Showing papers on "NS5B published in 1996"
TL;DR: Experimental evidence that an RNA‐dependent RNA polymerase is encoded by HCV and that this enzymatic activity is the function of the 65 kDa non‐structural protein 5B (NS5B) is presented, representing a first important step towards a better understanding of the life cycle of the HCV.
Abstract: Hepatitis C virus (HCV) is the major etiological agent of non-A, non-B post-transfusion hepatitis. Its genome, a (+)-stranded RNA molecule of approximately 9.4 kb, encodes a large polyprotein that is processed by viral and cellular proteases into at least nine different viral polypeptides. As with other (+)-strand RNA viruses, the replication of HCV is thought to proceed via the initial synthesis of a complementary (-) RNA strand, which serves, in turn, as a template for the production of progeny (+)-strand RNA molecules. An RNA-dependent RNA polymerase has been postulated to be involved in both of these steps. Using the heterologous expression of viral proteins in insect cells, we present experimental evidence that an RNA-dependent RNA polymerase is encoded by HCV and that this enzymatic activity is the function of the 65 kDa non-structural protein 5B (NS5B). The characterization of the HCV RNA-dependent RNA polymerase product revealed that dimer-sized hairpin-like RNA molecules are generated in vitro, indicating that NS5B-mediated RNA polymerization proceeds by priming on the template via a 'copy-back' mechanism. In addition, the purified HCV NS5B protein was shown to perform RNA- or DNA oligonucleotide primer-dependent RNA synthesis on templates with a blocked 3' end or on homopolymeric templates. These results represent a first important step towards a better understanding of the life cycle of the HCV.
756 citations
TL;DR: It is concluded that a benign course of HCV infection may be the consequence of the effective activation of T‐helper lymphocytes.
148 citations
TL;DR: Results do not directly support the hypothesis that increased intrahepatic HCV load is associated with more severe liver injury, and the multiple regression analysis showed no effect of length of HCV exposure, risk factors, degree of bile duct damage, steatosis, or total Scheuer or Knodell score on RNA levels.
118 citations
TL;DR: The potential utility of ribozyme therapy as a strategy for the treatment of hepatitis C virus infection is demonstrated, and six hammerhead ribozymes directed against a conserved region of the plus strand and minus strand of the HCV genome are demonstrated.
Abstract: Hepatitis C virus (HCV) infection is the cause of more than 90% of chronic non-A non-B hepatitis worldwide (2) It has been estimated that more than 1% of the world population is infected with HCV Although transmission is parenteral in the majority of cases, the incidence and mechanisms of nonparenteral transmission remain uncertain Viral infection is variable; persistent viremia occurs in about 90% of cases, chronic hepatitis occurs in 60 to 70% of cases, and 20 to 40% of infected individuals ultimately develop cirrhosis, end-stage liver disease,and/orhepatocellularcarcinoma(2)Accordingtorecent data, HCV infection is the leading indication for liver transplantation in the United States, and chronic hepatitis C is associated with more than 50% of hepatocellular carcinoma in the United States (6a) Orthotopic liver transplantation is performed in patients with end-stage liver disease, but viremia persists in 100% of patients, and ultimately, the transplanted organ becomes reinfected (11) HCV is an enveloped positive-strand RNA virus with a genome size of approximately 94 kb (5, 13) which encodes a single large open reading frame of ;3,010 amino acids The singlepolyproteingeneproductundergoesproteolyticprocessing to multiple gene products, including a capsid protein, two or more envelope glycoproteins (E1 and E2), and six or more nonstructural gene products (NS2, NS3, NS4a, NS4b, NS5a, and NS5b) which are thought to be involved in viral assembly and replication, respectively (10) After the infection of hepatocytes, the genomic, positive HCV RNA strand is translated The NS5 gene product, an RNA-dependent RNA polymerase, presumably catalyzes the synthesis of replicative, minus HCV RNAstrandswhichrepresentthetemplateforproducingmore
107 citations
Journal Article•
TL;DR: Modulation of the proteinase activity may be a way to regulate the expression and replication of the HCV genome.
103 citations
Patent•
24 May 1996
TL;DR: In this article, a method for reproducing RNA-dependent RNA polymerase activity associated with hepatitis C virus is described. But the method is restricted to a single cell and cannot be applied to other types of cells.
Abstract: This is a method for reproducing in vitro the RNA-dependent RNA polymerase activity associated with hepatitis C virus. The method is characterized in that sequences contained in NS5B are used in the reaction mixture. The terminal nucleotidyl transferase activity, a further property of the NS5B protein, can also be reproduced using this method. The method takes advantage of the fact that the NS5B protein, either purified to apparent homogeneity or present in extracts of overproducing organisms, can catalyse the addition of ribonucleotides to the 3'-termini of exogenous or endogenous RNA molecules. The invention also relates to a composition of matter that comprises sequences contained in NS5B, and to the use of these compositions for the set up of an enzymatic test capable of selecting, for therapeutic purposes, compounds that inhibit the enzymatic activity associated with NS5B. The figure shows plasmids used in the method to produce hepatitis C virus RNA-dependent RNA polymerase and terminal nucleotidyl transferase in cultivated eukaryotic and prokaryotic cells.
39 citations
TL;DR: A protease is obtained that is similar to the bacterial protein and unlike the wild-type enzyme, is able to cleave a substrate containing a phenylalanine in the P1 position, and represents one of the few examples of redesign of a serine protease substrate specificity directed by molecular modelling.
39 citations
TL;DR: The high variability of the HCV genome contributes greatly to the difficulty of designing potent vaccines, especially in the E2/NS1, E1, NS3 and NS5b regions.
Abstract: Hepatitis C virus (HCV) is an enveloped, single-stranded RNA virus that has been classified in the Flaviviridae family. The genome of 9400 nucleotides comprises two non-coding regions in 5' and 3' flanking a large reading frame which codes for a polyprotein of 3000 amino acids; this polyprotein is further cleaved into structural (C, E1, E2) and non-structural (NS1, NS2, NS3, NS4, NS5) proteins. The positive RNA acts as a cap-independent messenger; the transcription is mediated by the NS5 RNA polymerase. After the maturation step, the virion is liberated by budding through the cytoplasmic membrane. As for many other RNA viruses, the HCV genome exhibits a high degree of variability, especially in the E2/NS1, E1, NS3 and NS5b regions. Conversely the 5' non-coding region is highly conserved, at least in part, and can be used for diagnostic purposes by PCR technique. Six genotypes of HCV have already been reported, numbered from 1 to 6 in Simmonds' classification. The same genotype can be divided into subtypes (for instance, genotype 1 comprises three subtypes: 1a, 1b and 1c). Various minor variants of the same strain, called quasispecies, are commonly present in the blood of the same patient. Strains of genotype 1b--which is the most widespread worldwide--are correlated with more severe clinical manifestations, greater viral loads and lower response to interferon treatment. The high variability of the HCV genome contributes greatly to the difficulty of designing potent vaccines.
22 citations