Snijder E J, Wassenaar A L, Spaan W J, Gorbalenya A E
Department of Virology, Faculty of Medicine, Leiden University, The Netherlands.
J Biol Chem. 1995 Jul 14;270(28):16671-6. doi: 10.1074/jbc.270.28.16671.
The replicase ORF1a polyprotein of equine arteritis virus, a positive-stranded RNA virus, is proteolytically processed into (at least) six nonstructural proteins (Nsp). A papain-like Cys protease in Nsp1 and a chymotrypsin-like Ser protease in Nsp4 are involved in this process. In this paper we demonstrate that the Nsp2/3 junction is not cleaved by either of these previously described proteases. Comparative sequence analysis suggested that an additional Cys protease resided in the N-terminal Nsp2 domain. For equine arteritis virus, this domain was shown to induce Nsp2/3 cleavage in a trans-cleavage assay. Processing was abolished when the putative active site residues, Cys-270 and His-332, were replaced. Other Nsp2 domains and three other conserved Cys residues were also shown to be essential. The Nsp2 Cys protease displays sequence similarity with viral papain-like proteases. However, the presumed catalytic Cys-270 is followed by a conserved Gly rather than the characteristic Trp. Replacement of Gly-271 by Trp abolished the Nsp2/3 cleavage. Conservation of a Cys-Gly dipeptide is a hallmark of viral chymotrypsin-like Cys proteases. Thus, the arterivirus Nsp2 protease is an unusual Cys protease with amino acid sequence similarities to both papain-like and chymotrypsin-like proteases.
马动脉炎病毒是一种正链RNA病毒，其复制酶开放阅读框1a多聚蛋白经蛋白水解后被加工成（至少）六种非结构蛋白（Nsp）。Nsp1中的类木瓜蛋白酶半胱氨酸蛋白酶和Nsp4中的类胰凝乳蛋白酶丝氨酸蛋白酶参与了这一过程。在本文中，我们证明Nsp2/3连接处不会被上述任何一种蛋白酶切割。比较序列分析表明，在Nsp2结构域的N端存在一种额外的半胱氨酸蛋白酶。对于马动脉炎病毒，该结构域在反式切割试验中可诱导Nsp2/3的切割。当假定的活性位点残基Cys-270和His-332被替换时，切割作用消失。其他Nsp2结构域和另外三个保守的半胱氨酸残基也被证明是必不可少的。Nsp2半胱氨酸蛋白酶与病毒类木瓜蛋白酶具有序列相似性。然而，推测的催化性半胱氨酸Cys-270之后是一个保守的甘氨酸，而不是特征性的色氨酸。将甘氨酸-271替换为色氨酸可消除Nsp2/3的切割。半胱氨酸-甘氨酸二肽的保守性是病毒类胰凝乳蛋白酶样半胱氨酸蛋白酶的一个标志。因此，动脉炎病毒Nsp2蛋白酶是一种不寻常的半胱氨酸蛋白酶，其氨基酸序列与类木瓜蛋白酶和类胰凝乳蛋白酶均有相似性。
