Critical point mutations for hepatitis C virus NS3 proteinase.
Critical point mutations for hepatitis C virus NS3 proteinase.
复制标题
丙型肝炎病毒 NS3 蛋白酶的关键点突变。
作者：
Kazunori Yamada;Akiko Mori;M. Seki;J. Kimura;S. Yuasa;Y. Matsuura;T. Miyamura
丙型肝炎病毒NS3蛋白酶在丙型肝炎病毒非结构前体多蛋白的加工过程中起重要作用。为了检测其加工活性，我们开发了一种简单的反式裂解实验。将两个表达NS3蛋白酶区域的重组质粒和一个含有麦芽糖结合蛋白和蛋白a之间NS5A/5B切割位点的嵌合底物多蛋白共导入大肠杆菌细胞。在多蛋白表达过程中，蛋白酶在单位点处理底物。缺失分析表明，NS3蛋白酶的功能最小结构域由1059 ~ 1204个146个氨基酸组成。我们从慢性丙型肝炎患者的血清中分离了几个编码NS3蛋白酶功能域的cDNA克隆，并通过反式切割实验测定了它们的蛋白酶活性。活跃和不活跃的克隆都存在于同一患者中。这些克隆的比较序列分析表明，某些点突变似乎与蛋白质水解活性的丧失有关。反向突变实验证实了这一点。在关键突变中，对Thr的Pro-1168和对Gly的Arg-1135是有趣的。这些氨基酸位于氧阴离子孔附近，似乎对维持NS3蛋白酶活性中心的构象至关重要。
The hepatitis C virus NS3 proteinase plays an essential role in processing of HCV nonstructural precursor polyprotein. To detect its processing activity, we developed a simple trans-cleavage assay. Two recombinant plasmids expressing the NS3 proteinase region and a chimeric substrate polyprotein containing the NS5A/5B cleavage site between maltose binding protein and protein A were co-introduced into Escherichia coli cells. The proteinase processed the substrate at the single site during their polyprotein expression. Deletion analysis indicated that the functionally minimal domain of the NS3 proteinase was composed of 146 amino acids, 1059 to 1204. We isolated several cDNA clones encoding the functional domain of the NS3 proteinase from the sera of patients chronically infected with HCV and determined their proteinase activity by this trans-cleavage assay. Both active and inactive clones existed in the same patients. Comparative sequence analyses of these clones suggested that certain point mutations seemed to be related to the loss of proteolytic activity. This was confirmed by back mutation experiments. Among the critical mutations, Pro-1168 to Thr and Arg-1135 to Gly were intriguing. These amino acids, which are situated near the oxyanion hole, seem to be essential for maintaining the conformation of the active center of the NS3 proteinase.
影响因子：
3.5
作者：
HO, SN;HUNT, HD;PEASE, LR
通讯作者：
PEASE, LR
DOI：
10.1073/pnas.90.22.10583
发表时间：
1993-11-15
影响因子：
11.1
作者：
GRAKOUI, A;MCCOURT, DW;RICE, CM
通讯作者：
RICE, CM