To assess the RNA helicase activity of hepatitis C virus (HCV) nonstructural protein 3 (NS3), a polypeptide encompassing amino acids 1175 to 1657, which cover only the putative helicase domain, was expressed in Escherichia coli by a pET expression vector. The protein was purified to near homogeneity and assayed for RNA helicase activity in vitro with double-stranded RNA substrates prepared from a multiple cloning sequence and an HCV 5 nontranslated region (5-NTR) or 3-NTR. The enzyme acted successfully on substrates containing both 5 and 3 single-stranded regions (standard) or on substrates containing only the 3 single-stranded regions (3/3) but failed to act on substrates containing only the 5 single-stranded regions (5/5) or on substrates lacking the single-stranded regions (blunt). These results thus suggest 3 to 5 directionality for HCV RNA helicase activity. However, a 5/5 substrate derived from the HCV 5-NTR was also partially unwound by the enzyme, possibly because of unique properties inherent in the 5 single-stranded regions. Gel mobility shift analyses demonstrated that the HCV NS3 helicase could bind to either 5-or 3-tailed substrates but not to substrates lacking a single-stranded region, indicating that the polarity of the RNA strand to which the helicase bound was a more important enzymatic activity determinant. In addition to double-stranded RNA substrates, HCV NS3 helicase activity could displace both RNA and DNA oligonucleotides on a DNA template, suggesting that HCV NS3 too was disposed to DNA helicase activity. This study also demonstrated that RNA helicase activity was dramatically inhibited by the single-stranded polynucleotides. Taken altogether, our results indicate that the HCV NS3 helicase is unique among the RNA helicases characterized so far.

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