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Showing papers on "NS5B published in 1999"


Journal ArticleDOI
02 Jul 1999-Science
TL;DR: This work defines the structure of HCV replicons functional in cell culture and provides the basis for a long-sought cellular system that should allow detailed molecular studies ofHCV and the development of antiviral drugs.
Abstract: An estimated 170 million persons worldwide are infected with hepatitis C virus (HCV), a major cause of chronic liver disease. Despite increasing knowledge of genome structure and individual viral proteins, studies on virus replication and pathogenesis have been hampered by the lack of reliable and efficient cell culture systems. A full-length consensus genome was cloned from viral RNA isolated from an infected human liver and used to construct subgenomic selectable replicons. Upon transfection into a human hepatoma cell line, these RNAs were found to replicate to high levels, permitting metabolic radiolabeling of viral RNA and proteins. This work defines the structure of HCV replicons functional in cell culture and provides the basis for a long-sought cellular system that should allow detailed molecular studies of HCV and the development of antiviral drugs.

2,982 citations


Journal ArticleDOI
TL;DR: The HCV NS5B apoenzyme structure reported here can accommodate a template:primer duplex without global conformational changes, supporting the hypothesis that this structure is essentially preserved during the reaction pathway.
Abstract: Various classes of nucleotidyl polymerases with different transcriptional roles contain a conserved core structure. Less is known, however, about the distinguishing features of these enzymes, particularly those of the RNA-dependent RNA polymerase class. The 1. 9 A resolution crystal structure of hepatitis C virus (HCV) nonstructural protein 5B (NS5B) presented here provides the first complete and detailed view of an RNA-dependent RNA polymerase. While canonical polymerase features exist in the structure, NS5B adopts a unique shape due to extensive interactions between the fingers and thumb polymerase subdomains that serve to encircle the enzyme active site. Several insertions in the fingers subdomain account for intersubdomain linkages that include two extended loops and a pair of antiparallel alpha-helices. The HCV NS5B apoenzyme structure reported here can accommodate a template:primer duplex without global conformational changes, supporting the hypothesis that this structure is essentially preserved during the reaction pathway. This NS5B template:primer model also allows identification of a new structural motif involved in stabilizing the nascent base pair.

782 citations


Journal ArticleDOI
TL;DR: This superposition reveals the majority of the amino acid residues of the hepatitis C virus enzyme that are likely to be implicated in binding to the replicating RNA molecule and to the incoming NTP and suggests a rearrangement of the thumb domain as well as a possible concerted movement of thumb and fingertips during translocation of the RNA template-primer in successive polymerization rounds.
Abstract: We report the crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus, a major human pathogen, to 28-A resolution This enzyme is a key target for developing specific antiviral therapy The structure of the catalytic domain contains 531 residues folded in the characteristic fingers, palm, and thumb subdomains The fingers subdomain contains a region, the "fingertips," that shares the same fold with reverse transcriptases Superposition to the available structures of the latter shows that residues from the palm and fingertips are structurally equivalent In addition, it shows that the hepatitis C virus polymerase was crystallized in a closed fingers conformation, similar to HIV-1 reverse transcriptase in ternary complex with DNA and dTTP [Huang H, Chopra, R, Verdine, G L & Harrison, S C (1998) Science 282, 1669-1675] This superposition reveals the majority of the amino acid residues of the hepatitis C virus enzyme that are likely to be implicated in binding to the replicating RNA molecule and to the incoming NTP It also suggests a rearrangement of the thumb domain as well as a possible concerted movement of thumb and fingertips during translocation of the RNA template-primer in successive polymerization rounds

594 citations


Journal ArticleDOI
TL;DR: It is proposed that the unique alpha fingers might represent a common structural discriminator of the template-primer duplex that distinguishes between RNA and DNA during the replication of positive single-stranded RNA by viral RdRps.

478 citations


Journal ArticleDOI
TL;DR: Fine deletional analysis of this region revealed that a four-leucine motif (LLLL) in the hydrophobic domain is responsible for the solubility profile of the full-length NS5B.
Abstract: Production of soluble full-length nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) has been shown to be problematic and requires the addition of salts, glycerol, and detergents. In an effort to improve the solubility of NS5B, the hydrophobic C terminus containing 21 amino acids was removed, yielding a truncated NS5B (NS5BDeltaCT) which is highly soluble and monodispersed in the absence of detergents. Fine deletional analysis of this region revealed that a four-leucine motif (LLLL) in the hydrophobic domain is responsible for the solubility profile of the full-length NS5B. Enzymatic characterization revealed that the RNA-dependent RNA polymerase (RdRp) activity of this truncated NS5B was comparable to those reported previously by others. For optimal enzyme activity, divalent manganese ions (Mn2+) are preferred rather than magnesium ions (Mg2+), whereas zinc ions (Zn2+) inhibit the RdRp activity. Gliotoxin, a known poliovirus 3D RdRp inhibitor, inhibited HCV NS5B RdRp in a dose-dependent manner. Kinetic analysis revealed that HCV NS5B has a rather low processivity compared to those of other known polymerases.

292 citations


Journal ArticleDOI
10 Oct 1999-Virology
TL;DR: Cell fractionation analysis revealed that hVAP-33 is predominantly associated with the ER, the Golgi complex, and the prelysosomal membrane, consistent with its potential role in intracellular membrane trafficking.

245 citations


Journal ArticleDOI
TL;DR: This is the first recombinant HCV RdRp capable of copying the full-length HCV RNA in the primer-independent manner expected of the functional HCV RNAs, and it did not use the copy-back RNA as a primer.
Abstract: All of the previously reported recombinant RNA-dependent RNA polymerases (RdRp), the NS5B enzymes, of hepatitis C virus (HCV) could function only in a primer-dependent and template-nonspecific manner, which is different from the expected properties of the functional viral enzymes in the cells. We have now expressed a recombinant NS5B that is able to synthesize a full-length HCV genome in a template-dependent and primer-independent manner. The kinetics of RNA synthesis showed that this RdRp can initiate RNA synthesis de novo and yield a full-length RNA product of genomic size (9.5 kb), indicating that it did not use the copy-back RNA as a primer. This RdRp was also able to accept heterologous viral RNA templates, including poly(A)- and non-poly(A)-tailed RNA, in a primer-independent manner, but the products in these cases were heterogeneous. The RdRp used some homopolymeric RNA templates only in the presence of a primer. By using the 3′-end 98 nucleotides (nt) of HCV RNA, which is conserved in all genotypes of HCV, as a template, a distinct RNA product was generated. Truncation of 21 nt from the 5′ end or 45 nt from the 3′ end of the 98-nt RNA abolished almost completely its ability to serve as a template. Inclusion of the 3′-end variable sequence region and the U-rich tract upstream of the X region in the template significantly enhanced RNA synthesis. The 3′ end of minus-strand RNA of HCV genome also served as a template, and it required a minimum of 239 nt from the 3′ end. These data defined the cis-acting sequences for HCV RNA synthesis at the 3′ end of HCV RNA in both the plus and minus senses. This is the first recombinant HCV RdRp capable of copying the full-length HCV RNA in the primer-independent manner expected of the functional HCV RNA polymerase.

230 citations


Journal ArticleDOI
TL;DR: Deletion mutant analysis indicated that the N‐terminal region of NS5B protein was critical for the RNA binding, and this data provide not only important clues for understanding the mechanism of HCV replication, but also a new target of antiviral therapy.

118 citations


Journal ArticleDOI
TL;DR: Recombinant bovine viral diarrhea virus (BVDV) nonstructural protein 5B (NS5B) produced in insect cells has been shown to possess an RNA-dependent RNA polymerase (RdRp) activity and was found to utilize a circular single-stranded DNA as a template for RNA synthesis, suggesting that synthesis does not require ends in the template.
Abstract: Recombinant bovine viral diarrhea virus (BVDV) nonstructural protein 5B (NS5B) produced in insect cells has been shown to possess an RNA-dependent RNA polymerase (RdRp) activity. Our initial attempt to produce the full-length BVDV NS5B with a C-terminal hexahistidine tag in Escherichia coli failed due to the expression of insoluble products. Prompted by a recent report that removal of the C-terminal hydrophobic domain significantly improved the solubility of hepatitis C virus (HCV) NS5B, we constructed a similar deletion of 24 amino acids at the C terminus of BVDV NS5B. The resulting fusion protein, NS5BDeltaCT24-His, was purified to homogeneity and demonstrated to direct RNA replication via both primer-dependent (elongative) and primer-independent (de novo) mechanisms. Furthermore, BVDV RdRp was found to utilize a circular single-stranded DNA as a template for RNA synthesis, suggesting that synthesis does not require ends in the template. In addition to the previously described polymerase motifs A, B, C, and D, alignments with other flavivirus sequences revealed two additional motifs, one N-terminal to motif A and one C-terminal to motif D. Extensive alanine substitutions showed that while most mutations had similar effects on both elongative and de novo RNA syntheses, some had selective effects. Finally, deletions of up to 90 amino acids from the N terminus did not significantly affect RdRp activities, whereas deletions of more than 24 amino acids at the C terminus resulted in either insoluble products or soluble proteins (DeltaCT179 and DeltaCT218) that lacked RdRp activities.

116 citations


Journal ArticleDOI
TL;DR: The virus-encoded RNA-dependent RNA polymerase (RdRp), which is required for replication of the positive-strand RNA genome, is a key enzyme of members of the virus family Flaviviridae and is demonstrated to exhibit a common reactivity profile in vitro, typical of nucleic acid-polymerizing enzymes.
Abstract: The virus-encoded RNA-dependent RNA polymerase (RdRp), which is required for replication of the positive-strand RNA genome, is a key enzyme of members of the virus family Flaviviridae. By using heterologously expressed proteins, we demonstrate that the 77 kDa NS5B protein of two pestiviruses, bovine viral diarrhoea virus and classical swine fever virus, and the 100 kDa NS5 protein of the West Nile flavivirus possess RdRp activity in vitro. As originally shown for the RdRp of hepatitis C virus, RNA synthesis catalysed by the pestivirus and flavivirus enzymes is strictly primer- dependent in vitro. Accordingly, initiation of RNA polymerization on homopolymeric RNAs and heteropolymeric templates, the latter with a blocked 3′-hydroxyl group, was found to be dependent on the presence of complementary oligonucleotide primer molecules. On unblocked heteropolymeric templates, including authentic viral RNAs, the RdRps were shown to initiate RNA synthesis via intramolecular priming at the 3′-hydroxyl group of the template and ‘copy-back’ transcription, thus yielding RNase- resistant hairpin molecules. Taken together, the RdRps of different members of the Flaviviridae were demonstrated to exhibit a common reactivity profile in vitro, typical of nucleic acid- polymerizing enzymes.

112 citations


Journal ArticleDOI
TL;DR: In this study, a C-terminal hexahistidine-tagged helicase domain of the HCV NS3 protein was expressed in Escherichia coli and purified to homogeneity by conventional chromatography, resulting in enzymes that were indistinguishable from wild-type HCV helicase with regard to all four activities.
Abstract: Hepatitis C virus (HCV) is recognized as the main etiologic agent of parenterally transmitted non-A, non-B hepatitis (4, 24) and is responsible for a large proportion of cases of community-acquired hepatitis. The majority of HCV infections become persistent, leading to chronic hepatitis, liver cirrhosis, or even hepatocellular carcinoma (reviewed in reference 13). Development of an effective vaccine against HCV has been slow and difficult, in part due to the quasi-species nature of the virus. Approved therapies for viral hepatitis type C (hepatitis C) include alpha interferon and the combination of alpha interferon and ribavirin, which induce limited long-term response in hepatitis C patients and have relatively severe side effects. Therefore, it is of great interest to develop effective, HCV-specific therapeutic drugs. HCV is now classified in the Hepacivirus genus (reviewed in reference 13) of the Flaviviridae family (reviewed in reference 40), which includes two additional genera, Flavivirus and Pestivirus. The recently discovered hepatitis G virus (HGV or GBV-C) is closely related to HCV and has been proposed to be the fourth group in the Flaviviridae family. The majority of the 9.6-kb HCV genome encodes a large open reading frame corresponding to a polyprotein precursor of about 3,000 amino acids, which is flanked by 5′ and 3′ noncoding regions. The polyprotein precursor of the HCV H strain is proteolytically processed by cellular signal peptidase(s) and two HCV-encoded proteases into at least 10 distinct products, with the sequence NH2 - C - E1 - E2 - p7 - NS2 - NS3 - NS4A - NS4B - NS5A - NS5B - COOH (7–9, 27). The putative structural proteins include a core protein (C) and two envelope proteins (E1 and E2), whereas the nonstructural (NS) proteins, including two proteases, a combined helicase and nucleoside triphosphatase (NTPase), and an RNA-dependent RNA polymerase, are believed to be components of a complex responsible for viral RNA replication. The C-terminal 450 amino acids of the NS3 protein manifest a polynucleotide-stimulated NTPase activity (14, 45), a 3′-to-5′ unwinding activity (10, 14, 18, 46), and a single-stranded (ss) polynucleotide binding activity (10, 17, 46). Recently, elegant biochemical characterizations of this helicase have been presented by Porter and colleagues and Preugschat et al. (36–39). Helicases are enzymes that are able to separate the strands of duplex DNA or RNA by utilizing energy derived from hydrolysis of nucleoside 5′-triphosphates (NTPs, usually ATP) (reviewed in reference 30). Accordingly, the NTPase activity of helicases can be stimulated by polynucleotide. More than 200 proteins have been identified as putative helicases based on the presence of some or all of seven conserved amino acid motifs (for a recent review, see reference 6). Until now, only a handful of these putative helicases have been purified and shown to have duplex nucleic acid unwinding activity. Helicases have been shown to be involved in DNA metabolism (replication, repair, and genome recombination) (reviewed in references 30 and 32) and RNA metabolism (transcription, splicing or processing, transport, and translation initiation of RNA) (for a recent review, see reference 31). Mutations in several helicase genes have been associated with six human genetic disorders: Werner’s syndrome, Bloom’s syndrome, xeroderma pigmentosum, trichothiodystrophy, Cockayne’s syndrome, and α thalassemia-related mental retardation associated with the X chromosome (for a review, see reference 5). Helicases may also play critical roles in processes involved in the life cycles of many DNA and RNA viruses, including replication and recombination of viral DNA or RNA genome, RNA transcription, and translation (for a review, see references 16 and 21). Recently, four groups reported the X-ray crystallographic structures of three different helicases, i.e., the DNA helicases PcrA (44) and Rep (22) and the HCV NS3 RNA helicase (20, 48). These structures demonstrated that helicases have many common features in terms of protein folding and tertiary structure, although there is very limited homology in their primary amino acid sequences. For all three helicases, most of the six or seven conserved motifs (motifs I through VI) (see Fig. ​Fig.4A)4A) are located at similar positions within a cleft between two domains of the enzyme (see Fig. ​Fig.3A,3A, domains 1 and 2 of the HCV RNA helicase, and domains 1A and 2A of Rep or PcrA helicases (22, 44). The NTP-binding pocket, composed of residues of motifs I and II, is located on the side of domain 1 in this cleft. Our X-ray structural analysis of the HCV helicase bound to an ss (dU)8 oligonucleotide showed that the oligonucleotide binds in a groove that separates domain 3 from domains 1 and 2 (reference 20, and also see Fig. ​Fig.3A).3A). This nucleic acid-binding groove is oriented perpendicular to the NTP-binding cleft between domains 1 and 2. In this structure, most of the interactions between the enzyme and bound (dU)8 oligonucleotide involve hydrogen bonds with the phosphate backbone but not the bases of the oligonucleotide (20), which explains the sequence-independent nature of these helicases with regard to the duplex nucleic acid substrate. The most significant enzyme-base interaction involves a hydrophobic stacking interaction between Trp-501 of the HCV helicase and a base near the 3′ end of the bound (dU)8 oligonucleotide (see Fig. ​Fig.3B).3B). This structural analysis identifies several amino acids in this nucleic acid-binding groove that interact with the oligonucleotide (see Fig. ​Fig.3B).3B). Although some of these residues are conserved within various HCV strains, they are not a part of the previously reported conserved helicase motifs (motifs I through VI) (see Fig. ​Fig.4A).4A). In the present study, site-directed mutagenesis was used to investigate the role of these residues in the mechanism of action of the HCV NS3 helicase. FIG. 3 (A) Locations of conserved motifs in the ternary complex of HCV NS3 helicase, (dU)8 oligonucleotide, and ADP. The overall fold of the HCV NS3 helicase is illustrated by a green ribbon diagram. The HCV NS3 helicase consists of three domains: domain 1 (top ... FIG. 4 Alignment of functional conserved motifs or residues of selected helicases from SF1 and SF2. (A) Alignment of several conserved motifs and other functional conserved residues of two SF2 RNA helicases (HCV NS3 helicase and the eukaryotic translation factor ...

Journal ArticleDOI
TL;DR: The interaction between a partially purified recombinant NS5B protein and a 3′ viral genomic RNA with or without the conserved 98-nucleotide tail is demonstrated.
Abstract: Hepatitis C virus (HCV) NS5B protein is the viral RNA-dependent RNA polymerase capable of directing RNA synthesis In this study, an electrophoretic mobility shift assay demonstrated the interaction between a partially purified recombinant NS5B protein and a 3* viral genomic RNA with or without the conserved 98-nucleotide tail The NS5B-RNA complexes were specifically competed away by the unlabeled homologous RNA but not by the viral 5* noncoding region and very poorly by the 3* conserved 98-nucleotide tail A 3* coding region with conserved stem-loop structures rather than the 3* noncoding region of the HCV genome is critical for the specific binding of NS5B Nevertheless, no direct interaction between the 3* coding region and the HCV NS5A protein was detected Furthermore, two independent RNA-binding domains (RBDs) of NS5B were identified, RBD1, from amino acid residues 83 to 194, and RBD2, from residues 196 to 298 Interestingly, the conserved motifs of RNA-dependent RNA polymerase for putative RNA binding (220-DxxxxD-225) and template/primer position (282-S/TGxxxTxxxNS/T-292) are present in the RBD2 Nevertheless, the RNA-binding activity of RBD2 was abolished when it was linked to the carboxy-terminal half of the NS5B These results provide some clues to understanding the initiation of HCV replication Hepatitis C virus (HCV) is an enveloped virus that possesses a single-stranded positive-sense RNA genome encoding a polyprotein of approximately 3,000 amino acid residues (7, 20, 29) The conserved 59 noncoding region (59NCR) of the HCV genome (4, 13) is highly structured and contributes to the internal ribosome entry site important for the translation initiation of HCV RNA (14, 15, 25, 34, 35, 39) The 39 noncoding region (39NCR) consists of a short genotype-specific region and a poly(U)-C(U)n repeat stretch of variable length followed by a highly conserved tail of 98 nucleotides (nt) (12, 21, 30, 37) Studies to understand the molecular mechanism of HCV replication have been restricted by the lack of a well-established cell culture system, but studies from other positive-sense RNA viruses may provide some clues Upon flavivirus infection, translation of incoming viral genomic RNA occurs, and replication of the viral RNA begins with the synthesis of minus-strand RNA which then serves as the template for the synthesis of progeny genomic RNA The replication appears to take place at the perinuclear endoplasmic reticulum and requires virus-encoded proteins NS3 (proteinase/helicase) and NS5 (polymerase) as components of the presumed replicative complex (36) In addition, the 39 terminus of the flavivirus genomic RNA forms a conserved pseudoknot structure (26) It is generally believed that conserved sequences and structures at the 39 terminus of viral genomic RNA function as cis-acting signals that interact with viral and cellular proteins to initiate the synthesis of minus-strand RNA during viral replication The NS5B protein of HCV is a membrane-associated phosphoprotein (16) that possesses the conserved GDD motif of RNA-dependent RNA polymerase (RdRp) (19) RdRp activity of the HCV NS5B has been demonstrated in vitro, and

Journal ArticleDOI
TL;DR: It is suggested that NS2, although it is not essential for pestivirus RNA replication, has a regulatory function therein.
Abstract: Classical swine fever virus (CSFV) is the causative agent of a highly contagious disease in pigs. Virulent strains cause characteristic disease symptoms such as fever, neurological disorders, hemorrhages, and high mortality rates, whereas infection with avirulent strains remains clinically inapparent but induces a protective immunity. Furthermore, prenatal infection of fetuses can lead to persistently infected animals which shed virus over a long period, as do pigs which exhibit the chronic form of disease (3, 4, 29, 31). CSFV, bovine viral diarrhea virus (BVDV), and border disease virus form the pestivirus genus within the family Flaviviridae (33). CSFV is a small enveloped virus containing a positive-strand RNA genome of 12.3 kb, which consists of a 5′ untranslated region (5′UTR), a large open reading frame (ORF) encoding a single polyprotein, and a 3′ untranslated region (3′UTR) (16, 29). The 5′UTR contains an internal ribosomal entry site for cap-independent translation initiation of the viral polyprotein which is co- and posttranslationally processed by host cell and viral proteases (reviewed in reference 16). The cleavage sites of the polyprotein have been determined for BVDV, except for p7-NS2 (5, 16, 23, 26). C, Erns, E1, and E2 represent the structural proteins found in mature virions. As demonstrated for other flaviviruses (2), RNA replication of pestiviruses is thought to occur on cytoplasmatic membranes via the synthesis of a negative-stranded full-length genome (6, 7, 23), while the components of the viral replication complex have not been identified. It has been shown recently that the viral protein NS5B of BVDV has RNA-dependent RNA polymerase activity (37). The NS3 protease is responsible for the cleavage of the viral polyprotein downstream of NS3 and requires NS4A as a cofactor (28, 35). Furthermore, it possesses both helicase and NTPase activity (27, 32), suggesting a role in RNA replication. According to their behavior in tissue culture pestiviruses can be divided into two biotypes, noncytopathogenic (NCP) and cytopathogenic (CP). The mechanism responsible for the cytopathic effect (CPE) is poorly understood, but the overexpression of NS3 is a common feature of all CP pestiviruses (16). Furthermore, it has been shown that cells infected with CP BVDV undergo apoptosis (10, 36). CP pestiviruses represent mutants which arise from NCP viruses, presumably by RNA recombination during replication (16). Several mutants have been described for BVDV. Most of them contain rearrangements of viral sequences and/or insertions of host cell sequences (reviewed in reference 16). However, some CP BVDV and all CP CSFV isolates described so far are composed of defective interfering (DI) particles and NCP helper virus. CSFV DI genomes, with the identical deletion of 4,764 nucleotides (nt) corresponding to the genes encoding Npro through NS2, have been isolated from different sources (12, 14, 17). A genome with a similar deletion of 4,746 nt has been described recently (12). Furthermore, a defective genome was reported which lacks the 4,263 nt encoding C through NS2 but retains the foreign murine ubiquitin gene which had been used to replace Npro at the 5′ terminus of the ORF in the parental genome (30). After transfection into susceptible cells, such defective CSFV genomes, obtained either by extraction from infected cells or by transcription in vitro from the respective cloned cDNA, replicate and are packaged efficiently in the presence of helper virus RNA, thereby causing a CPE (14, 15). An autonomously replicating defective BVDV genome which lacks the genes encoding C, Erns, E1, E2, p7, and NS2 has been described recently by Behrens et al. (1). It demonstrates that none of these proteins is essential for RNA replication. In the same study it was suggested that the 5′ terminal region of the ORF contains a cis signal required for RNA replication, since the Npro gene could neither be deleted completely nor be replaced by a ubiquitin gene. However, the replacement of the Npro gene by ubiquitin in the CSFV Alfort/187 genome yielded infectious virus (30). In this report a series of in vitro-constructed defective CSFV genomes derived from strain Alfort/187 were analyzed with respect to autonomous RNA replication in SK-6 cells. The question of whether these defective genomes can be packaged in the presence of a helper virus and whether they are cytopathogenic was also addressed.

Journal ArticleDOI
TL;DR: Experimental data demonstrate that the diverse enzymatic activities of the NS3 protein—in particular the ATPase/RNA helicase—play a pivotal role even during early steps of the viral replication pathway, and indicate the C-terminal part of NS3 to be an important functional determinant of the RNA replication process.
Abstract: Studies on the replication of the pestivirus bovine viral diarrhea virus (BVDV) were considerably facilitated by the recent discovery of an autonomous subgenomic BVDV RNA replicon (DI9c). DI9c comprises mainly the untranslated regions of the viral genome and the coding region of the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. To assess the significance of the NS3-associated nucleoside triphosphatase/helicase activity during RNA replication and to explore other functional features of NS3, we generated a repertoire of DI9c derivatives bearing in-frame mutations in different parts of the NS3 coding unit. Most alterations resulted in deficient replicons, several of which encoded an NS3 protein with an inhibited protease function. Three lesions permitted replication, though at a lower level than that of the wild-type RNA, i.e., replacement of the third position of the DEYH helicase motif II by either T or F and an insertion of four amino acid residues in the C-terminal part of NS3. While polyprotein proteolysis was found to be almost unaffected in these latter replicons, in vitro studies with the purified mutant NS3 proteins revealed a significantly impaired helicase activity for the motif II substitutions. NS3 with a DEFH motif, moreover, showed a significantly lower ATPase activity. In contrast, the C-terminal insertion had no negative impact on the ATPase/RNA helicase activity of NS3. All three mutations affected the synthesis of both replication products—negative-strand intermediate and progeny positive-strand RNA—in a symmetric manner. Unexpectedly, various attempts to rescue or enhance the replication capability of nonfunctional or less functional DI9c NS3 derivatives, respectively, by providing intact NS3 in trans failed. Our experimental data thus demonstrate that the diverse enzymatic activities of the NS3 protein—in particular the ATPase/RNA helicase—play a pivotal role even during early steps of the viral replication pathway. They may further indicate the C-terminal part of NS3 to be an important functional determinant of the RNA replication process.

Journal ArticleDOI
TL;DR: The subcellular localization of hepatitis C viral proteins was determined as a first step towards the understanding of the functions of these proteins in the mammalian cell (CHO-K1) using fluorescence emitted from green fluorescent protein-fused to the viral proteins.
Abstract: We determined the subcellular localization of hepatitis C viral (HCV) proteins as a first step towards the understanding of the functions of these proteins in the mammalian cell (CHO-K1). We used fluorescence emitted from green fluorescent protein (GFP)-fused to the viral proteins to determine the subcellular localization of the viral proteins. We found that most of the viral proteins were excluded from the nucleus. Core exhibited a globular pattern near the nucleus. NS2 was concentrated in the perinuclear space. NS4A accumulated in the ER and the Golgi regions. NS3 was detected in the nucleus as well as the cytoplasm, when it was expressed by itself. However, NS3 became restricted to the cytoplasm, when it was produced together with NS4A. NS4B showed a spot-like pattern throughout the cytoplasm. NS5A and NS5B were distributed throughout the cytoplasm in a mesh-like pattern. These results can provide a basis for further investigations into the functions of the HCV proteins.

Journal ArticleDOI
TL;DR: It is reported here that an sg BVDV replicon which encodes from the viral proteins only the first three amino acids of the autoprotease Npro in addition to nonstructural (NS) proteins NS3 to NS5B replicates autonomously and also induces lysis of its host cells, demonstrating that the presence of a helper virus is not required for theLysis of the host cell.
Abstract: Defective interfering particles (DIs) of bovine viral diarrhea virus (BVDV) have been identified and shown to be cytopathogenic (cp) in the presence of noncytopathogenic (noncp) helper virus. Moreover, a subgenomic (sg) RNA corresponding in its genome structure to one of those BVDV DIs (DI9) was replication competent in the absence of helper virus. We report here that an sg BVDV replicon which encodes from the viral proteins only the first three amino acids of the autoprotease N(pro) in addition to nonstructural (NS) proteins NS3 to NS5B replicates autonomously and also induces lysis of its host cells. This demonstrates that the presence of a helper virus is not required for the lysis of the host cell. On the basis of two infectious BVDV cDNA clones, namely, BVDV CP7 (cp) and CP7ins- (noncp), bicistronic replicons expressing proteins NS2-3 to NS5B were established. These replicons express, in addition to the viral proteins, the reporter gene encoding beta-glucuronidase; the release of this enzyme from transfected culture cells was used to monitor cell lysis. Applying these tools, we were able to show that the replicon derived from CP7ins- does not induce cell lysis. Accordingly, neither N(pro) nor any of the structural proteins are necessary to maintain the noncp phenotype. Furthermore, these sg RNAs represent the first pair of cp and noncp replicons which mimic complete BVDV CP7 and CP7ins- with respect to cytopathogenicity. These replicons will facilitate future studies aimed at the determination of the molecular basis for the cytopathogenicity of BVDV.

Journal ArticleDOI
TL;DR: The NS5B protein has been shown to be the viral RNA-dependent RNA polymerase, which has been suggested that NS5A is involved in mediating the resistance of the hepatitis C virus to the action of interferon.

Journal ArticleDOI
TL;DR: The non‐structural (NS)5A protein of hepatitis C virus (HCV) is cleaved, after translation, by the NS3‐encoded zinc‐dependent serine proteinase, from the NS4B protein upstream and the NS5B protein downstream.
Abstract: The non-structural (NS)5A protein of hepatitis C virus (HCV) is cleaved, after translation, by the NS3-encoded zinc-dependent serine proteinase, from the NS4B protein upstream and the NS5B protein downstream. The released, mature NS5A protein is a 56 000 MW phosphoprotein (p56), which also exists within infected cells in a hyperphosphorylated form (p58). The NS5A gene has a quasispecies distribution, meaning that various NS5A sequences co-exist, in various proportions, in infected individuals. HCV NS5A appears to be located in cytoplasmic membranes surrounding the nucleus. Its precise functions are not known. HCV non-structural proteins, including NS5A, form a large multiprotein replication complex, which probably directs the replication of the HCV genome. HCV NS5A lacking the 146 N-terminal amino acids is a potent transcriptional activator in vitro. NS5A can also bind to single-strand RNA-dependent protein kinase (PKR) and inhibit its antiviral function. An 'interferon (IFN) sensitivity-determining region' has recently been postulated in the NS5A protein central region in hepatitis C virus (HCV) genotype 1b, but strongly conflicting evidence has been published. In fact, there would seem to be no such region in the NS5A protein, even though NS5A plays an important and complex role in HCV resistance to IFN. Structure-function studies are required to identify precisely how NS5A and IFN interact.

Patent
02 Feb 1999
TL;DR: In this article, peptides capable of inhibiting the serine-protease activity associated to the NS3 protein of HCV virus, their uses and a process for their production are described.
Abstract: Subject of the invention are peptides capable of inhibiting the serine-protease activity associated to the NS3 protein of HCV virus, their uses and a process for their production comprising the proteolysis of polypeptides containing at least one among the sequences of the NS3/NS4A, NS4A/NS4B, NS4B/NS5A and/or NS5A/NS5B junction sites of the polyprotein of HCV virus.

Journal ArticleDOI
TL;DR: Using chimeric viruses to select and characterize active variants of the NS3-NS4A protease revealed that all of the mutated proteases still efficiently processed the chimeric polyprotein in infected cells and also cleaved an HCV substrate in vitro.
Abstract: The major etiological agent of non-A, non-B hepatitis was identified in 1989 and named hepatitis C virus (HCV) (8, 23). Presently, it is estimated that approximately 1% of the human population is infected by HCV (42). Exposure to HCV results in an overt acute disease in only a small percentage of cases, while in most instances the virus establishes a chronic infection which causes liver inflammation and slowly progresses to liver failure and cirrhosis (24). In addition, seroepidemiological surveys have indicated an important role of HCV in the pathogenesis of hepatocellular carcinoma (27). The absence of a protective vaccine and the limited efficacy of alpha interferon treatment (55) have raised considerable interest in developing alternative anti-HCV therapies. The genetic organization of HCV is similar to that of flaviviruses and pestiviruses (9, 37), and therefore HCV was assigned to a separate genus of the family Flaviviridae (43). The HCV genome consists of a single-stranded RNA of about 9.5 kb in length encoding a precursor polyprotein of 3,010 to 3,033 amino acids (8, 9, 26, 50). Individual viral proteins are produced by proteolysis of the precursor: the putative structural proteins (C, E1, E2, and p7) span the amino-terminal third of the precursor and are generated by cleavages probably mediated by the endoplasmic reticulum signal peptidase (21, 44), and the remaining part of the precursor contains the nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B), which presumably form the virus replication machinery and are released from the nascent precursor by two virus-encoded proteases. A zinc-dependent protease associated with NS2 and the N terminus of NS3 is responsible for the cleavage between NS2 and NS3 (16, 19, 39). A distinct serine protease located in the N-terminal domain of NS3 is responsible for proteolytic cleavages at the NS3/NS4A, NS4A/NS4B, NS4B/NS5A, and NS5A/NS5B junctions (3, 17, 52). Substantial efforts have been devoted to the characterization of the HCV serine protease, which is contained within the amino-terminal 180 amino acids of NS3 (3, 13, 17, 20, 52). Although the NS3 protease domain possesses enzymatic activity, the 54-amino-acid NS4A protein is required for cleavage at the NS3/NS4A and NS4B/NS5A sites and increases cleavage efficiency at the NS4A/NS4B and NS5A/NS5B junctions (2, 14, 33, 51). The central domain of NS4A, encompassing amino acids 21 to 32, was shown to be sufficient for activation of the protease (30, 34, 46, 53). In transfected cells, NS3 and NS4A assemble into a stable heterodimeric complex whose formation requires both the amino-terminal and the central domains of NS4A, as well as about 30 amino acids at the amino terminus of NS3 (4, 14, 30, 34, 45, 51). The determination of the crystal structure of the NS3 protease domain uncomplexed and complexed with central domain of NS4A (29, 36, 56) has confirmed the characteristics of this enzyme predicted by molecular modelling and biochemical studies (12, 40). The enzyme adopts a chymotrypsin-like fold and features a tetrahedrally coordinated metal distal to the active site. The central domain of NS4A forms a β strand which contributes to the formation of an eight-stranded β barrel with the amino-terminal domain of NS3 and plays a significant role in stabilizing NS3. Thus, NS4A is considered an integral structural component of the enzyme. For this reason, we here refer to the HCV serine protease as the NS3-NS4A protease. This protease cleaves the viral polyprotein in a precise temporal order which is probably critical for virus replication: the NS3/NS4A cleavage is the first event and occurs only in cis, and this is followed by cleavage at the NS5A/NS5B, NS4A/NS4B, and NS4B/NS5A sites, which can also occur in trans (2, 14, 33). An additional peculiar feature of the protease domain of NS3 is that it is covalently attached to an RNA helicase possessing ATPase activity (18, 25, 28). This overwhelming amount of data makes the NS3 protease an attractive candidate for developing effective HCV therapies. Indeed, several in vitro assay systems have been developed and are being used for the identification of specific inhibitors. Protease inhibitors have proved to be good therapeutic agents in the case of the human immunodeficiency virus protease. However, the long-term clinical efficacy of these drugs is potentially limited by the existence of inhibitor-resistant protease variants which are found in untreated subjects and emerge both in vivo during treatment and during selection in culture (10, 22, 32). Apparently, the ability of the virus to produce inhibitor-resistant protease variants depends largely on the ability of the protease to tolerate substitutions in critical subsites. Thus, attempts to subvert viral resistance should take this feature in account and concentrate on the search for inhibitors active against a broad range of variants. These attempts greatly benefit from the possibility of using in vitro cell culture systems for the selection and characterization of virus variants with decreased sensitivity to inhibitors. Sequence analysis of several HCV isolates indicates that there are multiple HCV genotypes and subtypes and that even in the same individual the virus exists as quasispecies (6, 47). Accordingly, a number of sequence differences are found in the portion of the genome encoding the NS3-NS4A protease. Only a few variants have been characterized biochemically, and these show similar kinetic parameters. On the other hand, the lack of an efficient in vitro infection system prevents a large-scale comparison of the different protease variants present in various HCV isolates and also precludes testing the sensitivities to inhibitors of the different variants in cell culture. We have recently described the generation of stable Sindbis virus (SBV)-HCV chimeric viruses whose propagation depends on the activity of the serine protease of HCV (15). Here we report the use of these viruses as a genetic system for the identification of functional variants of the NS3-NS4A protease which could be used to identify and characterize protease variants with decreased sensitivity to inhibitors. Since there are no selective inhibitors of the NS3-NS4A protease presently available, we investigated whether variants of the NS3-NS4A protease could be selected in the absence of a specific selective pressure, taking advantage of the high rate of spontaneous mutations of the SBV replication machinery. We selected more-infectious virus mutants by serial passaging on BHK cells, and almost all of them produced an NS3-NS4A protease different from those encoded by the original chimeras. All of these mutant enzymes displayed a measurable activity when assayed in vitro and efficiently processed the chimeric polyprotein in infected cells. These results imply that HCV-SBV chimeric viruses can be used under the appropriate conditions of selective pressure as a surrogate system for the identification and characterization of inhibitor-resistant variants of the NS3-NS4A protease.

Journal Article
TL;DR: The results indicate that HCV type 4 may not be the only dominant genotype in SA and that the precise subtyping of these 42 type 3 variants awaits sequencing of longer HCV RNA stretches.
Abstract: Hepatitis C virus (HCV) genotype 4 is believed to be predominant in the Middle East including Saudi Arabia (SA). We attempted to genotype 80 HCV isolates from different parts of SA by direct sequencing of a variable 222bp fragment from the NS5B region. The phylogenetic analysis of the NS5B sequences was complemented by direct sequence analysis of the conserved 5'-NCR region for HCV type-specific polymorphism. All 80 NS5B sequences separated into 3 clades which comprised 6 type 1b variants, 30 type 4 variants (24 of type 4a and 6 of type 4c or d) and 44 type 3 variants. Apart from two definitive type 3b variants the other 42 type 3 NS5B sequences formed 4 clusters with low similarity to type 3a-f HCV sequences from the database. The precise subtyping of these 42 type 3 variants awaits sequencing of longer HCV RNA stretches. Our results indicate that HCV type 4 may not be the only dominant genotype in SA.

Book ChapterDOI
TL;DR: A method is presented here for assaying HCV RDRP activity based on this laboratory's experience with recombinant NS5B expressed in Escherichia coli and the experience of others studying NS5 B expressed in insect cells.
Abstract: The hepatitis C virus (HCV) chronically infects approx 4 million patients in the United States alone, and constitutes a major cause of chronic liver disease and hepatocellular carcinoma (1-3). Current antiviral therapies for chronic hepatitis C remain relatively ineffective and have significant side-effects in many patients. Moreover, the lack of an easily reproducible tissue-culture system to propagate HCV has hampered the development of new antiviral therapies. Although detailed studies of several recombinant HCV nonstructural proteins have been initiated, our knowledge of the HCV NS5B polymerase that encodes an RNA-dependent RNA polymerase (RDRP) is rudimentary (4-8). RNA-dependent RNA polymerases represent a class of viral enzymes that replicate the genomic RNA of plus strand RNA viruses (9-11). A model enzyme of this group, the poliovirus RDRP encoded by the 3D(pol) gene, has been extensively studied (12-17). However, studies of recombinant hepatitis C virus RDRP (or NS5B polymerase) have only recently begun, and there is much to learn. A method is presented here for assaying HCV RDRP activity based on this laboratory's experience with recombinant NS5B expressed in Escherichia coli and the experience of others studying NS5B expressed in insect cells (5-8).

01 Jan 1999
TL;DR: It is demonstrated that the BVDV RdRp can preferentially initiate RNA synthesis by a de novo mechanism from short templates containing the signals for the initiation of genomic positive strand synthesis.
Abstract: Recombinant RNA-dependent RNA polymerases have been reported to synthesize RNAs by extending from the 39 hydroxyl of a template or an oligonucleotide primer. De novo initiation has not been reported. Establishment of such an assay would facilitate the analysis of the initiation requirements and allow the testing of antiviral compounds specifically targeting initiation. Using chemically synthesized RNAs and DNAs, we demonstrate that the recombinant RNA-dependent RNA polymerase (NS5B) of bovine viral diarrhea virus initiates de novo RNA synthesis. Nucleotides required for efficient initiation of RNA synthesis and for stable interaction with NS5B were identified. © 1999 Academic Press Positive strand RNA viruses in the Flaviviridae family include human and animal pathogens such as flavivirus hepatitis C virus (HCV) and the pestivirus bovine viral diarrhea virus (BVDV) (1). After entry, the viral RNA directs the translation of a polyprotein that is proteolytically processed to produce individual structural and nonstructural proteins (2, 3). At the C-terminus of the polyprotein is the nonstructural protein 5B (NS5B), an RNA-dependent RNA polymerase that is a key subunit of the viral RNA replicase complex. Recombinant NS5B of HCV and BVDV has been previously reported to be able to catalyze nucleotidyl transfer by extending from the 39 hydroxyl of a template, an RNA or a DNA primer (4‐6). The ability of recombinant NS5B to initiate RNA synthesis by a primerindependent mechanism has not been previously reported. However, de novo initiation of RNA synthesis is likely to be the mode of initiation in an infected cell. In this report, we demonstrate that the BVDV RdRp can preferentially initiate RNA synthesis by a de novo mechanism from short templates containing the signals for the initiation of genomic positive strand synthesis. We also characterize the requirements for the interaction between RdRp and the initiation site. De novo initiation of RNA synthesis