The helicase activity associated with hepatitis C virus nonstructural protein 3 (NS3)
- 1 December 1996
- journal article
- research article
- Published by American Society for Microbiology in Journal of Virology
- Vol. 70 (12), 8477-8484
- https://doi.org/10.1128/jvi.70.12.8477-8484.1996
Abstract
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 59 nontranslated region (59-NTR) or 39-NTR. The enzyme acted successfully on substrates containing both 59 and 39 single-stranded regions (standard) or on substrates containing only the 39 single-stranded regions (39/39) but failed to act on substrates containing only the 59 single-stranded regions (59/59) or on substrates lacking the single-stranded regions (blunt). These results thus suggest 39 to 59 directionality for HCV RNA helicase activity. However, a 59/59 substrate derived from the HCV 59-NTR was also partially unwound by the enzyme, possibly because of unique properties inherent in the 59 single-stranded regions. Gel mobility shift analyses demonstrated that the HCV NS3 helicase could bind to either 59- or 39-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.Keywords
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