Processing of Norwalk virus nonstructural proteins by a 3C-like cysteine proteinase
Processing of Norwalk virus nonstructural proteins by a 3C-like cysteine proteinase
复制标题
DOI：
10.1016/s0042-6822(03)00004-7
复制
发表时间：
2003-04-10
期刊：
影响因子：
3.7
通讯作者：
Reilly, PA
中科院分区：
文献类型：
--
作者：
Blakeney, SJ;Cahill, A;Reilly, PA
诺瓦克病毒非结构多蛋白前体在体外的表达导致特异性位点的快速共译切割。裂解产物与先前鉴定的高度相关的南安普顿病毒相似。我们通过对假定的催化半胱氨酸残基的突变，灭活了负责非结构多蛋白切割的病毒编码蛋白酶，从而产生了全长多蛋白前体。NV蛋白酶在大肠杆菌中以谷胱甘肽s -转移酶融合表达，并通过gst亲和层析纯化。纯化后的蛋白酶与全长前体蛋白孵育证实了其活性。非结构蛋白前体的反式切割产生的切割产物与共翻译切割过程中观察到的相似，但效率较低。NV蛋白酶对半胱氨酸和丝氨酸蛋白酶抑制剂表现出与脊髓灰质炎病毒3C蛋白酶相似的敏感性，表明NV蛋白酶是病毒半胱氨酸蛋白酶家族的成员。我们认为该蛋白酶可能在病毒复制中起调节作用。(C) 2003 Elsevier Science（美国）版权所有。
Expression of Norwalk virus nonstructural polyprotein precursor in vitro resulted in rapid cotranslational cleavage at specific sites. The cleavage products were similar to those previously identified for Southampton virus, a highly related virus. We inactivated the virally encoded proteinase responsible for cleavage of the nonstructural polyprotein by mutation of the putative catalytic cysteine residue, which resulted in production of full-length polyprotein precursor. NV proteinase was expressed in Escherichia coli as a glutathione S-transferase fusion and purified by GST-affinity chromatography. Activity of the purified proteinase was demonstrated by incubation with the full-length precursor protein. trans cleavage of the nonstructural protein precursor resulted in cleavage products similar to those observed during cotranslational cleavage, however, at lesser efficiency. NV proteinase displayed sensitivities to cysteine and serine protease inhibitors similar to poliovirus 3C proteinase, suggesting that NV proteinase is a member of the viral cysteine proteinase family. We propose that the proteinase may play a regulatory role in viral replication. (C) 2003 Elsevier Science (USA). All rights reserved.