Characterization of ATPase activity of a hepatitis C virus NS3 helicase domain, and analysis involving mercuric reagents.

The C-terminal two-thirds of nonstructural protein 3 (NS3) of hepatitis C virus (HCV) exhibits RNA-dependent NTPase/helicase activity. This enzyme is considered to be involved in viral replication and is expected to be one of the target molecules of anti-HCV drugs. In a search for NTPase inhibitors specific to HCV, we expressed and purified the truncated NS3 NTPase/helicase domain. Here, we report the characterization of its RNA-dependent ATPase activity. This enzyme preferred Mg(2+) and the optimal pH was 7.0. We further investigated the effects of heavy metal ions on the ATPase activity. The mercuric ion inhibited it significantly, the 50% inhibitory concentration being 49 nM. The fact that the inhibitory profile was competitive and that this inhibition was blocked in the presence of a large excess of cysteine or dithiothreitol, suggested that a cysteine residue in the DECH box was the main target site of mercury.