Daimon Takaaki, Katsuma Susumu, Kang WonKyung, Shimada Toru
Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan.
Biochem Biophys Res Commun. 2006 Jun 30;345(2):825-33. doi: 10.1016/j.bbrc.2006.04.112. Epub 2006 May 2.
Baculovirus-encoded chitinases (V-CHIAs) were first proposed to be acquired from a bacterium via horizontal gene transfer. However, we have recently reported that lepidopteran hosts also encode v-chiA orthologs. Here we describe comparative studies of Bombyx mori nucleopolyhedrovirus (BmNPV) chitinase and its host ortholog, BmChi-h. We constructed recombinant BmNPVs in which native and modified forms of BmChi-h were driven under the polyhedrin promoter and the authentic v-chiA was deleted. Western blot analysis indicated that BmCHI-h was rapidly secreted from virus-infected BmN cells whereas BmNPV CHIA was localized within the virus-infected cells; probably because of the presence of a C-terminal endoplasmic reticulum retention motif on BmNPV CHIA. Enzymological studies showed that BmNPV CHIA was able to retain much higher chitinolytic activity under alkaline conditions. For B. mori larvae infected with v-chiA-deleted BmNPV, the terminal liquefaction of dead larvae and the activation of baculovirus-encoded cysteine protease were not observed, and the introduction of BmChi-h did not rescue these defects. Our findings show that BmNPV chiA possesses unique features that are not shared by host orthologs, which may reflect functional specialization of baculovirus chitinases.
杆状病毒编码的几丁质酶（V-CHIAs）最初被认为是通过水平基因转移从细菌中获得的。然而，我们最近报道鳞翅目宿主也编码v-chiA直系同源基因。在此，我们描述了家蚕核型多角体病毒（BmNPV）几丁质酶及其宿主直系同源基因BmChi-h的比较研究。我们构建了重组BmNPV，其中天然和修饰形式的BmChi-h在多角体蛋白启动子的驱动下表达，而真实的v-chiA被删除。蛋白质免疫印迹分析表明，BmCHI-h从病毒感染的BmN细胞中迅速分泌，而BmNPV CHIA定位于病毒感染的细胞内；这可能是因为BmNPV CHIA的C末端存在内质网滞留基序。酶学研究表明，BmNPV CHIA在碱性条件下能够保持更高的几丁质分解活性。对于感染了缺失v-chiA的BmNPV的家蚕幼虫，未观察到死亡幼虫的末期液化和杆状病毒编码的半胱氨酸蛋白酶的激活，并且引入BmChi-h并不能挽救这些缺陷。我们的研究结果表明，BmNPV chiA具有宿主直系同源基因所不具备的独特特征，这可能反映了杆状病毒几丁质酶的功能特化。
