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Altered Growth Characteristics of Recombinant Respiratory Syncytial Viruses Which Do Not Produce NS2 Protein

Teng, Michael N.; Collins, Peter L.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /01/1999 Português
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The second gene in the 3′-to-5′ gene order in respiratory syncytial virus (RSV) encodes the nonstructural protein NS2, for which there is no assigned function. To study the function of NS2, we have used a recently developed reverse genetics system to ablate expression of NS2 in recombinant RSV. A full-length cDNA copy of the antigenome of RSV A2 strain under the control of a T7 promoter was modified by introduction of tandem termination codons within the NS2 open reading frame (NS2stop) or by deletion of the entire NS2 gene (ΔNS2). The NS2 knockout antigenomic cDNAs were cotransfected with plasmids encoding the N, P, L, and M2-1 proteins of RSV, each controlled by the T7 promoter, into cells infected with a vaccinia virus recombinant expressing T7 RNA polymerase. Recombinant NS2stop and ΔNS2 RSVs were recovered and characterized. Both types of NS2 knockout virus displayed pinpoint plaque morphology and grew more slowly than wild-type RSV. The expression of monocistronic mRNAs for the five genes examined (NS1, NS2, N, F, and L) was unchanged in cells infected with either type of NS2 knockout virus, except that no NS2 mRNA was detected with the ΔNS2 virus. Synthesis of readthrough mRNAs was affected only for the ΔNS2 virus, where the NS1-NS2...

Correlation between Point Mutations in NS2 and the Viability and Cytopathogenicity of Bovine Viral Diarrhea Virus Strain Oregon Analyzed with an Infectious cDNA Clone

Kümmerer, Beate M.; Meyers, Gregor
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /01/2000 Português
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Cytopathogenicity of Bovine viral diarrhea virus (BVDV) is correlated with expression of the nonstructural protein NS3, which can be generated by processing of a fusion protein termed NS2-3. For the cytopathogenic (cp) BVDV strain Oregon, NS2-3 processing is based on a set of point mutations within NS2. To analyze the correlation between NS2-3 cleavage and cytopathogenicity, a full-length cDNA clone composed of cDNA from BVDV Oregon and the utmost 5′- and 3′-terminal sequences of a published infectious BVDV clone was established. After transfection of RNA transcribed from this cDNA clone, infectious virus with similar growth characteristics to wild-type BVDV Oregon could be recovered that also exhibited a cytopathic effect. Based on this cDNA construct and published cp and noncp infectious clones, chimeric full-length cDNA clones were constructed. Analysis of the recovered viruses demonstrated that the presence of the NS2 gene of BVDV Oregon in a chimeric construct is sufficient for NS2-3 processing and a cp phenotype. Since previous studies had revealed that the amino acid serine at position 1555 of BVDV Oregon plays an important role in efficient NS2-3 cleavage, mutants of BVDV Oregon with different amino acids at this position were constructed. Some of these mutants showed NS2-3 cleavage efficiencies in the range of the wild-type sequence and allowed the recovery of viruses that behaved similarly to wild-type virus with regard to growth characteristics and cytopathogenicity. In contrast...

Characterization of the Hepatitis C Virus NS2/3 Processing Reaction by Using a Purified Precursor Protein

Pallaoro, Michele; Lahm, Armin; Biasiol, Gabriella; Brunetti, Mirko; Nardella, Caterina; Orsatti, Laura; Bonelli, Fabio; Orrù, Stefania; Narjes, Frank; Steinkühler, Christian
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /10/2001 Português
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The NS2-NS3 region of the hepatitis C virus polyprotein encodes a proteolytic activity that is required for processing of the NS2/3 junction. Membrane association of NS2 and the autocatalytic nature of the NS2/3 processing event have so far constituted hurdles to the detailed investigation of this reaction. We now report the first biochemical characterization of the self-processing activity of a purified NS2/3 precursor. Using multiple sequence alignments, we were able to define a minimal domain, devoid of membrane-anchoring sequences, which was still capable of performing the processing reaction. This truncated protein was efficiently expressed and processed in Escherichia coli. The processing reaction could be significantly suppressed by growth in minimal medium in the absence of added zinc ions, leading to the accumulation of an unprocessed precursor protein in inclusion bodies. This protein was purified to homogeneity, refolded, and shown to undergo processing at the authentic NS2/NS3 cleavage site with rates comparable to those observed using an in vitro-translated full-length NS2/3 precursor. Size-exclusion chromatography and a dependence of the processing rate on the concentration of truncated NS2/3 suggested a functional multimerization of the precursor protein. However...

Nonstructural proteins NS2 of minute virus of mice associate in vivo with 14-3-3 protein family members.

Brockhaus, K; Plaza, S; Pintel, D J; Rommelaere, J; Salomé, N
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1996 Português
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The nonstructural NS2 proteins of the prototype strain of minute virus of mice (MVMp) were previously shown to be involved in parvoviral DNA amplification as well as in efficient virus production in a host cell-specific manner (L. K. Naeger, N. Salomé, and D. J. Pintel, J. Virol. 67:1034-1043, 1993). NS2 polypeptides were also reported to participate in the cytotoxic activity of parvoviruses (C. Legrand, J. Rommelaere, and P. Caillet-Fauquet, Virology 195:149-155, 1993), for which transformed cells are preferential targets. To identify cellular partners of NS2 proteins, coimmunoprecipitation experiments were performed with various antibodies directed against the parvoviral products. Two cellular proteins with molecular masses of 30 and 32 kDa were found to associate in vivo with the NS2 polypeptides. From amino acid sequence homology, these NS2 partners were assigned to the 14-3-3 family of cellular proteins, showing at least partial identity with the epsilon and beta or zeta 14-3-3 isoforms. In agreement with this assignment, NS2-30/32-kDa protein immune complexes displayed an activating function for exoenzyme S in vitro, a hallmark of 14-3-3 polypeptides. Interactions with 14-3-3 proteins did not appear sufficient for NS2 functions...

Uncleaved NS2-3 Is Required for Production of Infectious Bovine Viral Diarrhea Virus

Agapov, Eugene V.; Murray, Catherine L.; Frolov, Ilya; Qu, Lin; Myers, Tina M.; Rice, Charles M.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /03/2004 Português
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Despite increasing characterization of pestivirus-encoded proteins, functions for nonstructural (NS) proteins NS2, NS2-3, NS4B, and NS5A have not yet been reported. Here we investigated the function of bovine viral diarrhea virus (BVDV) uncleaved NS2-3. To test whether NS2-3 has a discrete function, the uncleaved protein was specifically abolished in two ways: first by inserting a ubiquitin monomer between NS2 and NS3, and second by placing an internal ribosome entry site between the two proteins (a bicistronic genome). In both cases, complete processing of NS2-3 prevented infectious virion formation without affecting RNA replication. We tested the hypothesis that uncleaved NS2-3 was involved in morphogenesis by creating a bicistronic genome in which NS2-3 was restored in the second cistron. With this genome, both uncleaved NS2-3 expression and particle production returned. We then investigated the minimal regions of the polyprotein that could rescue an NS2-3 defect by developing a trans-complementation assay. We determined that the expression of NS4A in cis with NS2-3 markedly increased its activity, while p7 could be supplied in trans. Based on these data, we propose a model for NS2-3 action in virion morphogenesis.

Hepatitis C Virus NS2 Protein Is Phosphorylated by the Protein Kinase CK2 and Targeted for Degradation to the Proteasome

Franck, Nathalie; Le Seyec, Jacques; Guguen-Guillouzo, Christiane; Erdtmann, Lars
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /03/2005 Português
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Hepatitis C virus (HCV) nonstructural 2 (NS2) protein is a hydrophobic transmembrane protein, described to be involved in different functions, such as apoptosis inhibition and gene transcription modulation. We investigated here NS2 protein turnover and found that NS2 was rapidly degraded by the proteasome in different cell lines, as in primary human hepatocytes. Since posttranslational modifications can influence protein turnover, we looked for potential phosphoacceptor sites in NS2. Computational sequence analysis in combination with screening of NS2 point mutants revealed that serine residue 168 was critical for degradation. In the quest of a protein kinase for NS2, we identified by sequence analysis that the serine residue 168 was part of a consensus casein kinase 2 (CK2) recognition site (S/TXXE). This motif was highly conserved since it could be found in the NS2 primary consensus sequences from all HCV genotypes. To verify whether CK2 is involved in NS2 phosphorylation, we showed by an in vitro kinase assay that CK2 phosphorylated NS2, as far as this CK2 motif was conserved. Interestingly, NS2 became resistant to protein degradation when the CK2 motif was modified by a single point mutation. Furthermore, inhibition of CK2 activity by curcumin decreased NS2 phosphorylation in vitro and stabilized NS2 expression in HepG2 cells. Finally...

Compensatory Mutations in E1, p7, NS2, and NS3 Enhance Yields of Cell Culture-Infectious Intergenotypic Chimeric Hepatitis C Virus▿

Yi, MinKyung; Ma, Yinghong; Yates, Jeremy; Lemon, Stanley M.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Português
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There is little understanding of mechanisms underlying the assembly and release of infectious hepatitis C virus (HCV) from cultured cells. Cells transfected with synthetic genomic RNA from a unique genotype 2a virus (JFH1) produce high titers of virus, while virus yields are much lower with a prototype genotype 1a RNA containing multiple cell culture-adaptive mutations (H77S). To characterize the basis for this difference in infectious particle production, we constructed chimeric genomes encoding the structural proteins of H77S within the background of JFH1. RNAs encoding polyproteins fused at the NS2/NS3 junction (“H-NS2/NS3-J”) and at a site of natural, intergenotypic recombination within NS2 [“H-(NS2)-J”] produced infectious virus. In contrast, no virus was produced by a chimera fused at the p7-NS2 junction. Chimera H-NS2/NS3-J virus (vH-NS2/NS3-J) recovered from transfected cultures contained compensatory mutations in E1 and NS3 that were essential for the production of infectious virus, while yields of infectious vH-(NS2)-J were enhanced by mutations within p7 and NS2. These compensatory mutations were chimera specific and did not enhance viral RNA replication or polyprotein processing; thus, they likely compensate for incompatibilities between proteins of different genotypes at sites of interactions essential for virus assembly and/or release. Mutations in p7 and NS2 acted additively and increased the specific infectivity of vH-(NS2)-J particles...

Organ-Specific Attenuation of Murine Hepatitis Virus Strain A59 by Replacement of Catalytic Residues in the Putative Viral Cyclic Phosphodiesterase ns2▿

Roth-Cross, Jessica K.; Stokes, Helen; Chang, Guohui; Chua, Ming Ming; Thiel, Volker; Weiss, Susan R.; Gorbalenya, Alexander E.; Siddell, Stuart G.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
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The Murine hepatitis virus (MHV) strain A59 ns2 protein is a 30-kDa nonstructural protein that is expressed from a subgenomic mRNA in the cytoplasm of virus-infected cells. Its homologs are also encoded in other closely related group 2a coronaviruses and more distantly related toroviruses. Together, these proteins comprise a subset of a large superfamily of 2H phosphoesterase proteins that are distinguished by a pair of conserved His-x-Thr/Ser motifs encompassing catalytically important residues. We have used a vaccinia virus-based reverse genetic system to produce recombinant viruses encoding ns2 proteins with single-amino-acid substitutions in, or adjacent to, these conserved motifs, namely, inf-ns2 H46A, inf-ns2 S48A, inf-ns2-S120A, and inf-ns2-H126R. All of the mutant viruses replicate in mouse 17 clone 1 fibroblast cells and mouse embryonic cells to the same extent as the parental wild-type recombinant virus, inf-MHV-A59. However, compared to inf-MHV-A59, the inf-ns2 H46A and inf-ns2-H126R mutants are highly attenuated for replication in mouse liver following intrahepatic inoculation. Interestingly, none of the mutant viruses were attenuated for replication in mouse brain following intracranial inoculation. These results show that the ns2 protein of MHV-A59 has an important role in virus pathogenicity and that a substitution of the histidine residues of the MHV-A59 ns2 His-x-Thr/Ser motifs is critical for virus virulence in the liver but not in the brain. This novel phenotype suggests a strategy to investigate the function of the MHV-A59 ns2 protein involving the search for organ-specific proteins or RNAs that react differentially to wild-type and mutant ns2 proteins.

Hepatitis C virus NS2 is a protease stimulated by cofactor domains in NS3

Schregel, V.; Jacobi, S.; Penin, F.; Tautz, N.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
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Chronic infection with hepatitis C virus (HCV) affects 130 million people worldwide and is a major cause of liver cirrhosis and liver cancer. After translation of the HCV RNA genome into a polyprotein, 2 viral proteases process its non-structural protein (NS) region. While the essential chymotrypsin-like serine protease NS3–4A mediates all cleavages downstream of NS3, the NS2–3 cysteine protease catalyzes a vital cleavage at the NS2/3 site. Protease activity of NS2–3 has been described to require, besides NS2, the N-terminal 181 aa of NS3. The latter domain corresponds to the NS3 serine protease domain and contains a structural Zn2+-binding site with functional importance for both viral proteases. The catalytic triad of the NS2–3 protease resides in NS2; the role of the NS3 part in proteolysis remained largely undefined. Here we report a basal proteolytic activity for NS2 followed by only 2 amino acids of NS3. Basal activity could be dramatically enhanced by the NS3 Zn2+-binding domain (NS3 amino acids 81–213) not only in cis but also in trans which, however, required a more extended N-terminal part of NS3 downstream of NS2 in cis. Thus, this study defines for the first time (i) NS2 as a bona fide protease, (ii) NS3 as its regulatory cofactor...

Hepatitis C Virus NS2 Protein Serves as a Scaffold for Virus Assembly by Interacting with both Structural and Nonstructural Proteins▿ †

Ma, Yinghong; Anantpadma, Manu; Timpe, Jennifer M.; Shanmugam, Saravanabalaji; Singh, Sher M.; Lemon, Stanley M.; Yi, MinKyung
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
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Many aspects of the assembly of hepatitis C virus (HCV) remain incompletely understood. To characterize the role of NS2 in the production of infectious virus, we determined NS2 interaction partners among other HCV proteins during productive infection. Pulldown assays showed that NS2 forms complexes with both structural and nonstructural proteins, including E1, E2, p7, NS3, and NS5A. Confocal microscopy also demonstrated that NS2 colocalizes with E1, E2, and NS5A in dot-like structures near lipid droplets. However, NS5A did not coprecipitate with E2 and interacted only weakly with NS3 in pulldown assays. Also, there was no demonstrable interaction between p7 and E2 or NS3 in such assays. Therefore, NS2 is uniquely capable of interacting with both structural and nonstructural proteins. Among mutations in p7, NS2, and NS3 that prevent production of infectious virus, only p7 mutations significantly reduced NS2-mediated protein interactions. These p7 mutations altered the intracellular distribution of NS2 and E2 and appeared to modulate the membrane topology of the C-terminal domain of NS2. These results suggest that NS2 acts to coordinate virus assembly by mediating interactions between envelope proteins and NS3 and NS5A within replication complexes adjacent to lipid droplets...

Hepatitis C Virus NS2 Coordinates Virus Particle Assembly through Physical Interactions with the E1-E2 Glycoprotein and NS3-NS4A Enzyme Complexes ▿

Stapleford, Kenneth A.; Lindenbach, Brett D.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
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The hepatitis C virus (HCV) NS2 protein is essential for particle assembly, but its function in this process is unknown. We previously identified critical genetic interactions between NS2 and the viral E1-E2 glycoprotein and NS3-NS4A enzyme complexes. Based on these data, we hypothesized that interactions between these viral proteins are essential for HCV particle assembly. To identify interaction partners of NS2, we developed methods to site-specifically biotinylate NS2 in vivo and affinity capture NS2-containing protein complexes from virus-producing cells with streptavidin magnetic beads. By using these methods, we confirmed that NS2 physically interacts with E1, E2, and NS3 but did not stably interact with viral core or NS5A proteins. We further characterized these protein complexes by blue native polyacrylamide gel electrophoresis and identified ≈520-kDa and ≈680-kDa complexes containing E2, NS2, and NS3. The formation of NS2 protein complexes was dependent on coexpression of the viral p7 protein and enhanced by cotranslation of viral proteins as a polyprotein. Further characterization indicated that the glycoprotein complex interacts with NS2 via E2, and the pattern of N-linked glycosylation on E1 and E2 suggested that these interactions occur in the early secretory pathway. Importantly...

NS2 Protein of Hepatitis C Virus Interacts with Structural and Non-Structural Proteins towards Virus Assembly

Popescu, Costin-Ioan; Callens, Nathalie; Trinel, Dave; Roingeard, Philippe; Moradpour, Darius; Descamps, Véronique; Duverlie, Gilles; Penin, François; Héliot, Laurent; Rouillé, Yves; Dubuisson, Jean
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Português
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Growing experimental evidence indicates that, in addition to the physical virion components, the non-structural proteins of hepatitis C virus (HCV) are intimately involved in orchestrating morphogenesis. Since it is dispensable for HCV RNA replication, the non-structural viral protein NS2 is suggested to play a central role in HCV particle assembly. However, despite genetic evidences, we have almost no understanding about NS2 protein-protein interactions and their role in the production of infectious particles. Here, we used co-immunoprecipitation and/or fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy analyses to study the interactions between NS2 and the viroporin p7 and the HCV glycoprotein E2. In addition, we used alanine scanning insertion mutagenesis as well as other mutations in the context of an infectious virus to investigate the functional role of NS2 in HCV assembly. Finally, the subcellular localization of NS2 and several mutants was analyzed by confocal microscopy. Our data demonstrate molecular interactions between NS2 and p7 and E2. Furthermore, we show that, in the context of an infectious virus, NS2 accumulates over time in endoplasmic reticulum-derived dotted structures and colocalizes with both the envelope glycoproteins and components of the replication complex in close proximity to the HCV core protein and lipid droplets...

Inhibition of Hepatitis C Virus Infection by DNA Aptamer against NS2 Protein

Gao, Yimin; Yu, Xiaoyan; Xue, Binbin; Zhou, Fei; Wang, Xiaohong; Yang, Darong; Liu, Nianli; Xu, Li; Fang, Xiaohong; Zhu, Haizhen
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 28/02/2014 Português
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NS2 protein is essential for hepatitis C virus (HCV) replication. NS2 protein was expressed and purified. Aptamers against NS2 protein were raised and antiviral effects of the aptamers were examined. The molecular mechanism through which the aptamers exert their anti-HCV activity was investigated. The data showed that aptamer NS2-3 inhibited HCV RNA replication in replicon cell line and infectious HCV cell culture system. NS2-3 and another aptamer NS2-2 were demonstrated to inhibit infectious virus production without cytotoxicity in vitro. They did not affect hepatitis B virus replication. Interferon beta (IFN-β) and interferon-stimulated genes (ISGs) were not induced by the aptamers in HCV-infected hepatocytes. Furthermore, our study showed that N-terminal region of NS2 protein is involved in the inhibition of HCV infection by NS2-2. I861T within NS2 is the major resistance mutation identified. Aptamer NS2-2 disrupts the interaction of NS2 with NS5A protein. The data suggest that NS2-2 aptamer against NS2 protein exerts its antiviral effects through binding to the N-terminal of NS2 and disrupting the interaction of NS2 with NS5A protein. NS2-specific aptamer is the first NS2 inhibitor and can be used to understand the mechanisms of virus replication and assembly. It may be served as attractive candidates for inclusion in the future HCV direct-acting antiviral combination therapies.

NS2 Proteins of GB Virus B and Hepatitis C Virus Share Common Protease Activities and Membrane Topologies

Boukadida, Célia; Marnata, Caroline; Montserret, Roland; Cohen, Lisette; Blumen, Brigitte; Gouttenoire, Jérôme; Moradpour, Darius; Penin, François; Martin, Annette
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /07/2014 Português
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GB virus B (GBV-B), which is hepatotropic in experimentally infected small New World primates, is a member of the Hepacivirus genus but phylogenetically relatively distant from hepatitis C virus (HCV). To gain insights into the role and specificity of hepaciviral nonstructural protein 2 (NS2), which is required for HCV polyprotein processing and particle morphogenesis, we investigated whether NS2 structural and functional features are conserved between HCV and GBV-B. We found that GBV-B NS2, like HCV NS2, has cysteine protease activity responsible for cleavage at the NS2/NS3 junction, and we experimentally confirmed the location of this junction within the viral polyprotein. A model for GBV-B NS2 membrane topology was experimentally established by determining the membrane association properties of NS2 segments fused to green fluorescent protein (GFP) and their nuclear magnetic resonance structures using synthetic peptides as well as by applying an N-glycosylation scanning approach. Similar glycosylation studies confirmed the HCV NS2 organization. Together, our data show that despite limited amino acid sequence similarity, GBV-B and HCV NS2 proteins share a membrane topology with 3 N-terminal transmembrane segments, which is also predicted to apply to other recently discovered hepaciviruses. Based on these data and using trans-complementation systems...

CHD3 facilitates vRNP nuclear export by interacting with NES1 of influenza A virus NS2

Hu, Yong; Liu, Xiaokun; Zhang, Anding; Zhou, Hongbo; Liu, Ziduo; Chen, Huanchun; Jin, Meilin
Fonte: Springer Basel Publicador: Springer Basel
Tipo: Artigo de Revista Científica
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NS2 from influenza A virus mediates Crm1-dependent vRNP nuclear export through interaction with Crm1. However, even though the nuclear export signal 1 (NES1) of NS2 does not play a requisite role in NS2–Crm1 interaction, there is no doubt that NES1 is crucial for vRNP nuclear export. While the mechanism of the NES1 is still unclear, it is speculated that certain host partners might mediate the NES1 function through their interaction with NES1. In the present study, chromodomain-helicase-DNA-binding protein 3 (CHD3) was identified as a novel host nuclear protein for locating NS2 and Crm1 on dense chromatin for NS2 and Crm1-dependent vRNP nuclear export. CHD3 was confirmed to interact with NES1 in NS2, and a disruption to this interaction by mutation in NES1 significantly delayed viral vRNPs export and viral propagation. Further, the knockdown of CHD3 would affect the propagation of the wild-type virus but not the mutant with the weakened NS2–CHD3 interaction. Therefore, this study demonstrates that NES1 is required for maximal binding of NS2 to CHD3, and that the NS2–CHD3 interaction on the dense chromatin contributed to the NS2-mediated vRNP nuclear export.

A Conserved NS3 Surface Patch Orchestrates NS2 Protease Stimulation, NS5A Hyperphosphorylation and HCV Genome Replication

Isken, Olaf; Langerwisch, Ulrike; Jirasko, Vlastimil; Rehders, Dirk; Redecke, Lars; Ramanathan, Harish; Lindenbach, Brett D.; Bartenschlager, Ralf; Tautz, Norbert
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 16/03/2015 Português
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Hepatitis C virus (HCV) infection is a leading cause of liver disease worldwide. The HCV RNA genome is translated into a single polyprotein. Most of the cleavage sites in the non-structural (NS) polyprotein region are processed by the NS3/NS4A serine protease. The vital NS2-NS3 cleavage is catalyzed by the NS2 autoprotease. For efficient processing at the NS2/NS3 site, the NS2 cysteine protease depends on the NS3 serine protease domain. Despite its importance for the viral life cycle, the molecular details of the NS2 autoprotease activation by NS3 are poorly understood. Here, we report the identification of a conserved hydrophobic NS3 surface patch that is essential for NS2 protease activation. One residue within this surface region is also critical for RNA replication and NS5A hyperphosphorylation, two processes known to depend on functional replicase assembly. This dual function of the NS3 surface patch prompted us to reinvestigate the impact of the NS2-NS3 cleavage on NS5A hyperphosphorylation. Interestingly, NS2-NS3 cleavage turned out to be a prerequisite for NS5A hyperphosphorylation, indicating that this cleavage has to occur prior to replicase assembly. Based on our data, we propose a sequential cascade of molecular events: in uncleaved NS2-NS3...

L’étude de l’impact des protéines non structurales NS1 et NS2 du virus respiratoire syncitial sur la réponse immunitaire innée

Yoboua, Fabrice Aman
Fonte: Université de Montréal Publicador: Université de Montréal
Tipo: Thèse ou Mémoire numérique / Electronic Thesis or Dissertation
Português
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Le virus respiratoire syncytial (RSV) est un virus à ARN de polarité négative. Les études démontrent que toute la population sera infectée par ce virus au moins deux fois avant l’âge de 3 ans. Le RSV peut provoquer plusieurs pathologies respiratoires telles que la bronchiolite aiguë et la pneumonie. Les infections sévères corrèlent avec le développement de l’asthme. Lors d’une infection virale, les particules du RSV sont détectées par le senseur RIG-I qui induit l’activation des facteurs de transcription NF-κB et IRF-3. Respectivement, les facteurs de transcription activeront les réponses inflammatoire et antivirale. Au coeur des pathologies induites par le RSV se trouve une réponse immunitaire mal adaptée. Plus précisément, par l’entremise de NF-κB, le RSV provoque une production exagérée de cytokines et chimiokines qui induisent une réponse inflammatoire démesurée provoquant du dommage tissulaire. Paradoxalement, le RSV est capable d’échapper à la réponse antivirale. Ces deux phénomènes sont contrôlés par l’entremise des protéines non structurales NS1 et NS2. Le mécanisme délimitant le mode d’action de NS1 et NS2 sur la réponse antivirale reste à être déterminé. Avec pour objectif d’élucider comment NS1 et NS2 inhibent la réponse antivirale...

Identification of residues involved in NS2 homodimerization and elucidation of their impact on the HCV life cycle

Gorzin, A.; Ramsland, P.; Tachedjian, G.; Gowans, E.
Fonte: Blackwell Science Ltd Publicador: Blackwell Science Ltd
Tipo: Artigo de Revista Científica
Publicado em //2012 Português
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The NS2 protein of hepatitis C virus (HCV) plays a critical role in virus morphogenesis and infectivity. The crystal structure of the C-terminus of the NS2 protein (NS2Pro) from the H77 strain indicates that NS2Pro forms a homodimer. In this study, using computational modelling, we identified residues at the NS2Pro dimer interface that have a role in dimerization and confirmed their capacity to influence dimerization by expression studies. Our modelling analysis identified 22 residues at the NS2Pro dimer interface that may be important for dimer formation. Based on the free binding energy, we selected the top five ranked mutations (V162A, M170A, I175A, D186A and I201A) for further study. Western blot analysis revealed that M170A, I175A, I201A, D186A and V162A resulted in a 4.0-, 3.2-, 3.0-, 2.8- and 1.5-fold increase, respectively, in the monomer/dimer ratio compared to wild type, confirming a role in homodimer formation or stability. Japanese Fulminant Hepatitis type 1 mutants expressing M170A, I175A, D186A and I201A demonstrated increasing defects in both RNA replication and the production of infectious virus compared to wild type. This study identified residues at the NS2Pro dimer interface that modulate NS2Pro homodimerization and demonstrated that abrogation of NS2Pro homodimerization results in defects in HCV replication and release of infectious virus.; A. A. Gorzin...

Identification of specific regions in hepatitis C virus core, NS2 and NS5A that genetically interact with p7 and co-ordinate infectious virus production

Gouklani, H.; Beyer, C.; Drummer, H.; Gowans, E.; Netter, H.; Haqshenas, G.
Fonte: Blackwell Publishing Publicador: Blackwell Publishing
Tipo: Artigo de Revista Científica
Publicado em //2013 Português
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The p7 protein of hepatitis C virus (HCV) is a small, integral membrane protein that plays a critical role in virus replication. Recently, we reported two intergenotypic JFH1 chimeric viruses encoding the partial or full-length p7 protein of the HCV-A strain of genotype 1b (GT1b; Virology; 2007; 360:134). In this study, we determined the consensus sequences of the entire polyprotein coding regions of the wild-type JFH1 and the revertant chimeric viruses and identified predominant amino acid substitutions in core (K74M), NS2 (T23N, H99P) and NS5A (D251G). Forward genetic analysis demonstrated that all single mutations restored the infectivity of the defective chimeric genomes suggesting that the infectious virus production involves the association of p7 with specific regions in core, NS2 and NS5A. In addition, it was demonstrated that the NS2 T23N facilitated the generation of infectious intergenotypic chimeric virus encoding p7 from GT6 of HCV.; H. Gouklani, C. Beyer, H. Drummer, E. J. Gowans, H. J. Netter, and G. Haqshenas

Einfluss von Mutationen auf die NS2/3-Prozessierung und die Zytopathogenität von Pestiviren; Infuence of mutations in the processing of NS2/3 and cytopathogenicity of Pestiviruses

Bernhard, Roger
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
Português
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Das Virus der bovinen viralen Diarrhöe (BVDV) und das Virus der klassischen Schweinepest (CSFV) sind umhüllte Viren, die dem Genus Pestivirus der Familie Flaviviridae angehören. Das Genom besteht aus einer einzelsträngigen RNA positiver Polarität und kodiert für ein etwa 4000 Aminosäuren langes Polyprotein, welches ko- und posttranslational in die reifen Virusproteine gespalten wird. BVD-Viren werden aufgrund ihres Verhaltens in der Zellkultur in zytopathogene (zp) und nicht zytopathogene (nzp) Viren unterteilt. Im Vergleich mit den nzp BVD-Viren weisen die bisher analysierten zp BVD-Viren verschiedene Genomveränderungen auf, die das Ergebnis von RNARekombinationsvorgängen sind. Zu diesen Veränderungen zählen: (1) Insertionen zellulärer Sequenzen, (2) Duplikationen viraler Sequenzen, (3) Insertion einer viralen Sequenz in einem anderen Leseraster und (4) Deletionen. Aufgrund dieser Genomveränderungen kommt es zur kostitutiven Expression eines für zp BVD-Viren spezifischen Proteins, des NS3, welches das carboxyterminale Spaltprodukt von NS2/3 darstellt und in nzp- Virus infizierten Zellen nur transient, im Frühstadium der Infektion auftritt. NS2/3 wird sowohl in zp- als auch in nzp-BVDV infizierten Zellen dauerhaft exprimiert. Viren der klassischen Schweinepest hingegen exprimieren sowohl das Nichtstrukturprotein NS2/3 als auch dessen Prozessierungsprodukte NS2 und NS3...