Showing papers on "NS5B published in 1998"

Characterization of an Autonomous Subgenomic Pestivirus RNA Replicon

Abstract: As an initial approach to define the requirements for the replication of bovine viral diarrhea virus (BVDV), a member of the Flaviviridae family with a positive-strand RNA genome, full-length genomic and subgenomic RNAs were originated by in vitro transcription of diverse BVDV cDNA constructs and transfected into eucaryotic host cells. RNA replication was measured either directly by an RNase protection method or by monitoring the synthesis of viral protein. When full-length BVDV cRNA was initially applied, the synthesis of negative-strand RNA intermediates as well as progeny positive-strand RNA was detected posttransfection in the cytoplasm of the host cells. Compared to the negative-strand RNA intermediate, an excess of positive-strand RNA was synthesized. Surprisingly, a subgenomic RNA molecule, DI9c, corresponding to a previously characterized defective interfering particle, was found to support both steps of RNA replication in the absence of a helper virus as well, thus functioning as an autonomous replicon. DI9c comprises the 5' and 3' untranslated regions of the BVDV genome and the coding regions of the autoprotease Npro and the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. Most interestingly, the NS2 polypeptide was thus determined to be nonessential for RNA replication. As expected, deletion of the genomic 3' end as well as abolition of the catalytic function of the virus-encoded serine protease resulted in DI9c molecules that were unable to replicate. Deletion of the entire Npro gene also destroyed the ability of DI9c molecules to replicate. On the other hand, DI9c derivatives in which the 5' third of the Npro gene was fused to a ubiquitin gene, allowing the proteolytic release of NS3 in trans, turned out to be replication competent. These results suggest that the RNA sequence located at the 5' end of the open reading frame exerts an essential role during BVDV replication. Replication of DI9c and DI9c derivatives was found not to be limited to host cells of bovine origin, indicating that cellular factors functioning as potential parts of the viral replication machinery are well conserved between different mammalian cells. Our data provide an important step toward the ready identification and characterization of viral factors and genomic elements involved in the life cycle of pestiviruses. The implications for other Flaviviridae and, in particular, the BVDV-related human hepatitis C virus are discussed.

Identification and Characterization of an RNA-Dependent RNA Polymerase Activity within the Nonstructural Protein 5B Region of Bovine Viral Diarrhea Virus

Abstract: Nonstructural protein 5B (NS5B) of bovine viral diarrhea virus (BVDV) contains sequence motifs that are predictive of an RNA-dependent RNA polymerase activity. We describe the expression and purification of the BVDV NS5B protein derived from an infectious cDNA clone of BVDV (NADL strain). BVDV NS5B protein was active in an in vitro RNA polymerase assay using homopolymeric RNA or BVDV minigenomic RNA templates. The major product was a covalently linked double-stranded molecule generated by a "copy-back" mechanism from the input template RNA. In addition, a nucleotide-nonspecific and template-independent terminal nucleotidyl transferase activity was observed with the BVDV NS5B preparation.

Molecular virology of hepatitis C virus: an update with respect to potential antiviral targets.

Abstract: Hepatitis C virus (HCV), a positive-strand enveloped RNA virus, is a major cause of chronic liver disease worldwide. Cis-acting RNA elements and virus-encoded polypeptides required for HCV replication represent attractive targets for the development of antiviral therapies. Internal ribosome entry site-directed translation of HCV genome RNA produces a long polyprotein which is co- and post-translationally processed to yield at least 10 viral proteins. A host signal peptidase is responsible for maturation of the structural proteins located in the N-terminal one-third of the polyprotein. Thus far, four enzymatic activities encoded by the non-structural (NS) proteins have been reported. The NS2-3 region encodes an autoproteinase responsible for cleavage at the 2/3 site. The N-terminal one-third of NS3 functions as the catalytic subunit of a serine proteinase which cleaves at the 3/4A, 4A/4B, 4B/5A and 5A/5B sites, and NS4A is an essential cofactor for some of these cleavages. NS3 also encodes an RNA-stimulated NTPase/RNA helicase at its C terminus, and NS5B has been shown to possess an RNA-dependent RNA polymerase activity. To date, no functions have been reported for NS4B or NS5A in RNA replication, however, NS5A has been implicated in modulating the sensitivity of HCV to interferon. Sequence and structural conservation within the 3' terminal 98 bases of genomic RNA suggest a functional importance in the virus life-cycle and hence another target for antiviral intervention. Recently, HCV infection was shown to be initiated in chimpanzees following intrahepatic inoculation of RNA transcribed from cloned HCV cDNA. The ability to generate large quantities of infectious HCV RNA may facilitate the development of reliable cell culture replication systems useful for the evaluation of antiviral drugs.

The hepatitis C virus NS3 proteinase: structure and function of a zinc-containing serine proteinase.

Abstract: The hepatitis C virus (HCV) NS3 protein contains a serine proteinase domain implicated in the maturation of the viral polyprotein. NS3 forms a stable heterodimer with NS4A, a viral membrane protein that acts as an activator of the NS3 proteinase. The three-dimensional structure of the NS3 proteinase complexed with an NS4A-derived peptide has been determined. The NS3 proteinase adopts a chymotrypsin-like fold. A beta-strand contributed by NS4A is clamped between two beta-strands within the N terminus of NS3. Consistent with the requirement for extraordinarily long peptide substrates (P6-P4'), the structure of the NS3 proteinase reveals a very long, solvent-exposed substrate-binding site. The primary specificity pocket of the enzyme is shallow and closed at its bottom by Phe-154, explaining the preference of the NS3 proteinase for cysteine residues in the substrate P1 position. Another important feature of the NS3 proteinase is the presence of a tetrahedral zinc-binding site formed by residues Cys-97, Cys-99, Cys-145 and His-149. The zinc-binding site has a role in maintaining the structural stability and guiding the folding of the NS3 serine proteinase domain. Inhibition of the NS3 proteinase activity is regarded as a promising strategy to control the disease caused by HCV. Remarkably, the NS3 proteinase is susceptible to inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A/NS4B, NS4B/NS5A and NS5A/NS5B cleavage sites. The Ki values of the inhibitory products are lower than the K(m) values of the respective substrates and follow the order NS4A < NS5A < NS4B. Starting from the observation that the NS3 proteinase undergoes product inhibition, very potent, active site-directed inhibitors have been generated using a combinatorial peptide chemistry approach.

Hepatitis C virus NS5B truncated protein and methods thereof to identify antiviral compounds

Abstract: The invention provides HCV NS5B polypeptides and DNA (RNA) encoding HCV NS5B polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing HCV NS5B polypeptides to screen for antiviral compounds.

Genetic variability and characterization of non-structural region 5 of hepatitis C virus genome from Chinese patients

Abstract: Sequence variation in the putative nonstructural region 5b (NS5b) of hepatitis C virus (HCV) was analyzed in China. Complementary DNA fragments from sera of 49 Chinese patients were amplified by polymerase chain reaction (PCR) and the products were cloned and sequenced. Based on the comparison in NS5b of 33 clones of genotype 1b and 16 clones of genotype 2a, Chinese isolates of HCV belong to the same subtype as HCV-J, and HC-J6 from Japan. There does exist, however, some heterogeneity in the primary structure of the nucleotide acid. Higher homology was found among Chinese isolates than among Chinese isolates and Japanese isolates. Furthermore, among Chinese isolates, we found some conserved nucleotide acid positions different from those of Japanese isolates. Comparison of average homology among the 33 clones of genotype 1b and the 16 clones of genotype 2a indicated that the average homology among genotype 2a was lower than that among genotype 1b. In addition, a deletion of three nucleotide acids and a frame-shift, resulting in the introduction of an in-frame stop codon, were first observed in the NS5b region. These results indicated geographical differences in the distribution of individual HCV isolates, and the existence of a local variant in the same subtype. Our findings also suggested the need for further study on the sequence of genotype 2a, to improve diagnosis and help to advance the development of a vaccine.

Molecular cloning and nucleotide sequence of 3'-terminal region of classical swine fever virus LPC vaccine strain.

Abstract: A cDNA of the 3′-terminus of classical swine fever virus (LPC vaccine strain) was cloned and sequenced. The 3431 nucleotides and deduced amino acid sequences were compared with those of other pestiviruses, and the similarity of nucleotide sequences and deduced amino acid sequences were found to be 84–95% and 95–98%, respectively. Similar to other isolates of classical swine fever virus, the sequenced region included the non-structural gene p58 (NS5A) and part of p76 (NS5B) gene. The p76 gene of LPC vaccine strain also contained a highly conserved motif G-D-D (Gly-Asp-Asp) that is present in the RNA replicase of positive-stranded RNA viruses. With the sequence data currently available, we carried out a phylogenetic analysis and obtained a genealogical relationship among members of the classical swine fever virus. The sequence has been submitted to GeneBank with an accession number AF001986.

HCV Genotyping by the Line Probe Assay INNO-LiPA HCV II.

Abstract: Hepatitis C viruses (HCVs) constitute a highly variable genus within the Flaviviridae, with closest homology to the hepatitis G and GB viruses, and Pestiviruses. The positive-stranded RNA genome encodes a polyprotein which is co- and posttranslationally cleaved into at least nine proteins. Core, E1, and E2 (the structural proteins) and NS2, NS3, NS4A, NS4B, NS5A, and NS5B (the nonstructural [NS] proteins). A theoretical protein of 7 kDa (tp7) may be processed from the carboxy terminal E2 region (1).

Expression and Dimerization of Hepatitis C Virus Core Protein in E. coli.

Abstract: Hepatitis C virus (HCV) is a positive-stranded RNA virus with a genome size of about 9-10 kb. The genome of this virus encodes a polyprotein with a length of over 3000 amino acids. This polyprotein is cleaved by cellular and viral proteases to generate at least 10 viral gene products. Recent reports have indicated that there are extensive interactions between various HCV proteins: the core (capsid) protein can interact with itself (1) and with the El envelope protein (2); El protein can interact with the E2 envelope protein (3,4) which in turn can be covalently linked to its following p7 protein and interact with another integral membrane protein named NS2 (5); NS2 can also interact with NS5A and NS5B nonstructural proteins (6); and NS3 proteinase/helicase has also been shown to complex with the NS4A protein (6,7). Thus, most of the known HCV proteins interact with at least another HCV protein. These interactions are presumably very important for morphogenesis and replication of HCV.

Confirmation of HCV Antibodies by the Line Immunoassay INNO-LIA HCV Ab III.

Abstract: HCVs constitute a genus within the Flaviviridae, with closest homology to the hepatitis G and GB viruses, and Pestiviruses. The positive-stranded RNA genome encodes at least nine proteins. Core, El, and E2 constitute the structural proteins; NS2, NS3, NS4A, NS4B, NS5A, and NS5B are nonstructural (NS) proteins. HCV isolates display high levels of sequence heterogeneity allowing classification into at least 11 types and 90 subtypes (1). HCV infection of the human liver is often clinically benign, with mild icterus in the acute phase, the disease may even go unnoticed in some cases of acute resolving hepatitis C. In the majority (>70%) of cases, however, HCV infection leads to chronic persistent or active infection, often with complications of liver cirrhosis and auto-immune disorders. Hepatocellular carcinoma may occur after about 20-35 yr (2); sometimes even without the intermediate phase of cirrhosis. No prophylaxis is available today and treatment with interferon-alpha (IFN-α) only leads to long-term resolution in about 4-36% of treated cases, depending on the HCV genotype (1).