The acidic sequence of the NS4A cofactor regulates ATP hydrolysis by the HCV NS3 helicase.

In flaviviruses and hepatitis C virus (HCV), the NS3 gene encodes the N-terminal protease (NS3pro) and the C-terminal helicase (NS3hel). In HCV, the downstream NS4A is required for the NS3pro activity and exhibits a conserved EFDEMEE motif. To identify the role of this motif, we compared the ATPase and helicase activities of NS3 alone with those of the NS3-NS4A constructs. Our results suggest that the EFDEMEE motif is essential for regulating the ATPase activity of NS3hel. It is likely that this motif interferes with the ATP-binding site of NS3hel. It is becoming clear that NS4A functions as a cofactor of both proteinase and helicase in HCV.