CHARACTERIZATION OF THE HEPATITIS-C VIRUS-ENCODED SERINE PROTEINASE - DETERMINATION OF PROTEINASE-DEPENDENT POLYPROTEIN CLEAVAGE SITES
CHARACTERIZATION OF THE HEPATITIS-C VIRUS-ENCODED SERINE PROTEINASE - DETERMINATION OF PROTEINASE-DEPENDENT POLYPROTEIN CLEAVAGE SITES
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DOI：
10.1128/jvi.67.5.2832-2843.1993
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发表时间：
1993-05-01
影响因子：
5.4
通讯作者：
RICE, CM
中科院分区：
文献类型：
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作者：
GRAKOUI, A;MCCOURT, DW;RICE, CM
丙型肝炎病毒 (HCV) H 株多蛋白的加工产生至少九种不同的裂解产物：NH2-C-E1-E2-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH。如本报告所述，使用定点诱变和瞬时表达分析来研究位于 NS3 蛋白 N 端三分之一处的假定丝氨酸蛋白酶结构域在 HCV 多蛋白的蛋白水解加工中的作用。通过用丙氨酸取代蛋白酶催化三联体中两个预测残基（His-1083 和 Ser-1165）中的任一个，消除了蛋白酶结构域 C 端发生的所有四个切割（3/4A、4A/4B、4B/5A 和 5A/5B）。然而，此类取代对结构区或 2/3 位点的切割没有可观察到的影响。删除分析表明多蛋白的结构和NS2区域对于HCV NS3蛋白酶活性来说不是必需的。通过 NS4A、NS4B、NS5A 和 NS5B 的 N 端序列分析定位 NS3 蛋白酶依赖性切割位点。对所有已测序的 HCV 菌株的这些切割位点两侧的残基进行序列比较，揭示了可能在确定 HCV NS3 蛋白酶底物特异性中发挥作用的保守残基。这些特征包括 P6 位点的酸性残基（Asp 或 Glu）、P1 位点的 Cys 或 Thr 残基以及 P1' 位点的 Ser 或 Ala 残基。
Processing of the hepatitis C virus (HCV) H strain polyprotein yields at least nine distinct cleavage products: NH2-C-E1-E2-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH. As described in this report, site-directed mutagenesis and transient expression analyses were used to study the role of a putative serine proteinase domain, located in the N-terminal one-third of the NS3 protein, in proteolytic processing of HCV polyproteins. All four cleavages which occur C terminal to the proteinase domain (3/4A, 4A/4B, 4B/5A, and 5A/5B) were abolished by substitution of alanine for either of two predicted residues (His-1083 and Ser-1165) in the proteinase catalytic triad. However, such substitutions have no observable effect on cleavages in the structural region or at the 2/3 site. Deletion analyses suggest that the structural and NS2 regions of the polyprotein are not required for the HCV NS3 proteinase activity. NS3 proteinase-dependent cleavage sites were localized by N-terminal sequence analysis of NS4A, NS4B, NS5A, and NS5B. Sequence comparison of the residues flanking these cleavage sites for all sequenced HCV strains reveals conserved residues which may play a role in determining HCV NS3 proteinase substrate specificity. These features include an acidic residue (Asp or Glu) at the P6 position, a Cys or Thr residue at the P1 position, and a Ser or Ala residue at the P1' position.