ANALYSIS OF NS3-MEDIATED PROCESSING OF THE HEPATITIS-C VIRUS NONSTRUCTURAL REGION IN-VITRO

被引:34
作者
DSOUZA, EDA [1 ]
OSULLIVAN, E [1 ]
AMPHLETT, EM [1 ]
ROWLANDS, DJ [1 ]
SANGAR, DV [1 ]
CLARKE, BE [1 ]
机构
[1] WELLCOME RES LABS, DEPT MOLEC SCI, BECKENHAM BR3 3BS, KENT, ENGLAND
关键词
D O I
10.1099/0022-1317-75-12-3469
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
The protease activity of the hepatitis C virus (HCV) NS3 protein has been investigated using transient expression methods in mammalian cells, as well as in vitro transcription/translation systems. We confirmed that expression of the NS3-5 polyprotein in rabbit reticulocyte lysates results in efficient cis processing at the NS3/NS4 junction. However, processing at the other predicted sites of NS3-mediated cleavage varied markedly in efficiency, the site most susceptible being that between NS5A and NS5B. Time-course analysis of the proteolytic processing of the HCV non-structural precursor showed that the cis cleavage between NS3 and NS4 occurred extremely rapidly. However, efficient cleavage at this position was dependent on the prior removal of the NS2 protein. Furthermore, the presence of uncleaved NS2 sequences on the enzyme severely impeded NS3-mediated proteolysis at downstream sites in the polyprotein. This suggests therefore that efficient cleavage at the NS2/NS3 junction is a pivotal event in HCV replication. During the course of this study a proteolytically inactive mutant of NS3 was characterized carrying a previously unreported amino acid substitution near the proposed active site of the enzyme. Molecular modelling suggested that the amino acid present at this position may influence the conformation of the active site of the enzyme. Recently a number of reports have described a second protease activity, located in the NS2/NS3 region, which is responsible for cleavage at the NS2/NS3 junction. We have identified an isolate of HCV, obtained from a U.K. patient, which has a virtually inactive NS2/NS3 protease. The possible implications of this observation are discussed.
引用
收藏
页码:3469 / 3476
页数:8
相关论文
共 40 条
  • [1] DETECTION OF ANTIBODY TO HEPATITIS-C VIRUS IN PROSPECTIVELY FOLLOWED TRANSFUSION RECIPIENTS WITH ACUTE AND CHRONIC NON-A-HEPATITIS, NON-B-HEPATITIS
    ALTER, HJ
    PURCELL, RH
    SHIH, JW
    MELPOLDER, JC
    HOUGHTON, M
    CHOO, QL
    KUO, G
    [J]. NEW ENGLAND JOURNAL OF MEDICINE, 1989, 321 (22) : 1494 - 1500
  • [2] ALTER HJ, 1989, CURRENT PERSPECTIVES, P83
  • [3] 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
  • [4] HEPATITIS-C VIRUS-RNA IN SOUTHERN AFRICAN BLACKS WITH HEPATOCELLULAR-CARCINOMA
    BUKH, J
    MILLER, RH
    KEW, MC
    PURCELL, RH
    [J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1993, 90 (05) : 1848 - 1851
  • [5] CLEAVAGE OF THE DENGUE VIRUS POLYPROTEIN AT THE NS3/NS4A AND NS4B/NS5 JUNCTIONS IS MEDIATED BY VIRAL PROTEASE NS2B-NS3, WHEREAS NS4A/NS4B MAY BE PROCESSED BY A CELLULAR PROTEASE
    CAHOUR, A
    FALGOUT, B
    LAI, CJ
    [J]. JOURNAL OF VIROLOGY, 1992, 66 (03) : 1535 - 1542
  • [6] EVIDENCE THAT THE N-TERMINAL DOMAIN OF NONSTRUCTURAL PROTEIN NS3 FROM YELLOW-FEVER VIRUS IS A SERINE PROTEASE RESPONSIBLE FOR SITE-SPECIFIC CLEAVAGES IN THE VIRAL POLYPROTEIN
    CHAMBERS, TJ
    WEIR, RC
    GRAKOUI, A
    MCCOURT, DW
    BAZAN, JF
    FLETTERICK, RJ
    RICE, CM
    [J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1990, 87 (22) : 8898 - 8902
  • [7] 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
  • [8] 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
  • [9] PROCESSING AND ASSEMBLY OF FOOT-AND-MOUTH-DISEASE VIRUS PROTEINS USING SUBGENOMIC RNA
    CLARKE, BE
    SANGAR, DV
    [J]. JOURNAL OF GENERAL VIROLOGY, 1988, 69 : 2313 - 2325
  • [10] TREATMENT OF CHRONIC HEPATITIS-C WITH RECOMBINANT INTERFERON-ALFA - A MULTICENTER RANDOMIZED, CONTROLLED TRIAL
    DAVIS, GL
    BALART, LA
    SCHIFF, ER
    LINDSAY, K
    BODENHEIMER, HC
    PERRILLO, RP
    CAREY, W
    JACOBSON, IM
    PAYNE, J
    DIENSTAG, JL
    VANTHIEL, DH
    TAMBURRO, C
    LEFKOWITCH, J
    ALBRECHT, J
    MESCHIEVITZ, C
    ORTEGO, TJ
    GIBAS, A
    [J]. NEW ENGLAND JOURNAL OF MEDICINE, 1989, 321 (22) : 1501 - 1506