BIOCHEMICAL AND KINETIC ANALYSES OF NS5B RNA-DEPENDENT RNA-POLYMERASEOF THE HEPATITIS-C VIRUS

The biochemical properties of the RNA-dependent RNA polymerase (RdRp) of the hepatitis C virus were analyzed. A hexahistidine affinity-tagge d NS5B fusion protein was expressed with recombinant baculoviruses in insect cells and purified to near homogeneity. Enzymatic activity of t he purified protein was inhibited by KCl or high concentrations of NaC l and was absolutely dependent on Mg2+, which could be replaced by Mn2 +. NS5B was found to be processive and able to copy long heteropolymer ic templates with an elongation rate of 150-200 nucleotides/min at 22 degrees C. Kinetic constants were determined for all four nucleoside t riphosphates and different templates. In case of a heteropolymeric RNA template corresponding to the last 319 nucleotides of the hepatitis C virus genome, K-m values for UTP, GTP, ATP, and CTP were similar to 1 .0, similar to 0.5, similar to 10, and similar to 0.3 mu M, respective ly. The profile of several inhibitors of RdRp activity and substrate a nalogs indicated that the enzyme has a strong preference for ribonucle oside 5'-triphosphates and that it closely resembles 3D(pol) of picorn aviruses. (C) 1998 Academic Press.