The NS3 ATPase/helicase was isolated and characterized from three different infectious clones of hepatitis C virus (HCV). One helicase was from a genotype that normally responds to therapy (Hel-2a), and the other two were from more resistant genotypes, 1a (Hel-1a) and 1b (Hel-1b). Although the differences among these helicases are generally minor, all three enzymes have distinct properties. Hel-1a is less selective for nucleoside triphosphates, Hel-1b hydrolyzes nucleoside triphosphates less rapidly, and Hel-2a unwinds DNA more rapidly and binds DNA more tightly than the other two enzymes. Unlike related proteins, different nucleic acid sequences stimulate ATP hydrolysis by HCV helicase at different maximum rates and with different apparent efficiencies. This nucleic acid stimulation profile is conserved among the enzymes, but it does not result entirely from differential DNA-binding affinities. Although the amino acid sequences of the three proteins differ by up to 15%, one variant amino acid that is critical for helicase action was identified. NS3 residue 450 is a threonine in Hel-1a and Hel-1b and is an isoleucine in Hel-2a. A mutant Hel-1a with an isoleucine substituted for threonine 450 unwinds DNA more rapidly and binds DNA more tightly than the parent protein.The hepatitis C virus (HCV) evolves so rapidly that, in individual patients infected with certain genotypes, HCV exists as a heterogeneous population of quasispecies. Both viral genotype and quasispecies diversity influence disease severity and treatment response (reviewed in references 9 and 45). Patients infected with genotype 1 often do not respond to antiviral therapy, whereas patients with other genotypes respond more favorably (25). The kinetics of viral load decreases after the initiation of interferon therapy suggested a high freevirion clearance rate, a low rate of virus production in infected cells, and a high rate of infected-cell death in patients infected with genotype 2 relative to those in patients infected with genotype 1 (28). Strains that more rapidly evolve into diverse quasispecies more readily evade the host immune system, leading to chronic hepatitis (10), and respond less well to therapeutic intervention (11). Exactly how HCV genetic variation leads to these clinically important viral phenotypes is still largely a mystery, however. This comparative study was therefore initiated to define the impact of genetic variation on the activity of one of the best characterized HCV proteins, the NS3 helicase.HCV is a positive-sense, single-stranded RNA (ssRNA), and as such, when it enters a cell, HCV genomic RNA is translated into a peptide over 3,000 amino acids long from a single open reading frame. Both the host and viral proteases process the HCV polyprotein into three structural proteins (core, E1, and E2) and seven nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Most studies dealing with the impact of genetic variation on the HCV life cycle have focused on linking heterogeneity in NS5A (7) and E2 (38) with the tre...