The helicase activity associated with hepatitis C virus nonstructural protein 3 (NS3)

被引:220
作者
Tai, CL
Chi, WK
Chen, DS
Hwang, LH
机构
[1] NATL TAIWAN UNIV HOSP, HEPATITIS RES CTR, TAIPEI 100, TAIWAN
[2] NATL TAIWAN UNIV HOSP, DEPT INTERNAL MED, TAIPEI 100, TAIWAN
[3] NATL TAIWAN UNIV HOSP, PROC DEV DIV, DEV CTR BIOTECHNOL, TAIPEI 100, TAIWAN
[4] NATL TAIWAN UNIV, GRAD INST MICROBIOL, TAIPEI 10764, TAIWAN
关键词
D O I
10.1128/JVI.70.12.8477-8484.1996
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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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页码:8477 / 8484
页数:8
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