RNA-dependent RNA polymerases from different hepatitis C virus genotypes reveal distinct biochemical properties and drug susceptibilities

The RNA-dependent RNA polymerase of the hepatitis C virus (HCV) is the key enzyme for viral replication, recognized as one of the promising targets for antiviral intervention. Several of the known non-nucleoside HCV polymerase inhibitors (NNIs) identified by screening approaches show limitations in the coverage of all six major HCV genotypes (GTs). Genotypic profiling therefore has to be implemented early in the screening cascade to discover new broadly active NNIs. This implies knowledge of the specific individual biochemical properties of polymerases from all GTs which is to date limited to GT 1 only. This work gives a comprehensive overview of the biochemical properties of HCV polymerases derived from all major GTs 1–6. Biochemical analysis of polymerases from 38 individual sequences revealed that the optima for monovalent cations, pH and temperature were similar between the GTs, whereas significant differences concerning concentration of the preferred cofactor Mg2+ were identified. Implementing the optimal requirements for the polymerases from each individual GT led to significant improvements in their enzymatic activities. However, the specific activity was distributed unequally across the GTs and could be ranked in the following descending order: 1b, 6a > 2a, 3a, 4a, 5a > 1a. Furthermore, the optimized assay conditions for genotypic profiling were confirmed by testing the inhibitory activity of 4 known prototype NNIs addressing the NNI binding sites 1 to 4.

research highlights
► Biochemical properties for optimal NS5B activity were investigated for all HCV GT.
► Significant differences in optimal cofactor Mg2+ concentration were observed.
► Substrate affinities of NS5B were found to be very similar across different GT.
► Differences in spec. act. of purified NS5Bs with highest activities for GT 1b and 6a.
► GT profiles of 4 NNIs were investigated. Only benzofuran stood out with a broad coverage.