Biochemical and pre-steady-state kinetic characterization of the hepatitis C virus RNA polymerase (NS5BA21, HC-J4)

Here we report a detailed characterization of the biochemical and kinetic properties of the hepatitis C virus (HCV, genotype-1b, J4 consensus) RNA-dependent RNA polymerase NS5B, by performing comprehensive RNA binding,nucleotide incorporation, and protein/protein oligomerization studies. By applying equilibrium fluorescence titrations, we determined a surprisingly high dissociation constant (K-d) of approximately 250 nM for single-stranded as well as for partially double-stranded RNA. A detailed analysis of the nucleic acid binding mechanism using pre-steady-state techniques revealed the association reaction to be nearly diffusion controlled. It occurs in a single step with a second-order rate constant (k(on)) of 0.273 nM(-1) s(-1). The dissociation of the nucleic acid-polymerase complex is fast with a dissociation rate constant (k(off)) of 59.3 s(-1). With short, partially double-stranded RNAs, no nucleotide incorporation could be observed, while de novo RNA synthesis with short RNA templates showed nucleotide incorporation and end-to-end template switching events. Sing le-turnover, single-nucleotide incorporation studies (representing here the initiation and not processive polymerization) using dinucleotide primers revealed a very slow incorporation rate (k(pol)) of 0.0007 s(-1) and a K-d of the binary enzyme-nucleic acid complex for the incoming ATP of 27.7 mu M. Using dynamic laser light scattering. it could be shown for the first time that oligomerization of HCV NS5B is a dynamic and monovalent salt concentration dependent process. While NS5B is highly oligomeric at low salt concentrations, monomers were only observed at NaCl concentrations above 300 mM. Binding of short RNA substrates led to a further increase in oligomerization, whereas GTP did not show any effect on protein/protein interactions. Furthermore., nucleotide incorporation studies indicate the oligomerization state does not correlate with enzymatic activities as previously proposed.