Biochemical and structural analysis of the NS5B RNA-dependent RNA polymerase of the hepatitis C virus

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 inte rmediates. Owing to the lack of reliable cell culture systems and convenien t animal models for HCV, the mechanisms governing RNA replication are not k nown. As a first step towards the development of appropriate in vitro syste ms, we expressed the NS5B RNA-dependent RNA polymerase (RdRp) in insect cel ls, purified the protein to near homogeneity and studied its biochemical pr operties. It is a primer- and RNA template-dependent RNA polymerase able to copy long heteropolymeric templates without additional viral or cellular c ofactors. We determined the optimal reaction parameters, the kinetic consta nts and the substrate specificity of the enzyme, which turned out to be sim ilar to those described for the 3D polymerase of poliovirus. By analysing a series of nucleosidic and non-nucleosidic compounds for their effect on Rd Rp activity, we found that ribavirin triphosphates have no inhibitory effec t, 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.