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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  • [1] NONSTRUCTURAL PROTEIN-3 OF THE HEPATITIS-C VIRUS ENCODES A SERINE-TYPE PROTEINASE REQUIRED FOR CLEAVAGE AT THE NS3/4 AND NS4/5 JUNCTIONS
    BARTENSCHLAGER, R
    AHLBORNLAAKE, L
    MOUS, J
    JACOBSEN, H
    [J]. JOURNAL OF VIROLOGY, 1993, 67 (07) : 3835 - 3844
  • [2] Vaccinia virion protein I8R has both DNA and RNA helicase activities: Implications for vaccinia virus transcription
    Bayliss, CD
    Smith, GL
    [J]. JOURNAL OF VIROLOGY, 1996, 70 (02) : 794 - 800
  • [3] DETECTION OF A TRYPSIN-LIKE SERINE PROTEASE DOMAIN IN FLAVIVIRUSES AND PESTIVIRUSES
    BAZAN, JF
    FLETTERICK, RJ
    [J]. VIROLOGY, 1989, 171 (02) : 637 - 639
  • [4] ASSOCIATION OF POLIOVIRAL PROTEINS OF THE P2-GENOMIC REGION WITH THE VIRAL REPLICATION COMPLEX AND VIRUS-INDUCED MEMBRANE SYNTHESIS AS VISUALIZED BY ELECTRON-MICROSCOPIC IMMUNOCYTOCHEMISTRY AND AUTORADIOGRAPHY
    BIENZ, K
    EGGER, D
    PASAMONTES, L
    [J]. VIROLOGY, 1987, 160 (01) : 220 - 226
  • [5] SECONDARY STRUCTURE OF THE 5' NONTRANSLATED REGIONS OF HEPATITIS-C VIRUS AND PESTIVIRUS GENOMIC RNAS
    BROWN, EA
    ZHANG, HC
    PING, LH
    LEMON, SM
    [J]. NUCLEIC ACIDS RESEARCH, 1992, 20 (19) : 5041 - 5045
  • [6] BURNETTE WN, 1981, ANAL BIOCHEM, V112, P195, DOI 10.1016/0003-2697(81)90281-5
  • [7] THE TAIWANESE HEPATITIS-C VIRUS GENOME - SEQUENCE DETERMINATION AND MAPPING THE 5' TERMINI OF VIRAL GENOMIC AND ANTIGENOMIC RNA
    CHEN, PJ
    LIN, MH
    TAI, KF
    LIU, PC
    LIN, CJ
    CHEN, DS
    [J]. VIROLOGY, 1992, 188 (01) : 102 - 113
  • [8] ISOLATION OF A CDNA CLONE DERIVED FROM A BLOOD-BORNE NON-A, NON-B VIRAL-HEPATITIS GENOME
    CHOO, QL
    KUO, G
    WEINER, AJ
    OVERBY, LR
    BRADLEY, DW
    HOUGHTON, M
    [J]. SCIENCE, 1989, 244 (4902) : 359 - 362
  • [9] GENETIC ORGANIZATION AND DIVERSITY OF THE HEPATITIS-C VIRUS
    CHOO, QL
    RICHMAN, KH
    HAN, JH
    BERGER, K
    LEE, C
    DONG, C
    GALLEGOS, C
    COIT, D
    MEDINASELBY, A
    BARR, PJ
    WEINER, AJ
    BRADLEY, DW
    KUO, G
    HOUGHTON, M
    [J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1991, 88 (06) : 2451 - 2455
  • [10] CLAUDE A, 1991, J BIOL CHEM, V266, P10358