title: Biochemical and structural analysis of the NS5B RNA-dependent RNA polymerase of the hepatitis C virus.
abstract: Hepatitis C virus (HCV), the major causative agent of chronic and sporadic non-A, non-B hepatitis worldwide, is a distinct member of the Flaviviridae virus family. These viruses have in common a plus-strand RNA genome that is replicated in the cytoplasm of the infected cell via minus-strand RNA intermediates. Owing to the lack of reliable cell culture systems and convenient animal models for HCV, the mechanisms governing RNA replication are not known. As a first step towards the development of appropriate in vitro systems, we expressed the NS5B RNA-dependent RNA polymerase (RdRp) in insect cells, purified the protein to near homogeneity and studied its biochemical properties. It is a primer- and RNA template-dependent RNA polymerase able to copy long heteropolymeric templates without additional viral or cellular cofactors. We determined the optimal reaction parameters, the kinetic constants and the substrate specificity of the enzyme, which turned out to be similar to those described for the 3D polymerase of poliovirus. By analysing a series of nucleosidic and non-nucleosidic compounds for their effect on RdRp activity, we found that ribavirin triphosphates have no inhibitory effect, providing direct experimental proof that the therapeutic effect observed in patients is not related to a direct inhibition of the viral polymerase. Finally, mutation analysis was performed to map the minimal NS5B sequence required for enzymatic activity and to identify the 'classical' polymerase motifs important for template and NTP binding and catalysis.
authors.affiliation: Institute for Virology, Johannes-Gutenberg University Mainz, Mainz, Germany.
authors.forename: V
authors.initials: V
authors.lastname: Lohmann
authors.forename: A
authors.initials: A
authors.lastname: Roos
authors.forename: F
authors.initials: F
authors.lastname: Körner
authors.forename: J O
authors.initials: JO
authors.lastname: Koch
authors.forename: R
authors.initials: R
authors.lastname: Bartenschlager
mesh_terms: D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D012321:DNA-Directed RNA Polymerases; D005822:Genetic Vectors; D016174:Hepacivirus / Q000201:enzymology*; D006801:Humans; D009154:Mutation; D009705:Nucleosides / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012254:Ribavirin / Q000494:pharmacology; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D009705:Nucleosides; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D012254:Ribavirin; D012313:RNA; C090443:polymerase 3Dpol, poliovirus; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases