Identification of the active site residues in the nsP2 proteinase of Sindbis virus.
Identification of the active site residues in the nsP2 proteinase of Sindbis virus.
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DOI：
10.1016/0042-6822(92)90268-t
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发表时间：
1992-12
期刊：
影响因子：
3.7
通讯作者：
Strauss JH
中科院分区：
文献类型：
--
作者：
Strauss EG;De Groot RJ;Levinson R;Strauss JH
辛德毕斯病毒的非结构多聚蛋白由病毒编码的蛋白酶加工，该蛋白酶位于nsP 2的C-末端结构域。在这里，我们进行了诱变分析，以确定该蛋白酶的活性位点残基。Cys-481或His-558的其他氨基酸的取代完全废除体外辛德毕斯病毒多蛋白的蛋白水解加工。甲病毒中保守的第二个半胱氨酸、其他四个保守的组氨酸或保守的丝氨酸在该结构域内的取代不影响酶的活性。这些结果表明nsP 2是一种木瓜蛋白酶样蛋白酶，其催化二联体由Cys-481和His-558组成。由于一个天冬酰胺残基已牵连在木瓜蛋白酶的活性位点，我们改变了四个保守的天冬酰胺残基的C-末端的一半nsP 2，并发现，所有可以取代没有完全丧失活性。在木瓜蛋白酶样蛋白酶中，在植物和动物酶中催化组氨酸之后的残基是丙氨酸或甘氨酸，并且在甲病毒中Trp-559的存在是不寻常的。含有Ala-559的突变酶完全失活，这意味着Trp-559是功能性蛋白酶所必需的。所有这些突变都被引入到辛德毕斯病毒的全长克隆中，感染性RNA可以在体外转录，并测试了这些变化对生存力的影响。在所有的情况下，它被发现，废除蛋白水解活性的突变是致命的，无论这些突变是否在催化残基，表明非结构多蛋白的蛋白水解是必不可少的辛德毕斯复制。
The nonstructural polyproteins of Sindbis virus are processed by a virus-encoded proteinase which is located in the C-terminal domain of nsP2. Here we have performed a mutagenic analysis to identify the active site residues of this proteinase. Substitution of other amino acids for either Cys-481 or His-558 completely abolished proteolytic processing of Sindbis virus polyproteins in vitro. Substitutions within this domain for a second cysteine conserved among alphaviruses, for four other conserved histidines, or for a conserved serine did not affect the activity of the enzyme. These results suggest that nsP2 is a papain-like proteinase whose catalytic dyad is composed of Cys-481 and His-558. Since an asparagine residue has been implicated in the active site of papain, we changed the four conserved asparagine residues in the C-terminal half of nsP2 and found that all could be substituted without total loss of activity. Among papain-like proteinases, the residue following the catalytic histidine is alanine or glycine in the plant and animal enzymes, and the presence of Trp-559 in alphaviruses is unusual. A mutant enzyme containing Ala-559 was completely inactive, implying that Trp-559 is essential for a functional proteinase. All of these mutations were introduced into a full-length clone of Sindbis virus from which infectious RNA could be transcribed in vitro, and the effects of these changes on viability were tested. In all cases it was found that mutations which abolished proteolytic activity were lethal, whether or not these mutations were in the catalytic residues, indicating that proteolysis of the nonstructural polyprotein is essential for Sindbis replication.
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影响因子：
2.9
作者：
GARAVITO, RM;ROSSMANN, MG;EVENTOFF, W
通讯作者：
EVENTOFF, W
影响因子：
3.5
作者：
Gorbalenya AE;Koonin EV;Lai MM
通讯作者：
Lai MM
影响因子：
3.5
作者：
GORBALENYA, AE;DONCHENKO, AP;KOONIN, EV
通讯作者：
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影响因子：
3.7
作者：
BOEGE, U;WENGLER, G;WITTMANNLIEBOLD, B
通讯作者：
WITTMANNLIEBOLD, B
影响因子：
3.7
作者：
FARAGHER, SG;MEEK, ADJ;DALGARNO, L
通讯作者：
DALGARNO, L