The hepatitis C virus NS5B RNA-dependent RNA polymerase activity and susceptibility to inhibitors is modulated by metal cations

Objectives: The aim of this study was to understand the effect of metal cat ions on the hepatitis C virus (HCV) NS5B in vitro RNA-dependent RNA polymer ase (RdRp) activity and its susceptibility to various inhibitors. Methods: A recombinant full-length HCV NS5B protein was expressed in insect cells and purified to homogeneity. RdRp activity was assessed using standa rd filtration or polyacrylamide gel-based assays. Results: Efficient inhibition of the HCV NS5B RdRp activity by gliotoxin, a s well as by various substrate analogs, occurs in the presence of Mn2+, but not of Mg2+. Assays performed in the presence of both cofactors suggest th at, in vitro, the enzyme's affinity for Mn2+ is higher than that for Mg2+. in addition, the RdRp activity, displayed in the presence of heteropolymeri c templates, is significantly increased when metal cofactor consists of Mn2 +. Finally, steady state kinetics showed that the velocity of the reaction, as well as the affinity of the enzyme for its substrate, could both be aff ected by the nature of the divalent metal cation used. (C) Lipponcott Willi ams & Wilkins, Inc.