Structure-based mutagenesis study of hepatitis C virus NS3 helicase

The NS3 protein of hepatitis C virus (HCV) is a bifunctional protein contai ning a serine protease in the N-terminal one-third, which is stimulated upo n binding of the NS4A cofactor, and an RNA helicase in the C-terminal two-t hirds. In this study, a C-terminal hexahistidine-tagged helicase domain of the HCN NS3 protein was expressed in Escherichia coli and purified to homog eneity by conventional chromatography, The purified HCV helicase domain has a basal ATPase activity, a polynucleotide-stimulated ATPase activity, and a nucleic acid unwinding activity and binds efficiently to single-stranded polynucleotide. Detailed characterization of the purified HCV helicase doma in with regard to all four activities is presented. Recently, we published an X-ray crystallographic structure of a binary complex of the HCV helicase with a (dU)(8) oligonucleotide, in which several conserved residues of the HCV helicase were shown to be involved in interactions between the HCV hel icase and oligonucleotide, Here, site-directed mutagenesis was used to eluc idate the roles of these residues in helicase function. Four individual mut ations, Thr to Ala at position 269, Thr to Ala at position 411, Trp to Leu at: position 501, and Trp to Ala at position 501, produced a severe reducti on of RNA binding and completely abolished unwinding activity and stimulati on of ATPase activity by poly(U), although the basal ATPase activity (activ ity in the absence of polynucleotide) of these mutants remained intact. Ala nine substitution at Ser-231 or Ser-370 resulted in enzymes that were indis tinguishable from wild-type HCV helicase with regard to all four activities . A mutant bearing Phe at Trp-501 showed wild-type levels of basal ATPase, unwinding activity, and single-stranded RNA binding activity. Interestingly , ATPase activity of this mutant became less responsive to stimulation by p oly(U) But not to stimulation by other polynucleotides, such as poly(C). Gi ven the conservation of some of these residues in other DNA and RNA helicas es, their role in the mechanism of unwinding of double-stranded nucleic aci d is discussed.