Mutational analysis of active site residues of chitinase from Bombyx mori nucleopolyhedrovirus
Mutational analysis of active site residues of chitinase from Bombyx mori nucleopolyhedrovirus
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DOI：
10.1016/j.virusres.2006.11.001
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发表时间：
2007-03-01
期刊：
影响因子：
5
通讯作者：
Shimada, Toru
中科院分区：
文献类型：
--
作者：
Daimon, Takaaki;Katsuma, Susumu;Shimada, Toru
家蚕核型多角体病毒(BmNPV)感染家蚕幼虫可引起宿主液化。这一过程归因于病毒编码的两个基因，几丁质酶(v-chia)和组织蛋白酶(v-cath)的协同作用。以前的研究表明，在没有AcMNPV chia的情况下，Autograph a calfornica核型多角体病毒(AcMNPV)Cath不能在感染细胞内被处理。为了研究V-chia和V-cath之间的相互作用，我们构建了一个重组BmNPV(103ChiAmut)，其中BmNPV chia活性部位的残基发生了突变(D302NE306Q)，该基因由其自身的启动子驱动。Bn-tNPV chia活性部位突变导致几丁质降解活性完全丧失。感染103ChiAmut的家蚕幼虫比感染野生型BmNPV的幼虫存活时间更长，死亡后不发生末端液化。半胱氨酸蛋白酶活性和Western印迹分析表明，在感染v-chia基因缺失的BmNPV(Chiad)的细胞中，BmNPV Cath没有被正确处理，并以不溶于洗涤剂的形式积累，这表明BmNPV chia在V-cath加工过程中起着关键作用。在感染103ChiAmut的细胞中，BtnNPV Cath形成了不可溶的聚集体，这表明活性位点突变的BmNPV chia在V-Cath处理过程中失去了作为分子伴侣的额外作用。(C)2006爱思唯尔B.V.保留所有权利。
Infection of Bombyx mori larvae with B. mori nucleopolyhedrovirus (BmNPV) results in liquefaction of the host. This process is attributed to the synergistic action of two virus-encoded genes, chitinase (v-chiA) and cathepsin (v-cath). Previous studies have suggested that Autographa californica nucleopolyhedrovirus (AcMNPV) CATH cannot be processed within infected cells in the absence of AcMNPV CHIA. To investigate the interactions between V-CHIA and V-CATH, we generated a recombinant BmNPV (103ChiAmut) in which the residues of the active site of BmNPV chiA were mutated (D302NE306Q) and the gene was driven by its own promoter at the native locus. Mutation at the active site of Bn-tNPV CHIA resulted in complete loss of chitinolytic activity. Bombyx mori larvae infected with 103ChiAmut survived longer than larvae infected with wild-type BmNPV and did not undergo terminal liquefaction after death. Cysteine protease activity and Western blot analysis showed that, in cells infected with v-chiA-deleted BmNPV (ChiAD), BmNPV CATH was not processed properly and was accumulated as a detergent-insoluble form, suggesting that BmNPV CHIA plays a crucial role in V-CATH processing. In cells infected with 103ChiAmut, BtnNPV CATH formed insoluble aggregates, suggesting that active site-mutated BmNPV CHIA loses its additional role as a molecular chaperon during V-CATH processing. (c) 2006 Elsevier B.V. All rights reserved.