To determine whether the two domains of hepatitis C Virus (HCV) NS3 and the NS4A interact with each other to regulate the RNA unwinding activity, this study compares the RNA unwinding, ATPase and RNA binding activities of three forms of NS3 proteins-the NS3H protein, containing only the helicase domain, the full-length NS3 protein, and the NS3-NS4A complex. The results revealed that NS3 displayed the weakest RNA helicase activity, not because it had lower ATPase or RNA binding activity than did NS3H or NS3-NS4A, but because it had the lowest RNA unwinding processivity. A mutant protein. R1487Q, which contained a mutation in the helicase domain, displayed a reduced protease activity as compared to the wild-type NS3-NS4A. Together, these results Suggest the existence of interactions between the two domains of NS3 and the NS4A, which regulates the HCV NS3 protease and RNA helicase activities. (C) 2004 Elsevier Inc. All rights reserved.