Mutational analysis of hepatitis C virus NS3-associated helicase

Nonstructural protein 3 (NS3) of hepatitis C virus contains a bipartite str ucture consisting of an N-terminal serine protease and a C-terminal DEXH bo x helicase. To investigate the roles of individual amino acid residues in t he overall mechanism of unwinding, a mutational-functional analysis was per formed based on a molecular model of the NS3 helicase domain bound to ssDNA , which has largely been confirmed by a recently published crystal structur e of the NS3 helicase-ssDNA complex. Three full-length mutated NS3 proteins containing Tyr(392)Ala, Val(432)Gly and Trp(501)Ala single substitutions, respectively, together with a Tyr(392)Ala/Trp(501)Ala double-substituted pr otein were expressed in Escherichia coli and purified to homogeneity, All i ndividually mutated forms showed a reduction in duplex unwinding activity, single-stranded polynucleotide binding capacity and polynucleotide-stimulat ed ATPase activity compared to wild-type, though to different extents. Simu ltaneous replacement of both Tyr(392) and Trp(501) with Ala completely abol ished all these enzymatic functions. On the other hand, the introduced amin o acid substitutions had no influence on NS3 intrinsic ATPase activity and proteolytic efficiency, The results obtained with Trp(501)Ala and Val(432)G ly single-substituted enzymes are in agreement with a recently proposed mod el for NS3 unwinding activity, The mutant phenotype of the Tyr(392)Ala and Tyr(392)Ala/Trp(501)Ala enzymes, however, represents a completely novel fin ding.