Processing of the Semliki Forest virus structural polyprotein: role of the capsid protease
Processing of the Semliki Forest virus structural polyprotein: role of the capsid protease
复制标题
塞姆利基森林病毒结构多蛋白的加工：衣壳蛋白酶的作用
作者：
Paul MELANCONt;Henrik Garoff
通过使用体外转录-翻译系统研究了负责塞姆利基森林病毒结构多蛋白的共翻译加工的蛋白酶活性。三次裂解从新生多蛋白中释放出单独的链，顺序为衣壳、p62、6K（一种非结构肽）和 E1。我们直接证明了负责衣壳蛋白释放的蛋白酶活性存在于衣壳本身中：通过逐步截断用于 SP6 转录的 cDNA，我们发现衣壳 C 末端仅含有 38 个 p62 蛋白残基的前体在体外仍能被非常有效地切割。我们进一步测试了衣壳的丝氨酸219通过定点体外诱变参与自蛋白水解的可能性。将序列 Gly-Asp-Ser(219)-Gly（几种动物丝氨酸蛋白酶中保守的四肽）更改为 Gly-Asp-Arg-Ser-Thr 可以完全消除体外裂解。这支持了衣壳是丝氨酸蛋白酶的观点。然后通过翻译杂合多蛋白来研究衣壳蛋白酶在 6K 连接处理中的作用，其中衣壳和大部分 p62 序列被分泌蛋白溶菌酶的序列取代。 6K 肽的裂解和伴随出现有效发生，并且表明需要膜的存在。这表明衣壳蛋白酶不是这些切割所必需的，并且表明膜相关宿主蛋白酶负责切割。
The protease activities responsible for the cotranslational processing of the Semliki Forest virus structural polyprotein were investigated by using an in vitro transcription-translation system. Three cleavages released the individual chains from the nascent polyprotein in the order capsid, p62, 6K (a nonstructural peptide), and E1. We showed directly that the protease activity responsible for the release of the capsid protein resides in the capsid itself: by progressive truncation of the cDNA used for the SP6 transcription, we showed that a precursor containing as few as 38 residues of the p62 protein left at the C terminus of the capsid was still very efficiently cleaved in vitro. We further tested the possibility that serine-219 of the capsid is involved in autoproteolysis by site-directed in vitro mutagenesis. A change in the sequence Gly-Asp-Ser(219)-Gly, a tetrapeptide conserved among several animal serine proteases, to Gly-Asp-Arg-Ser-Thr was shown to completely abolish in vitro cleavage. This supports the notion that the capsid is a serine protease. The role of the capsid protease in the processing of the 6K junctions was then investigated by translations of a hybrid polyprotein in which the capsid and most of the p62 sequences are replaced by those of the secretory protein lysozyme. The cleavages and concomitant appearance of the 6K peptide occurred efficiently and were shown to require the presence of membranes. This demonstrates that the capsid protease is not required for those cleavages and suggests that a membrane-associated host protease is responsible for the cleavage.
影响因子：
56.9
作者：
HOGLE, JM;CHOW, M;FILMAN, DJ
通讯作者：
FILMAN, DJ