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Charles M. Rice - One of the best experts on this subject based on the ideXlab platform.
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The C Terminus of Hepatitis C Virus NS4A Encodes an Electrostatic Switch That Regulates NS5A Hyperphosphorylation and Viral Replication
Journal of Virology, 2007Co-Authors: Brett D Lindenbach, Roland Montserret, Anna Marie Pyle, Béla M. Prágai, Rudolf K. F. Beran, Francois Penin, Charles M. RiceAbstract:Hepatitis C virus (HCV) nonstructural protein 4A (NS4A) is only 54 amino acids (aa) in length, yet it is a key regulator of the essential serine protease and RNA helicase activities of the NS3-4A complex, as well as a determinant of NS5A phosphorylation. Here we examine the structure and function of the C-terminal acidic region of NS4A through site-directed mutagenesis of a Con1 subgenomic replicon and through biophysical characterization of a synthetic peptide corresponding to this region. Our genetic studies revealed that in 8 of the 15 C-terminal residues of NS4A, individual Ala substitutions or charge reversal substitutions led to severe replication phenotypes, as well as decreased NS5A hyperphosphorylation. By selecting for replication-competent mutants, several second-site changes in NS3 were identified and shown to suppress these defects in replication and NS5A hyperphosphorylation. Circular-dichroism spectroscopy and nuclear magnetic resonance spectroscopy on a peptide corresponding to the C-terminal 19 aa of NS4A revealed that this region can adopt an alpha-helical conformation, but that this folding requires neutralization of a cluster of acidic residues. Taken together, these data suggest that the C terminus of NS4A acts as a dynamic regulator of NS3-4A interaction, NS5A hyperphosphorylation, and HCV replicase activity.
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uncleaved ns2 3 is required for production of infectious bovine viral diarrhea virus
Journal of Virology, 2004Co-Authors: Eugene Agapov, Charles M. Rice, Catherine L Murray, Ilya Frolov, Tina M MyersAbstract: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.
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mutations in the yellow fever virus nonstructural protein ns2a selectively block production of infectious particles
Journal of Virology, 2002Co-Authors: Beate M Kummerer, Charles M. RiceAbstract:The Yellow fever virus (YF) belongs to the genus Flavivirus within the family Flaviviridae. Members of the Flavivirus genus are typically transmitted to vertebrates by mosquitoes or ticks and frequently cause significant human morbidity and mortality (reviewed in reference 25). Human pathogens include dengue virus, Japanese encephalitis virus, tick-borne encephalitis virus, West Nile virus, and YF. The estimated 100 million cases of dengue virus infection per year worldwide (5) and the emergence and spread of West Nile virus in the Eastern United States underscore the need for continued efforts to develop effective and inexpensive flavivirus vaccines. For YF, a live attenuated vaccine strain (17D) has been used effectively for almost 65 years. However, YF remains an enduring global public health problem due to the endemic persistence of mosquito-borne disease in sub-Saharan Africa and South America and recent reports of six fatalities temporally associated with live-virus vaccination (22, 33). The YF genome is a positive-sense RNA approximately 11 kb in length that is capped at the 5′ end but lacks a 3′ poly(A) tract. The RNA contains a single large open reading frame that is cleaved co- and posttranslationally by host cell and viral proteases (reviewed in reference 16). The polyprotein is arranged with the structural proteins at the amino terminus (C-prM-E), followed by the nonstructural (NS) proteins (NS1 through NS5). The arrangement of the proteins is NH2-C-(pr)M-E-NS1-NS2A-NS2B-NS3-NS4A-2K-NS4B-NS5-COOH. Most of the cleavages releasing the structural proteins are mediated by host cell signal peptidase. Exceptions include prM cleavage into pr and M by the host cell enzyme furin shortly before virus release and the cleavage generating the C terminus of the virion C protein by the virus-encoded serine protease (NS2B-3 protease). This serine protease, consisting of NS2B and the N-terminal part of NS3, produces the N termini of NS2B, NS3, NS4A, 2K, and NS5. The enzyme responsible for cleavage at the NS1/2A site is unknown. Although all flavivirus proteins stem from a single polyprotein, the structural and NS proteins have been viewed as functionally distinct modules responsible for virion formation and RNA replication, respectively. For example, coexpression of prM and E is sufficient for secretion of subviral particles that mimic the subunit structure and fusogenic capabilities of the mature virion envelope (2, 29). Subgenomic replicons lacking the structural proteins replicate efficiently and can be packaged by trans expression of the structural proteins (11). Although initially thought to be involved in virion morphogenesis or release, secreted glycoprotein NS1 plays an essential role in RNA replication (18). These observations have reinforced a modular view of the flavivirus polyprotein with the dividing line between structural proteins and replicase drawn at the E/NS1 junction. The only known exception is the NS2B-3 serine protease-mediated cleavage at the C terminus of mature C, a cleavage that is a necessary prerequisite for signalase generation of the prM N terminus and virus production (4). Specific functions have been attributed to many flavivirus proteins (reviewed in reference 16), but little is known about the function of NS2A. NS2A is a small hydrophobic protein of about 22 kDa (8). Consistent with a role in RNA replication, studies with Kunjin virus (KUN) have shown that NS2A colocalizes with double-stranded RNA in discrete cytoplasmic foci and interacts with the 3′ untranslated region of KUN RNA, as well as NS3 and NS5 (20). NS2A has also been implicated in the Japanese encephalitis virus-induced cytopathic effect (CPE) (10). In YF-infected cells, 22- and 20-kDa forms of NS2A have been identified and both of these forms possess the same N-terminal sequence (8). The 20-kDa form (called NS2Aα) is believed to result from an additional internal cleavage by the NS2B-3 serine protease. Cleavage by the YF serine protease occurs at a consensus sequence consisting of two basic amino acids followed by an amino acid with a short side chain (RR↓S/G) (6). However, in the case of the NS4A/2K junction, the cleavage site is QR↓S (14). Based on the known cleavage sites, as well as substitutions at these sites that are tolerated (9, 15, 26), NS2A residues 189 to 191 (QK↓T) were identified as a possible target for the viral serine protease. Consistent with this hypothesis, replacement of Lys-190 with Ser resulted in loss of the 20-kDa protein (26). Inhibition of cleavage at the NS2A/2B site did not abrogate production of NS2Aα, indicating that processing at the NS2A/2B site was not a prerequisite for cleavage at the NS2Aα site (26). It was further shown that the NS2A Lys-190-Ser mutation blocked production of infectious virus (26). Although the roles of NS2A and this additional cleavage in YF replication were unknown, a block at the level of YF RNA replication seemed most likely. In this report, we show that NS2Aα cleavage site mutants have unimpaired RNA replication but are unable to produce infectious virus. This defect in virus production can be complemented by NS2A or NS2Aα supplied in trans and compensated for by mutations at the NS2Aα cleavage site or by second-site changes in the helicase domain of NS3. These results reveal a more complex interplay between the NS proteins and virus production than previously suspected.
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subcellular localization stability and trans cleavage competence of the hepatitis c virus ns3 NS4A complex expressed in tetracycline regulated cell lines
Journal of Virology, 2000Co-Authors: Benno Wolk, Charles M. Rice, Domenico Sansonno, Hansgeorg Krausslich, Franco Dammacco, Hubert E Blum, Darius MoradpourAbstract:A tetracycline-regulated gene expression system and a panel of novel monoclonal antibodies were used to examine the subcellular localization, stability, and trans-cleavage competence of the hepatitis C virus (HCV) NS3-NS4A complex in inducible cell lines. The NS3 serine protease domain and the full-length NS3 protein expressed in the absence of the NS4A cofactor were diffusely distributed in the cytoplasm and nucleus. Coexpression of NS4A, however, directed NS3 to the endoplasmic reticulum (ER) or an ER-like modified compartment, as demonstrated by colocalization with 3,3′-dihexyloxacarbocyanine iodide, protein disulfide isomerase, and calnexin, as well as subcellular fractionation analyses. In addition, coexpression with NS4A dramatically increased the intracellular stability of NS3 (mean protein half-life of 26 versus 3 h) and allowed for NS4A-dependent trans-cleavage at the NS4B-NS5A junction. Deletion analyses revealed that the hydrophobic amino-terminal domain of NS4A was required for ER targeting of NS3. These results demonstrate the importance of studying HCV proteins in their biological context and define a well-characterized cell culture system for further analyses of the NS3-NS4A complex and the evaluation of novel antiviral strategies against hepatitis C.
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identification of the major phosphorylation site of the hepatitis c virus h strain ns5a protein as serine 2321
Journal of Biological Chemistry, 1999Co-Authors: Karen E Reed, Charles M. RiceAbstract:Abstract The hepatitis C virus (HCV) NS5A protein is phosphorylated by a cellular, serine/threonine kinase. To identify the major site(s) of NS5A phosphorylation, radiolabeled HCV-H NS5A phosphopeptides were purified and subjected to phosphoamino acid analysis and Edman degradation. These data identified the major intracellular phosphorylation site in the HCV-H NS5A protein as Ser2321, a result verified by two additional, independent methods: (i) substitution of Ala for Ser2321and the concomitant disappearance of the major in vivophosphorylated peptides and corresponding in vitrophosphorylated peptides; and (ii) comigration of the digestion products of a synthetic peptide phosphorylated on Ser2321 with the major in vivo phosphorylated NS5A peptides. Site-directed mutagenesis of Ser2321 suggested that phosphorylation of NS5A is dispensable for previously described interactions with NS4A and PKR, a cellular, antiviral kinase that does not appear to catalyze NS5A phosphorylation. The proline-rich nature of the amino acid sequence flanking Ser2321 (PLPPPRS2321 PPVPPPR) suggests that a proline-directed kinase is responsible for the majority of HCV NS5A phosphorylation, consistent with previous kinase inhibitor studies.
Kunitada Shimotohno - One of the best experts on this subject based on the ideXlab platform.
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phosphorylation of nonstructural 5a protein of hepatitis c virus hcv group specific hyperphosphorylation
Virology, 1999Co-Authors: Masami Hirota, Shin Ichi Asabe, Shinya Satoh, Makoto Hijikata, Michinori Kohara, Kyoko Tsukiyamakohara, Nobuyuki Kato, Kunitada ShimotohnoAbstract:We previously showed that two proteins with molecular weights of 56 and 58 kDa are produced from nonstructural protein 5A (NS5A) derived from hepatitis C virus (HCV)-1b genotype. The 56-kDa protein is phosphorylated at serine residues in NS5A, including those located in the C-terminal region of NS5A, while the 58-kDa protein, the hyperphosphorylated form of the 56-kDa protein, is phosphorylated at serine residues in the central region. This hyperphosphorylation is dependent on the presence of HCV NS4A protein. To clarify whether NS4A-dependent phosphorylation also occurs in other HCV genotypes, phosphorylation of NS5A was analyzed by two-dimensional gel electrophoresis. Here, we report that NS5A from the HCV-2a genotype was phosphorylated. However, hyperphosphorylation of NS5A occurs in the HCV-1b genotype but not in the -2a genotype. This result suggests that modification of NS5A phosphorylation reflects the virological features of HCV and that there are physiological differences in the roles of differently phosphorylated NS5A between HCV genotypes.
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the n terminal region of hepatitis c virus nonstructural protein 3 ns3 is essential for stable complex formation with NS4A
Journal of Virology, 1995Co-Authors: Shinya Satoh, Makoto Hijikata, Yasunori Tanji, K Kimura, Kunitada ShimotohnoAbstract:Hepatitis C virus proteins are produced by proteolytic processing of the viral precursor polyprotein that is encoded in the largest open reading frame of the viral genome. Processing of the nonstructural viral polyprotein requires the viral serine-type proteinase present in nonstructural protein 3 (NS3). The cleavage of the junction between NS4B and NS5A is mediated by NS3 only when NS4A is present. NS4A is thought to be a cofactor that enhances the cleavage efficiency of NS3 in hepatitis C virus protein-producing cells. Stable NS3-NS4A complex formation required the N-terminal 22 amino acid residues of NS3. This interaction contributed to stabilization of the NS3 product as well as increased the efficiency of cleavage at the NS4B/5A site. The N-terminal 22 amino acid residues fused to Escherichia coli dihydrofolate reductase also formed a stable complex with NS4A. NS3 derivatives which lacked the N-terminal 22 amino acid residues showed drastically reduced cleavage activity at the NS4B/5A site even in the presence of NS4A. These data suggested that the interaction with NS4A through the 22 amino acid residues of NS3 is primarily important for the NS4A-dependent processing of the NS4B/5A site by NS3.
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hepatitis c virus encoded nonstructural protein NS4A has versatile functions in viral protein processing
Journal of Virology, 1995Co-Authors: Yasunori Tanji, Makoto Hijikata, Shinya Satoh, T. Kaneko, Kunitada ShimotohnoAbstract:A transient protein expression system in COS-1 cells was used to study the role of hepatitis C virus (HCV)-encoded NS4A protein on HCV nonstructural polyprotein processing. By analyzing the protein expression and processing of a deletion mutant polypeptide, NS delta 4A, which encodes the entire putative HCV nonstructural polyprotein except the region encoding NS4A, the versatile functions of NS4A were revealed. Most of the NS3 processed from NS delta 4A was localized in the cytosol fraction and was degraded promptly. Coproduction of NS4A stabilizes NS3 and assists in its localization in the membrane. NS4A was found to be indispensable for cleavage at the 4B/5A site but not essential for cleavage at the 5A/5B site in NS delta 4A. The functioning of NS4A as a cofactor for cleavage at the 4B/5A site was also observed when 30 amino acids around this site was used as a substrate and a serine proteinase domain of 167 amino acids, from Gly-1049 to Ser-1215, was used as an enzyme protein, suggesting that possible domains for the interaction of NS4A were in those regions of the enzyme protein (NS3) and/or the substrate protein. Two proteins, p58 and p56, were produced from NS5A. For the production of p58, equal or excess molar amounts of NS4A relative to NS delta 4A were required. Deletion analysis of NS4A revealed a minimum functional domain of NS4A of 10 amino acids, from Gly-1678 to Ile-1687.
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The N-Terminal Region of Hepatitis C Virus Nonstructural Protein 3 (NS3) Is Essential for Stable Complex
1995Co-Authors: Formation Nsa, Kunitada ShimotohnoAbstract:Hepatitis C virus proteins are produced by proteolytic processing of the viral precursor polyprotein that is encoded in the largest open reading frame of the viral genome. Processing of the nonstructural viral polyprotein requires the viral serine-type proteinase present in nonstructural protein 3 (NS3). The cleavage of the junction between NS4B and NS5A is mediated by NS3 only when NS4A is present. NS4A is thought to be a cofactor that enhances the cleavage efficiency of NS3 in hepatitis C virus protein-producing cells. Stable NS3-NS4A complex formation required the N-terminal 22 amino acid residues of NS3. This interaction contributed to stabilization of the NS3 product as well as increased the efficiency of cleavage at the NS4B/5A site. The N-terminal 22 amino acid residues fused to Escherichia coli dihydrofolate reductase also formed a stable complex with NS4A. NS3 derivatives which lacked the N-terminal 22 amino acid residues showed drastically reduced cleavage activity at the NS4B/5A site even in the presence of NS4A. These data suggested that the interaction with NS4A through the 22 amino acid residues of NS3 is primarily important for the NS4A-dependent processing of the NS4B/5A site by NS3. Hepatitis C virus (HCV) is a causative agent of posttransfusion non-A, non-B hepatitis (5, 18). From analysis of the viral genome and the putative viral proteins encoded in the genome
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Production of two phosphoproteins from the NS5A region of the hepatitis C viral genome.
Biochemical and Biophysical Research Communications, 1994Co-Authors: T. Kaneko, Shin Ichi Asabe, Shinya Satoh, Makoto Hijikata, Yasunori Tanji, K Kimura, Kunitada ShimotohnoAbstract:Abstract Hepatitis C virus produces about 12 viral proteins by proteolytic cleavage of the viral polyprotein precursor produced from the largest open reading frame in the viral genome. We have analyzed the production of viral nonstructural proteins with an in vivo transient expression system using COS-1 cells. Two proteins, a 56-kDa protein and a 58-kDa protein, were produced from the nonstructural region 5A (NS5A), which has the potential to produce a 49 kDa protein. We showed that these proteins are phosphorylated at the serine residues. The presence of the two proteins was reflected by different degrees of phosphorylation. Moreover, the hyper-phosphorylation of p58 was shown to depend on the presence of NS4A, another hepatitis C virus protein.
Ralf Bartenschlager - One of the best experts on this subject based on the ideXlab platform.
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Overview of amino acid residues in NS1 required for NS1 secretion, interaction with the NS4A-2K-4B precursor and RNA replication.
2019Co-Authors: Anna Płaszczyca, Pietro Scaturro, Christopher John Neufeldt, Mirko Cortese, Berati Cerikan, Salvatore Ferla, Andrea Brancale, Andreas Pichlmair, Ralf BartenschlagerAbstract:Replication-impairing mutations that (A) abolish NS1 secretion, (B) abrogate binding between NS1 and the NS4A-2K-4B precursor, and (C) can be complemented by second site mutations in NS4B. Upper panels show the linear map of NS1 with mutated residues indicated by red stars; bottom panels show the homology model of the 3D structure of NS1 based on PDB entries 4O6B and 5K6K as described in Fig 1(A) with mutated residues shown as van der Waal spheres in red. Wing, β-ladder and β-roll domains are shown in blue, turquoise and orange, respectively. Connector subdomains in Wing domain are shown in dark blue.
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a conserved ns3 surface patch orchestrates ns2 protease stimulation ns5a hyperphosphorylation and hcv genome replication
PLOS Pathogens, 2015Co-Authors: Olaf Isken, Harish N. Ramanathan, Brett D Lindenbach, Ralf Bartenschlager, Ulrike Langerwisch, Vlastimil Jirasko, Dirk Rehders, Lars Redecke, Norbert TautzAbstract: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, the hydrophobic NS3 surface patch promotes NS2 protease stimulation; upon NS2-NS3 cleavage, this surface region becomes available for functional replicase assembly. This model explains why efficient NS2-3 cleavage is pivotal for HCV RNA replication. According to our model, the hydrophobic surface patch on NS3 represents a module critically involved in the temporal coordination of HCV replicase assembly.
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production of infectious genotype 1b virus particles in cell culture and impairment by replication enhancing mutations
PLOS Pathogens, 2009Co-Authors: Thomas Pietschmann, Volker Lohmann, Nicole Appel, Margarita Zayas, Philip Meuleman, Gang Long, George Koutsoudakis, Stephanie Kallis, Geert Lerouxroels, Ralf BartenschlagerAbstract:With the advent of subgenomic hepatitis C virus (HCV) replicons, studies of the intracellular steps of the viral replication cycle became possible. These RNAs are capable of self-amplification in cultured human hepatoma cells, but save for the genotype 2a isolate JFH-1, efficient replication of these HCV RNAs requires replication enhancing mutations (REMs), previously also called cell culture adaptive mutations. These mutations cluster primarily in the central region of non-structural protein 5A (NS5A), but may also reside in the NS3 helicase domain or at a distinct position in NS4B. Most efficient replication has been achieved by combining REMs residing in NS3 with distinct REMs located in NS4B or NS5A. However, in spite of efficient replication of HCV genomes containing such mutations, they do not support production of infectious virus particles. By using the genotype 1b isolate Con1, in this study we show that REMs interfere with HCV assembly. Strongest impairment of virus formation was found with REMs located in the NS3 helicase (E1202G and T1280I) as well as NS5A (S2204R), whereas a highly adaptive REM in NS4B still allowed virus production although relative levels of core release were also reduced. We also show that cells transfected with the Con1 wild type genome or the genome containing the REM in NS4B release HCV particles that are infectious both in cell culture and in vivo. Our data provide an explanation for the in vitro and in vivo attenuation of cell culture adapted HCV genomes and may open new avenues for the development of fully competent culture systems covering the therapeutically most relevant HCV genotypes.
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the non structural protein 4a of dengue virus is an integral membrane protein inducing membrane alterations in a 2k regulated manner
Journal of Biological Chemistry, 2007Co-Authors: Sven Miller, Stefan Kastner, Jacomine Krijnselocker, Sandra Buhler, Ralf BartenschlagerAbstract:Dengue virus (DV) is a positive sense RNA virus replicating in the cytoplasm in membranous compartments that are induced by viral infection. The non-structural protein (NS) 4A is one of the least characterized DV proteins. It is highly hydrophobic with its C-terminal region (designated 2K fragment) serving as a signal sequence for the translocation of the adjacent NS4B into the endoplasmic reticulum (ER) lumen. In this report, we demonstrate that NS4A associates with membranes via 4 internal hydrophobic regions, which are all able to mediate membrane targeting of a cytosolic reporter protein. We also developed a model for the membrane topology of NS4A in which the N-terminal third of NS4A localizes to the cytoplasm, while the remaining part contains three transmembrane segments, with the C-terminal end localized in the ER lumen. Subcellular localization experiments in DV-infected cells revealed that NS4A resides primarily in ER-derived cytoplasmic dot-like structures that also contain dsRNA and other DV proteins, suggesting that NS4A is a component of the membrane-bound viral replication complex (RC). Interestingly, the individual expression of DV NS4A lacking the 2K fragment resulted in the induction of cytoplasmic membrane alterations resembling virus-induced structures, whereas expression of full-length NS4A does not induce comparable membrane alterations. Thus, proteolytic removal of the 2K peptide appears to be important for induction of membrane alterations that may harbor the viral RC. These results shed new light on the role of NS4A in the DV replication cycle and provide a model of how this protein induces membrane rearrangements and how this property may be regulated.
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characterization of cell lines carrying self replicating hepatitis c virus rnas
Journal of Virology, 2001Co-Authors: Thomas Pietschmann, Volker Lohmann, Gabriel Rutter, Katharina Kurpanek, Ralf BartenschlagerAbstract:The hepatitis C virus (HCV) is a major leading cause of chronic liver disease (reviewed in reference 45). Infections with HCV are usually subclinical, and most patients do not develop acute hepatitis or have only mild symptoms. However, most infected individuals are unable to eliminate the virus, resulting in a persistent infection in ∼80% of all cases, and these patients are at high risk to develop liver fibrosis, liver cirrhosis, or hepatocellular carcinoma. HCV was classified as the distinct genus Hepacivirus in the family Flaviviridae (38). Other members of this family are the pestiviruses, to which the Classical swine fever virus (CSFV) belongs, and the flaviviruses, with the prototype member Yellow fever virus. These viruses have in common a plus-strand RNA genome carrying a single long open reading frame (ORF) that is flanked at both termini by nontranslated regions (NTRs). In case of HCV, the 5′ NTR has a length of 341 nucleotides and carries an internal ribosome entry site (IRES) permitting the binding of ribosomes in close proximity of the start codon of the ORF (57, 58). The 3′ NTR has a tripartite structure composed of a variable region following the stop codon of the ORF, a polyuridine tract of variable length, and a 98-nucleotide sequence, designated the X-tail, that is highly conserved among all HCV genotypes and that is essential for replication in vivo (31, 32, 52, 53, 61). The ORF encodes a polyprotein of ∼3,000 amino acid residues and it is cleaved into at least 10 different products: core (C), envelope proteins E1 and E2, p7, and nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B (reviewed in references 9 and 47). C, E1, and E2 are the structural proteins that are processed by host cell signal peptidases (22). The function of the small hydrophobic polypeptide p7 so far is not known. NS2 and the amino-terminal domain of NS3 constitute the NS2-3 proteinase responsible for cleavage between NS2 and NS3 (20, 23). NS3 harbors three different enzymatic activities. The amino-terminal ∼180 residues constitute a chymotrypsin-like serine proteinase responsible for cleavage of the NS3-5B region (7, 21). The carboxy-terminal remainder possesses nucleoside triphosphatase and helicase activities (29, 51). NS4A is an NS3 proteinase cofactor forming a stable complex with this enzyme and enhancing its proteolytic activity (10, 16, 33, 54). The function of NS4B is currently unknown. NS5A is a highly phosphorylated protein that at least with some genotypes is produced in two phosphorylation states: a basal and a hyperphosphorylated form that can be separated because of their different apparent molecular weights (27, 48, 55). The requirements for hyperphosphorylation appear to differ with respect to HCV genotypes. For instance, in case of the genotype 1b HCV-J isolate NS5A hyperphosphorylation is increased upon coexpression of NS4A, and this increase depends on complex formation between both proteins (1). Recently, two groups have reported independently for two other genotype 1b isolates that a continuous NS3-5A region is required for hyperphosphorylation (30, 40). Whether NS5A is directly involved in RNA replication is not known. A more indirect role could be the inhibition of the effector proteins of the antiviral state induced in the cell after stimulation with alpha interferon. It was shown that NS5A of at least some HCV isolates can interact with the double-stranded RNA activated protein kinase R (18, 19). This enzyme normally induces a reduction of translation via the phosphorylation of translation initiation factor eIF-2α. Upon interaction of NS5A with protein kinase R, kinase activity is blocked, allowing continued translation in cells in the presence of alpha interferon. However, the in vivo relevance of this observation is not known (17). The most carboxy-terminal domain of the polyprotein is the RNA-dependent RNA polymerase NS5B (11, 34, 60). Despite great progress in understanding the genomic organization of the virus and the functions of viral proteins, fundamental aspects of HCV replication, pathogenesis, and persistence remain unknown. A major barrier in gaining experimental access to these issues is the lack of an efficient cell culture system allowing production of infectious virus particles. Although infection of primary cell cultures and certain human cell lines has been reported, the amounts of virus produced in these systems and the levels of HCV replication have been too low to permit detailed studies (for review, see reference 9). As a first step towards establishing a more productive system, we have recently described the construction of selectable subgenomic HCV RNAs that replicate to high levels in the human hepatoma cell line Huh-7 (35). These replicons were derived from a cloned full-length HCV consensus genome of genotype 1b by removing the C-p7 or C-NS2 region and insertion of the neomycin phosphotransferase gene (neo) downstream of the HCV IRES. Translation of the HCV NS2-5B or NS3-5B region was directed by the IRES of the encephalomyocarditis virus (EMCV) inserted downstream of neo. After transfection of Huh-7 cells only those supporting HCV RNA replication amplified neo and developed resistance against the drug G418. Cell lines derived from such G418-resistant colonies contained high levels of replicon RNAs and viral proteins (35). Since the availability of such a system for the first time allowed an analysis of the interplay between an autonomously replicating subgenomic HCV RNA and the host cell, we performed a detailed characterization of two of these cell lines carrying an NS3-5B replicon (cell lines 9-13 and 5-15 [35]). We analyzed the stabilities of HCV RNAs under different conditions of cell passage, polyprotein processing kinetics, the half-lives of the cleavage products, and their subcellular localization. A strong dependence of HCV RNA replication on cell growth was found, suggesting that cellular factors are limiting in resting cells. Finally, no ultrastructural changes or alterations of growth properties were found in cells with a replicon, suggesting that these HCV RNAs and the viral NS3-5B proteins are not cytopathogenic.
Raffaele De Francesco - One of the best experts on this subject based on the ideXlab platform.
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hyperphosphorylation of the hepatitis c virus ns5a protein requires an active ns3 protease NS4A ns4b and ns5a encoded on the same polyprotein
Journal of Virology, 1999Co-Authors: Petra Neddermann, Angelica Clementi, Raffaele De FrancescoAbstract:The nonstructural protein NS5A of hepatitis c virus (HCV) has been demonstrated to be a phosphoprotein with an apparent molecular mass of 56 kDa. In the presence of other viral proteins, p56 is converted into a slower-migrating form of NS5A (p58) by additional phosphorylation events. In this report, we show that the presence of NS3, NS4A, and NS4B together with NS5A is necessary and sufficient for the generation of the hyperphosphorylated form of NS5A (p58) and that all proteins must be encoded on the same polyprotein (in cis). Kinetic studies of NS5A synthesis and pulse-chase experiments demonstrate that fully processed NS5A is the substrate for the formation of p58 and that p56 is converted to p58. To investigate the role of NS3 in NS5A hyperphosphorylation, point and deletion mutations were introduced into NS3 in the context of a polyprotein containing the proteins from NS3 to NS5A. Mutation of the catalytic serine residue into alanine abolished protease activity of NS3 and resulted in total inhibition of NS5A hyperphosphorylation, even if polyprotein processing was allowed by addition of NS3 and NS4A in trans. The same result was obtained by deletion of the first 10 or 28 N-terminal amino acids of NS3, which are known to be important for the formation of a stable complex between NS3 and its cofactor NS4A. These data suggest that the formation of p58 is closely connected to HCV polyprotein processing events. Additional data obtained with NS3 containing the 34 C-terminal residues of NS2 provide evidence that in addition to NS3 protease activity the authentic N-terminal sequence is required for NS5A hyperphosphorylation.
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molecular virology of the hepatitis c virus
Journal of Hepatology, 1999Co-Authors: Raffaele De FrancescoAbstract:Infection with the hepatitis C virus (HCV) is the major cause of nonA-nonB hepatitis worldwide. Although this virus cannot be cultivated in vitro, several of its key features have been elucidated in the past few years. The viral genome is a positive-sense, single-stranded, 9.6 kb long RNA molecule. The viral genome is translated into a single polyprotein of about 3000 amino acids. The viral polyprotein is proteolytically processed by the combination of cellular and viral proteinases in order to yield all the mature viral gene products. The genomic order of HCV has been shown to be C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B. C, E1 and E2 are the virion.structural proteins. The function of p7 is currently unknown. These proteins have been shown to arise from the viral polyprotein via proteolytic processing by the host signal peptidases. Generation of the mature nonstructural proteins, NS2 to NS5B, relies on the activity of viral proteinases. Cleavage at the NS2/NS3 junction is accomplished by a metal-dependent autocatalytic proteinase encoded within NS2 and the N-terminus of NS3. The remaining cleavages downstream from this site are effected by a serine proteinase also contained within the N-terminal region of NS3. NS3 also contains an RNA helicase domain at its C-terminus. NS3 forms a heterodimeric complex with NS4A. The latter is a membrane protein that has been shown to act as a cofactor of the proteinase. While no function has yet been attributed to NS4B, it has recently been suggested that NS5A is involved in mediating the resistance of the hepatitis C virus to the action of interferon. Finally, the NS5B protein has been shown to be the viral RNA-dependent RNA polymerase.
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product inhibition of the hepatitis c virus ns3 protease
Biochemistry, 1998Co-Authors: Christian Steinkuhler, Gabriella Biasiol, Mirko Brunetti, Andrea Urbani, Uwe Koch, Riccardo Cortese, And Antonello Pessi, Raffaele De FrancescoAbstract:The nonstructural protein NS3 of the hepatitis C virus (HCV) harbors a serine protease domain that is responsible for most of the processing events of the nonstructural region of the polyprotein. Its inhibition is presently regarded as a promising strategy for coping with the disease caused by HCV. In this work, we show that the NS3 protease undergoes inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A−NS4B, NS4B−NS5A, and NS5A−NS5B cleavage sites, whereas no inhibition is observed with a cleavage product of the intramolecular NS3−NS4A junction. The Ki values of the hexamer inhibitory products [Ki(NS4A) = 0.6 μM, Ki(NS5A) = 1.4 μM, and Ki(NS4B) = 180 μM] are lower than the Km values of the respective substrate peptides [Km(NS4A−NS4B) = 10 μM, Km(NS5A−NS5B) = 3.8 μM, and Km(NS4B−NS5A) > 1000 μM]. Mutagenesis experiments have identified Lys136 as an important determinant for product binding. The phenomenon of product inhibition can be exploited to optimize peptide ...
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potent peptide inhibitors of human hepatitis c virus ns3 protease are obtained by optimizing the cleavage products
Biochemistry, 1998Co-Authors: Paolo Ingallinella, Raffaele De Francesco, Christian Steinkuhler, Riccardo Cortese, Sergio Altamura, Elisabetta Bianchi, Marina Taliani, Raffaele Ingenito, And Antonello PessiAbstract:In the absence of a broadly effective cure for hepatitis caused by hepatitis C virus (HCV), much effort is currently devoted to the search for inhibitors of the virally encoded protease NS3. This chymotrypsin-like serine protease is required for the maturation of the viral polyprotein, cleaving it at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B sites. In the course of our studies on the substrate specificity of NS3, we found that the products of cleavage corresponding to the P6-P1 region of the substrates act as competitive inhibitors of the enzyme, with IC50s ranging from 360 to 1 microM. A detailed study of product inhibition by the natural NS3 substrates is described in the preceding paper [Steinkuhler, C., et al. (1997) Biochemistry 37, 8899-8905]. Here we report the results of a study of the structure-activity relationship of the NS3 product inhibitors, which suggest that the mode of binding of the P region-derived products is similar to the ground-state binding of the corresponding substrates, with additional binding energy provided by the C-terminal carboxylate. Optimal binding requires a dual anchor: an "acid anchor" at the N terminus and a "P1 anchor" at the C-terminal part of the molecule. We have then optimized the sequence of the product inhibitors by using single mutations and combinatorial peptide libraries based on the most potent natural product, Ac-Asp-Glu-Met-Glu-Glu-Cys-OH (Ki = 0.6 microM), derived from cleavage at the NS4A-NS4B junction. By sequentially optimizing positions P2, P4, P3, and P5, we obtained several nanomolar inhibitors of the enzyme. These compounds are useful both as a starting point for the development of peptidomimetic drugs and as structural probes for investigating the substrate binding site of NS3 by modeling, NMR, and crystallography.
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in vitro activity of hepatitis c virus protease ns3 purified from recombinant baculovirus infected sf9 cells
Journal of Biological Chemistry, 1996Co-Authors: Christian Steinkuhler, Licia Tomei, Raffaele De FrancescoAbstract:A recombinant Baculovirus expression system was used for the production of a 20-kDa protein encompassing the hepatitis C virus NS3 protease domain. The protein was purified to apparent homogeneity after detergent extraction of cell homogenates. It was shown to be a monomer in solution and to cleave the in vitro translated precursor proteins NS4A-NS4B and NS5A-NS5B, but not the NS4B-NS5A or the NS3-NS4A precursors. The enzyme also cleaved a 20-mer peptide corresponding to the NS4A-NS4B junction with kcat/Km = 174 m(-1) s(-1). Peptides harboring NS4A sequences comprising amino acids 21-54 (Pep4A21-54) and 21-34 (Pep4A21-34) were found to induce an up to 2.8-fold acceleration of cleavage. Kinetic analysis revealed that this acceleration was due to an increase in kcat whereas no significant effect on Km could be detected. Pep4A21-54 was also an absolute requirement for cleavage of in vitro translated NS4B-NS5A by the purified protease. From these data we conclude that: (i) the purified protease domain shows substrate specificity and cleavage requirements similar to those previously reported on the basis of transfection experiments, (ii) activation of the purified protease by the NS4A co-factor can be mimicked by synthetic peptide analogs, and (iii) a central hydrophobic region of NS4A with a minimum core of 14 amino acids is responsible for the interaction with NS3.
Andreas Pichlmair - One of the best experts on this subject based on the ideXlab platform.
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a novel interaction between dengue virus nonstructural protein 1 and the NS4A 2k 4b precursor is required for viral rna replication but not for formation of the membranous replication organelle
PLOS Pathogens, 2019Co-Authors: Anna Plaszczyca, Pietro Scaturro, Christopher John Neufeldt, Mirko Cortese, Berati Cerikan, Salvatore Ferla, Andrea Brancale, Andreas PichlmairAbstract:Dengue virus (DENV) has emerged as major human pathogen. Despite the serious socio-economic impact of DENV-associated diseases, antiviral therapy is missing. DENV replicates in the cytoplasm of infected cells and induces a membranous replication organelle, formed by invaginations of the endoplasmic reticulum membrane and designated vesicle packets (VPs). Nonstructural protein 1 (NS1) of DENV is a multifunctional protein. It is secreted from cells to counteract antiviral immune responses, but also critically contributes to the severe clinical manifestations of dengue. In addition, NS1 is indispensable for viral RNA replication, but the underlying molecular mechanism remains elusive. In this study, we employed a combination of genetic, biochemical and imaging approaches to dissect the determinants in NS1 contributing to its various functions in the viral replication cycle. Several important observations were made. First, we identified a cluster of amino acid residues in the exposed region of the β-ladder domain of NS1 that are essential for NS1 secretion. Second, we revealed a novel interaction of NS1 with the NS4A-2K-4B cleavage intermediate, but not with mature NS4A or NS4B. This interaction is required for RNA replication, with two residues within the connector region of the NS1 “Wing” domain being crucial for binding of the NS4A-2K-4B precursor. By using a polyprotein expression system allowing the formation of VPs in the absence of viral RNA replication, we show that the NS1 –NS4A-2K-4B interaction is not required for VP formation, arguing that the association between these two proteins plays a more direct role in the RNA amplification process. Third, through analysis of polyproteins containing deletions in NS1, and employing a trans-complementation assay, we show that both cis and trans acting elements within NS1 contribute to VP formation, with the capability of NS1 mutants to form VPs correlating with their capability to support RNA replication. In conclusion, these results reveal a direct role of NS1 in VP formation that is independent from RNA replication, and argue for a critical function of a previously unrecognized NS4A-2K-NS4B precursor specifically interacting with NS1 and promoting viral RNA replication.
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Overview of amino acid residues in NS1 required for NS1 secretion, interaction with the NS4A-2K-4B precursor and RNA replication.
2019Co-Authors: Anna Płaszczyca, Pietro Scaturro, Christopher John Neufeldt, Mirko Cortese, Berati Cerikan, Salvatore Ferla, Andrea Brancale, Andreas Pichlmair, Ralf BartenschlagerAbstract:Replication-impairing mutations that (A) abolish NS1 secretion, (B) abrogate binding between NS1 and the NS4A-2K-4B precursor, and (C) can be complemented by second site mutations in NS4B. Upper panels show the linear map of NS1 with mutated residues indicated by red stars; bottom panels show the homology model of the 3D structure of NS1 based on PDB entries 4O6B and 5K6K as described in Fig 1(A) with mutated residues shown as van der Waal spheres in red. Wing, β-ladder and β-roll domains are shown in blue, turquoise and orange, respectively. Connector subdomains in Wing domain are shown in dark blue.
