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论文题目：水稻矮缩病毒外壳蛋白侵染昆虫宿主作用机制研究作者简介： 周锋，男， 1980年4月出生，2001年9月师从于北京大学李毅教授，于2007年1月获博士学位。中 文 摘 要大多数动、植物病毒需要通过昆虫宿主进行传播，对于有囊膜类动物病毒侵染昆虫宿主的研究了解的较多，但是对于植物病毒，尤其是没有囊膜类病毒如何进入昆虫宿主进而起始侵染几乎没有研究。水稻矮缩病毒（Rice Dwarf Virus, RDV）是跨越动、植物两界的双宿主病毒，可以在叶蝉和水稻体内复制。RDV病毒能够在叶蝉中无组织局限性地大量增殖但不致病，且能够借助虫卵传到后代；但它可以在水稻中引发水稻矮缩病且不能借助植物的种子进行传播。我们的研究主要集中在RDV外壳蛋白P2和P8在RDV侵染昆虫宿主过程中可能起的作用上。水稻矮缩病毒（RDV）必须通过其昆虫宿主叶蝉进行传播。明确病毒如何侵染昆虫、在昆虫宿主体内复制的过程，对于病毒防治具有非常重要的意义。早期研究发现P2蛋白对于RDV侵染昆虫宿主是必需的。本研究通过改进的载体系统将RDV各个结构蛋白在昆虫细胞表面表达并通过酸诱导处理模仿病毒进入细胞时在内含体的酸性环境进行，结果发现RDV基因组S2编码的蛋白P2具有膜融合蛋白功能，对P2蛋白的功能单位分析发现，该蛋白膜融合必须的功能单位如N端的融合肽，以及两个HR区，这些功能区对P2的膜融合功能是必须的。日本Omura教授在培养的感病叶蝉细胞系中证实上述结果。P8很早就被发现具有依赖于低pH的切割机制，在其N端和C端分别具有一个切割位点，天然病毒粒子中只存在C端的断裂，而大肠杆菌表达的蛋白两种切割同时存在。我们用MgCl2处理提纯的病毒粒子使病毒解壳继而获得纯化的P8蛋白。Western blotting结果表明N端和C端都发生了断裂，这说明P8在天然病毒粒子中不发生N端的断裂是由于病毒粒子的结构特点引起的。氨基酸序列分析表明P8的C端断裂产物P8N的C末端存在两性螺旋结构域。在细胞膜上表达这个结构域会破坏细胞膜的稳定性，导致细胞膜通透性增强，胞质大量流失；当把纯化的P8和P8N蛋白与培养的昆虫细胞共同孵育时，只有P8N能够进入细胞。这些结果说明P8在其C端的断裂有助于其改变细胞膜通透性，帮助病毒粒子进入宿主细胞。采用酵母双杂交技术，以P8蛋白为诱饵，从水稻的cDNA文库中筛选得到了与P8相互作用的宿主蛋白乙醇酸氧化酶（Glycolate oxidase, GOX）。免疫共沉淀实验证明P8与水稻和昆虫的GOX都有相互作用；对表达P8和GOX的昆虫细胞进行免疫荧光结果也表明，P8在昆虫细胞中的定位受到GOX的影响而向过氧化物酶体聚集。我们推测这种相互作用可能也影响了RDV在昆虫宿主细胞内的定位。综上所述，该结果对阐明RDV以及同属病毒进入和侵染宿主机制具有重要的推动作用，并提供了一个研究模型。这也是关于植物病毒蛋白介导宿主细胞膜融合的首次报道。关键词： 水稻矮缩病毒，外壳蛋白，膜融合，膜通透性，乙醇酸氧化酶，七肽重复序列Studies on the function of Rice Dwarf Virus outer-capsid proteins in virus entry into the vector insects cells Zhou Feng ABSTRACTInsect transmission is an essential process of infection for numerous plant and animal viruses. Howaninsect-transmissibleplantvirus enters an insect cell to initiate the infection cycle is poorly understood, especially for nonenveloped plant and animal viruses. Rice dwarf virus (RDV), the causal agent of rice dwarf disease, is a member of the genus Phytoreovirus in the family Reoviridae. It is not transmissible mechanically but is transmitted to rice exclusively by the leafhopper Nephotettix cincticeps and some other leafhopper species. RDV proliferates within the leafhopper vectors. An unusual biological characteristic of this virus is its ability to multiply in both plants and certain invertebrates. Investigations of the molecular features of the virus entry into the host cells are of great interest from a biological perspective. In the present study, we examined in detail the function of the P2 and P8 protein during the infection of cells of the insect vector.The capsid protein P2 of Rice dwarf virus (RDV), which is nonenveloped, is necessary for insect transmission. Here we present evidence that P2 shares structural features with membrane-fusogenic proteins encoded by enveloped animal viruses. When RDV P2 was ectopically expressed and displayed on the surface of insect Sf9 cells, it induced membrane fusion characterized by syncytium formation at low pH. Mutational analyses identified the N-terminal and a heptad repeat (HR1) as being critical for the membrane fusion-inducing activity. These results are corroborated with results from RDV-infected cells of the insect vector leafhopper. We propose that the RDV P2-induced membrane fusion plays a critical role in viral entry into insect cells. Previous studies showed that P8 auto-cleavages at the residues of Asp362/Pro363 in the C-terminus and the Asp56/Pro57 at the N-terminus are both low-pH dependent processes. Sequence analysis revealed that P8N, product of the C-terminus auto-cleavage, possesses characteristic structural features (including two amino-terminal amphipathic helices) compatible with virus penetration activity. When incubated with Sf9 cells, purified P8N can be internalized.This study suggests that P8 have a role in the membrane destabilization required for virion access to the cytoplasm.Using Yeast Two Hybrid system, we find that RDV P8 has interaction with Glycolate Oxidase of Rice. The interaction was confirmed by co-immunoprecipitation assays. We also examined the subcellular localization of P8 and GOX in Sf9 cells by confocal immunofluorescence microscopy. P8 was observed to be colocalized with GOX in the peroxisome of insect cells.These results support the hypothesis that, the P2 protein mediates the initial virion attachment to the host cell, as well as the auto cleavage products of P8 acts as a membrane permeabilization protein that mediates release of viral particles from endosomal compartments into the cytoplasm. After the enrty process, RDV virion localizes to the peroxisome because of the interaction of P8 and GOX, and initializes the replication This is the first report of a plant viral protein that can induce membrane fusion, which has broad significance i