Lim K P, Liu D X
Institute of Molecular Agrobiology, Singapore.
Adv Exp Med Biol. 1998;440:173-84. doi: 10.1007/978-1-4615-5331-1_22.
Our previous studies have shown that two overlapping papain-like proteinase domains (PLPDs) encoded by the IBV sequence from nucleotides 4155 to 5550 is responsible for cleavage of the ORF 1a polyprotein to an 87 kDa protein. In this study, we demonstrate that only the more 5' one of the two domains, PLPD-1 encoded between nucleotides 4155 and 5031, is required for processing to the 87 kDa protein. Site-directed mutagenesis studies have shown that the Cys1274 and His1435 residues are essential for the PLPD-1 activity, suggesting that they may be the components of the catalytic centre of this proteinase. Coexpression and immunoprecipitation studies have further revealed that PLPD can interact with the 87 kDa protein. Meanwhile, data obtained from the construction and expression of a series of deletion mutants have indicated that the 87 kDa protein is encoded by the 5'-most 2600 bp part of ORF1a. further deletion and mutagenesis studies are underway to determine precisely the C-terminal cleavage site of the 87 kDa protein.
我们之前的研究表明，传染性支气管炎病毒（IBV）序列中从核苷酸4155至5550编码的两个重叠的木瓜蛋白酶样蛋白酶结构域（PLPDs）负责将ORF 1a多聚蛋白切割成一个87 kDa的蛋白。在本研究中，我们证明，两个结构域中更靠近5'端的那个，即核苷酸4155至5031之间编码的PLPD-1，对于加工生成87 kDa蛋白是必需的。定点诱变研究表明，Cys1274和His1435残基对于PLPD-1的活性至关重要，这表明它们可能是该蛋白酶催化中心的组成部分。共表达和免疫沉淀研究进一步揭示，PLPD可与87 kDa蛋白相互作用。同时，从一系列缺失突变体的构建和表达获得的数据表明，87 kDa蛋白由ORF1a最5'端的2600 bp部分编码。正在进行进一步的缺失和诱变研究，以精确确定87 kDa蛋白的C端切割位点。
