IDENTIFICATION OF THE CATALYTIC SITES OF A PAPAIN-LIKE CYSTEINE PROTEINASE OF MURINE CORONAVIRUS
IDENTIFICATION OF THE CATALYTIC SITES OF A PAPAIN-LIKE CYSTEINE PROTEINASE OF MURINE CORONAVIRUS
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DOI：
10.1128/jvi.67.10.6056-6063.1993
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发表时间：
1993-10-01
影响因子：
5.4
通讯作者：
LAI, MMC
中科院分区：
文献类型：
--
作者：
BAKER, SC;YOKOMORI, K;LAI, MMC
鼠冠状病毒小鼠肝炎病毒基因1表达为多聚蛋白，其在感染后被切割成多个蛋白。其中一种蛋白质是p28，它代表多聚蛋白的氨基末端部分，推测是由多聚蛋白的自身蛋白酶结构域的活性产生的（S. C.贝克角，澳-地K.谢湖H. Soe，M.- F.张氏D. M. Vannier和M. M. C. Lai，J. Virol. 63：3693-3699，1989）。在这项研究中，该推定的蛋白酶结构域的边界和关键氨基酸残基的特点是通过缺失分析和定点突变。通过检测兔网织红细胞裂解物体外翻译过程中p28的产生来监测蛋白酶活性。缺失分析将蛋白酶结构域定义在从基因组5'端的3.6- 4.4-kb区域编码的序列内。底物（p28）和蛋白酶结构域之间的0.7-kb区域可以被删除，而不影响蛋白水解切割。然而，较大的缺失（1.6 kb）导致蛋白酶活性的丧失，这表明蛋白酶和底物之间的间隔序列的重要性。计算机辅助分析的蛋白酶结构域的氨基酸序列，确定潜在的催化半胱氨酸和组氨酸残基的延伸序列远相关的木瓜蛋白酶样半胱氨酸蛋白酶。这些推定的催化残基在蛋白酶活性的作用进行了研究，通过定点突变。Cys-1137或His-1288的突变导致蛋白酶活性的完全丧失，暗示这些残基是催化活性所必需的。相比之下，His-1317或Cys-1172的大多数突变对蛋白酶活性没有影响或只有很小的影响。本研究确定小鼠肝炎病毒基因1在基因组5'端3.6 - 4.4kb区域编码蛋白酶结构域，其类似于半胱氨酸蛋白酶的木瓜蛋白酶家族的成员，并且该蛋白酶结构域负责N-末端肽的切割。
The murine coronavirus mouse hepatitis virus gene 1 is expressed as a polyprotein, which is cleaved into multiple proteins posttranslationally. One of the proteins is p28, which represents the amino-terminal portion of the polyprotein and is presumably generated by the activity of an autoproteinase domain of the polyprotein (S. C. Baker, C. K. Shieh, L. H. Soe, M.-F. Chang, D. M. Vannier, and M. M. C. Lai, J. Virol. 63:3693-3699, 1989). In this study, the boundaries and the critical amino acid residues of this putative proteinase domain were characterized by deletion analysis and site-directed mutagenesis. Proteinase activity was monitored by examining the generation of p28 during in vitro translation in rabbit reticulocyte lysates. Deletion analysis defined the proteinase domain to be within the sequences encoded from the 3.6- to 4.4-kb region from the 5' end of the genome. A 0.7-kb region between the substrate (p28) and proteinase domain could be deleted without affecting the proteolytic cleavage. However, a larger deletion (1.6 kb) resulted in the loss of proteinase activity, suggesting the importance of spacing sequences between proteinase and substrate. Computer-assisted analysis of the amino acid sequence of the proteinase domain identified potential catalytic cysteine and histidine residues in a stretch of sequence distantly related to papain-like cysteine proteinases. The role of these putative catalytic residues in the proteinase activity was studied by site-specific mutagenesis. Mutations of Cys-1137 or His-1288 led to a complete loss of proteinase activity, implicating these residues as essential for the catalytic activity. In contrast, most mutations of His-1317 or Cys-1172 had no or only minor effects on proteinase activity. This study establishes that mouse hepatitis virus gene 1 encodes a proteinase domain, in the region from 3.6 to 4.4 kb from the 5' end of the genome, which resembles members of the papain family of cysteine proteinases and that this proteinase domain is responsible for the cleavage of the N-terminal peptide.