membrane. This loop further contains an 872GG873 dipeptide, and two aromatic residues (871W and 875W) accessible to solvent. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how amino acid substitutions within FPII may affect replication of BICv in vitro and virus virulence in swine. Recombinant CSFVs containing mutations in different residues of FPII were constructed. A particular construct, harboring amino acid residue substitutions W871T, W875D, and V878T (FPII.2), demonstrated a significantly decreased ability to replicate in a swine cell line (SK6) and swine macrophage primary cell cultures. Interestingly, mutated virus FPII.2 was completely attenuated in pigs. Also, animals infected with FPII.2 virus were protected against virulent challenge with Brescia virus at 21 days post-vaccination. Supporting a role for the E2 864-881 loop in membrane fusion, only synthetic peptides that were based on the native E2 functional sequence were competent for inserting into model membranes and perturbing their integrity and this functionality is lost in synthetic peptides harboring substitutions at FPII.2 amino acid residues W871T, W875D, and V878T.

    Importance This report constitutes the identification and characterization of a putative fusion peptide (FP) in the major structural protein E2 of classical swine fever virus (CSFV). The FP identified was performed by functional structural analysis of E2. We characterized the functional significance of this FP by using artificial membranes. Substitution of critical amino acid residues within the FP radically alters how it interacts with the artificial membranes. When we introduced these same mutations into the viral sequence there was a reduction in replication in cell cultures, and when we infected domestic swine, the natural host of CSFV host, we observed that the virus was now completely attenuated in swine. In addition, the virus mutant that was attenuated in vivo efficiently protects pigs against wild-type virus. These results provide the proof-of-principle to support as a strategy for vaccine development the discovery and manipulation of FPs.

  • Identification of essential genes in the Salmonella phage SPN3US reveals novel insights into giant phage head structure and assembly. [PublishAheadOfPrint]

  • Giant tailed bacterial viruses or phages, such as Pseudomonas aeruginosa phage KZ, have long genomes packaged into large, atypical virions. Many aspects of KZ and related phage biology are poorly understood mostly due to the fact that the functions of the majority of their proteins are unknown. We hypothesized that the Salmonella enterica phage SPN3US could be a useful model phage to address this gap in knowledge. The 240 kb SPN3US genome shares a core set of 91 genes with KZ and related phages, of which ~61 are virion genes, consistent with the expectation that virion complexity is an ancient, conserved feature. Nucleotide sequencing of eighteen mutants enabled assignment of thirteen genes as essential, information which could not have been determined by sequence-based searches for eleven genes. Proteome analyses of two SPN3US virion protein mutants with knockouts in 64 and 241 revealed new insight into the composition and assembly of giant phage heads. The 64 mutant analyses revealed all the genetic determinants required for assembly of the SPN3US head and a likely head-tail joining role for gp64, and its homologs in related phages, due to the tailless-particle phenotype produced. Analyses of the mutation in 241, which encodes a RNA polymerase bbeta; subunit, revealed that without this subunit, no other subunits are assembled into the head, and enabled identification of a "missing" bbeta;' subunit domain. These findings support SPN3US as an excellent model for giant phage research, laying the groundwork for future analyses of their highly unusual virions, host-interactions and evolution.

    Importance In recent years there has been a paradigm shift in virology with the realization that extremely large viruses infecting prokaryotes (giant phages) can be found in many environments. A group of phages related to the prototype giant phage KZ are of great interest due to their virions being amongst the most complex of prokaryotic viruses and their potential for biocontrol and phage therapy applications. Our understanding of the biology of these phages is limited as a large proportion of their proteins have either not been characterized and/or deemed putative without any experimental verification. In this study, we analyzed Salmonella phage SPN3US using a combination of genomics, genetics and proteomics and in doing so revealed new information regarding giant phage head structure and assembly, and virion RNA polymerase composition. Our findings demonstrate the suitability of SPN3US as model phage for the growing group of phages related to KZ.

  • Mitogen-activated Protein Kinase Kinase 2 (MEK2), a Novel E2-interacting Protein, Promotes the Growth of Classical Swine Fever Virus via Attenuation of the JAK-STAT Signaling Pathway [PublishAheadOfPrint]

  • Mitogen-activated protein kinase kinase/extracellular regulated kinase (MEK1/2/ERK1/2) cascade is involved in the replication of several members of the Flaviviridae family including hepatitis C virus and dengue virus. The effects of the cascade on the replication of classical swine fever virus (CSFV), a fatal pestivirus of pigs, remain unknown. In this study, MEK2 was identified as a novel binding partner of the E2 protein of CSFV using yeast two-hybrid screening. The E2-MEK2 interaction was confirmed by glutathione S-transferase pulldown, coimmunoprecipitation, and laser confocal microscopy assays. The C-termini of E2 [amino acids (aa) 890nndash;1053] and MEK2 (aa 266nndash;400) were mapped to be crucial for the interaction. Overexpression of MEK2 significantly promoted the replication of CSFV, whereas knockdown of MEK2 by lentivirus-mediated small hairpin RNAs dramatically inhibited CSFV replication. In addition, CSFV infection induced a biphasic activation of ERK1/2, the downstream signaling molecules of MEK2. Furthermore, the replication of CSFV was markedly inhibited in PK-15 cells treated with U0126, a specific inhibitor for MEK1/2/ERK1/2, whereas MEK2 did not affect CSFV replication after blocking the interferon-induced Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway by ruxolitinib, a JAK-STAT-specific inhibitor. Taken together, our results indicate that MEK2 positively regulates the replication of CSFV through inhibiting the JAK-STAT signaling pathway.

    IMPORTANCE Mitogen-activated protein kinase kinase 2 (MEK2) is a kinase that operates immediately upstream of extracellular regulated kinase 1/2 (ERK1/2) and links to Raf and ERK via phosphorylation. Currently, little is known about the role of MEK2 in the replication of classical swine fever virus (CSFV), a devastating porcine pestivirus. Here, we investigate the roles of MEK2 and the MEK2/ERK1/2 cascade in the growth of CSFV for the first time. We show that MEK2 positively regulates CSFV replication. Notably, we demonstrate that MEK2 promotes CSFV replication through inhibiting the interferon-induced JAK-STAT signaling pathway, a key antiviral pathway involved in the innate immunity. Our work reveals a novel role of MEK2 in CSFV infection and sheds light on the molecular basis by which pestiviruses interplay with the host cell.

  • Activation of the RLR/MAVS signaling pathway by the L protein of Mopeia virus [PublishAheadOfPrint]

  • The Arenaviridae family includes several important human pathogens that can cause severe hemorrhagic fever and greatly threaten public health. As a major component of the innate immune system, the RLR/MAVS signaling pathway is involved in recognizing viral components and initiating antiviral activity. It has been reported that arenavirus infection can suppress the innate immune response, and arenavirus NP and Z proteins of pathogenic arenaviruses can disrupt RLR/MAVS signaling, thus inhibiting production of IFN-I. However, recent studies have shown elevated IFN-I levels in certain arenavirus-infected cells. The mechanism by which arenavirus infection induces IFN-I responses remains unclear. In this study, we determined that the Lp of Mopeia virus (MOPV), an Old World (OW) arenavirus, can activate the RLR/MAVS pathway and thus induce the production of IFN-I. This activation is associated with the RNA-dependent RNA polymerase activity of Lp. This study provides a foundation for further studies of interactions between arenaviruses and the innate immune system and the elucidation of arenavirus pathogenesis.

    Importance Distinct innate immune responses are observed when hosts are infected with different arenaviruses. It has been widely accepted that NP and certain Z proteins of arenaviruses inhibit the RLR/MAVS signaling pathway. The viral components responsible for the activation of RLR/MAVS signaling pathway remain to be determined. In the current study, we demonstrate for the first time that the Lp of MOPV, an OW arenavirus, can activate the RLR/MAVS signaling pathway and thus induce the production of IFN-I. Based on our results, we proposed that dynamic interactions exist among Lp-produced RNA, NP and the RLR/MAVS signaling pathway, and the outcome of these interactions may determine the final IFN-I response pattern: elevated or declined. Our study here provided a possible explanation for how IFN-I can become activated during arenavirus infection and may help gain insights into the interactions that form between different arenavirus components and the innate immune system.

  • Canonical and variant forms of histone H3 are deposited onto the human cytomegalovirus genome during lytic and latent infections [PublishAheadOfPrint]

  • Chromatin is the nucleoprotein complex that protects and compacts eukaryotic genomes. It is responsible for a large part of the epigenetic control of transcription. The genomes of DNA viruses such as human cytomegalovirus (HCMV) are devoid of histones within virions but are chromatinized and epigenetically regulated following delivery to the host cell nucleus. How chromatin is initially assembled on viral genomes, and which variant forms of the core histone proteins are deposited is incompletely understood. We monitored the deposition of both ectopically expressed and endogenous histone H3.1/2 and H3.3 during lytic and latent HCMV infections. Here we show both are deposited on HCMV genomes during lytic and latent infections suggesting similar mechanisms of viral chromatin assembly during the different infection types and indicating both canonical and variant core histones may be important modulators of infecting viral genomes. We further show that association of both H3.1/2 and H3.3 occurs independent of viral DNA synthesis or de novo viral gene expression, implicating cellular factors and/or virion components in the formation of chromatin on virion-delivered genomes during both lytic and latent infections.

    Importance It is well established that infecting herpesvirus genomes are chromatinized upon entry into the host cell nucleus. Why or how this occurs is a mystery. It is important to know why they are chromatinized in order to better understand cellular pathogen recognition (DNA sensing) pathways, viral fate determinations (lytic or latent), and to anticipate how artificially modulating chromatinization may impact viral infections. It is important to know how they are chromatinized in order to potentially modulate the process for therapeutic effect. Our work showing HCMV genomes are loaded with canonical and variant H3 histones during both lytic and latent infections strengthens the hypothesis that chromatinization pathways are similar between the two infection types, implicates virion or cellular factors in this process, and exposes the possibility that histone variants, in addition to post-translational modification, may impact viral gene expression. These revelations are important to understanding and intelligently intervening in herpesvirus infections.

  • Increased Protein Degradation Improves Influenza Virus Nucleoprotein-specific CD8+ T cell Activation in vitro but not in C57BL/6 mice [PublishAheadOfPrint]

  • Due to antigenic drift of influenza viruses, seasonal influenza vaccines need to be updated annually. These vaccines are based on predictions of strains likely to circulate in the next season. However, vaccine efficacy is greatly reduced in case of a mismatch between circulating and vaccine strains. Furthermore, novel antigenically distinct influenza viruses are introduced into the human population from animal reservoirs occasionally and may cause pandemic outbreaks. To dampen the impact of seasonal and pandemic influenza, vaccines that induce broadly protective and long lasting immunity are preferred. Because influenza virus-specific CD8+ T cells are mainly directed against relatively conserved internal proteins, like nucleoprotein (NP), they are highly cross-reactive and afford protection against infection with antigenically distinct influenza virus strains, so-called heterosubtypic immunity. Here, we used modified vaccinia virus Ankara (MVA) as a vaccine vector for the induction of influenza virus NP-specific CD8+ T cells. To optimize induction of CD8+ T cell responses, we made several modifications to NP, which aimed at retaining the protein in the cytosol or targeting it to the proteasome. We hypothesized that these strategies would increase antigen processing and presentation and thus improve induction of CD8+ T cell responses. We showed that NP proteins with increased degradation rates improved CD8+ T cell activation in vitro, if the amount of antigen was limited or if CD8+ T cells were of low functional avidity. However, after immunization of C57BL/6 mice, no differences were detected between modified NP and NPwt, since NPwt already induced optimal CD8+ T cell responses.

    IMPORTANCE: Due to the continuous antigenic drift of seasonal influenza viruses and the threat of a novel pandemic, there is a great need for the development of novel influenza vaccines that offer broadly protective immunity against multiple subtypes. CD8+ T cells can provide immunity against multiple subtypes of influenza viruses by the recognition of relatively conserved internal antigens. In this study, we aimed at optimizing the CD8+ T cell response to influenza A virus by making modifications to influenza A virus nucleoprotein (NP) expressed from the vaccine vector modified vaccinia virus Ankara (MVA). These modifications resulted in increased antigen degradation, thereby producing elevated levels of peptides that can be presented on MHC class I molecules to CD8+ T cells. Although we were unable to increase the NP-specific immune response in the mouse strain used, this approach may have benefits for vaccine development using less immunogenic proteins.

  • Vif Proteins from Diverse HIV/SIV Lineages have Distinct Binding Sites in A3C [PublishAheadOfPrint]

  • Lentiviruses have evolved the Vif protein to counteract APOBEC3 (A3) restriction factors by targeting them for proteasomal degradation. Previous studies have identified important residues in the interface of HIV-1 Vif and human APOBEC3C (hA3C) or human APOBEC3F (hA3F). However, the interaction between primate A3C proteins with HIV-1 Vif or natural HIV-1 Vif variants is still poorly understood. Here, we report that HIV-1 Vif is inactive against A3Cs of rhesus macaques (rhA3C), sooty mangabey monkeys (smmA3C) and African green monkeys, while HIV-2, SIVagm and SIVmac Vif proteins efficiently mediate the depletion of all tested A3Cs. We identified that residues N/H130 and Q133 in rhA3C and smmA3C are determinants for this HIV-1 Vif triggered counteraction. We also found that the HIV-1 Vif interaction sites in helix 4 of hA3C and hA3F differ. Vif alleles from diverse HIV-1 subtypes were tested for degradation activities to hA3C. The subtype F-1 Vif was identified to be inactive to degrade hA3C and hA3F. The residues that determined F-1 Vif inactivity to A3C/A3F were located at the C-terminal region (K167 and D182). Structural analysis of F-1 Vif revealed that impairing the internal salt bridge of E171-K167 restored its reduction capacities to A3C/A3F. Furthermore, we found that D101 could also form an internal interaction with K167. Replacing D101 by glycine and R167 by lysine in NL4-3 Vif impaired its counteractivity to A3F and A3C. This finding indicates that internal interactions outside the A3 binding region in HIV-1 Vif influence the capacity to induce degradation of A3C/A3F.

    IMPORTANCE The APOBEC3 restriction factors can serve as potential barriers to lentiviral cross-species transmissions. Vif proteins from lentiviruses counteract APOBEC3 by proteasomal degradation. In this study, we found that monkey-derived APOBEC3C (A3C), rhA3C and smmA3C were resistant to HIV-1 Vif. This was determined by A3C residues N/H130 and Q133. However, HIV-2, SIVagm and SIVmac Vif proteins were found to be able to mediate the depletion of all tested primate A3C proteins. In addition, we identified a natural HIV-1 Vif (F-1 Vif) that was inactive to hA3C/hA3F. Here we provide for the first time a model that explains how an internal salt bridge of E171-K167-D101 influences Vif-mediated degradation of hA3C/hA3F. This finding provides a novel way to develop HIV-1 inhibitors by targeting the internal interactions of the Vif protein.

  • Sindbis virus can exploit a host antiviral protein to evade immune surveillance [PublishAheadOfPrint]

  • Viral infection induces production of type I interferons (IFNs), which stimulate the expression of a variety of antiviral factors to inhibit viral replication. To establish effective infection, viruses need to develop strategies to evade the immune responses. A neurovirulent Sindbis virus strain with neuroinvasive properties (SVNI) causes lethal encephalitis in mice, and its replication in cultured cells is inhibited by the zinc-finger antiviral protein (ZAP), a host factor that specifically inhibits the replication of certain viruses by binding to the viral mRNAs and repressing the translation and promoting the degradation of target mRNA. We report here that MEF cells from ZAP knockout mice supported more efficient SVNI replication than wildtype cells. SVNI infection of 10 day-old suckling mice led to reduced survival in the knockout mice. Unexpectedly, however, SVNI infection of 23 day-old weanling mice, whose immune system is more developed than the suckling mice, resulted in significantly improved survival in ZAP knockout mice. Further analyses revealed that in the weanling knockout mice, SVNI replicated more efficiently in lymphoid tissues at early times postinfection, and induced higher levels of IFN production, which restricted viral spread to the central nervous system. Blocking IFN activity through the use of receptor neutralizing antibodies rendered knockout mice more sensitive to SVNI infection than wildtype mice. These results uncover a mechanism by which SVNI exploits a host antiviral factor to evade innate immune surveillance.

    Importance Sindbis virus, a prototypic member of the Alphavirus genus, has been used to study the pathogenesis of acute viral encephalitis in mice for many years. How the virus evades immune surveillance to establish effective infection is largely unknown. ZAP is a host antiviral factor that potently inhibits Sindbis virus replication in cell culture. Here we show that infection of ZAP knockout suckling mice with a neuroinvasive Sindbis strain (SVNI) led to faster disease progression. However, SVNI infection of weanling mice led to slower disease progression in knockout mice. Further analyses revealed that in weanling knockout mice, SVNI replicated more efficiently in lymphoid tissues at early times postinfection, and induced higher levels of interferon production, which restricted viral spread to the central nervous system. These results uncover a mechanism by which SVNI exploits a host antiviral factor to evade innate immune surveillance and allow enhanced neuroinvasion.

  • Vaccinia virus uses retromer-independent cellular retrograde transport pathways to facilitate the wrapping of intracellular mature virions during viral morphogenesis [PublishAheadOfPrint]

  • Poxviruses such as Vaccinia virus (VACV) undertake a complex cytoplasmic replication cycle which involves morphogenesis through four distinct virion forms, and includes a crucial "wrapping" step whereby intracellular mature virions (IMVs) are wrapped in two additional membranes to form intracellular enveloped virions (IEVs). To determine if cellular retrograde transport pathways were required for this wrapping step we examined VACV morphogenesis in cells with reduced expression of the tetrameric tethering factor complex GARP (Golgi-associated retrograde pathway complex), a central component of retrograde transport. VACV multi-step replication was significantly impaired in cells transfected with siRNA targeting the GARP complex or in cells with a mutated GARP complex. Detailed analysis revealed that depletion of the GARP complex resulted in a reduction in the number of IEVs, thereby linking retrograde transport with the wrapping of IMVs. In addition foci of viral wrapping membrane proteins without an associated internal core accumulated in cells with a mutated GARP complex, suggesting that impaired retrograde transport uncouples nascent IMVs from the IEV membranes at the site of wrapping. Finally, small molecule inhibitors of retrograde transport strongly suppressed VACV multi-step growth in vitro and reduced weight loss and clinical signs in an in vivo murine model of systemic poxviral disease. This work links cellular retrograde transport pathways with morphogenesis of poxviruses and identifies a panel of novel inhibitors of poxvirus replication.

    Importance: Cellular retrograde transport pathways traffic cargo from endosomes to the trans-Golgi network and are a key part of the intracellular membrane network. This work reveals the prototypic poxvirus Vaccinia virus (VACV) exploits cellular retrograde transport pathways to facilitate the wrapping of intracellular mature virions and therefore promote the production of extracellular virus. Inhibition of retrograde transport by small molecule inhibitors reduced replication of VACV in cell culture and alleviated disease in mice experimentally infected with VACV. This research provides fundamental new knowledge about the "wrapping" step of poxvirus morphogenesis, furthers our knowledge of the complex cellular retrograde pathways, and identifies a new group of anti-poxvirus drugs.

  • Visualizing herpesvirus procapsids in living cells [PublishAheadOfPrint]

  • A complete understanding of herpesvirus morphogenesis requires studies of capsid assembly dynamics in living cells. Although fluorescent tags fused to the VP26 and pUL25 capsid proteins are available, neither of these components are present on the initial capsid assembly: the procapsid. To make procapsids accessible to live-cell imaging, we made a series of recombinant pseudorabies viruses that encoded GFP fused in-frame to the internal capsid scaffold and maturation protease. One recombinant, a GFP-VP24 fusion, maintained wild-type propagation kinetics in vitro and approximated wild-type virulence in vivo. The fusion also proved well tolerated in herpes simplex virus. Viruses encoding GFP-VP24 along with a traditional capsid reporter fusion (pUL25/mCherry) demonstrated that GFP-VP24 was a reliable capsid marker, and revealed that the protein remained capsid associated following entry into cells and upon nuclear docking. These dual-fluorescent viruses made possible the discrimination of procapsids during infection, and monitoring capsid shell maturation kinetics. The results demonstrate the feasibility of imaging herpesvirus procapsids and their morphogenesis in living cells, and indicate that the encapsidation machinery does not substantially help coordinate capsid shell maturation.

    IMPORTANCE The Herpesviridae family consists of human and veterinary pathogens that cause a wide range of diseases in their respective hosts. These viruses share a structurally-related icosahedral capsid that encases the dsDNA viral genome. The dynamics of capsid assembly and maturation has been inaccessible to examination in living cells. This study has overcome this technical hurdle and provides new insights into this fundamental stage of herpesvirus infection.

  • Releasing the genomic RNA sequestered in the mumps virus nucleocapsid [PublishAheadOfPrint]

  • In a negative strand RNA virus, the genomic RNA is sequestered inside the nucleocapsid when the viral RNA-dependent RNA polymerase uses it as the template for viral RNA synthesis. It must require a conformational change in the nucleocapsid protein (NP) to make the RNA accessible by the viral polymerase during this process. The structure of an empty mumps virus nucleocapsid-like particle is determined to 10.4 AAring; resolution by cryoEM image reconstruction. By modeling the crystal structure of parainfluenza virus 5 into the density, it is shown that the aalpha;-helix close to the RNA became flexible when RNA was removed. Point mutations in this helix resulted in loss of polymerase activities. Since the core of NP is rigid in the nucleocapsid, we suggest that interactions between this region of the mumps virus NP and its polymerase leads to exposure of the sequestered genomic RNA, instead of large NP domain rotations.

    Importance Mumps virus (MuV) infection may cause serious diseases including hearing loss, orchitis, oophoritis, mastitis, and pancreatitis. MuV is a negative strand RNA virus, similar to rabies virus or Ebola virus, that has a unique mechanism of viral RNA synthesis. They all make their own RNA-dependent RNA polymerase (RdRp). The viral RdRp uses the genomic RNA inside the viral nucleocapsid as the template to synthesize viral RNAs. Since the template RNA is always sequestered in the nucleocapsid, the viral RdRp must find a way to open it up in order to gain access to the covered template. Our work reported here shows that a helix structural element in the MuV nucleocapsid protein becomes open when the sequestered RNA is released. The amino acids related to this helix are required for RdRp to synthesize viral RNA. We propose that the viral RdRp pulls this helix open to release the genomic RNA.

  • Multiple Roles of the Cytoplasmic Domain of Herpes Simplex Virus 1 Envelope Glycoprotein D in Infected Cells [PublishAheadOfPrint]

  • Herpes simplex virus 1 (HSV-1) envelope glycoprotein D (gD) plays an essential role in viral entry. The functional regions of gD responsible for viral entry have been mapped to its extracellular domain, whereas the gD cytoplasmic domain plays no obvious role in viral entry. Thus far, the role(s) of the gD cytoplasmic domain in HSV-1 replication remained to be elucidated. In this study, we showed that ectopic expression of gD induced microvilli-like tubular structures at the plasma membrane, which resembled the reported projection structures of the plasma membrane induced in HSV-1-infected cells. Mutations in the arginine cluster (residues 365 to 367) in the gD cytoplasmic domain greatly reduced gD-induced plasma membrane remodeling. In agreement with this, the mutations in the arginine cluster in the gD cytoplasmic domain reduced the number of microvilli-like tubular structures at the plasma membrane in HSV-1-infected cells. In addition, the mutations produced an accumulation of unenveloped nucleocapsids in the cytoplasm, and reduced viral replication and cell-cell spread. These results suggested that the arginine cluster in the gD cytoplasmic domain was required for the efficient induction of plasma membrane projections and viral final envelopment, and these functions of the gD domain may lead to efficient viral replication and cell-cell spread.

    IMPORTANCE The cytoplasmic domain of HSV-1 gD, an essential envelope glycoprotein for viral entry, was reported to promote viral replication and cell-cell spread, but the role(s) of the domain during HSV-1 infection remained unknown. In this study, we clarified two functions of the arginine cluster in the HSV-1 gD cytoplasmic domain, both of which require host cell membrane remodeling; i.e., formation of microvilli-like projections at the plasma membrane and viral final envelopment in HSV-1-infected cells. We also showed that the gD arginine cluster was required for efficient HSV-1 replication and cell-cell spread. This is the first report clarifying not only the functions of the gD cytoplasmic domain but also identifying the gD arginine cluster as the HSV-1 factor responsible for the induction of plasma membrane projections in HSV-1-infected cells. Our results elucidate some of the functions of this multifunctional envelope glycoprotein during HSV-1 infection.

  • Structure of an N276-dependent HIV-1 Neutralizing Antibody Targeting a Rare V5 Glycan Hole adjacent to the CD4 Binding Site [PublishAheadOfPrint]

  • All HIV-1 infected individuals develop strain-specific neutralizing antibodies to their infecting virus, which in some cases mature into broadly neutralizing antibodies. Defining the epitopes of strain-specific antibodies that overlap with conserved sites of vulnerability might provide mechanistic insights into how broadly neutralizing antibodies arise. We have previously described an HIV-1 clade C infected donor, CAP257, who developed broadly neutralizing plasma antibodies targeting an N276 glycan-dependent epitope in the CD4 binding site. The initial CD4 binding site response potently neutralized the heterologous tier-2 clade B viral strain RHPA, which was used to design resurfaced gp120 antigens for single B cell sorting. We report the isolation and structural characterization of CAP257-RH1, an N276 glycan-dependent CD4 binding site antibody, representative of the early CD4 binding site plasma response in CAP257. A cocrystal structure of CAP257-RH1 bound to RHPA gp120 revealed critical interactions with the N276 glycan, loop D, and V5, but not with aspartic acid 368, similar to HJ16 and 179NC75. This antibody was derived from IGHV3-33 and IGLV3-10 genes, and neutralized RHPA but not the CAP257 transmitted/founder virus. Its narrow neutralization breadth was attributed to a binding angle that was incompatible with glycosylated V5 loops, present in almost all HIV-1 strains including the CAP257 transmitted/founder virus. Deep sequencing of autologous CAP257 viruses however, revealed minority variants early in infection that lacked V5 glycans. These glycan-free V5 loops likely resulted in unusual holes in the glycan shield that may have been necessary for initiating this N276 glycan-dependent CD4 binding site B cell lineage.

    Importance The conserved CD4 binding site on gp120 is a major target for HIV-1 vaccine design, but key events in the elicitation and maturation of different antibody lineages to this site remain elusive. Studies have shown that strain-specific antibodies can evolve into broadly neutralizing antibodies, or in some cases act as helper lineages. Therefore characterizing the epitopes of strain-specific antibodies will help to inform the design of HIV-1 immunogens to elicit broadly neutralizing antibodies. In this study we isolate a narrowly neutralizing N276 glycan-dependent antibody, and use x-ray crystallography and viral deep sequencing to describe how gp120 lacking glycans in V5 might have elicited these early glycan-dependent CD4 binding site antibodies. These data highlight the importance of glycan holes in the elicitation of B cell lineages targeting the CD4 binding site.

  • Efficiency in Complexity: Composition and Dynamic Nature of Mimivirus Replication Factories [PublishAheadOfPrint]

  • The recent discovery of multiple giant dsDNA viruses blurred the consensual distinction between viruses and cells due to their size, as well as their structural and genetic complexity. A dramatic feature revealed by these viruses as well as by many positive-strand RNA viruses is their ability to rapidly form elaborate intracellular organelles, coined viral factories where viral progeny are continuously generated. Here we report the first isolation of viral factories at progressive post-infection time-points. The isolated factories were subjected to mass spectrometry-based proteomics, bioinformatics and imaging analyses. These analyses reveal that numerous viral proteins are present in the factories but not in mature virions, thus implying that multiple and diverse proteins are required to promote the efficiency of viral factories as llsquo;production lines' of viral progeny. Moreover, our results highlight the dynamic and highly complex nature of viral factories, provide new and general insights into viral infection, and substantiate the intriguing notion that viral factories may represent the living state of viruses.

    IMPORTANCE Large dsDNA viruses such as Vaccinia and the giant Mimivirus, as well as many positive-strand RNA viruses, generate elaborate cytoplasmic organelles in which the multiple and diverse transactions required for viral replication and assembly occur. These organelles, which were coined llsquo;viral factories', are attracting much interest due to the increasing realization that the rapid and continuous production of viral progeny is a direct outcome of the elaborate structure and composition of the factories, which act as efficient llsquo;production lines'. To get new insights into the nature and function of viral factories, we devised a method that allows, for the first time, the isolation of these organelles. Analyses of the isolated factories generated at different times post-infection by mass spectrometry-based proteomics provide new perceptions on their role and reveal the highly dynamic nature of these organelles.

  • Residues K465 and G467 Within The Cytoplasmic Domain of GP2 Critically Contribute To LCMV Persistence In Mice [PublishAheadOfPrint]

  • Several arenaviruses, chiefly Lassa virus (LASV), cause hemorrhagic fever disease in humans and pose serious public health concerns in their endemic regions. Moreover, mounting evidence indicates that the worldwide-distributed, prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV), is a neglected human pathogen of clinical significance. We have documented that a recombinant LCMV containing the glycoprotein gene (GPC) of LASV within the backbone of the immunosuppressive Clone 13 (Cl-13) variant of Armstrong strain of LCMV (rCl-13/LASV-GPC) exhibited Cl-13 like growth properties in cultured cells but in contrast to Cl-13, rCl-13/LASV-GPC was unable to establish persistence in immune competent adult mice, which prevented its use for some in vivo experiments. Recently, V459K and K461G mutations within the GP2 cytoplasmic domain (CD) of rCl-13/LASV-GPC were shown to increase rCl-13/LASV-GPC infectivity in mice. Here we generated rCl-13(GPC/VGKS) by introducing the corresponding revertant mutations K465V and G467K within GP2 of rCl-13 and show that rCl-13(GPC/VGKS) was unable to persist in mice. K465V and G467K mutations did not affect GPC processing, virus RNA replication or gene expression. In addition, rCl-13(GPC/VGKS) grew to high titer in cultured cell lines and in immunodeficient mice. Further analysis revealed that rCl-13(GPC/VGKS) infected fewer splenic plasmacytoid dendritic cells when compared to rCl-13, yet both viruses there induced a similar type I interferon response in mice. Our findings have identified novel viral determinants of Cl-13 persistence and also revealed that virus GPC-host interactions yet to be elucidated critically contribute to Cl-13 persistence.

    IMPORTANCE The prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV) provides investigators with a superb experimental model system to investigate virus-host interactions. Armstrong strain (ARM) of LCMV causes an acute infection whereas its' derivative, Clone 13 (Cl-13), causes a persistent infection. Mutations F260L and K1079Q within GP1 and L polymerase, respectively, have been shown to play a critical role in Cl-13's ability to persist in mice. However, there is an overall lack of knowledge about other viral determinants required for Cl-13's persistence. Here we report that mutations K465V and G467K within the cytoplasmic domain of Cl-13 GP2 resulted in a virus, rCl-13(GPC/VGKS), that failed to persist in mice despite exhibiting Cl-13 wild type-like fitness in cultured cells and immune compromised mice. This finding has uncovered novel viral determinants of viral persistence, and a detailed characterization of rCl-13(GPC/VGKS) can provide novel insights about the mechanisms underlying virus persistent infection.

  • Effect of host species on the topography of fitness landscape for a plant RNA virus [PublishAheadOfPrint]

  • Adaptive fitness landscapes are a fundamental concept in evolutionary biology that relate the genotype of individuals with their fitness. At the end, the evolutionary fate of evolving populations depends on the topography of the landscape, that is, the number of accessible mutational pathways and of possible fitness peaks (i.e, adaptive solutions). For long time, fitness landscapes were only theoretical constructions due to a lack of precise information on the mapping between genotypes and phenotypes. In recent years, however, efforts have been devoted to characterize the properties of empirical fitness landscapes for individual proteins or for microbes adapting to artificial environments. In a previous study, we had characterized the properties of the empirical fitness landscape defined by the first five mutations fixed during adaptation of tobacco etch potyvirus (TEV) to a new experimental host, Arabidopsis thaliana. Here we evaluate the topography of this landscape in the ancestral host Nicotiana tabacum. Comparing the topographies of the landscape in the two hosts, we found that some features remain similar, such as the existence of fitness holes and the prevalence of epistasis, including cases of sign and of reciprocal sign that create rugged, uncorrelated and highly random topographies. However, we also observed significant differences in the fine-grained details among both landscapes due to changes in the fitness and epistatic interactions of some genotypes. Our results support the idea that not only fitness tradeoffs between hosts but also topographical incongruences among fitness landscapes in alternative hosts may contribute to virus specialization.

    IMPORTANCE Despite its importance for understanding virus' evolutionary dynamics, very little is known about the topography of virus adaptive fitness landscapes and even less is known about the effect that different host species and environmental conditions may have of this topography. To bring this gap, we have evaluated the topography of a small fitness landscape formed by all genotypes that result from every possible combination of the five mutations fixed during adaptation of TEV to its novel host A. thaliana. To assess the effect that host species may have on this topography, we evaluated the fitness of every genotype both in the ancestral and novel hosts. We found both landscapes share some macroscopic properties such as the existence of holes and being highly rugged and uncorrelated, yet they differ in microscopic details due to changes in the magnitude and sign of fitness and epistatic effects.

  • Virologic differences do not fully explain the diversification of swine influenza viruses in the United States [PublishAheadOfPrint]

  • Influenza A(H1N1) viruses entered the US swine population following the 1918 pandemic and remained genetically stable for roughly 80 years. In 1998, there was an outbreak of influenza-like illness among swine caused by A(H3N2) viruses containing the triple reassortant internal gene (TRIG) cassette. Following the TRIG cassette emergence, numerous reassortant viruses were isolated in nature suggesting the TRIG virus had an enhanced ability to reassort compared to the classical swine virus. This study was designed to quantify the relative reassortment capacity of classical and TRIG swine viruses. Reverse genetic viruses were generated from the classical H1N1 virus A/swine/MN/37866/1999 (MN/99), the TRIG virus A/swine/NC/18161/2002 (NC/02), and a seasonal human H3N2 virus A/TX/6/1996 (TX/96) to measure in vitro reassortment and growth potentials. After co-infection with NC/02 or MN/99 and TX/96, H1/H3 double positive cells were identified. Delayed TX/96 infection was fully excluded by both swine viruses equally. We then analyzed reassortant H3 viruses. 77 of 81 (95.1%) TX/96-NC/02 reassortants contained at least one polymerase gene segment from NC/02 whereas only 34 of 61 (55.7%) MN/99-TX/96 reassortants contained at least one polymerase gene segment from MN/99. Additionally, 38 of 81 (46.9%) NC/02-TX/96 reassortants contained all NC/02 polymerase gene segments while none of the MN/99-TX/96 reassortants contained all MN/99 polymerase genes. There were 21 H3 reassortants between MN/99-TX/96 compared to only 17 H3 reassortants between NC/02-TX/96. Overall, the results indicated that there are no distinct differences in the ability of the TRIG to reassort with a human virus compared to the classical swine virus.

    IMPORTANCE There appear to be no differences between the ability of classical swine and TRIG swine viruses to exclude a second virus suggesting that under the right circumstances both viruses have similar opportunities to reassort. The increased percentage of TRIG polymerase gene segments in reassortant H3 viruses indicate that they may be more compatible with gene segments from other viruses, however this needs to be further investigated. Nevertheless, the classical swine virus also showed the ability to reassort suggesting that factors other than reassortment capacity alone is responsible for the different epidemiology's of TRIG and classical swine viruses. The post-TRIG diversity was likely driven by increased intensive farming practices rather than virologic properties. Our results indicate that host ecology can be a significant factor in viral evolution.

  • Differential Control of BST2 restriction and Plasmacytoid Dendritic Cell Antiviral Response by Antagonists encoded by HIV-1 Group M and O Strains [PublishAheadOfPrint]

  • BST2/Tetherin is a type-I interferon (IFN-I) stimulated host factor that restricts the release of HIV-1 by entrapping budding virions at the cell surface. This membrane-associated protein can also engage and activate the plasmacytoid dendritic cell (pDC)-specific ILT7 inhibitory receptor to downregulate the IFN-I response by pDCs. Pandemic HIV-1 group M uses Vpu (M-Vpu) to counteract the two BST2 isoforms (long and short) that are expressed in human cells. M-Vpu efficiently downregulates surface long BST2, while it displaces short BST2 molecules away from viral assembly sites. We recently found that this attribute is used by M-Vpu to activate the BST2/ILT7-dependent negative-feedback pathway and to suppress pDC IFN-I responses during sensing of infected cells. However, whether this property is conserved with the endemic HIV-1 group O, which has evolved Nef (O-Nef) to counteract specifically the long BST2 isoform, remains unknown. In the present study, we validated that O-Nefs have the capacity to downregulate surface BST2 and enhance HIV-1 particle release, although less efficiently than M-Vpu. In contrast to M-Vpu, O-Nef did not enhance viral spread in T cell culture nor displace short BST2 from viral assembly sites to prevent its occlusion by tethered HIV-1 particles. Consequently, O-Nef impairs the ability of BST2 to activate negative ILT7 signaling to suppress the IFN-I response by pDC-containing PBMCs during sensing of infected cells. These distinctive features of BST2 counteraction by O-Nefs may in part explain the limited spread of the HIV-1 group O in the human population.

    Importance: Geographical distribution and prevalence of different HIV-1 groups show large variation. Understanding drivers of distinctive viral spread may aid the development of therapeutic strategies for controlling HIV-1 pandemic strains spread. Differential spread of HIV-1 groups appears linked to their capacities to antagonize the long and short isoforms of the BST2 restriction factor. We found that the endemic HIV-1 group O-encoded BST2 antagonist, Nef, is unable to counteract the restriction mediated by short BST2, a condition that impairs its ability to activate ILT7 and suppress pDC antiviral responses. This is in contrast to the pandemic HIV-1 group M-specified BST2 countermeasure, Vpu, which displays a diverse array of mechanisms to counteract short and long BST2, an attribute that allows an effective control of pDC antiviral responses. These findings may help explain the limited spread of HIV-1 group O as well as the continued predominance of HIV-1 group M throughout the world.

  • Dual-color Herpesvirus Capsids Discriminate Inoculum from Progeny and Reveal Axonal Transport Dynamics [PublishAheadOfPrint]

  • Alpha herpesviruses, such as herpes simplex virus and pseudorabies virus (PRV), are neuroinvasive dsDNA viruses that establish life-long latency in peripheral nervous system (PNS) neurons of their native hosts. Following reactivation, the infection can spread back to the initial mucosal site of infection or, in rare cases, to the central nervous system with usually serious outcomes. During entry and egress, viral capsids depend on microtubule-based molecular motors for efficient and fast transport. In axons of PNS neurons, cytoplasmic dynein provides force for retrograde movements towards the soma, and kinesins move cargo in the opposite, anterograde direction. The dynamic properties of virus particles in cells can be imaged by fluorescent protein fusions to the small capsid protein VP26, which are incorporated into capsids. However, single-color fluorescent protein tags fail to distinguish virus inoculum from progeny. Therefore, we established a dual-color system by growing a recombinant PRV expressing a red fluorescent VP26 fusion (PRV180) on a stable cell line expressing a green VP26 fusion (PK15-mNG-VP26). The resulting dual-color virus preparation (PRV180G) contains capsids tagged with both red and green fluorescent proteins, and 97% of particles contain detectable levels of mNG-VP26. After replication in neuronal cells, all PRV180G progeny exclusively contain mRFP-VP26 tagged capsids. We used PRV180G for an analysis of axonal capsid transport dynamics in PNS neurons. Fast dual-color total internal reflection fluorescence (TIRF) microscopy, single particle tracking and motility analyses reveal robust, bidirectional capsid motility mediated by cytoplasmic dynein and kinesin during entry, whereas egressing progeny particles are exclusively transported by kinesins.

    Importance: Alpha herpesviruses are neuroinvasive viruses that infect the peripheral nervous system (PNS) of infected hosts as an integral part of their life cycle. Establishment of a quiescent or latent infection in PNS neurons is a hallmark of most alpha herpesviruses. Spread of infection to the central nervous system is surprisingly rare in natural hosts, but can be fatal. Pseudorabies virus (PRV) is a broad host range, swine alpha herpesvirus that enters neuronal cells and utilizes intracellular transport processes to establish infection and to spread between cells. By using a virus preparation with fluorescent viral capsids that change color depending on the stage of the infectious cycle, we find that, during entry, axons of PNS neurons support robust, bidirectional capsid motility, similar to cellular cargo, towards the cell body. In contrast, progeny particles appear to be transported unidirectionally by kinesin motors towards distal egress sites.

  • Imperfect symmetry of Sp1 and core promoter elements regulates early and late viral gene expression of the bidirectional BK polyomavirus non-coding control region [PublishAheadOfPrint]

  • Rearrangements or point mutations in the non-coding control region (NCCR) of BK polyomavirus (BKPyV) have been associated with higher viral loads and more pronounced organ pathology in immunocompromised patients. The respective mutations affect a multitude of transcription factor binding sites (TFBS), but consistently cause increased expression of the early viral gene region (EVGR) at the expense of late viral gene region (LVGR) expression. By mutating TFBS, we identified three phenotypic groups leading to strong, intermediate, or impaired EVGR expression and corresponding BKPyV replication. Unexpectedly, Sp1 TFBS mutants either activated or inhibited EVGR expression, when located proximal to the LVGR (SP1-4) or the EVGR (SP1-2), respectively. We now demonstrate that the bidirectional balance of EVGR and LVGR expression is dependent on affinity, strand orientation, and number of Sp1 sites. Swapping the LVGR-proximal high-affinity SP1-4 with the EVGR-proximal low-affinity SP1-2, in-site strand flipping, or inserting an additional SP1-2 site caused a rearranged NCCR phenotype of increased EVGR expression and faster BKPyV replication. 5rrsquo; -RACE revealed an imperfect symmetry between the EVGR- and LVGR-proximal parts of the NCCR, consisting of TATA- and TATA-like elements, initiator elements, and downstream promoter elements. Mutation or deletion of the archetypal LVGR promoter, as found in activated NCCR variants, abrogated LVGR expression, which could be restored by providing large T-antigen in trans. Thus, whereas Sp1 sites control the initial EVGR-LVGR expression balance, LTag expression can override inactivation of the LVGR promoter and acts as a key driver of LVGR expression, independent of the Sp1 sites and core promoter elements.

    IMPORTANCE Polyomaviridae currently comprise more than 70 members including 13 human PyVs, all of which share the bidirectional genome organization mediated by the NCCR that determines species and host cell specificity, persistence, replication, and virulence. Here, we demonstrate that the BKPyV NCCR is fine-tuned by an imperfect symmetry of core promoter elements centering around TATA- and TATA-like sequences close to the EVGR and LVGR, respectively, which are governed by directionality and affinity of two Sp1 sites. The data indicated that the BKPyV NCCR is poised towards EVGR expression that can be readily unlatched by a simple switch affecting Sp1 binding. The resulting LTag as the major EVGR protein drives viral genome replication, renders subsequent LVGR expression independent of archetypal promoter elements, and can overcome enhancer/promoter mutations and deletions. The data are pivotal for understanding how human PyV NCCRs mediate secondary host cell specificity, reactivation and virulence in their natural hosts.

  • Non-coding subgenomic flavivirus RNA is processed by the mosquito RNAi machinery and determines West Nile virus transmission by Culex pipiens mosquitoes [PublishAheadOfPrint]

  • Flaviviruses, such as Zika, Yellow fever, dengue and West Nile (WNV) are a serious concern for human health. Flaviviruses produce an abundant non-coding subgenomic flavivirus RNA (sfRNA) in infected cells. SfRNA results from stalling of the host 5rrsquo; -3rrsquo; exoribonuclease XRN1/Pacman on conserved RNA structures in the 3rrsquo; UTR of the viral genomic RNA. SfRNA production is conserved in insect-specific, mosquito-borne, tick-borne and no-known-vector flaviviruses, suggesting a pivotal role for sfRNA in the flavivirus life cycle. Here we investigated the function of sfRNA during WNV infection of Culex pipiens mosquitoes and evaluated its role in determining vector competence. An sfRNA1-deficient WNV was generated that displayed similar growth kinetics as wildtype WNV in both RNAi-competent and -compromised mosquito cell lines. Small RNA deepsequencing of WNV-infected mosquitoes indicated an active siRNA-based antiviral response for both the wildtype and sfRNA1-deficient virus. Additionally, we provide the first evidence that sfRNA is an RNAi substrate in vivo. Two reproducible small RNA hotspots within the 3rrsquo; UTR/sfRNA of the wildtype virus mapped to RNA stem loops SL-III and 3'SL, which stick out of the 3D sfRNA structure model. Importantly, we demonstrate that sfRNA-deficient WNV displays significantly decreased infection and transmission rates in vivo when administrated via the blood meal. Finally, we show that transmission and infection rates are not affected by sfRNA after intrathoracic injection, thereby identifying sfRNA as a key driver to overcome the mosquito midgut infection barrier. This is the first report to describe a key biological function of sfRNA for flavivirus infection of the arthropod vector, providing an explanation for the strict conservation of sfRNA production.

    IMPORTANCE Understanding the flavivirus transmission cycle is important to identify novel targets to interfere with disease and to aid development of virus control strategies. Flaviviruses produce an abundant, non-coding viral RNA called sfRNA in both arthropod and mammalian cells. To evaluate the role of sfRNA in flavivirus transmission, we infected mosquitoes with the flavivirus West Nile and an sfRNA-deficient mutant West Nile virus. We demonstrate that sfRNA determines the infection and transmission rates of West Nile virus in Culex pipiens mosquitoes. Comparison of infection via the blood meal versus intrathoracic injection, which bypasses the midgut, revealed that sfRNA is important to overcome the mosquito midgut barrier. We also show that sfRNA is processed by the antiviral RNA interference machinery in mosquitoes. This is the first report to describe a pivotal biological function of sfRNA in arthropods. The results explain why sfRNA production is evolutionary conserved.

  • Intracerebral inoculation of mouse-passaged Saffold virus type 3 affects cerebellar development in neonatal mice [PublishAheadOfPrint]

  • Saffold virus (SAFV), a human cardiovirus, is occasionally detected in infants with neurological disorders, including meningitis and cerebellitis. We recently reported that SAFV type 3 isolates infect cerebellar glial cells, but not large neurons, in mice. However, the impact of this infection remained unclear. Here, we determined the neuropathogenesis of SAFV type 3 in the cerebella of neonatal ddY mice using SAFV passaged in the cerebellum of neonatal BALB/c mice. The virus titer in the cerebellum increased following inoculation of each of five passaged strains. The fifth passaged strain harbored amino acid substitutions in the VP2 (H160R and Q239R) and VP3 (K62M) capsid proteins. Molecular modeling of the capsid proteins suggested that the VP2-H160R and VP3-K62M mutations alter the structural dynamics of the receptor binding surface via formation of a novel hydrophobic interaction between the VP2 puff B and VP3 knob regions. When compared with the original strain, the passaged strain showed altered growth characteristics in human-derived astroglial cell lines and higher replication in the brains of neonatal mice. In addition, the passaged strain was more neurovirulent than the original strain, while both strains infected astroglial and neural progenitor cells in the mouse brain. Intracerebral inoculation of either the original or passaged strain affected brain Purkinje cell dendrites, and a high titer of the passaged strain induced cerebellar hypoplasia in neonatal mice. Thus, infection by mouse-passaged SAFV affected cerebellar development in neonatal mice. This animal model contributes to the understanding of the neuropathogenicity of SAFV infections in infants.

    IMPORTANCE Saffold virus (SAFV) is a candidate neuropathogenic agent in infants and children, but the neuropathogenicity of the virus has not been fully elucidated. Recently, we evaluated the pathogenicity of two clinical SAFV isolates in mice. Similar to other neurotropic picornaviruses, these isolates showed mild infectivity of glial and neural progenitor cells, but not of large neurons, in the cerebellum. However, the outcome of this viral infection in the cerebellum has not been clarified. Here, we examined the tropism of SAFV in the cerebellum. We obtained an in vivo-passaged strain from the cerebella of neonatal mice and examined its genome and its neurovirulence in the neonatal mouse brain. The passaged virus showed high infectivity and neurovirulence in the brain, especially the cerebellum, and affected cerebellar development. This unique neonatal mouse model will be helpful for elucidating the neuropathogenesis of SAFV infections occurring early in life.

  • Production of cytomegalovirus dense bodies by scalable bioprocess methods maintains immunogenicity and improves neutralizing antibody titers. [PublishAheadOfPrint]

  • With the goal of developing a virus-like particle-based vaccine based on dense bodies (DB) produced by human cytomegalovirus (HCMV) infections, we evaluated scalable culture, isolation, and inactivation methods, and applied technically advanced assays to determine relative purity, composition, and immunogenicity of DB particles. Our results increase our understanding on the benefits and disadvantages of methods to recover immunogenic DB and inactivate contaminating viral particles. Our results indicate: 1) HCMV strain Towne replicates in MRC-5 fibroblasts grown on microcarriers, 2) DB particles recovered from 2-bromo-5,6-dichloro-1-beta-d-ribofuranosyl benzimidazole riboside (BDCRB)-treated cultures and purified by tangential flow filtration (TFF-DB) or glycerol tartrate gradient sedimentation (GT-DB) constitute 92% or 98%, respectively, of all particles in the final product, 3) epithelial cell trophic DB particles are recovered from a single round of coinfection by AD169 and Towne strain viruses, consistent with complementation between UL130 and UL131A expressed by these strains and restoration of gH/gL/UL128-UL131A (gH-pentamer), 4) equivalent neutralizing antibody titers are induced in mice following immunization with epithelial cell tropic DB or gH-pentamer-deficient DB preparations, 5) UV-inactivation of residual virus in GT-DB or TFF-DB preparations retained immunogenicity and induction of neutralizing antibody preventing epithelial cell entry, 6) GT-DB and TFF-DB induced cellular immune responses to multiple HCMV peptides. Collectively, this work provides a foundation for future development of DB as an HCMV-based particle vaccine.

    Importance Development of a vaccine to prevent congenital HCMV infection remains a high priority. Human cytomegalovirus-derived noninfectious particles, or dense bodies, may constitute a safe vaccine strategy that mimics natural infection. The standard approach for purification of virus particles has been a multiple-step, complex gradient that presents a potential barrier to production scale-up and commercialization. Here we employ an approach that combines treatment with an anti-viral terminase inhibitor and purification by a simplified process to produce a vaccine candidate providing broad antiviral humoral and cellular immunity as a foundation for future development.

  • Heparan Sulfate Binding Promotes Accumulation of Intravitreally-Delivered Adeno-Associated Viral Vectors at the Retina for Enhanced Transduction but Weakly Influences Tropism [PublishAheadOfPrint]

  • Many adeno-associated virus (AAV) serotypes efficiently transduce the retina when delivered to the subretinal space, but show limited success when delivered to the vitreous due to the inner limiting membrane (ILM). Subretinal delivery of AAV2 and its HS-binding-deficient capsid lead to similar expression, indicating transduction of the outer retina occurred by HS-independent mechanisms. However, intravitreal delivery of HS-ablated rAAV2 lead to a 300-fold decrease in transduction ncompared to AAV2. Fluorescence in situ hybridization of AAV transgenes was used to identify differences in retinal trafficking and revealed HS-binding was responsible for AAV2 accumulation at the ILM. This mechanism was tested on human ex vivo retinas and showed similar accumulation with HS-binding AAV2 capsid only. To evaluate if HS binding could be applied to other AAV serotypes to enhanced their transduction, AAV1 and AAV8 were modified to bind HS with a single amino acid mutation and tested in mice. Both HS-binding mutants of AAV1 and AAV8 had higher intravitreal transduction over their non-HS-binding parent capsid due to increased retinal accumulation. To understand the influence that HS binding has on tropism, chimeric AAV2 capsids with dual glycan usage were tested intravitreally in mice. Compared to HS binding alone, these chimeric capsids displayed enhanced transduction that was correlated to a change in tropism. Taken together, this indicates that HS-binding serves to sequester AAV capsids from the vitreous to the ILM, but does not influence retinal tropism. The enhanced retinal transduction of HS-binding capsids provides a rational design strategy for engineering capsids for intravitreal delivery.

    IMPORTANCE Adeno-associated virus (AAV) has become the vector of choice for viral gene transfer and has shown great promise in clinical trials. Development of an easy, less invasive injection route for ocular gene therapy is met with intravitreal delivery, but delivery of AAV by this route results in poor transduction outcomes. The inner limiting membrane (ILM) creates a barrier separating the vitreous and the retina. Binding of AAV to heparan sulfate proteoglycan (HSPG) at the ILM may allow the virus to traverse this barrier for better retinal transduction. We show that HSPG binding is correlated in greater accumulation and penetration of AAV in the retina. We validated that this accumulation is conserved across mouse and human retinas and that the addition of HSPG binding to other AAV capsids can increase the number of vector accumulating at the ILM without dictating tropism.

  • Adoptive Transfer of Engineered Rhesus SIV-Specific CD8+ T Cells Reduces the Number of Transmitted/Founder Viruses Established in Rhesus Macaques [PublishAheadOfPrint]

  • AIDS virus infections are rarely controlled by cell-mediated immunity, in part due to viral immune evasion and immunodeficiency resulting from CD4+ T-cell infection. One likely aspect of this failure is that antiviral cellular immune responses are either absent or present at low levels during the initial establishment of infection. To test whether a large, timely, and effective response could potentially reduce the establishment of infection from a high dose inoculum, we adoptively transferred large numbers of T cells that were molecularly engineered with anti-SIV activity into rhesus macaques three days following an intrarectal SIV inoculation. To measure in vivo antiviral activity, we assessed the number of viruses transmitted using SIVmac239X, a molecularly-tagged viral stock containing 10 genotypic variants, at a dose calculated to transmit 12 founder viruses. Single genome sequencing of plasma virus revealed that the two animals receiving T cells expressing SIV-specific TCRs had significantly fewer viral genotypes than the two control animals receiving non-SIV-specific T cells (mean 4.0 versus 7.5 transmitted viral genotypes, p=0.044). Accounting for the likelihood of transmission of multiple viruses of a particular genotype, the calculated mean of total founder viruses transmitted is 4.5 versus 14.5 in the experimental and control groups respectively, p=0.021. Thus, a large antiviral T-cell response temporally timed with virus exposure can limit viral transmission. The presence of strong, preexisting T-cell responses, including those induced by vaccines, might help prevent the establishment of infection at the lower exposure doses in humans that typically transmit only a single virus.

    IMPORTANCE The establishment of AIDS virus infection in an individual is essentially a race between the spreading virus and host immune defenses. Cell-mediated immune responses to infection or induced by vaccination are important contributors in limiting viral replication. However, in HIV/SIV infection, the virus usually wins the race, irreversibly crippling the immune system before an effective cellular immune response is developed and active. We found that providing an accelerated response by adoptively transferring large numbers of antiviral T cells shortly after a high-dose mucosal inoculation, while not preventing infection altogether, limited the number of individual viruses transmitted. Thus the presence of strong, preexisting T-cell responses, including those induced by vaccines, might prevent infection in humans where the virus exposure is considerably lower.

  • Enterovirus 71 2B induces cell apoptosis by directly inducing the conformational activation of the pro-apoptotic protein Bax [PublishAheadOfPrint]

  • To survive and replicate within a host, many viruses have evolved strategies that target crucial components within the apoptotic cascade, leading to either an inhibition or induction of cell apoptosis. Enterovirus 71 (EV71) infections have been demonstrated to impact the mitochondrial apoptotic pathway and induce apoptosis in many cell lines. However, the detailed mechanism of EV71-induced apoptosis remains to be elucidated. In this study, we report that EV71 2B protein (2B) localized to the mitochondria and induced cell apoptosis by interacting directly with and activating the pro-apoptotic protein Bax. 2B recruited Bax to the mitochondria and induced Bax conformational activation. In addition, mitochondria isolated from 2B expressing cells that were treated with a recombinant Bax showed increased Bax interaction and Cyt c release. Importantly, apoptosis in cells with either EV71 infection or 2B expression was dramatically reduced in Bax but not in Bak knockdown cells, suggesting that Bax played a pivotal role in EV71 or 2B-induced apoptosis. Further studies indicate that a hydrophobic region of 18 amino acids (aa) in the C-terminal (63-80) region of 2B was responsible for the location of 2B in the mitochondria. A hydrophilic region of 14 aa in the N-terminal region of 2B was functional in Bax interaction and its subsequent activation. Moreover, over-expression of the anti-apoptotic protein Bcl-XL abrogates 2B-induced release of Cyt c and caspase activation. Therefore, this study provides direct evidence that EV71 2B induces cell apoptosis and impacts the mitochondrial apoptotic pathway by directly modulating the redistribution and activation of pro-apoptotic protein Bax.

    Importance EV71 infections are usually accompanied by severe neurological complications. It has also been postulated that the induction of cell apoptosis resulting from tissue damage, are possible processes of EV71-related pathogenesis. In this study, we report that EV71 2B protein (2B) localized to the mitochondria and induced cell apoptosis by interacting directly with and activating the pro-apoptotic protein Bax. This study provides the evidence that EV71 induces cell apoptosis by modulating Bax activation and reveals important clues regarding the mechanism of Cyt c release and mitochondria permeabilization during EV71 infection.

  • HIV-1-neutralizing IgA is detected in genital secretions of highly HIV-1 exposed seronegative women on oral pre-exposure prophylaxis [PublishAheadOfPrint]

  • Although non-human primate studies have shown that SIV/SHIV exposure during pre-exposure prophylaxis (PrEP) with oral tenofovir can induce SIV-immunity without productive infection, this has not been documented in humans. We evaluated cervicovaginal IgA in Partners PrEP Study participants using a subtype C primary isolate, and found that women on PrEP had IgA with higher average HIV-1-neutralizing magnitude than women on placebo (33% versus 7%, p=0.008). Using a cut-off of gge;90% HIV-1 neutralization, 19% of women on-PrEP had HIV-1 neutralizing IgA compared to 0% of women on placebo, p=0.09. We also estimated HIV-1 exposure and found that the proportion of women with HIV-1-neutralizing IgA was associated with the level of HIV-1 exposure (p=0.04). Taken together, our data suggest that PrEP and high levels of exposure to HIV may each enhance mucosal HIV-1-specific humoral immune responses in sexually exposed but HIV-1 uninfected individuals.

    Importance Although there is not yet an effective HIV-1 vaccine, PrEP for at-risk HIV-1-uninfected individuals is a highly efficacious intervention to prevent HIV-1 acquisition, and is currently being recommended by the CDC and WHO for all individuals at high risk of HIV-1 acquisition. We previously demonstrated that PrEP use does not enhance peripheral blood HIV-1-specific T-cell responses in HIV-exposed individuals. Here, we evaluate for cervicovaginal HIV-neutralizing IgA responses in genital mucosal secretions of HIV-exposed women, which is likely a more relevant site than peripheral blood for observation of potentially protective immune events occurring in response to time-varying sexual HIV-1 exposure. Furthermore, we assess for host response in the context of longitudinal quantification of HIV-1 exposure. We report that HIV-neutralizing IgA is significantly correlated with higher HIV-1 exposure, and further, that there are more women with HIV-1-neutralizing IgA in the on-PrEP group as compared to placebo.

  • Antagonism of RNase L is required for murine coronavirus replication in Kupffer cells and liver sinusoidal endothelial cells but not in hepatocytes [PublishAheadOfPrint]

  • Mouse hepatitis virus strain A59 infection of mice is a useful tool for studying virus-host interaction during hepatitis development. The NS2H126R mutant is attenuated in liver replication due to loss of phosphodiesterase activity, which the wild-type virus uses to block the 2rrsquo;,5rrsquo;-oligoadenylate synthetase (OAS)-ribonuclease L (RNase L) antiviral pathway. The activation of RNase L by NS2H126R is cell-type dependent and correlates with high basal expression levels of OAS, as found in myeloid cells. We tested the hypothesis that resident liver macrophages, Kupffer cells (KC), are the most likely cell type to restrict NS2H126R and prevent hepatitis. As found previously, A59 and NS2H126R replicate similarly in hepatocytes and neither activates RNase L, as assessed by an rRNA degradation assay. In contrast, in KCs, A59 exhibited a 100-fold higher titer than NS2H126R and NS2H126R induced rRNA degradation. Interestingly, in liver sinusoidal endothelial cells (LSEC), the cells that form a barrier between blood and liver parenchymal cells, NS2H126R activates RNase L, which limits viral replication. Similar growth kinetics were observed for both viruses in KC and LSEC from RNase L-/- mice, demonstrating that both use RNase L to limit NS2H126R replication. Depletion of KC by gadolinium(III) chloride or LSEC by cyclophosphamide partially restores liver replication of NS2H126R, leading to hepatitis. Thus, during MHV infection, hepatitis, which damages the parenchyma, is prevented by RNase L activity in both KC and LSEC but not in hepatocytes. This may be explained by the undetectable levels of RNase L as well as OASs expressed in hepatocytes.

    IMPORTANCE Mouse hepatitis virus infection of mice provides a useful tool for studying virus-host interactions during hepatitis development. The NS2H126R mutant is attenuated in liver replication due to loss of phosphodiesterase activity, with which the wild-type virus blocks the potent OAS-RNase L antiviral pathway. RNase L activation by NS2H126R is cell-type dependent and correlates with high basal expression levels of OAS, as found in myeloid cells. We showed that hepatocytes that comprise the liver parenchyma do not activate RNase L when infected with NS2H126R nor do they restrict replication. However, both Kupffer cells (KC), liver resident macrophages and liver sinusoidal endothelial cells (LSEC) which line the sinusoids activate RNase L in response to NS2H126R. These data suggest that KC and LSEC prevent viral spread into the parenchyma, preventing hepatitis. Furthermore, hepatocytes express undetectable levels of OASs and RNase L, which likely explains the lack of RNase L activation during NS2H126R infection.

  • Reduction of neuraminidase activity exacerbates disease in 2009 pandemic influenza virus-infected mice [PublishAheadOfPrint]

  • During the first wave of the 2009 pandemic, caused by a H1N1 influenza virus (pH1N1) of swine origin, antivirals were the only form of therapeutic available to control proliferation of disease until the conventional strain-matched vaccine was produced. Oseltamivir is an antiviral that inhibits the sialidase activity of the viral neuraminidase (NA) protein and was shown to be effective against pH1N1 viruses in ferrets. Furthermore, it was used in humans to treat infections during the pandemic and is still used for current infections without reported complication or exacerbation of illness. However, in an evaluation of the effectiveness of oseltamivir against pH1N1 infection, we unexpectedly observed exacerbation of disease in virus-infected mice treated with oseltamivir, transforming an otherwise mild illness into one with high morbidity and mortality. By contrast, an identical treatment regime alleviated all signs of illness in mice infected with the pathogenic mouse-adapted virus A/WSN/33 (H1N1). The worsened clinical outcome with pH1N1 viruses occurred over a range of oseltamivir doses and treatment schedules, and was directly linked to a reduction in NA-enzymatic activity. Our results suggest that the suppression of NA activity with antivirals may exacerbate disease in a host dependent manner by increasing replicative fitness in viruses that are not optimally adapted for replication in that host.

    Importance Here we report that treatment of pH1N1-infected mice with oseltamivir enhanced disease progression, transforming a mild illness into a lethal infection. This raises a potential pitfall of using the mouse model for evaluation of the therapeutic efficacy of neuraminidase inhibitors. We show that antiviral efficacy determined in a single animal species may not represent treatment in humans and that caution should be used when interpreting the outcome. Furthermore, increased virulence due to oseltamivir treatment was the effect of a shift in the hemagglutinin (HA) and neuramindase (NA) activity balance. This is the first study that has demonstrated that altering the HA/NA activity balance by reduction in NA activity can result in an increase in virulence in any animal model from non-pathogenic to lethal and the first to demonstrate a situation in which treatment with a NA activity inhibitor has opposite to the intended therapeutic effect of ameliorating the infection.

  • Pathogenicity and transmission of H5 and H7 highly pathogenic avian influenza viruses in mallards [PublishAheadOfPrint]

  • Wild aquatic birds have been associated with the intercontinental spread of H5 subtype highly pathogenic avian influenza (HPAI) viruses of the A/goose/Guangdong/1/96 (Gs/GD) lineage during 2005, 2008 and 2014, but dispersion by wild waterfowl has not been implicated with spread of other HPAI viruses. To better understand why Gs/GD H5 HPAI viruses infect and transmit more efficiently in waterfowl than other HPAI viruses, groups of mallard ducks were challenged with one of fourteen different H5 and H7 HPAI viruses, including a Gs/GD lineage H5N1 (clade 2.2) virus from Mongolia, part of the 2005 dispersion, and the H5N8 and H5N2 index HPAI viruses (clade 2.3.4.4) from the United States, part of the 2014 dispersion. All virus-inoculated ducks and contact exposed ducks became infected and shed moderate to high titers of the viruses, with the exception that mallards were resistant to Ck/Pennsylvania/83 and Ck/Queretaro/95 H5N2 HPAI virus infection. Clinical signs were only observed in ducks challenged with the H5N1 2005 virus, which all died, and the H5N8 and H5N2 2014 viruses, which had decreased weight gain and fever. These three viruses were also shed in higher titers by the ducks, which could facilitate virus transmission and spread. This study highlights the possible role of wild waterfowl in the spread of HPAI viruses.

    Importance The spread of H5 subtype highly pathogenic avian influenza (HPAI) viruses of the Gs/GD lineage by migratory waterfowl is a serious concern for animal and public health. H5 and H7 HPAI viruses are considered to be adapted to gallinaceous species (chickens, turkeys, quail, etc.) and less likely to infect and transmit in wild ducks. In order to understand why this is different with certain Gs/GD lineage H5 HPAI viruses, we compared the pathogenicity and transmission of several H5 and H7 HPAI viruses from previous poultry outbreaks to Gs/GD lineage H5 viruses, including H5N1 (clade 2.2), H5N8 and H5N2 (clade 2.3.4.4) viruses, in mallards as a representative wild duck species. Surprisingly, most HPAI viruses used in this study replicated well and transmitted among mallards, however the three Gs/GD lineage H5 HPAI viruses replicated to higher titers which could explain the transmission of these viruses in susceptible wild duck populations.

  • LFA-1 engagement triggers T cell polarization at the HIV-1 virological synapse [PublishAheadOfPrint]

  • HIV-1 efficiently disseminates by cell-cell spread at intercellular contacts called virological synapses (VS) where the virus preferentially assembles and buds. Cell-cell contact triggers active polarization of organelles and viral proteins within infected cells to the contact site to support efficient VS formation and HIV-1 spread; critically however, which cell surface protein triggers contact-induced polarization at the VS remains unclear. Additionally, the mechanism by which the HIV-1 envelope glycoprotein (Env) is recruited to the VS remains ill-defined. Here we have used a reductionist bead-coupled antibody assay as a model of the VS and show that cross-linking the integrin LFA-1 is alone sufficient to induce active T cell polarization and recruitment of the MTOC in HIV-1 infected cells. Mutant cell lines coupled with inhibitors demonstrated that LFA-1 induced polarization was dependent on the T cell kinase ZAP70. Notably, immunofluorescence staining of viral proteins revealed an accumulation of surface Env at sites of LFA-1 engagement with intracellular Env localized to a Golgi-compartment proximal to the polarized MTOC. Furthermore, blocking LFA-1 induced MTOC polarization through ZAP70 inhibition prevented intracellular Env polarization. Taken together these data reveal that LFA-1 is a key determinant in inducing dynamic T cell remodeling to the VS and suggest a model in which LFA-1 engagement triggers active polarization of the MTOC and the associated Env-containing secretory apparatus to sites of cell-cell contact to support polarized viral assembly and egress for efficient cell-cell spread.

    IMPORTANCE HIV-1 causes AIDS by spread within immune cells and depletion of CD4 T lymphocytes. Rapid spread between these cells occurs by highly efficient cell-cell transmission that takes place at the Virological Synapse (VS). VS are characterized by striking T cell remodeling that is spatially associated with polarized virus assembly and budding at sites of cell contact. Here we show that the integrin LFA-1 triggers organelle polarization and viral protein recruitment, facilitating formation of the VS and that this requires the T cell kinase ZAP70. Taken together, these data suggest a mechanism for how HIV-1 infected T cells sense and respond to cell contact to polarize viral egress and promote cell-cell spread. Understanding how cell-cell spread is regulated may help reveal therapeutic targets to specifically block this mode of HIV-1 dissemination.

  • A Single Mutation at Position 190 in Hemagglutinin Enhances Binding Affinity for Human Type Sialic Acid Receptor and Replication of H9N2 Avian Influenza Virus in Mice [PublishAheadOfPrint]

  • H9N2 avian influenza virus (AIV) has an extended host range but the molecular basis underlying H9N2 AIV transmission to mammals remains unclear. We isolated more than 900 H9N2 AIVs in our 3-year surveillance in live bird markets in China from 2009 to 2012. Thirty-seven representative isolates were selected for further detailed characterization. These isolates were categorized into 8 genotypes (B64nndash;B71), and formed a distinct antigenic subgroup. Three isolates belonging to the genotype B69, which is a predominant genotype circulating in China, replicated efficiently in mice, while the viruses in parallel tested in other genotypes replicated poorly although they have a same leucine at position 226 in the hemagglutinin (HA) receptor binding site critical for binding human-like sialic acid receptors as these three viruses. Further molecular and single mutation analysis revealed that a valine (V) residue at position 190 in HA is responsible for efficient replication of these H9N2 viruses in mice. The 190V in HA does not affect virus receptor binding specificity, but enhances binding affinity to human cells and lung tissues from mouse and human. All these data indicate that the 190V in HA is one of the important determinants for H9N2 AIVs to cross species barrier to infect mammals despite multiple genes conferring adaptation and replication of H9N2 viruses in mammals. Our findings provide novel insight on understanding host range expansion of H9N2 AIVs.

    IMPORTANCE Influenza hemagglutinin (HA) is responsible for binding to host cell receptors and therefore influences viral host range and pathogenicity in different species. We showed that the H9N2 avian influenza viruses harboring an amino acid 190V in the HA exhibit enhanced virus replication in mice. Further studies demonstrate that amino acid 190V in the HA does not change virus receptor binding specificity, but enhances virus binding affinity of the H9N2 virus to human cells and attachment to lung tissues from human and mouse. Our findings suggest that more attention should be given to the H9N2 AIVs with HA-190V during surveillance, which have a potential threat to mammals including humans.

  • Affinity purification of the Hepatitis C virus replicase identifies Valosin-containing protein (VCP) AAA+ATPase as an active viral replication modulator [PublishAheadOfPrint]

  • Like almost all the positive-strand RNA viruses, Hepatitis C virus (HCV) induces host intracellular membrane modification to form the membrane-bound viral replication complex (RC), within which viral replicase amplify the viral RNA genome. Despite accumulated information about HCV co-opts host factors for viral replication, our knowledge of the molecular mechanisms of how viral proteins, hijack host factors for replicase assembly has only begun to emerge. Purification of the viral replicase and identification of the replicase associated host factors to dissect their roles in RC biogenesis will shed light on the molecular mechanisms of RC assembly. Sought to purify the viral replicase in the context of genuine viral replication, we developed a HCV subgenomic replicon system in which two different affinity tags were simultaneously in-frame inserted into the HCV NS5A and NS5B. After solubilizing the replicon cells, we purified the viral replicase by two-step-affinity purification and identified the associated host factors by mass spectrometry (MS). We identified Valosin-containing protein (VCP) AAA+ATPase as an active viral replication modulator and its ATPase Activity is required for viral replication. Transient replication assay indicated VCP is mainly involved in viral genome amplification. VCP associated with viral replicase and co-localized with viral RC marker. Further, In a HCV replicase formation surrogate system, abolishing VCP function resulted in aberrant distribution of HCV NS5A. We propose HCV may co-opt a host AAA+ATPase for its replicase assembly.

    Importance Almost all the positive-strand RNA viruses share a common replication strategy, in which viral proteins modify host membranes to from the membrane-associated viral replicase. Viruses hijack host factors to facilitate this energy unfavorable process. Understanding this fundamental process is hampered by the challenges to purify the replicase due to the technical difficulties. In this study, we developed a HCV subgenomic replicon system in which two different affinity tags were simultaneously in-frame inserted into two replicase components. Using this dual-affinity-tagged replicon system, we purified the viral replicase and identified Valosin-containing protein (VCP) AAA+ATPase as a pivotal viral replicase associated host factor that is required for viral genome replication. Abolishing VCP function resulted in aberrant viral protein distribution. We propose HCV hijack a host AAA+ATPase for its replicase assembly. Understanding the molecular mechanism of VCP regulates viral replicase assembly may lead to novel anti-viral strategies targeting the most conserved viral replication step.

  • Cell Surface THY-1 Contributes to Human Cytomegalovirus Entry via a Macropinocytosis-like Process [PublishAheadOfPrint]

  • Previously we showed that THY-1 has a critical role in the initial stage of infection of certain cell types with human cytomegalovirus (HCMV) and that THY-1 is important for HCMV-mediated activation of PI3K/Akt during virus entry. THY-1 is known to interact with integrins and is a major cargo protein of clathrin-independent endocytic vesicles. Since macropinocytosis involves integrin signaling, is PI3K/Akt-dependent, and is a clathrin-independent endocytic process, we determined if THY-1 has a role in HCMV entry by macropinocytosis. Using electron microscopy in two cell lines that support HCMV infection in a THY-1 dependent manner, we found that HCMV enters these cells by a macropinocytosis-like process. THY-1 associated with HCMV virions on the cell surface and colocalized with virus inside macropinosomes. EIPA and soluble THY-1 blocked HCMV infection in the cell lines by gge; 80% and 60%, respectively. HCMV entry into the cells triggered increased influx of extracellular fluid, a marker of macropinocytosis, and this increased fluid uptake was inhibited by EIPA and by soluble THY-1. Blocking actin depolymerization, Na+/H+ exchange, PI3K, and Pak1 kinase, which are critical molecules for macropinocytosis, impaired HCMV infection. Neither internalized HCMV virions nor THY-1 in virus-infected cells colocalized with transferrin by confocal microscopy, indicating that clathrin-mediated endocytosis was not involved in THY-1 associated virus entry. These results suggest that HCMV has adopted to utilize THY-1, a cargo protein of clathrin-independent endocytotic vesicles to facilitate efficient entry into certain cell types by a macropinocytosis-like process.

    Importance: Human cytomegalovirus (HCMV) infects over half of the population and is the most common infectious cause of birth defects. The virus is the most important infection occurring in transplant recipients. The mechanism of how HCMV enters cells is controversial. In this study we show that THY-1, a cell surface protein that is critical for the early stage of entry of HCMV into certain cell types, contributes to virus entry by macropinocytosis. Our findings suggest that HCMV has adopted to utilize THY-1 to facilitate entry of HCMV into macropinosomes in certain cell types. Further knowledge into the mechanism of HCMV entry into cells may facilitate development novel inhibitors of virus infection.

  • Protein primary structure of the Vaccinia virion at increased resolution [PublishAheadOfPrint]

  • Here we examine the protein covalent structure of the Vaccinia virion. Within two virion preparations, ggt;88% of the theoretical Vaccinia-encoded proteome was detected with high confidence, including the first-time detection of products from 27 Open reading frames (ORFs) previously designated llsquo;predicted', llsquo;uncharacterized', llsquo;inferred' or llsquo;hypothetical, polypeptides as short as 39 aa, and six proteins whose detection required non-tryptic proteolysis. We also detected the expression of four short "ORF-within-ORF"s including one not previously recognized or known to be expressed. Using quantitative MS, between 58 and 72 proteins were determined to be packaged. 63 host proteins were also identified as candidates for packaging. Evidence is provided that some portion of virion proteins are llsquo;nicked' via a combination of endoproteoylysis and concerted exoproteolysis in a manner, and at sites, independent of virus origin or laboratory procedures. The size of the characterized virion phosphoproteome was doubled from 189 (1) to 396 confident, unique phosphorylation sites, 268 of which were within the packaged proteome. This included the unambiguous identification of phosphorylation "hotspots" within virion proteins. Using isotopically enriched ATP, 23 sites of intravirion kinase phosphorylation were detected within nine virion proteins, all at sites already partially occupied within the virion preparations. The clear phosphorylation of proteins RAP94 and RP19 was consistent with the roles of these proteins in intravirion early gene transcription. In a blind search for protein modifications, cysteine glutathionylation and O-linked glycosylation featured prominently. We provide evidence for the phosphoglycosylation of Vaccinia proteins.

    Importance: Poxviruses have among the most complex and irregular virions, about whose structure little is known. To better understand pox virion structure, imaging should be supplemented with other tools. Here, we provide a deep study of the covalent structure of the Vaccinia virion using the various tools of contemporary mass spectrometry.

  • Characterization of Clade 7.2 H5 Avian Influenza Viruses That Continue to Circulate in Chickens in China [PublishAheadOfPrint]

  • The H5N1 avian influenza viruses emerged in Southeast Asia in the later 20th century and have evolved into multiple phylogenetic clades based on their hemagglutinin (HA)-encoding genes. The clade 7.2 viruses were first detected in chickens in northern China in 2006, and vaccines specifically targeted to this clade were developed and have been used in poultry in China since 2006. During routine surveillance and disease diagnosis, we isolated seven H5 viruses between 2011 and 2014 that bear the clade 7.2 HA genes. Here we performed extensive studies to understand how the clade 7.2 H5 viruses have evolved in chickens in China. Full genome sequence analysis revealed that the seven viruses formed two subtypes (four H5N1 viruses and three H5N2 viruses) and four genotypes by deriving genes from other influenza viruses. All of the viruses were antigenically drifted from the clade 7.2 viruses that were isolated in 2006. Pathogenicity studies of four viruses, one from each genotype, revealed that all of the viruses are highly pathogenic in chickens, but none of them could replicate in ducks. The four viruses exclusively bound to avian-type receptors and replicated only in the turbinates and/or lungs of mice; none of them were lethal to mice at the 106EID50 dosage. Our study indicates that although the clade 7.2 viruses have not been eradicated from poultry through vaccination, they have not become more dangerous to other animals (e.g., ducks and mice) and humans.

    IMPORTANCE Animal influenza viruses can acquire the ability to infect and kill humans. The H5N1 viruses have been a concern in recent decades because of their clear pandemic potential. We sorted H5N1 influenza viruses to different phylogenetic clades based on their HA genes. The clade 7.2 viruses were detected in chickens in several provinces of northern China in 2006. Vaccines to these viruses were subsequently developed and have been used ever since to control infection of poultry. Here, we analyzed the genetic and biologic properties of seven clade 7.2 viruses that were isolated from chickens between 2011 and 2014. We found that after nearly nine years of circulation in chickens, the clade 7.2 viruses still exclusively bind to avian-type receptors and are of low pathogenicity to mice, suggesting that these H5 viruses pose a low risk to human public health.

  • Monocytes but not lymphocytes carrying HIV-1 on their surface transmit infection to human tissue ex vivo [PublishAheadOfPrint]

  • Unprotected sexual intercourse with HIV-infected men is the major cause of new infections. HIV virions are released into semen by various cells of the male genital tract as well as by infected monocytes and lymphocytes present in semen. Some of these virions may attach to the surface of cells, infected or uninfected. We investigated whether cells carrying attached HIV on their surface can transmit infection. We addressed this question in a model system of human tissue ex vivo exposed to monocytes and lymphocytes carrying HIV on their surfaces. We gamma-irradiated these cells to prevent their productive infection. In spite of comparable amounts of HIV attached to monocytes and lymphocytes, only monocytes were capable of transmitting infection and triggering productive infection in tissue. This HIV-1 transmission was mediated by cellnndash;cell contacts. Our experiments suggest that in vivo, HIV attached to infected or uninfected monocytes, which far outnumber lymphocytes in HIV-infected semen, may contribute to sexual transmission of HIV from men to their partners.

    IMPORTANCE The vast majority of new HIV infections occur through sexual transmission in which HIV is transferred from semen of an infected male to an uninfected partner. In semen, HIV-1 particles may exist as free floating virions, inside infected cells or attached to the surface of cells, whether they are infected or not. Here we investigated whether HIV attached at the surface of monocytes or lymphocytes could transmit infection to human tissue. Incubation of human tissue with monocyte-attached HIV resulted in tissue productive infection. In contrast, there was no infection of tissues when they were incubated with lymphocyte-attached HIV-1. Our results highlight the important role that seminal monocytes may play in HIV transmission in vivo, especially since monocytes far outnumber lymphocytes in semen of HIV-infected individuals.

  • CD95 signaling inhibits B cell receptor-mediated gammaherpesviral replication in apoptosis-resistant B lymphoma cells [PublishAheadOfPrint]

  • While CD95 is an apoptosis-inducing receptor and emerges as a potential anticancer therapy target, mounting evidence shows that CD95 is also emerging as a tumor promoter by activating nonapoptotic signaling pathways. Gammaherpesviral infection is tightly associated with lymphoproliferative diseases, including B cell lymphomas. The non-apoptotic function of CD95 in gammaherpesvirus-associated lymphomas is largely unknown. Here we showed that stimulation of CD95 agonist antibody drove the majority of sensitive gammaherpesvirus-transformed B cells to undergo caspase-dependent apoptosis and promoted the survival and proliferation of a subpopulation of apoptosis-resistant B cells. Surprisingly, CD95-mediated nonapoptotic signaling induced IFN-bbeta; expression, and correlatively inhibited B cell receptor (BCR)-mediated gammaherpesviral replication in those apoptosis-resistant lymphoma cells without influencing BCR signaling. Further analysis showed that IFN-bbeta; alone or synergizing with CD95 blocked the activation of lytic switch proteins and the gene expression of gammaherpesviruses. Our findings indicate that independent of its apoptotic activity, CD95 signaling activity plays an important role in blocking viral replication in apoptosis-resistant, gammaherpesvirus-associated lymphoma B cells, suggesting a novel mechanism which indicates how host CD95 prototype death receptor controls the life cycle of gammaherpesviruses independent of its apoptotic activity.

    IMPORTANCE Gammaherpesviruses are tightly associated with lymphoid malignancies and other cancers. Viral replication and persistence strategies leading to cancer involve the activation of anti-apoptotic and proliferation program as well as evasion of host immune response. Here we provide evidence that the stimulation of CD95 agonist antibody, mimicking one of major mechanisms of cytotoxic T cell killing, inhibits B cell receptor-mediated gammaherpesviral replication in CD95 apoptosis-resistant lymphoma cells. CD95-induced type I interferon IFN-bbeta; contributes to the inhibition of gammaherpesviral replication. This finding sheds new light on CD95 nonapoptotic function and provides a novel mechanism for gammaherpesviruses that help to escape host immune surveillance.

  • Exploitation of IL-10 Signaling Pathways: The Alternate Roles of Viral and Cellular IL-10 in Rhesus Cytomegalovirus Infection [PublishAheadOfPrint]

  • There is accumulating evidence that the viral interleukin-10 (vIL-10) ortholog of both human and rhesus cytomegalovirus (HCMV and RhCMV, respectively) suppresses the functionality of cell types that would be critical to contain virus dissemination and help shape long-term immunity during the earliest virus-host interactions. In particular, exposure of macrophages, peripheral blood mononuclear cells, monocyte-derived dendritic cells, and plasmacytoid dendritic cells to vIL-10 suppresses multiple effector functions, including notably, those that link innate and adaptive immune responses. Further, vaccination of RhCMV-uninfected rhesus macaques with non-functional forms of RhCMV vIL-10 greatly restricts parameters of RhCMV infection following RhCMV challenge of the vaccines. Vaccinees exhibited significantly reduced shedding of RhCMV in saliva and urine following RhCMV challenge compared to unvaccinated controls. Based on the evidence that vIL-10 is critical during acute infection, the role of vIL-10 during persistent infection was analyzed in rhesus macaques infected long-term with RhCMV to determine whether post-infection vaccination against vIL-10 could change the virus-host balance. RhCMV-seropositive macaques, which were shedding RhCMV in saliva, were vaccinated with non-functional RhCMV vIL-10, and shedding levels of RhCMV in saliva were evaluated. Following robust increases in vIL-10-binding and vIL-10-neutralizing antibodies, shedding levels of RhCMV modestly declined, consistent with the interpretation that vIL-10 may play a functional role during persistent infection. However, a more significant association was observed between the levels of cellular IL-10 secreted in PBMC exposed to RhCMV antigens and shedding of RhCMV in saliva. This result implies that RhCMV persistence is associated with the induction of cIL-10 receptor-mediated signaling pathways.

    IMPORTANCE Human health is adversely impacted by viruses that establish lifelong infections that are often accompanied with increased morbidity and mortality (e.g., HIV, hepatitis C virus, human cytomegalovirus). A longstanding but unfulfilled goal has been to develop post-infection vaccine strategies that could llsquo;reboot' the immune system of an infected individual in ways that enable the infected host to develop immune responses that clear reservoirs of persistent virus infection, effectively curing the host of infection. This concept was evaluated in rhesus macaques infected long-term with rhesus cytomegalovirus by repeatedly immunizing infected animals with non-functional versions of the rhesus cytomegalovirus-encoded, viral interleukin-10 immune modulating protein. Following vaccine-mediated boosting of antibody titers to viral interleukin-10, there was modest evidence for increased immunological control of the virus following vaccination. More significantly, data were also obtained indicating that rhesus cytomegalovirus is able to persist due to upregulation of the cellular interleukin-10 signaling pathway.

  • An essential role of INI1/hSNF5 chromatin remodeling protein in HIV-1 post-transcriptional events and Gag/GagPol stability [PublishAheadOfPrint]

  • INI1/hSNF5/SMARCB1/BAF47 is an HIV-specific integrase (IN)-binding protein, which influences HIV-1 transcription and particle production. INI1 binds to SAP18 (Sin3a associated protein 18 kDa) and both INI1 and SAP18 are incorporated into HIV-1 virions. To determine the significance of INI1 and the INI1-SAP18 interaction during HIV-1 replication, we isolated a panel of SAP18-Interaction-Defective (SID) INI1 mutants using a yeast reverse two-hybrid screen. SID-INI1 mutants, which retained the ability to bind to IN, cMYC and INI1, but impaired for binding to SAP18, were tested for their effect on HIV-1 particle production. SID-INI1 dramatically reduced intracellular Gag/Gag-Pol protein levels and additionally decreased viral particle production. SID-INI1-mediated effects were less dramatic in trans-complementation assays using IN deleted viruses with Vpr-RT-IN. SID-INI1 did not inhibit LTR-mediated transcription, but marginally decreased steady state gag RNA levels, suggesting a post-transcriptional effect. Pulse chase analysis indicated that in SID-INI1 expressing cells the pr55Gag levels rapidly decreased. RNA interference analysis indicated that shRNA-mediated knockdown of INI1 reduced intracellular Gag/Gag-Pol levels, and further inhibited HIV-1 particle production. These results suggest that SID-INI1 mutants inhibit multiple stages of post-transcriptional events of HIV-1 replication, including intracellular Gag/Gag-Pol RNA and protein levels, which in turn inhibit assembly and particle production. Interfering with INI1 leads to a decrease in particle production and Gag/GagPol protein levels. Understanding the role of INI1 and SAP18 in HIV-1 replication is likely to provide novel insight into the stability of Gag/Gag-Pol, which may lead to the development of novel therapeutic strategies to inhibit HIV-1 late events.

    Importance Significant gaps exist in our current understanding of the mechanism and host factors that influence HIV-1 post-transcriptional events including gag RNA levels, Gag/Gag-Pol protein levels, assembly and particle production. Our previous studies suggested that IN-binding host factor INI1 plays a role in HIV-1 assembly. An ectopically expressed minimal IN-binding domain of INI1, S6, potently and selectively inhibited HIV-1 Gag/Gag-Pol trafficking and particle production. However, whether or not endogenous INI1 and its interacting partners, such as SAP18, are required for late events was unknown. Here we report that endogenous INI1 as well as its interaction with SAP18 are necessary to maintain intracellular levels of Gag/Gag-Pol and for particle production. Interfering with INI1 or INI1-SAP18 interaction leads to the impairment of these processes, suggesting a novel strategy for inhibiting post-transcriptional events of HIV-1 replication.

  • Functional and structural characterization of P[19] rotavirus VP8* interaction with histo-blood group antigens [PublishAheadOfPrint]

  • Rotaviruses of species A (RVA) are a major causative agent of acute gastroenteritis. Recently, histo-blood group antigens (HBGAs) have been reported to interact with human RVA VP8* proteins. Human P[19] is a rare P-genotype of porcine origin that infects humans sporadically. The functional and structural characteristics of P[19] VP8* interaction with HBGAs are unknown. In this study, we expressed and purified the VP8* proteins of human and porcine P[19] RV. In oligosaccharide and saliva binding assays, P[19] VP8* proteins showed obvious binding to A, B, O type saliva samples irrespective of the secretor status, implying broad binding patterns. However, they did not display specific binding to any of the oligosaccharides tested. In addition, we solved the structure of human P[19] VP8* at 2.4 AAring;, which revealed a typical galectin-like fold. The structural alignment demonstrated that P[19] VP8* was most similar to that of P[8], which was consistent with the phylogenetic analysis. Structure superimposition revealed the basis for the lack of binding to the oligosaccharides. Our study indicates that P[19] RVs may bind to other oligosaccharides or ligands and have the potential to spread widely among humans. Thus, it is necessary to place the prevalence and evolution of P[19] RVs under surveillance.

    IMPORTANCE Human P[19] is a rare P-genotype of porcine origin. Based on phylogenetic analysis of VP8* sequences, P[19] was classified into the P[II] genogroup, together with P[4], P[6], and P[8], which have been reported to interact with HBGAs in a genotype-dependent manner. In this study, we explored the functional and structural characteristics of P[19] VP8* interaction with HBGAs. P[19] VP8* showed binding to A, B, O type saliva samples as well as saliva of non-secretors. This implies that P[19] has the potential to spread among humans with a broad binding range. Careful attention should be paid to the evolution and prevalence of P[19] RVs. Furthermore, we solved the structure of P[19] VP8*. Structure superimposition indicates that P[19] may bind to other oligosaccharides or ligands using potential binding sites, suggesting that further investigation of the specific cell attachment factors is warranted.

  • Evidence for the introduction, reassortment and persistence of diverse influenza A viruses in Antarctica [PublishAheadOfPrint]

  • Avian influenza virus (AIV) surveillance in Antarctica during 2013 revealed the prevalence of evolutionarily distinct influenza viruses of H11N2 subtype in Adeeacute;lie penguins. Here we present results from the continued surveillance of AIV on the Antarctic Peninsula during 2014 and 2015. In addition to the continued detection of H11 subtype viruses during 2014 in a snowy sheathbill, we isolated a novel H5N5 subtype virus during 2015 in a chinstrap penguin. Gene sequencing and phylogenetic analysis revealed that the H11 virus detected in 2014 had a ggt;99.1% nucleotide similarity to the H11N2 viruses isolated in 2013, suggesting continued prevalence of this virus over multiple years in Antarctica. However, phylogenetic analysis of the H5N5 virus showed that their genome segments were recently introduced into the continent, except for the NP gene that was similar to that in the endemic H11N2 viruses. Our analysis indicates geographically diverse origins for the H5N5 virus genes; with the majority of its genome segments derived from North American lineage viruses, but the neuraminidase gene derived from a Eurasian lineage virus. In summary, we show the persistence of AIV lineages over multiple years in Antarctica; recent introduction of gene segments from diverse regions; and reassortment between different AIV lineages in Antarctica, which together, significantly increases our understanding of AIV ecology in this fragile and pristine environment.

    IMPORTANCE Analysis of avian influenza viruses (AIVs) detected in Antarctica reveals both the relatively recent introduction of an H5N5 AIV predominantly of North American-like origin, as well as the persistence of an evolutionarily divergent H11 AIV. These data demonstrate that the flow of viruses from North America may be more common than initially thought, and that once introduced, these AIVs have the potential to be maintained within Antarctica. The future introduction of AIVs from North America into the Antarctic Peninsula is of particular concern given that highly pathogenic H5Nx viruses have recently been circulating amongst wild birds in parts of Canada and the Unites States following the movement of these viruses from Eurasia via migratory birds. The introduction of a highly pathogenic influenza virus into penguin colonies within Antarctica might have devastating consequences.

  • The antiviral alkaloid berberine reduces chikungunya virus-induced mitogen-activated protein kinase (MAPK) signaling [PublishAheadOfPrint]

  • Chikungunya virus (CHIKV) has infected millions of people in the tropical and sub-tropical regions since its re-emergence in the last decade. We recently identified the non-toxic plant alkaloid berberine as an antiviral substance against CHIKV in a high-throughput screen. Here, we show that berberine is effective in multiple cell types against a variety of CHIKV strains, also at a high multiplicity of infection, consolidating the potential of berberine as an antiviral drug. We excluded any effect of this compound on virus entry or on the activity of the viral replicase. A human phosphokinase array revealed that CHIKV infection specifically activated the major mitogen-activated protein kinase (MAPK) signaling pathways: ERK, p38 and JNK. Upon treatment with berberine, this virus-induced MAPK activation was markedly reduced. Subsequent analyses with specific inhibitors of these kinases indicated that the ERK and JNK signaling cascades are important for the generation of progeny virions. In contrast to specific MAPK inhibitors, berberine lowered virus-induced activation of all major MAPK pathways and resulted in stronger reduction in viral titers. Further, we assessed the in vivo efficacy of berberine in a mouse model and measured a significant reduction of CHIKV-induced inflammatory disease. In summary, we demonstrate the efficacy of berberine as a drug against CHIKV and highlight the importance of the MAPK signaling pathways in the alphavirus infectious cycle.

    IMPORTANCE Chikungunya virus (CHIKV) is a mosquito-borne virus that causes severe and persistent muscle and joint pain and has recently spread to the Americas. No licensed drug exists to counter this virus. In this study we report that the alkaloid berberine is antiviral against different CHIKV strains and in multiple human cell lines. We demonstrate that berberine collectively reduced the virus-induced activation of cellular mitogen-activated protein kinase signaling. The relevance of these signaling cascades in the viral life cycle was emphasized by specific inhibitors of these kinase pathways, which decreased the production of progeny virions. Berberine significantly reduced CHIKV-induced inflammatory disease in a mouse model, demonstrating efficacy of the drug in vivo. Overall, this work makes a strong case for pursuing berberine as a potential anti-CHIKV therapeutic compound and for exploring the MAPK signaling pathways as antiviral targets against alphavirus infections.

  • Neglected but Important Role of Apolipoprotein E Exchange in Hepatitis C Virus Infection [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) is a major cause of chronic liver disease infecting approximately 170 million people worldwide. HCV assembly is tightly associated with the lipoprotein pathway. Exchangeable apolipoprotein E (apoE) is incorporated on infectious HCV virions and it is important for infectious HCV virion morphogenesis and entry. Moreover, virion apoE level is positively correlated with its ability to escape E2 antibody neutralization. However, the role of apoE exchange in HCV life cycle is unclear. In this study, the relationship between apoE expression and cell permissiveness to HCV infection was assessed by infecting apoE knock-down and derived apoE rescue cell lines with HCV. Exchange of apoE between lipoproteins and HCV lipo-viral particle (LVP) was evaluated by immunoprecipitation, infectivity test and viral genome quantification. Cell and heparin column binding assays were applied to determine the attachment efficiency of LVP with different level of incorporated apoE. The results showed that cell permissiveness for HCV infection was determined by exogenous apoE associated lipoproteins. Furthermore, apoE exchange did occur between HCV LVP and lipoproteins, which was important to maintain a high apoE level on LVP. Lipid free apoE was capable of enhancing HCV infectivity to apoE knock-down cells but not apoE rescue cells. Higher apoE level on LVP conferred more efficient LVP attachment to both cell surface and heparin beads. This study revealed that exogenous apoE incorporated lipoproteins from uninfected hepatocytes safeguarded apoE level of LVP for more efficient attachment during HCV infection.

    IMPORTANCE With this study, a neglected but important role of apoE exchange in HCV LVP infectivity post virus assembly and release is identified. The data indicated that apoE expression level in uninfected cell is important for high permissiveness to HCV infection. Secreted apoE associated lipoprotein specifically enhances infection of HCV LVP. ApoE exchange between HCV LVP and lipoproteins is important to maintain an adequate apoE level on LVPs for their efficient attachment to cell surface. These data defined for the first time an extracellular role of exchangeable apoE in HCV infection and suggested that exchangeable apolipoproteins reach a natural equilibrium between HCV LVPs and lipoprotein particles, which provides a new perspective to the understanding of the heterogeneity of HCV LVPs in composition.

  • Inhibition of polyamine biosynthesis is a broad-spectrum strategy against RNA viruses [PublishAheadOfPrint]

  • RNA viruses present an extraordinary threat to human health, given their sudden and unpredictable appearance, the potential for rapid spread among the human population, and their ability to evolve resistance to antiviral therapies. Recent emergence of chikungunya virus, Middle East respiratory syndrome coronavirus, Zika virus and Ebola virus highlight the struggles to contain outbreaks. A significant hurdle is the availability of antivirals to treat the infected or protect at-risk populations. While several compounds show promise in vitro and in vivo, these outbreaks underscore the need to accelerate drug discovery. The replication of several viruses has been described to rely on host polyamines, small and abundant positively-charged molecules found in the cell. Here, we describe the antiviral effects of two molecules that alter polyamine levels: difluoromethylornithine (DFMO, eflornithine), a suicide inhibitor of ornithine decarboxylase (ODC1), and diethylnorspermine (DENSpm), an activator of spermidine/spermine N1-acetyltransferase (SAT1). We show that reducing polyamine levels negatively impacts diverse RNA viruses, including several viruses involved in recent outbreaks, in vitro and in vivo. These findings highlight the importance of the polyamine biosynthetic pathway to viral replication as well as its potential as a target in the development of further antivirals or currently available molecules, such as DFMO.

    IMPORTANCE RNA viruses present a significant hazard to human health and combatting these viruses requires the exploration of new avenues for targeting viral replication. Polyamines, small positively-charged molecules within the cell, have been demonstrated to facilitate infection of a handful of different viruses. Our study demonstrates that diverse RNA viruses rely on the polyamine pathway for replication and highlights polyamine biosynthesis as a promising drug target.

  • RSV Infection Triggers Epithelial HMGB1 Release as a Damage-Associated Molecular Pattern Promoting a Monocytic Inflammatory Response [PublishAheadOfPrint]

  • Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infant and elderly populations worldwide. Currently, there is no efficacious vaccine or therapy available for RSV infection. The molecular mechanisms underlying RSV-induced acute airway disease and associated long-term consequences remain largely unknown; however, experimental evidence suggests that the lung inflammatory response plays a fundamental role in the outcome of RSV infection. High Mobility Group Box 1 (HMGB1) is a nuclear protein that triggers inflammation when released from activated immune or necrotic cells and drives the pathogenesis of various infectious agents. Although HMGB1 has been implicated in many inflammatory diseases, its role in RSV-induced airway inflammation has not been investigated. This study investigates the molecular mechanism of action of extracellularly released HMGB1 in airway epithelial cells (A549 and small airway epithelial cells) to establish its role in RSV infection. Immunofluorescence microscopy and Western blotting results showed that RSV infection of human airway epithelial cells induced a significant release of HMGB1 as a result of translocation of HMGB1 from the cell nuclei to the cytoplasm and subsequent release into the extracellular space. Treating RSV-infected A549 cells with antioxidants significantly inhibited RSV-induced HMGB1 extracellular release. Studies using recombinant HMGB1 triggered immune responses by activating primary human monocytes. Finally, HMGB1 released by airway epithelial cells due to RSV infection appears to function as a paracrine factor priming epithelial cells and monocytes to inflammatory stimuli in the airways.

    IMPORTANCE RSV is a major cause of serious lower respiratory tract infections in young children and causes severe respiratory morbidity and mortality in the elderly. In addition, to date there is no effective treatment or vaccine available for RSV infection. The mechanisms responsible for RSV-induced acute airway disease and associated long-term consequences remain largely unknown. The oxidative stress response in the airways plays a major role in the pathogenesis of RSV. High Mobility Group Box 1 (HMGB1) is a ubiquitous redox-sensitive multifunctional protein that serves as both a DNA regulatory protein and an extracellular cytokine signaling molecule that promotes airway inflammation as a damage-associated molecular pattern. This study investigated the mechanism of action of HMGB1 in RSV infection with the aim to identify new inflammatory pathways at the molecular level that may be amenable to therapeutic interventions.

  • Stimulation of the replication of ICP0-null mutant HSV-1 and pp71-deficient HCMV by EBV tegument protein BNRF1 [PublishAheadOfPrint]

  • It is now well established that several cellular proteins that are components of promyelocytic leukaemia nuclear bodies (PML-NBs, also known as ND10) have restrictive effects on herpesvirus infections that are countered by viral proteins that are either present in the virion particle or are expressed during the earliest stages of infection. For example, herpes simplex virus 1 (HSV-1) immediate-early (IE) protein ICP0 overcomes the restrictive effects of PML-NB components PML, Sp100, hDaxx and ATRX while human cytomegalovirus IE protein IE1 and tegument protein pp71 target PML and Sp100, and hDaxx and ATRX, respectively. The functions of these viral regulatory proteins are in part interchangeable, thus both IE1 and pp71 stimulate the replication of ICP0-null mutant HSV-1, while ICP0 increases plaque formation by pp71-deficient HCMV. Here, we extend these studies by examining proteins that are expressed by Epstein-Barr virus (EBV). We report that EBV tegument protein BNRF1, discovered by others to target the hDaxx/ATRX complex, increases the replication of both ICP0-null mutant HSV-1 and pp71-deficient HCMV. In addition, EBV protein EBNA-LP, which targets Sp100, also augments ICP0-null mutant HSV-1 replication. The combination of these two EBV regulatory proteins had a greater effect than each one individually. These findings enhance the concept that disruption of the functions of PML-NB proteins is important for efficient herpesvirus infections.

    IMPORTANCE Whether a herpesvirus initiates a lytic infection in a host cell or establishes quiescence or latency is influenced by events that occur soon after the viral genome has entered the host cell nucleus. Certain cellular proteins respond in a restrictive manner to the invading pathogen DNA, while viral functions are expressed that counteract the cell mediated repression. One aspect of cellular restriction of herpesvirus infections is mediated by components of nuclear structures known as PML Nuclear Bodies (PML NBs) or ND10. Members of the alpha, beta and gamma herpesvirus families all express proteins that interact with, degrade or otherwise counteract the inhibitory effects of various PML NB components. Previous work has shown that there is the potential of functional interchange between these viral proteins expressed by alpha and beta herpesviruses, despite a lack of obvious sequence similarity. Here this concept is extended to include a member of the gamma herpesviruses.

  • Lineage-specific differences in the gp120 Inner Domain Layer 3 of Human and Simian Immunodeficiency Viruses [PublishAheadOfPrint]

  • Binding of HIV-1 and SIV gp120 exterior envelope glycoprotein to CD4 triggers conformational changes in gp120 that promote its interaction with one of the chemokine receptors, usually CCR5, ultimately leading to gp41-mediated virus-cell membrane fusion and entry. We previously described that topological Layers (Layer 1, Layer 2 and Layer 3) in the gp120 inner domain contribute to gp120-trimer association in the unliganded state but also help secure CD4 binding. Relative to Layer 1 of HIV-1 gp120, the SIVmac239 gp120 Layer 1 plays a more prominent role in maintaining gp120-trimer association but is minimally involved in promoting CD4 binding, which could be explained by the existence of a well-conserved Tryptophan 375 (Trp 375) in HIV-2/SIVsmm. Here we investigated the role of SIV Layer 3 on viral entry, cell-to-cell fusion and CD4 binding. We observed that a network of interactions involving some residues of the bbeta;8-aalpha;5 region in SIVmac239 Layer 3 may contribute to CD4 binding by helping shape the nearby Phe 43 cavity which directly contacts CD4. In summary, our results suggest that SIV Layer 3 has a greater impact on CD4 binding than in HIV-1. This work defines lineage-specific differences in Layer 3 from HIV-1 and SIV.

    IMPORTANCE CD4-induced conformational changes in the gp120 inner domain involve rearrangements between three topological layers. While the role of Layers 1-3 for HIV-1 and 1-2 for SIV on gp120 transition to the CD4-bound conformation has been reported, the role of SIV Layer 3 remains unknown. Here we report that SIV Layer 3 has a greater impact on CD4 binding than in HIV-1 gp120. This work defines lineage specific differences in Layer 3 from HIV-1 and SIV.

  • Human Herpesvirus 6 downregulates the expression of activating ligands during lytic infection to escape elimination by natural killer cells [PublishAheadOfPrint]

  • The herpesviridae family consists of eight viruses, most of which infect a majority in the human population. One of the less-studied members is Human Herpesvirus 6 (HHV-6, Roseolovirus) that causes a mild, well-characterized childhood disease. Primary HHV-6 infection is followed by life-long latency. Reactivation frequently occurs in immune-compromised patients, such as those suffering from HIV infection, cancer or following transplantation, and causes potentially life-threatening complications. In this study, we investigated the mechanisms HHV-6 utilizes to remain undetected by natural killer (NK) cells, key participants in the innate immune response to infections. We revealed viral mechanisms which downregulate ligands for two powerful activating NK cell receptors: ULBP1, ULBP3 and MICB that trigger NKG2D, and B7-H6 that activates NKp30. Accordingly, this downregulation impaired NK cells' ability to recognize HHV-6 infected cells. Thus, we describe for the first time immune evasion mechanisms of HHV-6 that protect lytically infected cells from NK elimination.

    IMPORTANCE Human Herpesvirus 6 (HHV-6) latently infects a large portion of the human population and can reactivate in humans lacking a functional immune system, like cancer or AIDS patients. Under these conditions, it can cause life-threatening diseases. To date, actions and interplay of immune cells, and particularly cells of the innate immune system, during HHV-6 infection are poorly defined. Therefore, we aimed to understand how cells undergoing lytic HHV-6 infection are interacting with natural killer (NK) cells, an innate lymphocyte constituting the first line of defense against viral intruders. We show that HHV-6 suppresses the expression of surface proteins that alert the immune cells by triggering two major receptors on natural killer (NK) cells, NKG2D and NKp30. In consequence, HHV-6 can replicate undetected by innate immune system and potentially spread infection throughout the body.

    This study advances the understanding of HHV-6 biology and its measures used to successfully escape immune elimination.

  • The near-atomic resolution structure of a highly neutralizing Fab bound to canine parvovirus [PublishAheadOfPrint]

  • Canine parvovirus (CPV) is a highly contagious pathogen that causes severe disease in dogs and wildlife. Previously, a panel of neutralizing monoclonal antibodies (MAb) raised against CPV was characterized. An antibody fragment (Fab) of MAb E was found to neutralize the virus at low molar ratios. Using recent advances in cryo-electron microscopy we determined the structure of CPV in complex with Fab E to 4.1 AAring; resolution, which allowed de novo building of the Fab structure. The footprint identified was significantly different than the footprint obtained previously from models fitted into lower resolution maps. Using single chain variable fragments (scFv) we tested antibody residues that control capsid binding. The near atomic structure also revealed that Fab binding had caused capsid destabilization in regions containing key residues conferring receptor binding and tropism, which suggests a mechanism for efficient virus neutralization by antibody. Furthermore, a general technical approach for solving the structures of small molecules is demonstrated, as binding the Fab to the capsid allowed us to determine the 50kDa Fab structure by cryo-EM.

    Significance Using cryo-electron microscopy and new direct electron detector technology we have solved the 4AAring; resolution structure of a Fab molecule bound to a picornavirus capsid. The Fab induced conformational changes to regions of the virus capsid that control receptor binding. The antibody footprint is markedly different from the previous one identified using a 12AAring; structure. This work emphasizes the need for a high-resolution structure to guide mutational analysis and cautions against relying on older low-resolution structures even though they were interpreted with the best methodology possible at the time.

  • High Glucose Induces Reactivation of Latent Kaposi's Sarcoma-Associated Herpesvirus [PublishAheadOfPrint]

  • High prevalence of Kaposi's sarcoma (KS) is seen in diabetic patients. It is unknown if the physiological condition of diabetes contributes to KS development. We found elevated levels of viral lytic gene expression when Kaposi's sarcoma-associated herpesvirus (KSHV) infected cells were cultured in high glucose medium. To demonstrate the association between high glucose and KSHV replication, we xeno-grafted telomerase-immortalized human umbilical vein endothelial cells that are infected with KSHV (TIVE-KSHV) into hyperglycemic and normal nude mice. The injected cells expressed significantly higher levels of KSHV lytic genes in hyperglycemic mice than in normal mice. We further demonstrated that high glucose induced production of hydrogen peroxide (H2O2), which down regulated silent information regulator 1 (SIRT1), a class-III histone deacetylase (HDAC), resulting in epigenetic transactivation of KSHV lytic genes. These results suggest that high blood glucose in diabetic patients contributes to development of KS by promoting KSHV lytic replication and infection.

    AUTHORS' SUMMARY Multiple epidemiological studies have reported a higher prevalence of classic KS in diabetic patients. By using both in vitro and in vivo models, we demonstrated an association between high glucose and KSHV lytic replication. High glucose induces oxidative stress and production of H2O2, which mediates reactivation of latent KSHV through multiple mechanisms. Our results provide the first experimental evidence and mechanistic support for the association of classic KS with diabetes.

  • Equine rhinitis A virus mutants with altered acid resistance unveil a key role of VP3 and intra-subunit interactions in the control of the pH stability of Aphthovirus capsid [PublishAheadOfPrint]

  • Equine rhinitis A virus (ERAV) is a picornavirus associated with respiratory disease in horses and genetically closely related to foot-and-mouth disease virus (FMDV), the prototype aphthovirus. ERAV has recently gained interest as an FMDV alternative for the study of aphthovirus biology, including cell entry and uncoating or antiviral testing. As described for FMDV, current data support that acidic pH inside cellular endosomes triggers ERAV uncoating. In order to provide further insights into aphthovirus uncoating mechanism, we have isolated a panel of ERAV mutants with altered acid sensitivity and that differed on their degree of sensitivity to the inhibition of endosome acidification. These results provide functional evidence of the involvement of acidic pH on ERAV uncoating within endosomes. Remarkably, all amino acid substitutions found in acid-labile or acid-resistant ERAVs were located in the capsid protein VP3, indicating that this protein plays a pivotal role for the control of pH stability of the ERAV capsid. Moreover, all amino acid substitutions mapped at the intra-protomer interface between VP3 and VP2, or VP3 and the N terminus of VP1. These results expand our knowledge on the regions that regulate the acid stability of aphthovirus capsid and should be taken into account when using ERAV as a surrogate of FMDV.

    IMPORTANCE The viral capsid constitutes a sort of dynamic nanomachine that protects the viral genome against environmental assaults while accomplishing important functions such as receptor attachment for viral entry or genome release. We have explored the molecular determinants of aphthovirus capsid stability by isolating and characterizing a panel of equine rhinitis A virus mutants that differed on their acid sensitivity. All the mutations were located within a specific region of the capsid, the intra-protomer interface among capsid proteins, thus providing new insights into the regions that control the acid stability of aphthovirus capsid. These findings could positively contribute to the development of antiviral approaches targeting aphthovirus uncoating or the refinement of vaccine strategies based on capsid stabilization.

  • An NS1 protein mutation (I64T) affects interferon responses and virulence of circulating H3N2 human influenza A viruses [PublishAheadOfPrint]

  • Influenza NS1 protein is the main viral protein counteracting host innate immune responses, allowing the virus to efficiently replicate in interferon (IFN)-competent systems. In this manuscript, we analyzed NS1 protein variability within influenza A (IAV) H3N2 viruses infecting humans during the 2012/2013 season. We also evaluated the impact of the mutations on the ability of NS1 proteins to inhibit the host innate immune responses and general gene expression. Surprisingly, a previously unidentified mutation in the double stranded (ds)RNA-binding domain (I64T) decreased NS1-mediated general inhibition of host protein synthesis, by decreasing its interaction with the cleavage and polyadenylation specificity factor 30 (CPSF30), leading to increased innate immune responses after viral infection. Notably, a recombinant A/Puerto Rico/8/34 H1N1 virus encoding the H3N2 NS1-T64 protein was highly attenuated in mice, most likely because of its ability to induce higher antiviral IFN responses at early times after infection, and because this virus is highly sensitive to the IFN-induced antiviral state. Interestingly, using Peripheral Blood Mononuclear Cells (PBMCs) collected at the acute visit (days 2-3 after infection), we show that the subject infected with the NS1-T64 attenuated virus has diminished responses to interferon and to interferon induction, suggesting why this subject could be infected with this highly IFN-sensitive virus. These data demonstrate the importance of influenza virus surveillance to identify new mutations in the NS1 protein affecting its ability to inhibit innate immune responses, and as a consequence, the pathogenicity of the virus.

    Importance Influenza A and B viruses are one of the most common causes of respiratory infections in humans, causing 1 billion infections, and between 300,000 and 500,000 deaths annually. Influenza virus surveillance to identify new mutations in the NS1 protein affecting innate immune responses, and as a consequence, the pathogenicity of the circulating viruses is highly relevant. Here, we analyzed amino acid variability in the NS1 proteins from human seasonal viruses, and the effect of the mutations in innate immune responses and virus pathogenesis. A previously unidentified mutation in the dsRNA-binding domain decreased NS1-mediated general inhibition of host protein synthesis and the interaction of the protein with CPSF30. This mutation led to increased innate immune responses after viral infection, augmented IFN-sensitivity and virus attenuation in mice. Interestingly, using PBMCs, the subject infected with the virus encoding the attenuating mutation induced decreased antiviral responses, suggesting why this subject could be infected with this virus.

  • DDB1 stimulates the viral transcription of hepatitis B virus via HBx-independent mechanisms [PublishAheadOfPrint]

  • HBx, a small regulatory protein of the hepatitis B virus (HBV), augments viral DNA replication by stimulating viral transcription. Among numerous HBx-binding proteins reported, DDB1 has drawn attention, because DDB1 acts as a substrate receptor of Cul4-DDB1 ubiquitin E3 ligase. Earlier work reported that the DDB1-HBx interaction is indispensable for HBx-stimulated viral DNA replication, suggesting that the Cul4-DDB1 ubiquitin E3 ligase might target cellular restriction factors for ubiquitination and proteasomal degradation. To gain further insight into the DDB1-HBx interaction, we generated HBx mutants deficient for DDB1 binding (i.e., R96A, L98A, and G99A) and examined whether they support HBx-stimulated viral DNA replication. In contrast to previous reports, our results showed that the HBx mutants deficient for DDB1 binding supported viral DNA replication to nearly wild-type level, revealing that the DDB1-HBx interaction is largely dispensable for HBx-stimulated viral DNA replication. Instead, we found that DDB1 directly stimulates viral transcription, regardless of HBx expression. Through an HBV infection study, importantly, we demonstrated that DDB1 stimulates viral transcription from covalently closed circular DNA, a physiological template for viral transcription. Overall, we concluded that DDB1 stimulates viral transcription via a mechanism that does not involve interaction with HBx.

    IMPORTANCE DDB1 constitutes a cullin-based ubiquitin E3 ligase, where DDB1 serves as an adaptor linking the cullin scaffold to the substrate receptor. Earlier findings that the DDB1 binding ability of HBx is essential for the HBx-stimulated viral DNA replication led to a hypothesis that HBx could downregulate host restriction factors that limit HBV replication through cullin ubiquitin E3 ligase that requires the DDB1-HBx interaction. Consistent with the hypothesis, a recent work identified Smc5/6 as a host restriction factor that is regulated by the viral cullin ubiquitin E3 ligase. In contrast, we here found that the DDB1-HBx interaction is largely dispensable for the HBx-stimulated viral DNA replication. Instead, our results clearly showed that DDB1, regardless of HBx expression, enhances viral transcription. Overall, besides its role in the viral cullin ubiquitin E3 ligase, DDB1 itself stimulates the viral transcription via HBx-independent mechanisms.

  • CAR-T Cells Guided by the Single-Chain Fv of a Broadly Neutralizing Antibody Specifically and Effectively Eradicate the Reactivated Virus-latently-infected CD4+ T-lymphocytes Isolated from HIV-1-infected Individuals Receiving Suppressive cART [PublishAheadOfPrint]

  • Despite the advent of combined antiretroviral therapy (cART), the persistence of viral reservoirs remains a major barrier to curing Human Immunodeficiency Virus Type 1 (HIV-1) infection. Recently, the llsquo;shock and kill' strategy, by which such reservoirs are eradicated following reactivation of latent HIV-1 by latency-reversing agents (LRAs), has been extensively practiced. It is important to re-establish the virus-specific and reliable immune surveillance to eradicate the reactivated virus-harboring cells. In this report, we attempted to reach this goal by using newly-developed chimeric antigen receptor (CAR)-T cell technology. To generate anti-HIV-1 CAR-T cells, we connected the single-chain variable fragment of the broadly neutralizing HIV-1-specific antibody VRC01 to a llsquo;third generation' CAR moiety, as the extracellular and intracellular domains, respectively, and subsequently transduced this into primary CD8+ T lymphocytes. We demonstrated that the resulting VC-CAR-T cells induced T cell-mediated cytolysis of cells expressing HIV-1 Env proteins and significantly inhibited HIV-1 rebound after removal of antiviral inhibitors in a viral infectivity model in cell culture which mimics the termination of the cART in clinic. Importantly, the VC-CAR-T cells also effectively induced the cytolysis of LRA-reactivated HIV-1-infected CD4+ T lymphocytes isolated from infected individuals receiving suppressive cART. Our data demonstrate that the special features of genetically engineered CAR-T cells make them a particularly suitable candidate for therapeutic application in efforts to reach a functional HIV cure.

    Importance The presence of latently infected cells remains a key obstacle to the development of a functional HIV-1 cure. Reactivation of dormant viruses is possible with latency-reversing agents, but the effectiveness of these compounds and the subsequent immune response require optimization if the eradication of HIV-1-infected cells is to be achieved. Here, we describe the use of a chimeric antigen receptor (CAR), comprising T cell activation domains and a broadly neutralizing antibody VRC01 targeting HIV-1, to treat the infected cells. T cells expressing this construct exerted specific cytotoxic activity against wild-type HIV-1-infected cells, resulting in a dramatic reduction in viral rebound in vitro, and showed persistent effectiveness against reactivated latently-infected T lymphocytes from HIV-1 patients receiving combined antiretroviral therapy. The methods used in this study constitute an improvement over existing CD4-based CAR-T technology, and offer a promising approach to HIV-1 immunotherapy.

  • ARID3B: A novel regulator of the Kaposi's sarcoma-associated herpesvirus lytic cycle [PublishAheadOfPrint]

  • KSHV is the causative agent of commonly fatal malignancies of immuno-compromised individuals, including primary effusion lymphoma (PEL) and Kaposi's sarcoma (KS). A hallmark of all herpesviruses is their biphasic lifecycle nndash; viral latency and the productive lytic cycle, and it is well established that reactivation of the KSHV lytic cycle is associated with KS pathogenesis. Therefore, a thorough appreciation of the mechanisms that govern reactivation is required to better understand disease progression. The viral protein, replication and transcription activator (RTA), is the KSHV lytic switch protein due to its ability to drive the expression of various lytic genes, leading to reactivation of the entire lytic cycle. While the mechanisms for activating lytic gene expression have received much attention, how RTA impacts on cellular function is less well understood. To address this, we developed a cell line with doxycycline-inducible RTA expression and applied SILAC-based quantitative proteomics. Using this methodology, we have identified a novel cellular protein (AT-rich interacting domain containing 3B, ARID3B) whose expression was enhanced by RTA and that relocalised to replication compartments upon lytic reactivation. We also show that siRNA knockdown or overexpression of ARID3B led to an enhancement or inhibition of lytic reactivation, respectively. Furthermore, DNA affinity and chromatin immunoprecipitation assays demonstrated that ARID3B specifically interacts with A/T-rich elements in the KSHV origin of lytic replication (oriLyt), and this was dependent on lytic cycle reactivation. Therefore, we have identified a novel cellular protein whose expression is enhanced by KSHV RTA with the ability to inhibit KSHV reactivation.

    Importance Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of fatal malignancies of immunocompromised individuals, including Kaposi's sarcoma (KS). Herpesviruses are able to establish a latent infection, where they escape immune detection by restricting viral gene expression. Importantly however, reactivation of productive viral replication (the lytic cycle) is necessary for the pathogenesis of KS. Therefore, it is important that we comprehensively understand the mechanisms that govern lytic reactivation, to better understand disease progression. In this study, we have identified a novel cellular protein (AT-rich interacting domain protein 3B, ARID3B) that we show is able to temper lytic reactivation. We showed that the master lytic switch protein, RTA, enhanced ARID3B levels, which then interacted with viral DNA in a lytic cycle dependent manner. Therefore, we have added a new factor to the list of cellular proteins that regulate the KSHV lytic cycle, which has implications for our understanding of KSHV biology.

  • Long and short isoforms of the human cytomegalovirus UL138 protein silence IE1 transcription and promote latency. [PublishAheadOfPrint]

  • The UL133-138 locus present in clinical strains of human cytomegalovirus (HCMV) encodes genes required for latency and reactivation in CD34+ hematopoietic progenitor cells and virion maturation in endothelial cells. The encoded proteins form multiple homo- and hetero-interactions and localize within secretory membranes. One of these genes, UL136, is expressed as at least five different protein isoforms with overlapping and unique functions. Here we show that another gene from this locus, UL138, also generates more than one protein isoform. A long form of UL138 (pUL138-L) initiates translation from codon 1, possesses an amino terminal signal sequence, and is a type one integral membrane protein. Here we identify a short protein isoform (pUL138-S) initiating from codon 16 that displays a similar subcellular localization as pUL138-L. Reporter, short-term transcription, and long-term virus production assays revealed that both pUL138-L and nndash;S are able to suppress major immediate early (IE) gene transcription and the generation of infectious virions in cells where HCMV latency is studied. The long form appears more potent at silencing IE1 transcription shortly after infection, while the short form seems more potent at restricting progeny virion production at later times, indicating that both isoforms of UL138 likely cooperate to promote HCMV latency.

    Importance Latency allows herpesviruses to persist for the lives of their hosts in the face of effective immune control measures for productively infected cells. Controlling latent reservoirs is an attractive antiviral approach complicated by knowledge deficits for how latently infected cells are established, maintained, and reactivated. This is especially true for betaherpesviruses. Functional consequences of HCMV UL138 protein expression during latency include repression of viral IE1 transcription and suppression of virus replication. Here we show that short and long isoforms of UL138 exist and can themselves support latency, but may do so in a temporally distinct manner. Understanding the complexity of gene expression and its impact on latency is important for considering potential antivirals targeting latent reservoirs.

  • NF-{kappa}B and IRF1 Induce Endogenous Retrovirus-K Expression via Interferon-Stimulated Response Elements in its 5 Long Terminal Repeat. [PublishAheadOfPrint]

  • Within the human genome reside thousands of endogenous retroviruses (ERV), viral fossils of ancient germ-line infections. Evidence of ERV activity has been widely observed in health and disease. Most often cited as a bystander effect of cell culture or disease states, it is unclear as to which signals control ERV transcription. Bioinformatic analysis suggests that the viral promoter of endogenous retrovirus-K (ERVK) is responsive to inflammatory transcription factors. Here, we show that one reason ERVK is upregulated in Amyotrophic Lateral Sclerosis (ALS) is because of functional interferon-stimulated response elements (ISREs) in the viral promoter. Transcription factor over-expression assays revealed independent and synergistic up-regulation of ERVK by IRF1 and NF-B isoforms. TNFaalpha; and LIGHT cytokine treatments of human astrocytes and neurons enhanced ERVK transcription and protein levels through IRF1 and NF-B binding to the ISREs. We further show that in ALS brain tissue, neuronal ERVK re-activation is associated with the nuclear translocation of IRF1 and NF-B isoforms p50 and p65. ERVK overexpression can cause motor neuron pathology in murine models. Our results implicate neuroinflammation as a key trigger of ERVK provirus reactivation in ALS. These molecular mechanisms may also extend to the pathobiology of other ERVK-associated inflammatory diseases, such as cancers, HIV infection, rheumatoid arthritis and schizophrenia.

    IMPORTANCE It has been well established that inflammatory signaling pathways in ALS converge at NF-B to promote neuronal damage. Our findings suggest that inflammation-driven IRF1 and NF-B activity promotes ERVK re-activation in neurons of the motor cortex in ALS. Thus, quenching ERVK activity through anti-retroviral or immunomodulatory regimens may hinder virus-mediated neuropathology and improve the symptoms of ALS or other ERVK-associated diseases.

  • Graf1 controls the growth of human parainfluenza virus type 2 through inactivation of RhoA signaling. [PublishAheadOfPrint]

  • Rho GTPases are involved in a variety of cellular activities and are regulated by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs). We found that the activation of Rho GTPases by lysophosphatidic acid promotes the growth of human parainfluenza virus type 2 (hPIV-2). Furthermore, hPIV-2 infection causes RhoA activation, a Rho GTPase. We hypothesized that Graf1 (also known as ARHGAP26), a GAP, regulates hPIV-2 growth by controlling RhoA signaling. Immunofluorescence analysis showed that hPIV-2 infection altered Graf1 localization from a homogenous distribution within the cytoplasm to granules. Graf1 co-localized with hPIV-2 P, NP, and L proteins. Graf1 interacts with P and V proteins via their N-terminal common region, and the C-terminal Src homology 3 domain-containing region of Graf1 is important for these interactions. In HEK293 cells constitutively expressing Graf1, hPIV-2 growth was inhibited and RhoA activation was not observed during hPIV-2 infection. In contrast, Graf1 knockdown restored the hPIV-2 growth and RhoA activation. Overexpression of hPIV-2 P and V proteins enhanced hPIV-2-induced RhoA activation. These results collectively suggested that hPIV-2 P and V proteins enhanced hPIV-2 growth by binding to Graf1, and that Graf1 inhibits the hPIV-2 growth through RhoA inactivation.

    Importance Robust growth of hPIV-2 requires Rho activation. hPIV-2 infection causes RhoA activation which is suppressed by Graf1. Graf1 co-localizes with vRNP in hPIV-2-infected cells. We found that Graf1 interacts with hPIV-2 P and V proteins. We also identified regions in these proteins which are important for this interaction. hPIV-2 P and V proteins enhanced the hPIV-2 growth via binding to Graf1, while Graf1 inhibited the hPIV-2 growth through RhoA inactivation.

  • Antigenic fingerprinting of antibody response following highly pathogenic H7N7 avian influenza virus exposure in humans: Evidence for anti-PA-X antibodies [PublishAheadOfPrint]

  • Infections with H7 highly pathogenic avian influenza (HPAI) viruses remain a major public health concern. Adaptation of low pathogenic H7N7 to highly pathogenic H7N7 in Europe in 2015 raised further alarm for a potential pandemic. In-depth understanding of antibody responses to HPAI H7 virus following infection in humans could provide important insight into virus gene expression as well as define key protective and serodiagnostic targets. Here we used whole genome phage display libraries (GFPDL) expressing peptides of 15-350 amino acids across the complete genome of HPAI H7N7-A/Netherlands/33/03 virus. The hemagglutinin (HA) antibody epitope repertoires of fifteen H7N7 exposed humans identified clear differences between individuals with no hemagglutination inhibition (HI) titers (llt; 1:10) vs. those with HI ggt;1:40. Several potentially protective H7N7 epitopes close to the HA receptor binding domain (RBD) and neuraminidase (NA) catalytic site were identified. Surface Plasmon Resonance (SPR) analysis identified a strong correlation between HA1 (but not HA2) binding antibodies and H7N7 HI titers. A proportion of HA1 binding in plasma was contributed by IgA antibodies. Antibodies against the N7 neuraminidase were less frequent but targeted sites close to the sialic acid binding site. Importantly, we identified strong antibody reactivity against PA-X, a putative virulence factor, in most H7N7 exposed individuals, providing the first evidence for in vivo expression of PA-X and its recognition by the immune system during human influenza A infection. This knowledge can help inform in the development and selection of the most effective countermeasures for prophylactic as well as therapeutic treatments of HPAI H7N7 avian influenza.

    Importance An outbreak of pathogenic H7N7 virus occurred in poultry farms in the Netherlands in 2003. Severe outcome included conjunctivitis, influenza-like illness, and one lethality. In the current study, we investigated convalescent sera from H7N7-exposed individuals using whole genome phage display library (H7N7-GFPDL) to explore the complete repertoire of post-H7N7 exposure antibodies.

    PA-X is a recently identified influenza virulence protein, generated by ribosomal frame shifting in segment 3 of influenza virus coding for PA. But PA-X expression during influenza infection in humans is unknown. We identified strong antibody reactivity against PA-X in most H7N7 exposed individuals (but not in unexposed adults), providing the first evidence for in vivo expression of PA-X and its recognition by the immune system during human infection with pathogenic H7N7 avian influenza.

  • Interrelationship of primary virus replication, level of latency and time to reactivate in the trigeminal ganglia of latently infected mice [PublishAheadOfPrint]

  • We sought to determine the possibility of an inter-relationship between primary virus replication in the eye the level of viral DNA in the trigeminal ganglia (TG) during latency and the amount of virus reactivation following ocular herpes simplex virus type 1 (HSV-1) infection. Mice were infected with virulent (McKrae) or avirulent (KOS, RE) strains of HSV-1 and virus titers in the eyes and TG during primary infection, level of viral gB DNA in TG on day 28 post-infection (PI) and virus reactivation on day 28 PI as measured by explant reactivation were calculated. Our results suggest that the avirulent strains of HSV-1 even after corneal scarification had lower virus titers in the eye, less latency in the TG, and took a longer time to reactivate compared to virulent strains of HSV-1. The time to explant reactivation of avirulent strains of HSV-1 was similar to that of the virulent LAT(-) McKrae derived mutant. The viral dose with the McKrae strains of HSV-1 affected the level of viral DNA and time to explant reactivation. Overall, our results suggest that there is no absolute correlation between primary virus titer in the eye and TG with the level of viral DNA in latent TG and time to reactivation.

    Importance Very little is known regarding the inter-relationship between primary virus replication in the eye, the level of latency in TG and the time to reactive in the mouse model. This study was designed to answers these questions. Our results point to the absence of any correlation between the level of primary virus replication with the level of viral DNA during latency and neither were an indicator of how rapidly the virus reactivated following explant TG induced reactivation.

  • Epitope-independent purification of native-like envelope trimers from diverse HIV-1 isolates [PublishAheadOfPrint]

  • Soluble forms of trimeric HIV-1 envelope glycoprotein (Env) have long been sought as immunogens and as reagents for analysis of Env structure and function. Isolation of trimers that mimic native Env, derived from diverse viruses, however, represents a major challenge. Thus far, the most promising "native-like" (NL) structures have been obtained by engineering trimer-stabilizing mutations, termed "SOSIP," into truncated Env sequences. However, the abundance of NL trimeric conformers varies among Envs, necessitating purification by monoclonal antibodies (Mabs) like PGT145, which target specific epitopes. To surmount this inherent limitation, we developed an approach that uses lectin affinity, ion exchange, hydrophobic interaction, and size exclusion chromatography to isolate NL trimers from non-native Env species. We validated this method with SOSIP trimers from HIV-1 clades A and B. Analyses by SEC, blue native- and SDS-PAGE, and dynamic light scattering indicated that the resulting material was homogeneous (ggt;95% pure), fully-cleaved, and of the appropriate molecular weight and size for SOSIP trimers. Negative stain-electron microscopy further demonstrated that our preparations were composed of NL trimeric structures. By hydrogen/deuterium-exchange mass spectrometry, these HIC-pure trimers exhibited structural organization consistent with NL trimers and inconsistent with profiles seen in non-native Envs. Screened for antigenicity, some Envs like BS208.b1 and KNH1144 T162A, did not present the glycan/quaternary structure-dependent epitope for PGT145 binding, suggesting that these SOSIPs would be challenging to isolate by existing Mab affinity methods. By selecting based on biochemical rather than antigenic properties, our method offers an epitope-independent alternative to Mabs for isolation of NL Env trimers.

    IMPORTANCE The production and purification of diverse soluble Env trimers that maintain native-like (NL) structure present technical challenges that must be overcome in order to advance vaccine development and provide reagents for HIV research. Low levels of NL trimer expression amid heteroeneous Env conformers, even with the addition of stabilizing mutations, has presented a major challenge. In addition, it has been difficult to separate the NL trimers from these heterogeneous mixtures. While Mabs with specificity for quaternary NL trimer epitopes have provided one approach to purifying the desirable species, such methods are dependent on the Env displaying the proper epitope. In addition, Mab affinity chromatography can be expensive, the necessary Mab may be in limited supply, and large-scale purification may not be feasible. Our method based on biochemical separation techniques offers an epitope-independent approach to purification of NL trimers with general application to diverse Envs.

  • Identification of residues that affect oligomerization and/or enzymatic activity of influenza virus H5N1 neuraminidase proteins [PublishAheadOfPrint]

  • Influenza A virus attachment to and release from sialoside receptors is determined by the balance between hemagglutinin and neuraminidase (NA). The molecular determinants that mediate the specificity and activity of NA are still poorly understood. In this study, we aimed to design the optimal recombinant soluble NA protein to identify residues that affect NA enzymatic activity. To this end, recombinant soluble versions of four different NA proteins from H5N1 viruses were compared with their full-length counterparts. The soluble NA ectodomains were fused to three commonly used tetramerization domains. Our results indicate that the particular oligomerization domain used does not affect the Km value, but may affect the specific enzymatic activity. This particularly holds true when the stalk domain is included and for NA ectodomains that display a low intrinsic ability to oligomerize. NA ectodomains extended with a Tetrabrachion domain, which forms a nearly parallel four-helix bundle, better mimicked the enzymatic properties of full-length proteins than when other coiled coil tetramerization domains were used, which probably distort the stalk domain. Comparison of different NA proteins and mutagenic analysis of recombinant soluble versions thereof resulted in the identification of several residues that affected oligomerization of the NA head domain (position 95), and therefore the specific activity, or sialic acid binding affinity (Km value; positions 252 and 347). This study demonstrates the potential of using recombinant soluble NA proteins to reveal determinants of NA assembly and enzymatic activity.

    IMPORTANCE The IAV hemagglutinin (HA) and neuraminidase (NA) glycoproteins are important determinants of host tropism and pathogenicity. However, NA is relatively understudied compared to HA. Analysis of soluble versions of these glycoproteins is an attractive way to study their activities as they are easily purified from cell culture media and applied in downstream assays. In the present study, we analyzed the enzymatic activity of different NA ectodomains with three commonly used tetramerization domains and compared them with full-length NA proteins. By performing a mutagenic analysis, we identified several residues that affected NA assembly, activity and/or substrate binding. In addition, our results indicate that the design of the recombinant soluble NA protein including the particular tetramerization domain is an important determinant for maintaining the enzymatic properties within the head domain. NA ectodomains extended with a Tetrabrachion domain better mimicked that of full-length proteins compared to the other tetramerization domains used.

  • In vitro studies show that sequence variability contributes to marked variation in Hepatitis B virus replication, protein expression and function observed across genotypes. [PublishAheadOfPrint]

  • The hepatitis B virus (HBV) exists as 9 major genotypes (A to I), one minor strain (designated J) and multiple subtypes. Marked differences in HBV natural history, disease progression and treatment response are exhibited by many of these genotypes and subtypes. For example, HBV genotype C is associated with later HBeAg seroconversion and high rates of liver cancer compared to other HBV genotypes, whereas genotype A2 is rarely associated with HBeAg negative disease or liver cancer. The reasons for these and other differences in HBV natural history are yet to be determined but could in part be due to sequence differences in the HBV genome that alter replicative capacity and / or gene expression. Direct comparative studies on HBV replication and protein expression have been limited to date, due largely to the absence of infectious HBV cDNA clones for each of the HBV genotypes present in the same genetic arrangement. We have produced replication competent infectious cDNA clones of the most common subtypes of genotypes A to D, namely A2, B2, C2, D3 and the minor strain J and compared their HBV replication phenotype using transient transfection models. We identified striking differences in HBV replicative capacity as well as hepatitis B e antigen (HBeAg) and surface (HBsAg) protein expression across genotypes, which may in part be due to sequence variability in regulatory regions of the HBV genome. Functional analysis showed that sequence differences in the major upstream regulatory region across genotypes impacted promoter activity.

    Importance There have been very few studies directly comparing the replication phenotype of different HBV genotypes, for which there are marked differences in natural history and disease progression worldwide. We have generated replication competent 1.3 mer cDNA clones of the major genotypes A2, B2, C2, D3, as well as a recently identified strain J, and identified striking differences in replicative capacity and protein expression that may contribute to some of the observed differences in HBV natural history observed globally.

  • Baculovirus FP25K Localization: Role of the Coiled-Coil Domain [PublishAheadOfPrint]

  • Two types of viruses are produced during the baculovirus life cycle: budded virus (BV) and occlusion-derived virus (ODV). A particular baculovirus protein, FP25K, is involved in the switch from BV to ODV production. Previously, FP25K from the model alphabaculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), was shown to traffic ODV envelope proteins. However, FP25K localization and the domains involved are inconclusive. Here we used a quantitative approach to study FP25K subcellular localization during infection using an AcMNPV bacmid virus that produces a functional AcMNPV FP25K-GFP fusion protein. During cell infection, FP25K-GFP localized primarily to the cytoplasm, particularly amorphous structures, with a small fraction in the nucleus. To investigate the sequences involved in FP25K localization, an alignment of baculovirus FP25K sequences revealed that the N-terminal putative coiled-coil domain is present in all alphabaculoviruses, but absent in betabaculoviruses. Structural prediction indicated strong relatedness of AcMNPV FP25K to LINE-1 ORF1p, which contains an N-terminal coiled-coil domain responsible for cytoplasmic retention. Point mutations and deletions of this domain lead to a change in AcMNPV FP25K localization from cytoplasmic to nuclear. The coiled-coil and C-terminal deletion viruses increased BV production. Furthermore, a betabaculovirus FP25K, lacking this N-terminal coiled-coil domain, localized predominantly to the nucleus and exhibited increased BV production. These data suggest that acquisition of this N-terminal coiled-coil domain in FP25K is important to the evolution of alphabaculoviruses. Moreover, with the divergence of pre-occlusion nuclear membrane breakdown in betabaculoviruses and membrane integrity in alphabaculoviruses, this domain represents an alphabaculovirus adaptation for nuclear trafficking of occlusion-associated proteins.

    Importance Baculovirus infection produces two forms of viruses; BV and ODV. Manufacturing ODV involves trafficking of envelope proteins to the inner nuclear membrane mediated partly through the protein FP25K. Since FP25K is present in alpha-, beta-, and gammabaculoviruses, it is uncertain if this trafficking function is conserved. In this study, we looked at alpha- and betabaculovirus FP25K trafficking by its localization. Alphabaculovirus FP25K localized primarily to the cytoplasm, whereas betabaculovirus FP25K localized to the nucleus. We found that an N-terminal coiled-coil domain present in all alphabaculovirus FP25K, but absent in betabaculovirus FP25K, was critical to alphabaculovirus FP25K cytoplasmic localization. We believe this represents an evolutionary process that partly led to the gain-of-function of this N-terminal coiled-coil domain in alphabaculovirus FP25K to aid in nuclear trafficking of occlusion-associated proteins. Due to betabaculovirus breakdown of the nuclear membrane before occlusion, this function is not needed and the domain was lost or never acquired.

  • Benign Rabbit Caliciviruses Exhibit Similar Evolutionary Dynamics to their Virulent Relatives [PublishAheadOfPrint]

  • Two closely related caliciviruses co-circulate in Australia: rabbit hemorrhagic disease virus (RHDV) and rabbit calicivirus Australia 1 (RCV-A1). RCV-A1 causes benign enteric infections in the European rabbit (Oryctolagus cuniculus) in Australia and New Zealand, while its close relative RHDV causes a highly pathogenic infection of the liver in the same host. The comparison of these viruses provides important information on the nature and trajectory of virulence evolution, particularly as highly virulent strains of RHDV may have evolved from non-pathogenic ancestors such as RCV-A1. To determine the evolution of RCV-A1 we sequenced the full length genomes of 44 RCV-A1 samples isolated from healthy rabbits and compared key evolutionary parameters to those of its virulent relative, RHDV. Despite their marked differences in pathogenicity and tissue tropism, RCV-A1 and RHDV have evolved in a very similar manner. Both viruses have evolved at broadly similar rates, suggesting that their dynamics are largely shaped by high background mutation rates, and both exhibit occasional recombination and an evolutionary environment dominated by purifying selection. In addition, our comparative analysis revealed that there have been multiple changes in both virulence and tissue tropism in the evolutionary history of these and related viruses. Finally, these new genomic data suggest that RCV-A1 was either introduced into Australia after the introduction of myxoma virus as a biocontrol agent in 1950, or that there was drastic reduction of the rabbit population, and hence of RCV-A1 genetic diversity, perhaps co-incident with the emergence of myxoma virus.

    IMPORTANCE The comparison of closely related viruses that differ profoundly in propensity to cause disease in their hosts offers a powerful opportunity to reveal the causes of changes in virulence, and to study how such changes alter the evolutionary dynamics of these pathogens. Herein, we describe such a novel comparison involving two closely related RNA viruses that co-circulate in Australia; the highly virulent rabbit hemorrhagic disease virus (RHDV) and the non-pathogenic rabbit calicivirus Australia 1 (RCV-A1). Both viruses infect the European rabbit, but they differ in virulence, tissue tropism and mechanisms of transmission. Surprisingly, and despite these fundamental differences, RCV-A1 and RHDV have evolved at very similar (high) rates and with strong purifying selection. Furthermore, candidate key mutations were identified that may play a role in virulence and/or tissue tropism and therefore warrant further investigation.

  • Influenza A virus infection in pigs attracts multifunctional and cross-reactive T cells to the lung [PublishAheadOfPrint]

  • Pigs are natural hosts for influenza A viruses and play a critical role in influenza epidemiology. However, little is known about their influenza-evoked T-cell response. We performed a thorough analysis of both the local and systemic T-cell response in influenza-infected pigs, addressing kinetics and phenotype, as well as multifunctionality (IFN-, TNF-aalpha;, IL-2) and cross-reactivity. A total of 31 pigs were intratracheally infected with an H1N2 swine influenza A virus (FLUAVsw) and consecutively euthanized. Lung, tracheobronchial lymph nodes and blood were sampled during the first 15 days post infection (pi) and at six weeks pi. Ex vivo flow cytometry of lung lymphocytes revealed an increase in proliferating (Ki-67+) CD8+ T cells with an early effector phenotype (perforin+CD27+) at day 6 pi. Low frequencies of influenza-specific IFN- producing CD4+ and CD8+ T cells could be detected in the lung as early as 4 days pi. On consecutive days, influenza-specific CD4+ and CD8+ T cells produced mainly IFN- and/or TNF-aalpha;, reaching peak frequencies around day 9 pi, which were up to 30-fold higher in the lung compared with tracheobronchial lymph nodes or blood. Six weeks pi, CD4+ and CD8+ memory T cells had accumulated in lung tissue. These cells showed diverse cytokine profiles and in vitro reactivity against heterologous influenza strains, all of which supports their potential to combat heterologous influenza infections in pigs.

    IMPORTANCE Pigs are not only a suitable large-animal model for human influenza infection and vaccine development, but also play a central role in the emergence of new pandemic strains. Although promising candidate universal vaccines are tested in pigs, and local T cells are the major correlate of heterologous control, detailed and targeted analyses of T-cell responses at the site of infection are scarce. With the present study, we provide the first detailed characterization of magnitude, kinetics, and phenotype of specific T cells recruited to the lungs of influenza-infected pigs, and could demonstrate multifunctionality, cross-reactivity, and memory formation of these cells. This, and ensuing work in the pig, will strengthen the position of this species as a large-animal model for human influenza infection and will immediately benefit vaccine development for improved control of influenza infections in pigs.

  • Avian leukosis virus activation of an antisense RNA upstream of TERT in B-cell lymphomas [PublishAheadOfPrint]

  • Avian leukosis virus (ALV) induces tumors by integrating its proviral DNA into the chicken genome and altering expression of nearby genes via strong promoter and enhancer elements. Viral integration sites that contribute to oncogenesis are selected in tumor cells. Deep sequencing analysis of B-cell lymphoma DNA confirmed that the telomerase reverse transcriptase (TERT) promoter is a common ALV integration target. Twenty-six unique proviral integration sites were mapped between 46 and 3552 nt upstream of the TERT transcription start site, predominantly in the opposite transcriptional orientation of TERT. RNA-seq analysis of normal bursa revealed a transcribed region upstream of TERT in the opposite orientation, suggesting the TERT promoter is bidirectional. This transcript appears to be an uncharacterized antisense RNA. We have previously shown that TERT expression is up regulated in tumors with integrations in the TERT promoter region. We now report that the viral promoter drives expression of a chimeric transcript, containing viral sequences spliced to exons 4 through 7 of this antisense RNA. Clonal expansion of cells with ALV integrations driving over expression of this TERT antisense RNA suggest it may have a role in tumorigenesis.

    Importance: These data suggest that ALV integrations in the TERT promoter region drive overexpression of a novel antisense RNA and contribute to the development of lymphomas.

  • Japanese Macaque Rhadinovirus Encodes a Viral microRNA Mimic of the miR-17 Family [PublishAheadOfPrint]

  • Japanese macaque rhadinovirus (JMRV) is a novel, gamma-2 herpesvirus that was recently isolated from Japanese macaques (JM) with inflammatory demyelinating encephalomyelitis (JME). JME is a spontaneous and chronic disease with clinical characteristics and immuno-histopathology comparable to Multiple Sclerosis in humans. Little is known about the molecular biology of JMRV. Here, we sought to identify and characterize the small RNAs expressed during lytic JMRV infection using deep sequencing. 15 novel viral microRNAs (miRNAs) were identified in JMRV-infected fibroblasts, all of which were readily detectable by 24 hrs post infection and accumulated to high levels by 72 hrs. Sequence comparisons to the human Kaposi's sarcoma-associated herpesvirus (KSHV) miRNAs revealed several viral miRNA homologs. To functionally characterize JMRV miRNAs, we screened for their effects on nuclear factor kappa B (NF-kB) signaling in the presence of two pro-inflammatory cytokines, tumor necrosis factor alpha (TNFa) or interleukin 1 beta (IL-1b). Multiple JMRV miRNAs suppressed cytokine-induced NF-kB activation. One of these miRNAs, miR-J8, has seed sequence homology to members of the cellular miR-17/20/106 and miR-373 families, which are key players in cell cycle regulation as well as inflammation. Using reporters, we show that miR-J8 can target 3rrsquo; UTRs with miR-17-5p or miR-20a cognate sites. Our studies implicate JMRV miRNAs in suppression of innate antiviral immune responses, which is an emerging feature of many viral miRNAs.

    Importance Gamma-herpesviruses are associated with multiple diseases linked to immunosuppression and inflammation, including AIDS-related cancers and autoimmune diseases. JMRV is a recently identified herpesvirus that has been linked to JME, an inflammatory demyelinating disease in Japanese macaques that mimics MS. Few large animal models exist for gamma-herpesvirus-associated pathogenesis. Here, we provide the first experimental evidence of JMRV miRNAs in vitro and demonstrate that one of these viral miRNAs can mimic activity of the cellular miR-17/20/106 family. Our work sheds unique insight into the roles of viral miRNAs during rhadinovirus infection and provides an important step towards understanding viral miRNA function in a non-human primate model system.

  • A novel acylguanidine-based inhibitor of HIV-1 [PublishAheadOfPrint]

  • The emergence of transmissible HIV-1 strains with resistance to antiretroviral drugs highlights a continual need for new therapies. Here we describe a novel acylguanidine-containing compound, 1-(2-(azepan-1-yl)nicotinoyl)guanidine (or SM111), that inhibits in vitro replication of HIV-1, including strains resistant to licensed protease, reverse transcriptase and integrase inhibitors, without major cellular toxicity. At inhibitory concentrations, intracellular p24Gag production was unaffected, but virion release (measured as extracellular p24Gag) was reduced and virion infectivity was substantially impaired, suggesting that SM111 acts at a late stage of viral replication. SM111-mediated inhibition of HIV-1 was partially overcome by a Vpu I17R mutation alone or a Vpu W22* truncation in combination with Env N136Y. These mutations enhanced virion infectivity and Env expression on the surface of infected cells in the absence and presence of SM111, but also impaired Vpu's ability to downregulate CD4 and BST2/tetherin. Taken together, our results support acylguanidines as a class of HIV-1 inhibitors with a distinct mechanism of action compared to licensed antiretrovirals. Further research on SM111 and similar compounds may help to elucidate knowledge gaps related to Vpu's role in promoting viral egress and infectivity.

    Importance New inhibitors of HIV-1 replication may be useful as therapeutics to counteract drug resistance and as reagents to perform more detailed studies of viral pathogenesis. SM111 is a small molecule that blocks the replication of wild type and drug-resistant HIV-1 strains by impairing viral release and substantially reducing virion infectivity, most likely through its ability to prevent Env expression at the infected cell surface. Partial resistance to SM111 is mediated by mutations in Vpu and/or Env, suggesting that the compound affects host/viral protein interactions that are important during viral egress. Further characterization of SM111 and similar compounds may allow more detailed pharmacological studies of HIV-1 egress and provide opportunities to develop new treatments for HIV-1.

  • Effects of membrane charge and order on membrane binding of the retroviral structural protein Gag [PublishAheadOfPrint]

  • The retroviral structural protein Gag binds to the inner leaflet of the plasma membrane (PM), and many cellular proteins do so as well. We used Rous Sarcoma Virus (RSV) Gag together with membrane sensors to study the principles governing peripheral protein membrane binding, including electrostatics, specific recognition of phospholipid headgroups, sensitivity to phospholipid acyl chain compositions, preference for membrane order, and protein multimerization. We used an in vitro liposome-pelleting assay to test protein membrane binding properties of Gag, the well-characterized MARCKS peptide, a series of fluorescent electrostatic sensor proteins (mNG-KRn), and the specific phosphatidylserine (PS) binding protein Evectin2. RSV Gag and mNG-KRn bound well to membranes with saturated and unsaturated acyl chains, whereas the MARCKS peptide and Evectin2 preferentially bound to membranes with unsaturated acyl chains. To further discriminate whether the primary driving force for Gag membrane binding is electrostatic interactions or preference for membrane order, we measured protein binding to giant unilamellar vesicles (GUVs) containing the same PS concentration in both disordered (Ld) and ordered (Lo) phases. RSV Gag and mNG-KRn membrane association followed membrane charge, independent of membrane order. Consistent with pelleting data, the MARCKS peptide showed preference for the Ld domain. Surprisingly, the PS sensor Evectin2 bound to the PS-rich Ld domain with ten-fold greater affinity than to the PS-rich Lo domain. In summary, we found that RSV Gag shows no preference for membrane order, while proteins with reported membrane penetrating domains show preference for disordered membranes.

    IMPORTANCE Retroviral particles assemble on the PM and bud from infected cells. Our understanding of how Gag interacts with the PM and how different membrane properties contribute to overall Gag assembly is incomplete. This study examined how membrane charge and membrane order influence Gag membrane association. Consistent with previous work on RSV Gag, we report here that electrostatic interactions provide the primary driving force for RSV Gag membrane association. Using phase-separated GUVs with known lipid composition of the Ld and Lo phases, we demonstrate for the first time that RSV Gag is sensitive to membrane charge but not membrane order. In contrast, the cellular protein domain MARCKS and the PS sensor Evectin2 show preference for disordered membranes. We also demonstrate how to define GUV phase composition, which could serve as a tool in future studies of protein membrane interactions.

  • Human IFIT1 inhibits mRNA translation of rubulaviruses but not other members of the Paramyxoviridae family. [PublishAheadOfPrint]

  • We have previously shown that IFIT1 is primarily responsible for the antiviral action of interferon (IFN) alpha/beta against parainfluenza virus (PIV) type 5, selectively inhibiting the translation of PIV5 mRNAs. Here we report that whilst PIV2, PIV5 and mumps virus (MuV) are sensitive to IFIT1, non-rubulavirus members of the paramyxoviridae such as PIV3, Sendai virus (SeV) and canine distemper virus (CDV) are resistant. The IFIT1-sensitivity of PIV5 was not rescued by co-infection with an IFIT1-resistant virus (PIV3), demonstrating that PIV3 does not specifically inhibit the antiviral activity of IFIT1 and that the inhibition of PIV5 mRNAs is regulated by cis-acting elements. We developed an in vitro translation system using purified human IFIT1 to further investigate the mechanism of action of IFIT1. Whilst the translation of PIV2, PIV5 and MuV mRNAs were directly inhibited by IFIT1, the translation of PIV3, SeV and CDV mRNAs were not. Using purified human mRNA capping enzymes we show biochemically that efficient inhibition by IFIT1 is dependent upon a 5rrsquo; guanosine nucleoside cap (which need not be N7-methylated) and that this sensitivity is partly abrogated by 2rrsquo; O methylation of the cap 1 ribose. Intriguingly, PIV5 M mRNA, in contrast to NP mRNA, remained sensitive to inhibition by IFIT1 following in vitro 2rrsquo; O methylation, suggesting that other structural features of mRNAs may influence their sensitivity to IFIT1. Thus, surprisingly, the viral polymerases (which have 2rrsquo; -O-methyltransferase activity) of rubulaviruses do not protect these viruses from inhibition by IFIT1. Possible biological consequences of this are discussed.

    Importance Paramyxoviruses cause a wide variety of diseases and yet most of their genes encode for structural proteins and proteins involved in their replication cycle. Thus the amount of genetic information that determines the type of disease paramyxoviruses cause is relatively small. One factor that will influence disease outcomes is how they interact with innate host cell defences, including the interferon (IFN) system. Here we show that different paramyxoviruses interact in distinct ways with cells in a pre-existing IFN-induced antiviral state. Strikingly, all the rubulaviruses tested were sensitive to the antiviral action of ISG56/IFIT1, whilst all the other paramyxoviruses tested were resistant. We developed novel in vitro biochemical assays to investigate the mechanism of action of IFIT1, demonstrating that the mRNAs of rubulaviruses can be directly inhibited by IFIT1 and that this is at least partially because their mRNAs are not correctly methylated.

  • Selection pressure in the human adenovirus fiber knob drives cell specificity in epidemic keratoconjunctivitis [PublishAheadOfPrint]

  • Human adenoviruses (HAdVs) contain seven species (A-G), each associated with specific disease conditions. Among these, HAdV-D includes those viruses associated with epidemic keratoconjunctivitis (EKC), a severe ocular surface infection. The reasons for corneal tropism for some but not all HAdV-Ds are not known. The fiber protein is a major capsid protein; its C-terminal "knob" mediates binding with host cell receptors to facilitate subsequent viral entry. In a comprehensive phylogenetic analysis of HAdV-D capsid genes, fiber knob gene sequences of HAdV-D types associated with EKC formed a unique clade. By proteotyping analysis, EKC virus-associated fiber knobs were uniquely shared. Comparative structural modeling showed no distinct variations in fiber knobs of EKC types, but did show variation among HAdV-Ds in a region overlapping with the known CD46 binding site in HAdV species B. We also found signature amino acid positions that distinguish EKC from non-EKC types, and by in-vitro studies show that corneal epithelial cell tropism can be predicted by the presence of a lysine or alanine at residue 240. This same amino acid residue in EKC viruses shows evidence for positive selection, suggesting evolutionary pressure enhances fitness in corneal infection, and may be a molecular determinant in EKC pathogenesis.

    Importance: Viruses adapt various survival strategies to gain entry into target host cells. Human adenovirus (HAdV) types are associated with distinct disease conditions, yet evidence for connections between genotype and cellular tropism is generally lacking. Here, we provide a structural and evolutionary basis for the association between specific genotypes within HAdV species D and epidemic keratoconjunctivitis, a severe ocular surface infection. We find that HAdV-D fiber genes of major EKC pathogens, specifically the fiber knob region, share a distinct phylogenetic clade. Deeper analysis of the fiber gene revealed that evolutionary pressure at crucial amino acid sites have a significant impact on its structural conformation, likely important in host cell binding and entry. Specific amino acids in hotspot residues provide a link to ocular cell tropism and possibly to corneal pathogenesis.

  • Activation of the Chicken Anemia Virus Apoptin protein by Chk1/2 phosphorylation is required for apoptotic activity and efficient viral replication [PublishAheadOfPrint]

  • The chicken anemia virus is a single-stranded circular DNA virus that encodes 3 genes, of which the most studied is VP3, also known as Apoptin. This protein has been demonstrated to specifically kill transformed cells while leaving normal cells unharmed in a manner that is independent of p53 status. Although the mechanistic basis for this differential activity is unclear, it is evident that the subcellular localization of this protein is important for this difference. In normal cells, Apoptin exists in filamentous networks in the cytoplasm, whereas in transformed cells Apoptin is present in the nucleus and appears as distinct foci. We have previously demonstrated that DNA damage signaling through the ATM pathway induces the translocation of Apoptin from the cytoplasm to the nucleus where it induces apoptosis. We found that Apoptin contains four checkpoint kinase consensus sites and that mutation of either threonine 56 or 61 to alanine restricts Apoptin to the cytoplasm. Furthermore, treatment of tumor cells expressing Apoptin with inhibitors of Chk1 and Chk2 causes Apoptin to localize to the cytoplasm. Importantly, silencing of Chk2 rescues cancer cells from the cytotoxic effects of Apoptin. Finally, treatment of virus producing cells with Chk inhibitor protects them from virus-mediated toxicity and reduces the titer of progeny virus. Taken together, our results indicate that Apoptin is a sensor of DNA damage signaling through the ATM-Chk2 pathway, which induces it to migrate to the nucleus during viral replication.

    Importance The chicken anemia virus (CAV) protein Apoptin is known to induce tumor cell specific death when expressed. Therefore, understanding its regulation and mechanism of action could reveal new insights into tumor cell biology. We have determined that Checkpoint kinase 1 and 2 signaling is important for Apoptin regulation and is a likely feature of both tumor cells and host cells producing virus progeny. Inhibition of checkpoint signaling prevents Apoptin toxicity in tumor cells and attenuates CAV replication, suggesting it may be a future target for anti-viral therapy.

  • Inhibition of the membrane attack complex by dengue NS1 through interaction with vitronectin and terminal complement proteins [PublishAheadOfPrint]

  • Dengue virus (DENV) infects millions of people worldwide and is a major public health problem. DENV nonstructural protein 1 (NS1) is a conserved glycoprotein that associates with membranes and is also secreted into the plasma of DENV-infected patients. The present study describes a novel mechanism by which NS1 inhibits the terminal complement pathway. First, we identified the terminal complement regulator vitronectin (VN) as a novel DENV2 NS1-binding partner using the yeast two-hybrid system. This interaction was further assessed by ELISA and SPR assays. The NS1-VN complex was also detected in plasma from DENV-infected patients, suggesting that this interaction occurs during dengue infection. We also demonstrated that DENV2 NS1 protein either by itself or by interacting with VN hinders the formation of the membrane attack complex (MAC) and C9 polymerization. Finally, we showed that DENV2, West Nile virus (WNV), and Zika virus (ZIKV) NS1 produced in mammalian cells inhibited C9 polymerization. Taken together, our results points to a role for NS1 as a terminal pathway inhibitor of the complement system.

    Importance Dengue is the most important arthropod-borne viral disease nowadays and is caused by dengue virus (DENV). The flavivirus NS1 glycoprotein has been functionally characterized as a complement evasion protein that can attenuate the activation of the classical, the lectin and the alternative pathways. The present study describes a novel mechanism by which DENV NS1 inhibits the terminal complement pathway. We identified the terminal complement regulator vitronectin (VN) as a novel DENV NS1-binding partner, and its complex was detected in plasma from DENV-infected patients, suggesting that this interaction occurs during dengue infection. We also demonstrated that the NS1-VN complex inhibited the membrane attack complex (MAC) formation thus interfering with the complement terminal pathway. Interestingly, NS1 itself also inhibited MAC activity, suggesting a direct role in inhibition of this protein in this process. Our findings imply a role for NS1 as a terminal pathway inhibitor of the complement system.

  • B Virus (Macacine Herpesvirus 1) Divergence: Variations in Glycoprotein D from Clinical and Laboratory Isolates Diversify Virus Entry Strategies [PublishAheadOfPrint]

  • B virus (Macacine herpesvirus 1) can cause deadly zoonotic disease in humans. Molecular mechanisms of B virus cell entry are poorly understood both in macaques and humans. Here we investigated the abilities of clinical B virus isolates to use entry receptors of herpes simplex viruses (HSV). We showed that resistant B78H1 cells became susceptible to B virus clinical strains upon expression of either human nectin-2 or nectin-1. Antibody against glycoprotein D (gD) protected these nectin-bearing cells from B virus infection and a gD-negative recombinant B virus failed to enter these cells indicating that the nectin-mediated B virus entry depends on gD. We observed that infectivity of B virus isolates with a single-amino-acid substitution D122N in the IgV-core of gD ectodomain was impaired on nectin-1-bearing cells. Computational homology-based modeling of the B virus gD:nectin-1 complex revealed conformational differences between the structures of gD-122N and gD-122D variants that impacted the gD:nectin-1 protein-protein interface and binding affinity. Unlike HSV, B virus clinical strains were unable to use HVEM as a receptor regardless of conservation of the gD- amino acid residues essential for HSV-1 entry via HVEM. Based on the model of the B virus gD-HVEM interface we predict that the residues R7, R11, and G15 are largely responsible for the inability of B virus to utilize HVEM for entry. The ability of B virus to enter cells of a human host by using a combination of receptors distinct from HSV-1 or HSV-2 suggests a possible mechanism of enhanced neuropathogenicity associated with zoonotic infections.

    IMPORTANCE B virus causes brainstem destruction in zoonotically infected humans in the absence of timely diagnosis and intervention. Nectins are cell adhesion molecules that are widely expressed in human tissues, including neurons and neuronal synapses. Here we report that human nectin-2 is a target receptor for B virus entry in addition to reported human nectin-1. Similar to B virus lab strain, B virus clinical strains can effectively use both nectin-1 and nectin-2 as cellular receptors for entry into human cells. But, unlike HSV-1 and HSV-2, none of the clinical strains uses a HVEM-mediated entry pathway. Ultimately, these differences between B virus and HSV-1 and HSV-2 may provide insight into the neuropathogencity of B virus during zoonotic infections.

  • RIG-I Mediated STING Up-Regulation Restricts HSV-1 Infection [PublishAheadOfPrint]

  • STING has emerged in recent years as a key player in orchestrating innate immune responses to cytosolic DNA and RNA derived from pathogens. However, the regulation of STING still remains poorly defined. In the present study, we investigated the mechanism of the regulation of STING expression in relation to the RIG-I pathway. Our data show that signalling through RIG-I induces STING expression at both the transcriptional and protein levels in various cell types. STING induction by the RIG-I agonist 5rrsquo; pppRNA was recognized as a delayed event resulting from an autocrine/paracrine mechanism. Indeed, co-treatment with TNFaalpha; and IFNaalpha; was found to have a synergistic effect on the regulation of STING expression, and could be potently decreased by impairing NF-B and/or STAT1/2 signalling. STING induction significantly contributed to sustaining the immune signalling cascade, following 5rrsquo; pppRNA treatment. Physiologically, this cross-talk between the RNA and DNA sensing pathways allowed 5rrsquo; pppRNA to efficiently block infection by herpes simplex virus type 1 (HSV-1) both in vitro and in vivo in a STING-dependent fashion. These observations demonstrate that STING induction by RIG-I signalling through the NF-B and STAT1/2 cascades, is essential for RIG-I agonist-mediated HSV-1 restriction.

    IMPORTANCE The innate immune system represents the first line of defense against invading pathogens. The dysregulation of this system can result in failure to combat pathogens, inflammation, and autoimmune diseases. Thus, precise regulation at each level of the innate immune system is crucial. Recently, a number of studies have established STING as a central molecule in the innate immune response to cytosolic DNA and RNA derived from pathogens. Here, we describe the regulation of STING via RIG-I mediated innate immune sensing. We found that STING is synergistically induced via proinflammatory and antiviral cytokine cascades. In addition, we showed that in vivo protection against the herpes simplex virus type 1 (HSV-1) by a RIG-I agonist required STING. Our study provides new insights into the cross-talk between DNA and RNA pathogen sensing systems via the control of STING.

  • In vivo conditions enable IFNAR-independent type I interferon production by peritoneal CD11b+ cells upon Thogoto virus infection [PublishAheadOfPrint]

  • Type I interferons (IFNs) crucially contribute to host survival upon viral infections. Robust expression of type I IFNs (IFN-aalpha;/bbeta;) and induction of an antiviral state critically depend on amplification of the IFN signal via the type I IFN receptor (IFNAR): A small amount of type I IFN produced early upon virus infection binds the IFNAR, and activates a self-enhancing positive feedback loop, resulting in induction of large, protective amounts of IFN-aalpha;. Unexpectedly, we found robust, systemic IFN-aalpha; expression upon infection with the orthomyxovirus Thogoto virus (THOV) in IFNAR knock-out mice. The IFNAR-independent IFN-aalpha; production required in vivo conditions within infected animals and was not achieved during in vitro infection. Using replication incompetent THOV-derived virus-like particles we demonstrate that IFNAR-independent type I IFN induction depends on viral polymerase activity but is largely independent of viral replication. To uncover the cell type responsible for this effect, we used type I IFN reporter mice and identified CD11b+F4/80+ myeloid cells within the peritoneal cavity of infected animals as the main source of IFNAR-independent type I IFN, corresponding to the particular tropism of THOV for this cell type.

    Importance Type I IFNs are crucial for the survival of a host upon most viral infections and, moreover, they shape subsequent adaptive immune responses. Production of protective amounts of type I IFN critically depends on the positive feedback amplification via the IFNAR. Unexpectedly, we observed robust IFNAR-independent type I IFN expression upon THOV infection and unraveled molecular mechanisms as well as the tissue and cell type involved. Our data indicate that the host can effectively use alternative pathways to induce type I IFN responses if the classical feedback amplification is not available. Understanding how type I IFN can be produced in large amounts independently of the IFNAR-dependent enhancement, will identify mechanisms which might contribute to novel therapeutic strategies to fight viral pathogens.

  • Heat Shock Protein 70 family members interact with Crimean-Congo hemorrhagic fever virus and Hazara virus nucleocapsid proteins and perform a functional role in the nairovirus replication cycle [PublishAheadOfPrint]

  • The Nairovirus genus of the Bunyaviridae family contains serious human and animal pathogens classified within multiple serogroups and species. Of these serogroups, the Crimean-Congo hemorrhagic fever virus (CCHFV) serogroup comprises sole members CCHFV and Hazara virus (HAZV). CCHFV is an emerging zoonotic virus that causes often-fatal hemorrhagic fever in infected humans for which preventative or therapeutic strategies are not available. In contrast HAZV is non-pathogenic to humans, and thus represents an excellent model to study aspects of CCHFV biology under more accessible biological containment. The three RNA segments that form the nairovirus genome are encapsidated by the viral nucleocapsid protein (N) to form ribonucleoprotein (RNP) complexes that are substrates for RNA synthesis and packaging into virus particles. We used quantitative proteomics to identify cellular interaction partners of CCHFV N, and identified robust interactions with cellular chaperones. These interactions were validated using immunological methods, and the specific interaction between native CCHFV N and cellular chaperones of the HSP70 family was confirmed during live CCHFV infection. Using infectious HAZV we showed for the first time that the nairovirus N-HSP70 association was maintained within both infected cells and virus particles, where N is assembled as RNPs. Reduction of active HSP70 levels in cells using small molecule inhibitors significantly reduced HAZV titres, and a model for chaperone function in the context of high genetic variability is proposed. These results suggest chaperones of the HSP70 family are required for nairovirus replication and thus represent a genetically stable cellular therapeutic target for preventing nairovirus-mediated disease.

    Importance Nairoviruses are a group of human and animal viruses that are transmitted by ticks and associated with serious or fatal disease. One member is Crimean-Congo hemorrhagic fever virus (CCHFV) that is responsible for fatal human disease and is recognized as an emerging threat within Europe in response to climate change. No preventative or therapeutic strategies against nairovirus-mediated disease are currently available. Here we show that the N protein of CCHFV and the related Hazara virus interact with a cellular protein, HSP70, during both the intracellular and extracellular stages of the virus lifecycle. The use of inhibitors that block HSP70 function reduces virus titres by up to 1000-fold suggesting this interaction is important within the context of the nairovirus life cycle and may represent a potent target for anti-nairoviral therapies, against which the virus cannot easily develop resistance.

  • Neuroinvasion of {alpha}-Synuclein Prionoids after Intraperitoneal and Intraglossal Inoculation [PublishAheadOfPrint]

  • aalpha;-Synuclein is a soluble, cellular protein that in a number of neurodegenerative diseases, including Parkinson's disease and multiple system atrophy, forms pathologic deposits of protein aggregates. Because misfolded aalpha;-synuclein has some characteristics that resemble prions, we investigated its potential to induce disease after intraperitoneal and intraglossal challenge injection in bigenic Tg(M83+/-:Gfap-luc+/-) mice expressing the A53T-mutant of human aalpha;-synuclein and firefly luciferase. After a single intraperitoneal injection with aalpha;-synuclein fibrils four out of five mice developed paralysis and aalpha;-synuclein pathology in their central nervous system with a median incubation time of 229 pplusmn; 17 d. Diseased mice accumulated aggregates of sarkosyl-insoluble and phosphorylated aalpha;-synuclein in brain and spinal cord, which colocalized with ubiquitin and p62 and were accompanied by gliosis. In contrast, only one mouse out of five developed aalpha;-synuclein pathology in its central nervous system after intraglossal injection with aalpha;-synuclein fibrils after 285 d. These findings are novel and important because they show that similar to prions, aalpha;-synuclein prionoids can neuroinvade the central nervous system after intraperitoneal and intraglossal injection and cause neuropathology and disease.

    Importance Synucleinopathies are neurodegenerative diseases that are characterized by the pathologic presence of aggregated aalpha;-synuclein in cells of the nervous system. Previous studies have shown that aalpha;-synuclein aggregates made from recombinant protein or derived from brains of patients can spread in the central nervous system in a spatiotemporal manner when inoculated into the brain of animals and induce pathology and neurologic disease suggesting that misfolded aalpha;-synuclein can behave similar to prions. Here we show that inoculation into the peritoneal cavity or tongue of mice overexpressing aalpha;-synuclein causes neurodegeneration after neuroinvasion from the periphery, which further corroborates the prionoid character of misfolded aalpha;-synuclein.

  • Inhibition of human metapneumovirus binding to heparan sulfate blocks infection in human lung cells and airway tissues [PublishAheadOfPrint]

  • Human metapneumovirus (HMPV), a recently discovered paramyxovirus, infects nearly 100% of the world population and causes severe respiratory disease in infants, the elderly, and immunocompromised patients. We previously showed that HMPV binds heparan sulfate proteoglycans (HSPGs) and that HMPV binding requires only the viral fusion (F) protein. To characterize the features of this interaction critical for HMPV binding and the role of this interaction in infection in relevant models, we utilized sulfated polysaccharides, HS mimetics and occluding compounds. Iota-carrageenan had potent anti-HMPV activity by inhibiting binding to lung cells mediated by the F protein. Furthermore, analysis of a minilibrary of variably sulfated derivatives of Escherichia coli K5 polysaccharide mimicking HS structure revealed that the highly O-sulfated K5 polysaccharides inhibited HMPV infection, identifying a potential feature of HS critical for HMPV binding. The peptide dendrimer SB105-A10, which binds HS, reduced binding and infection in an F-dependent manner, suggesting occlusion of HS at the target cell surface is sufficient to prevent infection. HMPV infection was also inhibited by these compounds during apical infection of polarized airway tissues, suggesting these interactions take place during HMPV infection in a physiologically relevant model. These results reveal key features of the interaction between HMPV and HS, supporting the hypothesis that apical HS in the airway serves as a binding factor during infection, and HS modulating compounds may serve as a platform for potential antiviral development.

    Importance Human metapneumovirus (HMPV) is a paramyxovirus that causes respiratory disease worldwide. It has been previously shown that HMPV requires binding to heparan sulfate on the surface of target cells for attachment and infection. In this study, we characterize the key features of this binding interaction using heparan sulfate mimetics, identify an important sulfate modification, and demonstrate that these interactions occur at the apical surface of polarize airways tissues. These findings provide insights into the initial binding step of HMPV infection that has potential for antiviral development.

  • Infectious Progression of Canine Distemper Virus in Circulating Cerebrospinal Fluid to the Central Nervous System [PublishAheadOfPrint]

  • In the current study, we generated recombinant chimeric CDVs by replacing the hemagglutinin (H) and/or phosphoprotein (P) genes in an avirulent strain expressing enhanced green fluorescent protein (EGFP) with those of a mouse-adapted neurovirulent strain.

    An in vitro experimental infection indicated that the chimeric CDVs possessing the H gene derived from the mouse-adapted CDV acquired infectivity to neural cells. These cells lack the CDV receptors that have been identified to date (SLAM and nectin-4), indicating that the H protein defines infectivity in various cell lines. These recombinant viruses were administered intracerebrally to 1-week-old mice. Fatal neurological signs of disease were observed only in a recombinant CDV that possesses both the H and P genes of the mouse-adapted strain, similar to the parental mouse-adapted strain, suggesting that the both genes are important to drive the virulence of CDV in mice.

    Using this recombinant CDV, we traced the intracerebral propagation of CDV by detecting EGFP. Widespread infection was observed in the cerebral hemispheres and brainstems of the infected mice. In addition, EGFP fluorescence in the brain slices demonstrated a sequential infectious progression in the central nervous system: CDV primarily infected the neuroependymal cells lining the ventricular wall and the neurons of the hippocampus and cortex adjacent to the ventricle, and it then progressed to an extensive infection of the brain surface, followed by the parenchyma and cortex. In the hippocampal formation, CDV spread in a unidirectional retrograde pattern along neuronal processes in the hippocampal formation, from the CA1 region to the CA3 region and the dentate gyrus. Our mouse model demonstrated that the main target cells of CDV are neurons in the acute phase and that the virus spreads via neuronal transmission pathways in the hippocampal formation.

    IMPORTANCE CDV is the etiological agent of distemper in dogs and other carnivores, and in many respects, the pathogenesis of CDV infection in animals resembles that of measles virus infection in humans. We successfully generated a recombinant CDV containing the H and P genes from a mouse-adapted neurovirulent strain and expressing EGFP. The recombinant CDV exhibited severe neurovirulence with high mortality, comparable to the parental mouse-adapted strain. The mouse-infectious model could become a useful tool for analyzing CDV infection of the central nervous systems subsequent to passing through the blood-cerebrospinal fluid barrier and infectious progression in the target cells in acute disease.

  • Global transcriptional analysis of virus-host interactions between phage {varphi}29 and Bacillus subtilis [PublishAheadOfPrint]

  • The study of phage-host relationships is essential to understanding the dynamic of microbial systems. Here, we analyse genome-wide interactions of Bacillus subtilis and its lytic phage 29 during the early stage of infection. Simultaneous high-resolution analysis of virus and host transcriptomes by deep RNA sequencing allowed us to identify differentially expressed bacterial genes. Phage 29 induces significant transcriptional changes in about 0.9% (38/4,242) and 1.8% (76/4,242) of the host protein-coding genes after 8 and 16 min of infection, respectively. Gene ontology (GO) enrichment analysis clustered up-regulated genes into several functional categories such as nucleic acid metabolism (including DNA replication) and protein metabolism (including translation). Surprisingly, most of the transcriptional repressed genes were involved in the utilization of specific carbon sources such as ribose and inositol, and many contained promoter binding-sites for the catabolite control protein A (CcpA). Another interesting finding is the presence of previously uncharacterised antisense transcripts complementary to the well-known phage 29 messenger RNAs that adds an additional layer to the viral transcriptome complexity.

    IMPORTANCE The specific virus-host interactions that allow phages to redirect cellular machineries and energy resources to support the viral progeny production are poorly understood. This work provides, for the first time, an insight into the genome-wide transcriptional response of the Gram-positive model B. subtilis to phage 29 infection.

  • The Role of the B-Allele of the Influenza A Virus Segment 8 in Setting Mammalian Host Range and Pathogenicity. [PublishAheadOfPrint]

  • Two llsquo;allelesrrsquo; of segment 8 (NS) circulate in non-chiropteran influenza A viruses. The A-allele is found in avian and mammalian viruses, but the B-allele is viewed as almost exclusively avian. This might reflect that one or both of its encoded proteins (NS1 and NEP) are maladapted for replication in mammalian hosts. To test this, a number of clade A and B avian NS segments were introduced into human H1N1 and H3N2 viruses. In no case was peak virus titre substantially reduced following infection of various mammalian cell types. Exemplar reassortant viruses also replicated to similar titres in mice, although the avian segment 8s reduced weight-loss compared to the PR8 parent. In vitro, the viruses coped similarly with type I interferons. Temporal proteomics analysis of cellular responses to infection showed that the avian NS segments provoked lower expression of IFN-stimulated genes in cells than the WT. Thus, neither A- nor B-alleles of avian virus-derived NS segments necessarily attenuate virus replication in a mammalian host although they can attenuate disease. Phylogenetic analyses identified 32 independent incursions of an avian-derived A-allele into mammals compared to 6 introductions of a B-allele. However, A-allele isolates from birds outnumber B-allele samples and the relative rates of Aves to Mammalia transmission are not significantly different. We conclude that while the introduction of an avian virus segment 8 into mammals is a relatively rare event, the dogma of the B-allele being especially restricted is misleading nndash; with implications in the assessment of pandemic potential of avian influenza viruses.

    Importance Influenza A virus (IAV) can adapt to poultry and mammalian species, inflicting a great socioeconomic burden on farming and healthcare sectors. Host adaptation likely involves multiple viral factors. Here, we investigated the role of IAV segment 8. Segment 8 has evolved into two distinct clades nndash; the llsquo;Arrsquo; and llsquo;Brrsquo; alleles. The B-allele genes have previously been suggested to be avian-restricted. We introduced a selection of avian A- and B-allele segment 8s into human H1N1 and H3N2 backgrounds, and found that these reassortant viruses were fully competent in mammalian host systems. We also analysed the currently available public data on segment 8 gene distribution, finding surprisingly little evidence for specific avian-host restriction of the B clade segment. We conclude that B-allele segment 8 genes are in fact capable of supporting infection in mammals and that they should be considered during the assessment of pandemic risk of zoonotic influenza A viruses.

  • Flexibility of the head-stalk linker domain of paramyxovirus HN glycoprotein is essential for triggering virus fusion [PublishAheadOfPrint]

  • The Paramyxoviridae comprise a large family of enveloped, negative sense, single stranded RNA viruses with significant economic and public health implications. For nearly all paramyxoviruses, infection is initiated by fusion of the viral and host cell plasma membranes in a pH-independent fashion. Fusion is orchestrated by the receptor binding protein hemagglutinin-neuraminidase (HN; also called H or G depending on the virus type) protein and a fusion (F) protein, the latter undergoing a major refolding process to merge the two membranes. Mechanistic details regarding the coupling of receptor binding to F-activation are not fully understood. Here, we have identified the flexible loop region connecting the bulky enzymatically active head and the 4-helix bundle stalk to be essential for fusion promotion. Proline substitution in this region of HN of parainfluenza virus 5 (PIV5) and Newcastle disease virus (NDV) HN abolishes cell-cell fusion while HN retains receptor binding and neuraminidase activity. By using reverse genetics, we engineered recombinant PIV5-EGFP viruses with mutations in the head-stalk linker region of HN. Mutations in this region abolished virus recovery and infectivity. In sum, our data suggest that the loop region acts as a llsquo;hingerrsquo; around which the bulky head of HN swings to-and-fro to facilitate timely HN-mediate F-triggering, a notion consistent with the stalk-mediated activation model of paramyxovirus fusion.

    SIGNIFICANCE Paramyxovirus fusion with the host cell plasma membrane is essential for virus infection. Membrane fusion is orchestrated via interaction of the receptor binding protein (HN, H or G) with the viral fusion glycoprotein (F). Two distinct models have been suggested to describe the mechanism of fusion: these include llsquo;the clamprrsquo; and the llsquo;provocateurrrsquo; model of activation. By using biochemical and reverse genetics tools, we have obtained strong evidence in favor of the HN stalk-mediated activation of paramyxovirus fusion. Specifically, our data strongly support the notion that the short linker between the head and stalk plays a role in llsquo;conformational switchingrrsquo; of the head group to facilitate F-HN interaction and triggering.

  • Carcinoembryonic Antigen-related Cell Adhesion Molecule 5 (CEACAM5) Is an Important Surface Attachment Factor Facilitating the Entry of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) [PublishAheadOfPrint]

  • The spike proteins of coronaviruses are capable of binding to a wide range of cellular targets, which contribute to the broad species tropism of coronaviruses. Previous reports have demonstrated that Middle East respiratory syndrome coronavirus (MERS-CoV) predominantly utilizes dipeptidyl peptidase-4 (DPP4) for cell entry. However, additional cellular binding targets of the MERS-CoV spike protein that may augment MERS-CoV infection have not been further explored. In the current study, using the virus overlay protein binding assay (VOPBA), we identified carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) as a novel cell surface binding target of MERS-CoV. CEACAM5 co-immunoprecipitated with the spike protein of MERS-CoV in both overexpressed and endogenous settings. Disrupting the interaction between CEACAM5 and MERS-CoV spike with anti-CEACAM5 antibody, recombinant CEACAM5 protein, or siRNA knockdown of CEACAM5 significantly inhibited the entry of MERS-CoV. Recombinant expression of CEACAM5 did not render the non-permissive baby hamster kidney (BHK21) cells susceptible to MERS-CoV infection. Instead, CEACAM5 overexpression significantly enhanced the attachment of MERS-CoV to the BHK21 cells. More importantly, the entry of MERS-CoV was increased when CEACAM5 was overexpressed in permissive cells, which suggested that CEACAM5 could facilitate MERS-CoV entry in conjunction with DPP4 despite not being able to support MERS-CoV entry independently. Taken together, our study identified CEACAM5 as a novel cell surface binding target of MERS-CoV that facilitates MERS-CoV infection through augmenting the attachment of the virus to the host cell surface.

    Importance Infection with the Middle East respiratory syndrome coronavirus (MERS-CoV) is associated with the highest mortality rate among all known human-pathogenic coronaviruses. Currently, there are no approved vaccines or therapeutics against MERS-CoV infection. The identification of carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) as a novel cell surface binding target of MERS-CoV advanced our knowledge on the cell binding biology of MERS-CoV. Importantly, CEACAM5 could potentiate the entry of MERS-CoV through functioning as an attachment factor. In this regard, CEACAM5 could serve as a novel target in addition to dipeptidyl peptidase-4 (DPP4) in the development of antiviral strategies for MERS-CoV.

  • Changes in structure and antigenicity of HIV-1 Env trimers resulting from removal of a conserved CD4 binding site-proximal glycan [PublishAheadOfPrint]

  • The envelope glycoprotein (Env) is the major target for HIV-1 broadly neutralizing antibodies (bNAbs). One of the mechanisms HIV has evolved to escape the host's immune response is to mask conserved epitopes on Env with dense glycosylation. Previous studies have shown that removal of a particular conserved glycan at N197 increases neutralization sensitivity of the virus to antibodies targeting the CD4 binding site (CD4bs), making it a site of significant interest from the perspective of vaccine design. At present, the structural consequences that result from removal of the N197 glycan have not been characterized. Using native-like SOSIP trimers, we examine the effects on the antigenicity and local structural dynamics resulting from removal of this glycan. A large increase in the binding of CD4bs and V3-targeting antibodies is observed in the N197Q mutant in trimeric Env while no changes are observed with monomeric gp120. While the overall structure and thermo-stability are not altered, a subtle increase in flexibility of the variable loops at the trimeric interface of adjacent protomers is evident in the N197Q mutant by Hydrogen/Deuterium-exchange mass spectrometry. Structural modelling of the glycan chains suggests that the spatial occupancy of the N197 glycan leads to steric clashes with CD4bs antibodies in the Env trimer, but not monomeric gp120. Our results indicate that removal of the N197 glycan enhances exposure of relevant bNAb epitopes on Env with a minimal impact on the overall trimeric structure. These findings present a simple modification for enhancing trimeric Env immunogens in vaccines.

    IMPORTANCE The HIV-1 Env glycoprotein presents a dense patchwork of host-cell-derived N-linked glycans. This so-called glycan shield is considered to be a major protective mechanism from immune recognition. While positions of many N-linked glycans are isolate-specific, some are highly conserved and are believed to play key functional roles. In this study, we examine the conserved, CD4 binding site-proximal N197 glycan and demonstrate that its removal both facilitates neutralizing antibody access to the CD4 binding site and modestly impacts the structural dynamics at the trimer crown, without drastically altering global Env trimer stability. This indicates that surgical glycosylation site modification may be an effective way of sculpting epitope presentation in Env-based vaccines.

  • ITCH E3 Ubiquitin Ligase Interacts with Ebola Virus VP40 to Regulate Budding [PublishAheadOfPrint]

  • Ebola (EBOV) and Marburg (MARV) viruses belong to the Filoviridae family and can cause outbreaks of severe hemorrhagic fever with high mortality rates in humans. The EBOV VP40 (eVP40) and MARV VP40 (mVP40) matrix proteins play a central role in virion assembly and egress, such that independent expression of VP40 leads to the production and egress of virus-like particles (VLPs) that accurately mimic budding of infectious virus. Late (L) budding domains of eVP40 recruit host proteins (e.g. Tsg101, Nedd4, and Alix) that are important for efficient virus egress and spread. For example, the PPxY-type L-domain of eVP40 and mVP40 recruits host Nedd4 E3 ubiquitin ligase via its WW-domains to facilitate budding. Here we sought to identify additional WW-domain host interactors and demonstrate that the PPxY L-domain motif of eVP40 interacts specifically with the WW-domain of host E3 ubiquitin ligase ITCH. ITCH, like Nedd4, is a member of the HECT class of E3 ubiquitin ligases, and the resultant physical and functional interaction with eVP40 facilitates VLP and virus budding. Identification of this novel eVP40 interactor highlights the functional interplay between cellular E3 ligases, ubiquitination, and regulation of VP40-mediated egress.

    Importance. The unprecedented magnitude and scope of the recent 2014-2015 EBOV outbreak in West Africa, and its emergence here in the United States and other countries underscores the critical need for a better understanding of the biology and pathogenesis of this emerging pathogen. We have identified a novel and functional EBOV VP40 interactor, ITCH, that regulates VP40-mediated egress. This virus-host interaction may represent a new target for our previously identified small molecule inhibitors of virus egress.

  • MOUSE HEPATITIS VIRUS INFECTION INDUCES A TLR2-DEPENDENT ACTIVATION OF INFLAMMATORY FUNCTIONS IN LIVER SINUSOIDAL ENDOTHELIAL CELLS DURING ACUTE HEPATITIS [PublishAheadOfPrint]

  • Under physiological conditions, the liver sinusoidal endothelial cells (LSECs) mediate hepatic immune tolerance towards self or foreign antigens through constitutive expression of anti-inflammatory mediators. However, upon viral infection or TLR2 activation, LSECs can achieve proinflammatory functions but their role in hepatic inflammation during acute viral hepatitis is unknown. Using the highly virulent mouse hepatitis virus (MHV) type 3 and the attenuated variants 51.6-MHV3 and YAC-MHV3, exhibiting lower tropism for LSECs, we investigated in vivo and in vitro the consequence of LSEC infection on their pro-inflammatory profile and the aggravation of acute hepatitis process. In vivo infection with virulent MHV3, in comparison to attenuated strains, resulted in fulminant hepatitis associated with higher hepatic viral load, tissue necrosis, levels of inflammatory mediators and earlier recuitement of inflammatory cells. Such hepatic inflammatory disorders correlated with disturbed production of IL-10 and vascular factors by LSECs. We next showed in vitro that infection of LSECs by the virulent MHV3 strain altered their production of anti-inflammatory cytokines and promoted higher release of pro-inflammatory and procoagulant factors and earlier cell damage in comparison to attenuated strains. This higher replication and pro-inflammatory activation in LSECs by the virulent MHV3 strain was associated with a specific activation of TLR2 signalling by the virus. We provided evidence that TLR2 activation of LSCEs by MHV3 is an aggravating factor of hepatic inflammation and correlates with the severity of hepatitis. Taken together, these results indicate that preservation of immunotolerant properties of LSECs during acute viral hepatitis is an imperative factor to limit hepatic inflammation and damages.

    IMPORTANCE Viral hepatitis B and C infections are serious health problem infecting over 350 million and 170 million people worldwide respectively. It has been suggested that a balance between protection and liver damage mediated by the host's immune response during the acute phase of infection would be determinant in hepatitis outcome. Thus, it appears crucial to identify the factors that predispose in exacerbating liver inflammation to limit hepatocyte injury. Liver sinusoidal endothelial cells (LSECs) can express both anti- and pro-inflammatory functions but their role in acute viral hepatitis has never been investigated. Using the mouse hepatitis virus (MHV) infections as animal models of viral hepatitis, we report for the first time that in vitro and in vivo infection of LSECs by the pathogenic MHV3 serotype leads to a reversion of their intrinsic anti-inflammatory phenotype towards a pro-inflammatory profile as well as disorders in vascular factors, correlating with the severity of hepatitis. These results highlight a new viral-promoted mechanism of exacerbation of liver inflammatory response during acute hepatitis.

  • Acute liver damage associated with innate immune activation in a small nonhuman primate model of Hepacivirus infection [PublishAheadOfPrint]

  • Despite its importance in shaping adaptive immune responses, viral clearance and immune-based inflammation, tissue-specific innate immunity remains poorly characterized in Hepatitis C virus (HCV) infection due to lack of access to acute tissues. In this study we evaluated the impact of natural killer (NK), myeloid (mDC) and plasmacytoid dendritic cells (pDC) on control of virus replication and virus-induced pathology caused by another more rapidly resolving Hepacivirus, GBV-B infection of common marmosets. High plasma and liver viral loads and robust hepatitis characterized acute GBV-B infection, and while viremia was generally cleared by 2-3 months post-infection, hepatitis and liver fibrosis persisted after clearance. Coinciding with peak viral loads and liver pathology, NK cells, mDCs and pDCs increased up to 3-fold in the liver. Although, no obvious numerical changes occurred in peripheral innate cells, circulating NK cells exhibited increased perforin, Ki67, and surface expression of CXCR3. These data suggested increased NK cell arming and proliferation, as well as tissue trafficking may be associated with influx into the liver during acute infection. Indeed NK cell frequencies in the liver positively correlated with plasma (R= 0.698, p = 0.015) and liver viral load (R = 0.567, p = 0.057). Finally, soluble factors associated with NK and DCs including IFN- and RANTES were increased in acute infection and also associated with viral loads and hepatitis. Collectively, the mobilization of local and circulating innate immune responses was linked to acute virus-induced hepatitis and potentially resolution of GBV-B infection, and could provide insight into similar mechanisms in HCV.

    Importance Hepatitis C virus (HCV) infection has created a global health crisis and, despite new effective antivirals, is still a leading cause of liver disease and death worldwide. Recent evidence suggests innate immunity could be a potential therapeutic target for HCV, but may also be a correlate of increased disease. Due to a lack of access to human tissues in acute HCV infection, in this study we evaluated the role of innate immunity in a resolving Hepacivirus, GBV-B infection of common marmosets. Collectively our data suggest that NK and DC mobilization in acute Hepacivirus infection can dampen virus replication, but also regulate acute and chronic liver damage. How these two opposing effects on the host could be modulated in future therapeutic and vaccine approaches warrants further study.

  • Protective effects of glutamine antagonist DON in mice with alphaviral encephalomyelitis [PublishAheadOfPrint]

  • Inflammation is a necessary part of the response to infection, but can also cause neuronal injury in both infectious and autoimmune diseases of the central nervous system (CNS). A neurovirulent strain of Sindbis virus (NSV) causes fatal paralysis in adult C57BL/6 mice during clearance of infectious virus from the CNS, and the virus-specific immune response is implicated as a mediator of neuronal damage. Previous studies have shown that survival is improved in T-cell deficient mice and in mice with pharmacologic inhibition of the inflammatory response and glutamate excitotoxicity. Because glutamine metabolism is important in the CNS for the generation of glutamate and in the immune system for lymphocyte proliferation, we tested the effect of glutamine antagonist DON (6-diazo-5-oxo-l-norleucine) on the outcome of NSV infection in mice. DON treatment for 7 days from the time of infection delayed onset of paralysis and death. Protection was associated with reduced lymphocyte proliferation in the draining cervical lymph nodes, decreased leukocyte infiltration into the CNS, lower levels of inflammatory cytokines and delayed viral clearance. In vitro studies showed that DON inhibited stimulus-induced proliferation of lymphocytes. When in vivo treatment with DON was stopped, paralytic disease developed along with the inflammatory response and viral clearance. These studies show that fatal NSV-induced encephalomyelitis is immune-mediated and that antagonists of glutamine metabolism can modulate the immune response and protect against virus-induced neuroinflammatory disease.

    IMPORTANCE Encephalomyelitis due to infection with mosquito-borne alphaviuses is an important cause of death and of long-term neurological disability in those that survive infection. This study demonstrates the role of the virus-induced immune response in the generation of neurologic disease. DON, a glutamine antagonist, inhibited proliferation of lymphocytes in response to infection, prevented development of brain inflammation and protected mice from paralysis and death during treatment. However, because DON inhibited the immune response to infection, clearance of virus from the brain was also prevented. When treatment was stopped the immune response was generated, brain inflammation occurred, virus was cleared and mice developed paralysis and died. Therefore, more definitive treatment for alphaviral encephalomyelitis should inhibit virus replication as well as neuroinflammatory damage.

  • Ferrets infected with Bundibugyo virus or Ebola virus recapitulate important aspects of human filoviral disease [PublishAheadOfPrint]

  • Bundibugyo virus (BDBV) is the etiological agent of a severe hemorrhagic fever in humans with a case-fatality rate ranging from 25-36%. Despite being known to the scientific and medical communities for almost a decade, there is a dearth of studies on this pathogen due to the lack of a small animal model. Domestic ferrets are commonly used to study other RNA viruses, including members of the order Mononegavirales. To investigate whether ferrets were susceptible to filovirus infections, ferrets were challenged with a clinical isolate of BDBV. Animals became viremic within 4 days and succumbed to infection between 8-9 days, and petechial rash was observed with moribund ferrets. Furthermore, several hallmarks of human filoviral disease were recapitulated in the ferret model, including substantial decreases in lymphocyte and platelet counts, dysregulation of key biochemical markers related to hepatic/renal function, as well as coagulation abnormalities. Virological, histopathological and immunohistochemical analyses confirmed uncontrolled BDBV replication in the major organs. Ferrets were also infected with Ebola virus (EBOV) to confirm their susceptibility to another filovirus species and to potentially establish a virus transmission model. Similar to BDBV, important hallmarks of human filoviral disease were observed in EBOV-infected ferrets. This study demonstrates the potential of this small animal model for studying BDBV and EBOV using wild-type isolates, and will accelerate efforts to understand filovirus pathogenesis and transmission, as well as the development of specific vaccines and antivirals.

    Importance The 2013-2016 outbreak of Ebola virus in West Africa has highlighted the threat posed by filoviruses to global public health. Bundibugyo virus (BDBV) is a member of the genus Ebolavirus, and has caused outbreaks in the past, but is relatively understudied, likely due to the lack of a suitable small animal model. Such a model for BDBV is crucial to evaluating vaccines, therapies, and potentially understanding transmission. To address this, we demonstrated that ferrets are susceptible models to BDBV infection, as well as Ebola virus, and that no virus adaptation is required. Moreover, these animals develop a disease that is similar to human and non-human primates. We believe this will improve the ability to study BDBV, and provide a platform to test vaccines and therapeutics.

  • SRSF3 and hnRNP A1 regulate alternative RNA splicing and gene expression of human papillomavirus type 18 through two functionally distinguishable cis-elements [PublishAheadOfPrint]

  • Human papillomavirus type 18 (HPV18) is the second most common oncogenic HPV type associated with cervical, anogenital and oropharyngeal cancers. Like other oncogenic HPVs, HPV18 encodes two major (one early and one late) polycistronic pre-mRNAs which are regulated by alternative RNA splicing to produce a repertoire of viral transcripts for the expression of individual viral genes. However, RNA cis-regulatory elements and trans-acting factors contributing to HPV18 alternative RNA splicing remain unknown. In this study, an exonic splicing enhancer (ESE) at the nt 3520-3550 region in the HPV18 genome was identified and characterized for promotion of HPV18 929^3434 splicing and E1^E4 production through interaction with SRSF3, a host oncogenic splicing factor differentially expressed in epithelial cells and keratinocytes. Introduction of point mutations in the SRSF3-binding site or knockdown of SRSF3 expression in cells reduces the 929^3434 splicing and E1^E4 production, but activates other minor 929^3465 and 929^3506 splicing. Knockdown of SRSF3 expression also enhances the expression of E2 and L1 mRNA. An exonic splicing silencer (ESS) in the HPV18 612-639 region was identified being inhibitory to the 233^416 splicing of HPV18 E6E7 pre-mRNAs via binding to hnRNP A1, a well characterized, abundantly and ubiquitously expressed RNA-binding protein. Introduction of point-mutations into the hnRNP A1-binding site or knockdown of hnRNP A1 expression promotes the 233^416 splicing and reduces E6 expression. These data provide the first evidence that the alternative RNA splicing of HPV18 pre-mRNAs subjects to regulation by viral RNA cis-elements and host trans-acting splicing factors.

    IMPORTANCE Expression of HPV18 genes are regulated by alternative RNA splicing of vial polycistronic pre-mRNAs to produce a repertoire of viral early and late transcripts. RNA cis-elements and trans-acting factors contributing to HPV18 alternative RNA splicing have been discovered in this report for the first time. The identified ESS at the E7 ORF prevents HPV18 233^416 splicing in the E6 ORF through interaction with a host splicing factor hnRNP A1 and regulates E6 and E7 expression of the early E6E7 polycistronic pre-mRNA. The identified ESE at the E1^E4 ORF promotes HPV18 929^3434 splicing of both viral early and late pre-mRNAs and E1^E4 production through interaction with SRSF3. This study provides the important observations on how alternative RNA splicing of HPV18 pre-mRNAs subjects to regulation by viral RNA cis-elements and host splicing factors and offers potential therapeutic targets to overcome HPV-related cancer.

  • Human Choline Kinase-{alpha} Promotes Hepatitis C Virus RNA Replication through Modulating Membranous Viral Replication Complex Formation [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) infection reorganizes cellular membranes to create an active viral replication site named the "membranous web" (MW). The role that human choline kinase-aalpha; (hCKaalpha;) plays in HCV replication remains elusive. Here, we first showed that hCKaalpha; activity, not the CDP-choline pathway, promoted viral RNA replication. Confocal microscopy and subcellular fractionation of HCV-infected cells revealed that a small fraction of hCKaalpha; colocalized with the viral replication complex (RC) on the endoplasmic reticulum (ER) and that HCV infection increased hCKaalpha; localization to the ER. In the pTM-NS3-NS5B model, NS3-NS5B expression increased the localization of the wild-type, not the inactive D288A mutant, hCKaalpha; on the ER, and hCKaalpha; activity was required for effective trafficking of hCKaalpha; and NS5A to the ER. Coimmunoprecipitation showed that hCKaalpha; was recruited onto the viral RC presumably through its binding to NS5A domain 1. hCKaalpha; silencing or treatment with CK37, an hCKaalpha; activity inhibitor, abolished HCV-induced MW formation. In addition, hCKaalpha; depletion hindered NS5A localization on the ER, interfered with NS5A and NS5B colocalization, and mitigated NS5A-NS5B interactions but had no apparent effect on NS5A-NS4B and NS4B-NS5B interactions. Nevertheless, hCKaalpha; activity was not essential for the binding of NS5A to hCKaalpha; or NS5B. These findings demonstrate that hCKaalpha; forms a complex with NS5A and that hCKaalpha; activity enhances the targeting of the complex to the ER, where hCKaalpha; protein, not activity, mediates NS5A binding to NS5B, thereby promoting functional membranous viral RC assembly and viral RNA replication.

    Importance HCV infection reorganizes the cellular membrane to create an active viral replication site named the "membranous web" (MW). Here, we report that human choline kinase-aalpha; (hCKaalpha;) acts as an essential host factor for HCV RNA replication. A fraction of hCKaalpha; colocalizes with the viral replication complex (RC) on the endoplasmic reticulum (ER) in HCV-infected cells. NS3-NS5B expression increases ER localization of wild-type, but not D288A mutant, hCKaalpha;, and hCKaalpha; activity facilitates the transport of itself and NS5A to the ER. Silencing or inactivation of hCKaalpha; abrogates MW formation. Moreover, hCKaalpha; is recruited by NS5A independent of hCKaalpha; activity presumably through binding to NS5A D1. hCKaalpha; activity then mediates the ER targeting of the hCKaalpha;-NS5A complex. On the ER membrane, hCKaalpha; protein, per se, induces NS5A binding to NS5B, thereby promoting membranous RC formation and viral RNA replication. Our study may benefit the development of hCKaalpha;-targeted anti-HCV therapeutics.

  • Fungal-derived neoechinulin B as a novel antagonist of liver X receptor, identified by chemical genetics using hepatitis C virus cell culture system [PublishAheadOfPrint]

  • Cell culture systems reproducing virus replication can serve as unique models for the discovery of novel bioactive molecules. Here, using a hepatitis C virus (HCV) cell culture system, we identified neoechinulin B (NeoB), a fungal-derived compound, as an inhibitor of the liver X receptor (LXR). NeoB was initially identified by chemical screening as a compound that impeded the production of infectious HCV. Genome-wide transcriptome analysis and reporter assays revealed that NeoB specifically inhibits LXR-mediated transcription. NeoB was also shown to interact directly with LXRs. Analysis of structural analogs suggested that the molecular interaction of NeoB with LXR correlated with the capacity to inactivate LXR-mediated transcription and to modulate lipid metabolism in hepatocytes. Our data strongly suggested that NeoB is a novel LXR antagonist. Analysis using NeoB as a bio-probe revealed that LXRs support HCV replication: LXR inactivation resulted in dispersion of double-membrane vesicles, putative viral replication sites. Indeed, cells treated with NeoB showed decreased replicative permissiveness for poliovirus, which also replicates in double-membrane vesicles, but not for dengue virus, which replicates via a distinct membrane compartment. Together, our data suggest that LXR-mediated transcription regulates the formation of virus-associated membrane compartments. Significantly, inhibition of LXRs by NeoB enhanced the activity of all known classes of anti-HCV agents, showing especially strong synergy when combined with interferon or an HCV NS5A inhibitor. Thus, our chemical genetics analysis demonstrates the utility of the HCV cell culture system for identifying novel bioactive molecules and characterizing the virus-host interaction machinery.

    Importance Hepatitis C virus (HCV) is highly dependent on host factors for efficient replication. In the present study, we used a HCV cell culture system to screen an uncharacterized chemical library. Our results identified neoechinulin B (NeoB) as a novel inhibitor of the liver X receptor (LXR). NeoB inhibited the induction of LXR-regulated genes and altered lipid metabolism. Intriguingly, our results indicated that LXRs are critical to the process of HCV replication: LXR inactivation by NeoB disrupted double-membrane vesicles, putative sites of viral replication. Moreover, NeoB augmented the antiviral activity of all known classes of currently approved anti-HCV agents without increasing cytotoxicity. Thus, our strategy directly links the identification of novel bioactive compounds to basic virology and the development of new antiviral agents.

  • Mutations determining resistance-breaking in a plant RNA virus have pleiotropic effects on its fitness that depend on the host environment and on the type, single or mixed, of infection [PublishAheadOfPrint]

  • Overcoming host resistance in gene-for-gene host-virus interactions is an important instance of host-range expansion, which can be hindered by across-host fitness trade-offs. Trade-offs are generated by negative effects of host-range mutations on the virus fitness in the original host, i.e., by antagonistic pleiotropy. It has been reported that different mutations in Pepper mild mottle virus (PMMoV) coat protein result in overcoming L-gene resistance in pepper. To analyze if resistance-breaking mutations in PMMoV result in antagonistic pleiotropy, all reported mutations determining the overcoming of L3 and L4 alleles were introduced in biologically active cDNA clones. Then, the parental and mutant virus genotypes were assayed in susceptible pepper genotypes with alleles L+, L1 or L2, in single and in mixed infection. Resistance-breaking mutations had pleiotropic effects on the virus fitness that, according to the specific mutation, the host genotype and the type of infection, single or mixed with other virus genotypes, were antagonistic or positive. Thus, resistance-breaking mutations can generate fitness trade-offs both across hosts and across types of infection, and the frequency of host-range mutants will depend on the genetic structure of the host population and on the frequency of mixed infections by different virus genotypes. Also, resistance-breaking mutations variously affected virulence, which may further influence the evolution of host-range expansion.

    IMPORTANCE A major cause of virus emergence is host-range expansion, which may be hindered by across-host fitness trade-offs caused by negative pleiotropy of host-range mutations. An important instance of host-range expansion is overcoming host resistance in gene-for-gene plant-virus interactions. We analyze here if mutations in the coat protein of Pepper mild mottle virus determining L-gene resistance-breaking in pepper, have associated fitness penalties in susceptible host genotypes. Results show that pleiotropic effects of resistance-breaking mutations on the virus fitness depend on the specific mutation, the susceptible host genotype and the type of infection, single or mixed with other virus genotypes. Accordingly, resistance-breaking mutations can have negative, positive or no pleiotropic effects on the virus fitness. These results underscore the complexity of host-range expansion evolution and, specifically, the difficulty of predicting the overcoming of resistance factors in crops.

  • Entry of Classical Swine Fever Virus into PK-15 cells via a pH-, dynamin- and cholesterol-dependent, clathrin-mediated endocytic pathway that requires Rab5 and Rab7 [PublishAheadOfPrint]

  • Classical swine fever virus (CSFV), a member of the genus Pestivirus within the family Flaviviridae, is a small enveloped positive strand RNA virus. Due to its economic importance to the pig industry, CSFV biology and pathogenesis have been investigated extensively. However, the mechanisms of CSFV entry into cell are not well characterized. In this study, we used systematic approaches to dissect CSFV cell entry. We first observed that CSFV infection was inhibited by chloroquine and NH4Cl, suggesting that viral entry required a low pH enviroment. By using the specific inhibitor dynasore, or by expressing the dominant-negative mutant K44A, we verified that dynamine is required for CSFV entry. CSFV particles were observed to colocalize with clathrin 5 minutes post internalization, and CSFV infection was significantly reduced by chlorpromazine treatment, overexpression of a dominant-negative form of EPS15 protein, or knock down of the clathrin heavy chain by RNA interference. These results suggested that CSFV entry depends on clathrin. Additionally, we found that endocytosis of CSFV was dependent on membrane cholesterol while neither the overexpression of the dominant-negative caveolin mutant, nor the knock down of caveolin had an effect. These results further suggested that CSFV entry required cholesterol, not caveolae. Importantly, the effect on CSFV infection by dominant-negative (DN) mutants of three Rab proteins that regulate endosomal traffic was examined. Expression of DN mutants Rab5 and Rab7, but not DN mutant Rab11, significantly inhibited CSFV replication. These results were confirmed by silencing Rab5 and Rab7. Confocal microscopy showed that virus particles colocalized with Rab5 or Rab7 during the early phase of infection within 45 minutes after virus entry. These results indicated that after internalization, CSFV moved to early and late endosomes before releasing its RNA. Taken together, our findings for the first time demonstrate that CSFV enters cells through the endocytic pathway, providing new insights into the life cycle of pestiviruses. .

    Importance Bovine viral diarrhoea virus (BVDV), a single-stranded positive-sense pestivirus within the family Flaviviridae, is internalized by clathrin-dependent receptor-mediated endocytosis. However, the detailed mechanism of cell entry is unknown for other pestiviruses, such as classical swine fever virus (CSFV). CSFV is the etiological agent of classical swine fever (CSF), a highly contagious disease of swine, causing numerous deaths in pigs and enormous economic losses in China. Understanding the entry pathway of CSFV will not only advance our knowledge of CSFV infection and pathogenesis but also provide novel drug targets for antiviral intervention. Based on this objective, we used systematic approaches to dissect the entry pathway of CSFV into PK-15 cells. This is the first report to show that entry of CSFV into PK-15 cells requires a low pH environment, and involves a dynamin- and cholesterol-dependent, clathrin-mediated endocytosis that requires Rab5 and Rab7.

  • Kinetics of HIV-1 latency reversal quantified on the single cell level using a novel flow-based technique [PublishAheadOfPrint]

  • HIV-1 establishes a pool of latently infected cells early following infection. New therapeutic approaches aiming at diminishing this persisting reservoir by reactivation of latently infected cells are currently being developed and tested. However, the reactivation kinetics of viral mRNA and viral protein production, and their respective consequences for phenotypical changes of infected cells that might enable immune recognition, remain poorly understood. We adapted a novel approach to assess the dynamics of HIV-1 mRNA and protein expression in latently and newly infected cells on the single cell level by flow cytometry. This technique allowed for simultaneous detection of gagpol mRNA, intracellular p24 Gag protein and cell surface markers. Following stimulation of latently HIV-1-infected J89 cells with hTNFaalpha;/RMD or HIV-1 infection of primary CD4+ T cells, four cell populations according to their expression levels of viral mRNA and protein were detected. Gagpol mRNA was quantifiable for the first time in J89 cells 3 hrs post-stimulation with hTNFaalpha; and 12 hrs post-stimulation with RMD, while p24 Gag protein was detected for the first time after 18 hrs post-stimulation. HIV-1-infected primary CD4+ T cells downregulated CD4, BST-2 and HLA class I expression at early stages of infection, proceeding Gag protein detection. In conclusion, we here describe a novel approach allowing to quantify the kinetics of HIV-1 mRNA and protein synthesis on the single cells level and to phenotypically characterize HIV-1-infected cells at different stages of the viral life cycle.

    Importance Early after infection, HIV-1 establishes a pool of latently infected cells, which are hiding from the immune system. Latency reversal and immune-mediated elimination of these latently infected cells is one of the goals of current HIV-1 cure approaches, however little is known about the HIV-1 reactivation kinetics following stimulation with latency reversing agents. Here we describe a novel approach allowing for the first time to quantify the kinetics of HIV-1 mRNA and protein synthesis after latency reactivation or de novo infection on the single cells level using flow cytometry. This new technique furthermore enabled the phenotypic characterization of latently and de novo infected cells dependent on the presence of viral RNA or protein.

  • The Ubiquitin Ligase Itch and Ubiquitination Regulate BFRF1-mediated Nuclear Envelope Modification for EBV Maturation [PublishAheadOfPrint]

  • The cellular endosomal sorting complex required for transport (ESCRT) was recently found to mediate important morphogenesis processes at the nuclear envelope (NE). We previously showed that the Epstein-Barr virus (EBV) BFRF1 protein recruits the ESCRT-associated protein Alix to modulate NE structure and promote EBV nuclear egress. Here, we uncover new cellular factors and mechanisms involved in this process. BFRF1-induced NE vesicles are similar to those observed following EBV reactivation. BFRF1 is ubiquitinated and elimination of possible ubiquitination with either lysine mutations or fusion of a de-ubiquitinase hampers NE-derived vesicle formation and virus maturation. While interacting with multiple Nedd4-like ubiquitin ligases, BFRF1 binds Itch ligase preferably. We show that Itch associates with Alix and BFRF1, and is required for BFRF1-induced NE vesicle formation. Our data demonstrate that Itch, ubiquitin and Alix control the BFRF1-mediated modulation of the NE and EBV maturation, uncovering novel regulatory mechanisms of nuclear egress of viral nucleocapsids.

    IMPORTANCE The nuclear envelope (NE) of eukaryotic cells not only serves as a transverse scaffold for cellular processes, but also a natural barrier for most DNA viruses that assemble their nucleocapsids in the nucleus. Previously, we showed the cellular endosomal sorting complex required for transport (ESCRT) machinery is required for the nuclear egress of EBV. Here, we further report the molecular interplays among viral BFRF1, ESCRT-adaptor Alix and ubiquitin ligase Itch. We found that BFRF1-induced NE vesicles are similar to those observed following EBV reactivation. The lysine residues and the ubiquitination of BFRF1 regulate the formation of BFRF1-induced NE-derived vesicles and EBV maturation. During the process, an ubiquitin ligase Itch associates preferably with BFRF1 and is required for BFRF1-induced NE vesicle formation. Therefore, our data indicate that Itch, ubiquitin and Alix control the BFRF1-mediated modulation of the NE, suggesting novel regulatory mechanisms for ESCRT-mediated NE modulation.

  • Type 1 interferons and NK cells restrict gamma-herpesvirus lymph node infection [PublishAheadOfPrint]

  • Gamma-herpesviruses establish persistent, systemic infections and cause cancers. Murid Herpesvirus-4 (MuHV-4) provides a unique window onto the early events of host colonization. It spreads via lymph nodes. While dendritic cells (DC) pass MuHV-4 to lymph node B cells, subcapsular sinus macrophages (SSM), which capture virions from the afferent lymph, restrict its spread. Understanding how this restriction works offers potential clues to a more comprehensive defence. Type I interferons (IFN-I) blocked SSM lytic infection and reduced lytic cycle-independent viral reporter gene expression. Plasmacytoid DC were not required; but neither were SSM the only source of IFN-I, as IFN-I blockade increased infection in both intact and SSM-depleted mice. NK cells restricted lytic SSM infection independently of IFN-I, and SSM-derived virions spread to the spleen only when IFN-I responses and NK cells were both lacking. Thus, multiple innate defences allowed SSM to adsorb virions from the afferent lymph with relative impunity. Enhancing IFN-I and NK cell recruitment could potentially also restrict DC infection and so improve infection control.

    Importance Human gamma-herpesviruses cause cancers by infecting B cells. However vaccines designed to block virus binding to B cells have not stopped infection. Using a related gamma-herpesvirus of mice, we showed that B cells are infected not via cell-free virus but via infected myeloid cells. This suggests a different strategy to stop B cell infection: stop virus production by myeloid cells. Not all myeloid infection is productive. We show that subcapsular sinus macrophages, which do not pass infection to B cells, restrict gamma-herpesvirus production by recruiting type 1 interferons and natural killer cells. Therefore a vaccine that speeds the recruitment of these defences might stop B cell infection.

  • Existence of two distinct infectious ERVs in domestic cats and their different strategies for adaptation to transcriptional regulation [PublishAheadOfPrint]

  • Endogenous retroviruses (ERVs) are the remnants of ancient retroviral infections of germ cells. Earlier work identified one of the youngest feline ERV groups, ERV-DC, and reported that two ERV-DC loci, ERV-DC10 and ERV-DC18 (ERV-DC10/DC18), can replicate in cultured cells. Here, we identified another replication-competent provirus, ERV-DC14, on chromosome C1q32. ERV-DC14 differs from ERV-DC10/DC18 in its phylogeny, receptor usage, and, most notably, in its transcriptional activities; although ERV-DC14 can replicate in cultured cells, it cannot establish a persistent infection owing to its low transcriptional activity. Furthermore, we examined ERV-DC transcription and its regulation in feline tissues. Quantitative RT-PCR detected extremely low ERV-DC10 expression levels in feline tissues, and bisulfite sequencing showed that 5'-long terminal repeats (LTRs) of ERV-DC10/DC18 are significantly hyper-methylated in feline blood cells. Reporter assays found that the 5'-LTR promoter activities of ERV-DC10/DC18 are high, whereas that of ERV-DC14 is low. This difference in promoter activity is due to a single substitution from A to T in the LTR, and reverse mutation at this nucleotide in ERV-DC14 enhanced its replication and enabled its persistent infection of cultured cells. Therefore, ERV-DC LTRs can be divided into two types based on this nucleotide, A-type or T-type, which have strong or attenuated promoter activity, respectively. Notably, ERV-DCs with T-type LTRs, such as ERV-DC14, have expanded in the cat genome significantly more than A-type ERV-DCs, despite their low promoter activities. Our results provide insights into how the host controls potentially infectious ERVs and, conversely, how ERVs adapt to and invade the host genome.

    IMPORTANCE The domestic cat genome contains many endogenous retroviruses, including ERV-DCs. These ERV-DCs have been acquired through germ cell infections with exogenous retroviruses. Some of these ERV-DCs are still capable of producing infectious virions. Hosts must tightly control these ERVs because replication-competent viruses in the genome pose a risk to the host. Here, we investigated how ERV-DCs are adapted by their hosts. Replication-competent viruses with strong promoter activity such as ERV-DC10 and ERV-DC18 were suppressed by promoter methylation in LTRs. On the other hand, replication-competent virus with weak promoter activity such as ERV-DC14 seemed to escape from strict control via promoter methylation by the host. Interestingly, ERV-DCs with weak promoter activity such as ERV-DC14 have expanded in the cat genome significantly more than ERV-DCs with strong promoter activity. Our results improve understanding of the host-virus conflict and how ERVs adapt in their hosts over time.

  • Crystal Structure of the Human Astrovirus Capsid Protein [PublishAheadOfPrint]

  • Human astrovirus (HAstV) is a leading cause of viral diarrhea in infants and young children worldwide. HAstV is a non-enveloped virus with a T = 3 capsid and a positive-sense RNA genome. The capsid protein (CP) of HAstV is synthesized as a 90 kDa precursor (VP90) that can be divided into three linear domains: a conserved N-terminal domain, a hypervariable domain, and an acidic C-terminal domain. Maturation of the HAstV requires proteolytic processing of the astrovirus CP both inside and outside of the host cell, resulting in the removal of the C-terminal domain and the breakdown of the rest of the CP into three predominant protein species with molecular weights of approximately 34, 27/29, and 25/26 kDa, respectively. We have now solved the crystal structure of VP9071-415 (aa71-415 of VP90) of the human astrovirus serotype 8 at 2.15 AAring; resolution. VP9071-415 encompasses the conserved N-terminal domain of the VP90, but lacks the hypervariable domain, which forms the capsid surface spikes. The structure of VP9071-415 is comprised of two domains: an S domain which adopts the typical jelly-roll bbeta;-barrel fold, and a P1 domain which forms a squashed bbeta;-barrel consisting of six anti-parallel bbeta;-strands similar to what was observed in the hepatitis E virus (HEV) capsid structure. Fitting the VP9071-415 structure into the cryo-EM maps of the HAstV produced an atomic model for the continuous, T = 3 icosahedral capsid shell. Our pseudo-atomic model of the human HAstV capsid shell provides valuable insights into intermolecular interactions required for capsid assembly and trypsin-mediated proteolytic maturation needed for virus infectivity. Such information has potential applications in the development of VLP vaccine as well as small molecule drugs targeting astrovirus assembly/maturation.

    IMPORTANCE Human astrovirus (HAstV) is a leading cause of viral diarrhea in infants and young children worldwide. As a non-enveloped virus, HAstV exhibits an intriguing feature in that its maturation requires extensive proteolytic processing of the astrovirus capsid protein (CP) both inside and outside of the host cell. Mature HAstV contains three predominant protein species, but the mechanism for the acquired infectivity upon maturation is unclear. We have solved the crystal structure of VP9071-415 of the human astrovirus serotype 8. VP9071-415 encompasses the conserved N-terminal domain of the viral CP. Fitting the VP9071-415 structure into the cryo-EM maps of the HAstV produced an atomic model for the T=3 icosahedral capsid. Our model of the HAstV capsid provides valuable insights into intermolecular interactions required for capsid assembly and trypsin-mediated proteolytic maturation. Such information has potential applications in the development of VLP vaccine as well as small molecule drugs targeting astrovirus assembly/maturation.

  • HIV maintains an evolving and dispersed population among multiple tissues during suppressive cART with periods of rapid expansion corresponding to the onset of cancer [PublishAheadOfPrint]

  • While combined antiretroviral therapy (cART) can result in undetectable plasma viral loads, it does not eradicate HIV infection. Furthermore, HIV-infected individuals while on cART remain at an increased risk of developing serious co-morbidities, such as cancer, neurological disease, and atherosclerosis, suggesting that during cART, tissue-based HIV may contribute to such pathologies.

    We obtained DNA and RNA env, nef and pol sequences using single genome sequencing from post mortem tissues of three HIV+/cART+ individuals with undetectable viral load and metastatic cancer at death, and performed time-scaled Bayesian evolutionary analyses. We used a sensitive in situ hybridization technique to visualize HIV gag-pol mRNA transcripts in cerebellum and lymph node tissues from one patient.

    Tissue-associated virus evolved at a similar rate in cART+ and cART- patients. Phylogenetic trees were characterized by two distinct features: 1) branching patterns consistent with constant viral evolution and dispersal amongst tissues; and 2) very recently derived clades containing both DNA and RNA sequences from multiple tissues. Cancer diagnoses were temporally associated with diversification of viral lineages. Rapid expansion of virus near death corresponded to wide-spread metastasis. HIV RNA+ cells clustered in cerebellum tissue but were dispersed in lymph node tissue, mirroring the evolutionary patterns observed for that patient. Activated, infiltrating macrophages were associated with HIV-expressing cells.

    Our data provide evidence that tissues serve as a sanctuary for wild-type HIV during cART and suggest the importance of macrophages as an alternative reservoir and mechanism of virus spread.

    IMPORTANCE Combined anti-retroviral therapy (cART) reduces plasma HIV to undetectable levels; however, removal of cART results in plasma HIV rebound, thus highlighting its inability to entirely rid the body of infection. Additionally, HIV-infected individuals on cART remain at high risk of serious diseases, which suggests a contribution from residual HIV. Here, we isolated and sequenced HIV from post mortem tissues from three HIV+/cART+ individuals who died with metastatic cancer and had no detectable plasma viral load. Using high-resolution evolutionary analyses, we found that tissue-based HIV continues to replicate, evolve and migrate among tissues during cART. Furthermore, cancer onset and metastasis coincided with increased HIV diversity, suggesting a linked mechanism. HIV-expressing cells were associated with tissue macrophages, a target of HIV infection. Our results suggest the importance of tissues, and macrophages in particular, as a target for novel anti-HIV therapies.

  • HIV DNA is frequently present within pathologic tissues evaluated at autopsy from cART-treated patients with undetectable viral load [PublishAheadOfPrint]

  • HIV infection treatment strategies have historically defined effectiveness through measuring patient plasma HIV RNA. While combined antiretroviral therapy (cART) can reduce plasma viral load (pVL) to undetectable levels, the degree that HIV is eliminated from other anatomical sites remains unclear. We investigated the HIV DNA levels in 229 varied autopsy tissues from 20 HIV+/cART-treated study participants with low or undetectable plasma VL and cerebrospinal fluid (CSF) VL prior to death, who were enrolled in the National Neurological AIDS Bank (NNAB) longitudinal study and autopsy cohort. Extensive medical histories were obtained for each participant. Autopsy specimens, including at least six brain and non-brain tissues per participant, were reviewed by study pathologists. HIV DNA, measured in tissues by quantitative and droplet digital PCR, was identified in 48/87 brain tissues and 82/142 non-brain tissues at levels ggt;200 HIV copies/million cell equivalents. No participant was found to be completely free of tissue HIV. Parallel sequencing studies from some tissues recovered intact HIV DNA and RNA. Abnormal histological findings were identified in all participants, especially in brain, spleen, lung, lymph node, liver, aorta and kidney. All brain tissues demonstrated some degree of pathology. 95% of participants had some degree of atherosclerosis and 75% of participants died with cancer. This study assists in characterizing the anatomic locations of HIV, in particular, macrophage-rich tissues, such as CNS and testis. Additional studies are needed to determine if the HIV recovered from tissues promotes the pathogenesis of inflammatory diseases, such as HIV-associated neurocognitive disorders, cancer, and atherosclerosis.

    IMPORTANCE It is well-known that combined antiretroviral therapy (cART) can reduce plasma HIV to undetectable levels; however, cART cannot completely clear HIV infection. An on-going question is, "Where is HIV hiding?" A well-studied HIV reservoir are "resting" T-cells, which can be isolated from blood products and succumb to cART once activated. Less studied reservoirs are anatomical tissue samples, which have unknown cART penetration, contain a comparably diverse spectrum of potentially HIV-infected immune cells, and are important since llt; 2% of body lymphocytes actually reside in blood. We examined 229 varied autopsy specimens from 20 HIV+ participants who died while on cART and identified that ggt;50% of tissues were HIV-infected. Additionally, we identified considerable pathology in participants' tissues, especially in brain, spleen, lung, lymph node, liver, aorta and kidney. This study substantiates that tissue-associated HIV is present despite cART and can inform future studies into HIV persistence.

  • Guinea pig prion protein supports rapid propagation of bovine spongiform encephalopathy and variant Creutzfeldt-Jakob disease prions [PublishAheadOfPrint]

  • The properties of bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (vCJD) prions are faithfully maintained upon transmission to guinea pigs. However, primary and secondary transmission of BSE and vCJD in guinea pigs results in long incubation periods of ~450 and ~350 days, respectively. To determine if the incubation periods for BSE and vCJD prions could be shortened, we generated transgenic (Tg) mice expressing guinea pig prion protein (GPPrP). Inoculation of Tg(GPPrP) mice with BSE and vCJD prions resulted in mean incubation periods of 210 and 199 days, respectively, which shortened to 137 and 122 days upon serial transmission. In contrast, three different isolates of sporadic CJD prions failed to transmit disease to Tg(GPPrP mice). Many of the strain-specified biochemical and neuropathological properties of BSE and vCJD prions, including the presence of type-2 protease-resistant PrPSc, were preserved upon propagation in Tg(GPPrP) mice. Structural modeling revealed that two residues near the N-terminal region of aalpha;-helix 1 in GPPrP might mediate its susceptibility to BSE and vCJD prions. Our results demonstrate that expression of GPPrP in Tg mice supports the rapid propagation of BSE and vCJD prions and suggest that Tg(GPPrP) mice may serve as a useful paradigm for bioassaying these prion isolates.

    IMPORTANCE Variant Creutzfeldt-Jakob disease (vCJD) and bovine spongiform encephalopathy (BSE) prions are two of the most relevant prion strains to human health. However, propagating these strains in mice expressing human or bovine prion protein has been difficult due to prolonged incubation periods or inefficient transmission. Here, we show that transgenic mice expressing guinea pig prion protein are fully susceptible to vCJD and BSE prions, but not to sporadic CJD prions. Our results suggest that the guinea pig prion protein is a better, more rapid substrate than either bovine or human prion protein for propagating BSE and vCJD prions.

  • Characterization of Viral Populations using Circular Sequencing [PublishAheadOfPrint]

  • With the enormous sizes viral populations reach, many variants are at too low a frequency to be detected by conventional next generation sequencing methods. Circular Sequencing (CirSeq) is a method by which the error rate of NGS is decreased so that even low frequency viral variants can be accurately detected. The ability to visualize almost the entire genetic makeup of a viral swarm has implications in epidemiology, viral evolution and vaccine design. Here we discuss experimental planning, analysis, and recent insights using CirSeq.

  • CD25+FoxP3+ memory CD4 T cells are frequent targets of HIV infection in vivo [PublishAheadOfPrint]

  • Interleukin 2 (IL2) signaling through the IL2 receptor alpha chain+ (CD25) facilitates HIV replication in vitro and facilitates homeostatic proliferation of CD25+FoxP3+CD4+ T cells. CD25+FoxP3+CD4+ T cells may therefore constitute a suitable subset for HIV infection and plasma virion production.

    CD25+FoxP3+CD4+ T cell frequencies, absolute numbers and the expression of CCR5 and cell cycle marker Ki67 were studied in peripheral blood from HIV+ and HIV- study volunteers. Different memory CD4+ T cell subsets were then sorted for quantification of cell-associated HIV-DNA and phylogenetic analyses of the highly variable EnvV1V3 region in comparison to plasma-derived virus sequences.

    In HIV+ subjects, 51% (median) of CD25+FoxP3+CD4+ T cells expressed the HIV co-receptor CCR5. Very high frequencies of Ki67+ cells were detected in CD25+FoxP3+ (median, 27.6%) in comparison to memory CD25-FoxP3- memory CD4+ T cells (median, 4.1%, pllt;0.0001). HIV-DNA content was 15-fold higher in CD25+FoxP3+ compared to CD25-FoxP3- memory CD4+ T cells (p=0.003). EnvV1V3 sequences derived from CD25+FoxP3+ memory CD4+ T cells did not preferentially cluster with plasma-derived sequences. Quasi-identical cell-plasma-sequence pairs were rare and their proportion further decreased with the estimated HIV infection duration.

    These data suggest that specific cellular characteristics of CD25+FoxP3+ memory CD4+ T cell might facilitate efficient HIV infection in vivo and passage of HIV DNA to cell progeny in the absence of active viral replication. Contribution of this cell population to plasma virion production remains unclear.

    Importance: Despite recent advances in the understanding of AIDS virus pathogenesis, it is incompletely understood, which cell subsets support HIV infection and replication in vivo. In vitro, the IL2 signaling pathway and IL2 dependent cell cycle induction are essential for HIV infection of stimulated T cells. CD25+FoxP3+ memory CD4 T cells - often referred to as regulatory CD4 T cells nndash; depend on IL2 signaling for homeostatic proliferation in vivo. Our results show that CD25+FoxP3+ memory CD4+ T cells often express the HIV co-receptor CCR5, are significantly more proliferative and contain more HIV-DNA compared to CD25-FoxP3- memory CD4 T cell subsets. The specific cellular characteristics of CD25+FoxP3+ memory CD4+ T cell probably facilitate efficient HIV infection in vivo and passage of HIV DNA to cell progeny in the absence of active viral replication. However contribution of this cell subset to plasma viremia remains unclear.

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