In silico analysis of ORF1ab in coronavirus HKU1 genome reveals a unique putative cleavage site of coronavirus HKU13C-like protease
In silico analysis of ORF1ab in coronavirus HKU1 genome reveals a unique putative cleavage site of coronavirus HKU13C-like protease
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DOI：
10.1111/j.1348-0421.2005.tb03681.x
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发表时间：
2005-01-01
影响因子：
2.6
通讯作者：
Yuen, KY
中科院分区：
文献类型：
--
作者：
Woo, PCY;Huang, Y;Yuen, KY
最近，我们描述了一种与肺炎相关的新型冠状病毒，冠状病毒HKU 1（CoV-HKU 1）的发现和全基因组序列。在这项研究中，对编码CoV-HKU 1基因组中7，182个氨基酸的复制酶多聚蛋白的ORF 1ab进行了详细的计算机分析，结果表明CoV-HKU 1的复制酶多聚蛋白被其木瓜蛋白酶样蛋白酶和3C样蛋白酶（3CL（pro））切割成16条与其他冠状病毒中相应多肽同源的多肽。令人惊讶的是，对3CL（pro）的推定切割位点的分析揭示了独特的推定切割位点。在所有已知的冠状病毒中，3CL（pro）切割位点的P1位置被谷氨酰胺占据。在CoV-HKU 1中也观察到这一点，除了nsp 10（解旋酶）和nsp 11（核酸外切酶家族成员）之间连接处的一个位点，其中P1位置被组氨酸占据。这种氨基酸替换是由于CoV-HKU 1基因组中的单核苷酸突变，CAG/A到CAT。这可能代表了一个新的切割位点，因为在来自不同患者的多个标本的CoV-HKU 1序列中一致地观察到相同的突变;该切割位点的P2和P1 '-P12'位置在CoV-HKU 1和其他冠状病毒之间是一致的;由于解旋酶是冠状病毒中最保守的蛋白质之一，所以nsp 10和nsp 11之间的切割应该是产生成熟功能性解旋酶的必要步骤。包括CoV-HKU 1解旋酶的纯化和C-末端氨基酸测序以及CoV-HKU 1 3CL（pro）的反式切割测定的实验将证实该新切割位点的存在。
Recently we have described the discovery and complete genome sequence of a novel coronavirus associated with pneumonia, coronavirus HKU1 (CoV-HKU1). In this study, a detailed in silico analysis of the ORF1ab, encoding the 7,182-amino acid replicase polyprotein in the CoV-HKU1 genome showed that the replicase polyprotein of CoV-HKU1 is cleaved by its papain-like proteases and 3C-like protease (3CL(pro)) into 16 polypeptides homologous to the corresponding polypeptides in other coronaviruses. Surprisingly, analysis of the putative cleavage sites of the 3CL(pro) revealed a unique putative cleavage site. In all known coronaviruses, the P1 positions at the cleavage sites of the 3CL(pro) are occupied by glutamine. This is also observed in CoV-HKU1, except for one site at the junction between nsp10 (helicase) and nsp11 (member of exonuclease family), where the P1 position is occupied by histidine. This amino acid substitution is due to a single nucleotide mutation in the CoV-HKU1 genome, CAG/A to CAT. This probably represents a novel cleavage site because the same mutation was consistently observed in CoV-HKU1 sequences from multiple specimens of different patients; the P2 and P1'-P12' positions of this cleavage site are consistent between CoV-HKU1 and other coronaviruses; and as the helicase is one of the most conserved proteins in coronaviruses, cleavage between nsp10 and nsp11 should be an essential step for the generation of the mature functional helicase. Experiments, including purification and C-terminal amino acid sequencing of the CoV-HKU1 helicase and trans-cleavage assays of the CoV-HKU1 3CL(pro) will confirm the presence of this novel cleavage site.