Structure and cleavage specificity of the chymotrypsin-like serine protease (3CLSP/nsp4) of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV).
Structure and cleavage specificity of the chymotrypsin-like serine protease (3CLSP/nsp4) of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV).
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牛乳糖蛋白酶样丝氨酸蛋白酶（3clsp/nsp4）生殖和呼吸综合征病毒（PRRSV）的结构和切割特异性。
DOI：
10.1016/j.jmb.2009.07.062
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发表时间：
2009-10-02
影响因子：
5.6
通讯作者：
Gao GF
中科院分区：
文献类型：
--
作者：
Tian X;Lu G;Gao F;Peng H;Feng Y;Ma G;Bartlam M;Tian K;Yan J;Hilgenfeld R;Gao GF
猪繁殖与呼吸综合征病毒(PRRSV)的生物发生和复制包括利用自身编码的蛋白酶处理复制性多蛋白的关键步骤。全基因组生物信息学分析表明，非结构蛋白4(NSP4)是一种类似3C的丝氨酸蛋白酶(3CLSP)，负责大多数非结构蛋白的加工。为了证实这一预测，我们克隆了该酶的编码基因，并在大肠杆菌中进行了表达。纯化的蛋白质被结晶，并在1.9°分辨率下解析其结构。此外，还测定了Ser118Ala突变体的晶体结构，其分辨率为2.0。该单体酶折叠成三个结构域，类似于马动脉炎病毒的同源蛋白酶，马动脉炎病毒与PRRSV一样，是Nidovirales目的动脉病毒科的成员。PRRSV 3CLSP的活性部位位于结构域I和II之间，含有一个典型的催化三联体，由Ser118、His39和Asp64组成。该结构还显示了一个非典型的氧阴离子空洞和一个部分折叠的S1特异性口袋。对纯化蛋白的体外蛋白水解性进行了测定。在PRRSV复制多蛋白中，三个连接非结构蛋白结构域的位置被证实被该酶处理。其中两个接头nsp3/nsp4和nsp11/nsp12在反式中被切割，而在nsp4/nsp5连接子中被顺式切割。因此，我们提供了NSP4蛋白是一个功能性3CLSP的结构证据和酶证据。我们还发现该酶在底物的P1位对谷氨酸有很强的选择性。
Biogenesis and replication of the porcine reproductive and respiratory syndrome virus (PRRSV) include the crucial step of replicative polyprotein processing by self-encoded proteases. Whole genome bioinformatics analysis suggests that nonstructural protein 4 (nsp4) is a 3C-like serine protease (3CLSP), responsible for most of the nonstructural protein processing. The gene encoding this protease was cloned and expressed in Escherichia coli in order to confirm this prediction. The purified protein was crystallized, and the structure was solved at 1.9 Å resolution. In addition, the crystal structure of the Ser118Ala mutant was determined at 2.0 Å resolution. The monomeric enzyme folds into three domains, similar to that of the homologous protease of equine arteritis virus, which, like PRRSV, is a member of the family Arteriviridae in the order of Nidovirales. The active site of the PRRSV 3CLSP is located between domains I and II and harbors a canonical catalytic triad comprising Ser118, His39, and Asp64. The structure also shows an atypical oxyanion hole and a partially collapsed S1 specificity pocket. The proteolytic activity of the purified protein was assessed in vitro. Three sites joining nonstructural protein domains in the PRRSV replicative polyprotein are confirmed to be processed by the enzyme. Two of them, the nsp3/nsp4 and nsp11/nsp12 junctions, are shown to be cleaved in trans, while cis cleavage is demonstrated for the nsp4/nsp5 linker. Thus, we provide structural evidence as well as enzymatic proof of the nsp4 protein being a functional 3CLSP. We also show that the enzyme has a strong preference for glutamic acid at the P1 position of the substrate.
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影响因子：
4.4
作者：
Blaha, T
通讯作者：
Blaha, T
冠状病毒主蛋白酶的结构揭示了甲over依蛋白酶折叠与额外的α-螺旋结构域的组合。
影响因子：
11.4
作者：
Anand, Kanchan;Palm, Gottfried J;Mesters, Jeroen R;Siddell, Stuart G;Ziebuhr, John;Hilgenfeld, Rolf
通讯作者：
Hilgenfeld, Rolf
猪呼吸与繁殖综合征病毒变种，越南和中国，2007 年。
影响因子：
11.8
作者：
Feng Y;Zhao T;Nguyen T;Inui K;Ma Y;Nguyen TH;Nguyen VC;Liu D;Bui QA;To LT;Wang C;Tian K;Gao GF
通讯作者：
Gao GF
影响因子：
5.8
作者：
Gouet, P;Courcelle, E;Métoz, F
通讯作者：
Métoz, F
影响因子：
3.8
作者：
Hegyi, A;Ziebuhr, J
通讯作者：
Ziebuhr, J