title: RNA-dependent RNA polymerases from different hepatitis C virus genotypes reveal distinct biochemical properties and drug susceptibilities.
abstract: 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 Mg(2+) 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.
authors.affiliation: AiCuris GmbH and Co. Wuppertal, Germany.
authors.forename: Marina M
authors.initials: MM
authors.lastname: May
authors.forename: Heike
authors.initials: H
authors.lastname: Lorengel
authors.forename: Joerg
authors.initials: J
authors.lastname: Kreuter
authors.forename: Holger
authors.initials: H
authors.lastname: Zimmermann
authors.forename: Helga
authors.initials: H
authors.lastname: Ruebsamen-Schaeff
authors.forename: Andreas
authors.initials: A
authors.lastname: Urban
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D001669:Biochemical Phenomena / Q000502:physiology; D002414:Cations, Monovalent / Q000494:pharmacology; D024882:Drug Resistance, Viral / Q000187:drug effects / Q000235:genetics*; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D005838:Genotype; D016174:Hepacivirus / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D015636:Magnesium Chloride / Q000494:pharmacology; D008954:Models, Biological; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013696:Temperature
chemical_list: D000998:Antiviral Agents; D002414:Cations, Monovalent; D004791:Enzyme Inhibitors; D015636:Magnesium Chloride; D012324:RNA-Dependent RNA Polymerase