CHARACTERIZATION OF A HUMAN CORONAVIRUS (STRAIN 229E) 3C-LIKE PROTEINASE ACTIVITY
CHARACTERIZATION OF A HUMAN CORONAVIRUS (STRAIN 229E) 3C-LIKE PROTEINASE ACTIVITY
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DOI：
10.1128/jvi.69.7.4331-4338.1995
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发表时间：
1995-07-01
影响因子：
5.4
通讯作者：
SIDDELL, SG
中科院分区：
文献类型：
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作者：
ZIEBUHR, J;HEROLD, J;SIDDELL, SG
人冠状病毒（HCV）229 E的RNA聚合酶基因编码一个大的多聚蛋白，其包含具有木瓜蛋白酶样半胱氨酸蛋白酶和与小核糖核酸病毒的3C蛋白酶具有同源性的蛋白酶的特征性基序的结构域。在这项研究中，我们，首先，表达了推定的HCV 229 E 3C样蛋白酶结构域的一部分，在大肠杆菌中的β-半乳糖苷酶融合蛋白，并已表明，表达的蛋白质具有蛋白水解活性。在预测的蛋白酶结构域中替换一个氨基酸（His-3006->Asp-3006）消除或至少显著降低了该活性。纯化的34-kDa切割产物的氨基末端序列分析表明，细菌融合蛋白在二肽Gln-2965-Ala-2966处被切割，这是推定的3C样蛋白酶结构域的预测氨基末端。我们已经证实了具有预测的HCV 229 E 3C-1的氨基酸序列的细菌表达的多肽的蛋白水解活性。如蛋白酶，通过RNA聚合酶基因的开放阅读框1b内编码的体外翻译多肽的反式切割。最后，利用融合蛋白特异性抗血清，我们在HCV 229 E感染的MRC-5细胞中鉴定出一种34-kDa的3C样蛋白酶多肽，这种多肽在感染后3 - 5小时就可以检测到，但在感染细胞中的含量很低。这些数据有助于HCV 229 E的3C样蛋白酶活性的表征。
The RNA polymerase gene of human coronavirus (HCV) 229E encodes a large polyprotein that contains domains with motifs characteristic of both papain-like cysteine proteinases and proteinases with homology to the 3C proteinase of picornaviruses. In this study, we have, first, expressed the putative HCV 229E 3C-like proteinase domain as part of a beta-galactosidase fusion protein in Escherichia coli and have shown that the expressed protein has proteolytic activity. The substitution of one amino acid within the predicted proteinase domain (His-3006-->Asp-3006) abolishes, or at least significantly reduces, this activity. Amino-terminal sequence analysis of a purified, 34-kDa cleavage product shows that the bacterial fusion protein is cleaved at the dipeptide Gln-2965-Ala-2966, which is the predicted amino-terminal end of the putative 3C-like proteinase domain, Second, we have confirmed the proteolytic activity of a bacterially expressed polypeptide with the amino acid sequence of the predicted HCV 229E 3C-like proteinase by trans cleavage of an in vitro translated polypeptide encoded within open reading frame 1b of the RNA polymerase gene. Finally, using fusion protein-specific antiserum, we have identified a 34-kDa, 3C-like proteinase polypeptide in HCV 229E-infected MRC-5 cells, This polypeptide can be detected as early as 3 to 5 h postinfection but is present in the infected cell in very low amounts. These data contribute to the characterization of the 3C-like proteinase activity of HCV 229E.