The Hepatitis C Virus (HCV) NS3 protein has RNA binding activity, RNA-stimulated NTPase activity, and RNA helicase activity. The RNA binding activity of the C-terminal domain of the HCV NS3 protein is less sensitive to pH, KCl, and MgCl2, than NTPase and the RNA helicase activity. The overall order of the binding of homoribopolymer for the NS3 protein was poly(U) much greater than poly(A) > poly(G), poly(C). The minimal RNA binding size of the HCV NS3 protein was determined using a gel retardation assay and is estimated between 7 nt and 20 nr. The HCV RNA helicase unwinds RNA/DNA heteroduplexes as well as RNA/RNA duplexes and it catalytically translocates in the 3' to 5' direction. (C) 1996 Academic Press, Inc.