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論文題目：水稻矮縮病毒外殼蛋白侵染昆蟲宿主作用機(jī)制研究作者簡介： 周鋒，男， 1980年4月出生，2001年9月師從于北京大學(xué)李毅教授，于2007年1月獲博士學(xué)位。中 文 摘 要大多數(shù)動(dòng)、植物病毒需要通過昆蟲宿主進(jìn)行傳播，對(duì)于有囊膜類動(dòng)物病毒侵染昆蟲宿主的研究了解的較多，但是對(duì)于植物病毒，尤其是沒有囊膜類病毒如何進(jìn)入昆蟲宿主進(jìn)而起始侵染幾乎沒有研究。水稻矮縮病毒（Rice Dwarf Virus, RDV）是跨越動(dòng)、植物兩界的雙宿主病毒，可以在葉蟬和水稻體內(nèi)復(fù)制。RDV病毒能夠在葉蟬中無組織局限性地大量增殖但不致病，且能夠借助蟲卵傳到后代；但它可以在水稻中引發(fā)水稻矮縮病且不能借助植物的種子進(jìn)行傳播。我們的研究主要集中在RDV外殼蛋白P2和P8在RDV侵染昆蟲宿主過程中可能起的作用上。水稻矮縮病毒（RDV）必須通過其昆蟲宿主葉蟬進(jìn)行傳播。明確病毒如何侵染昆蟲、在昆蟲宿主體內(nèi)復(fù)制的過程，對(duì)于病毒防治具有非常重要的意義。早期研究發(fā)現(xiàn)P2蛋白對(duì)于RDV侵染昆蟲宿主是必需的。本研究通過改進(jìn)的載體系統(tǒng)將RDV各個(gè)結(jié)構(gòu)蛋白在昆蟲細(xì)胞表面表達(dá)并通過酸誘導(dǎo)處理模仿病毒進(jìn)入細(xì)胞時(shí)在內(nèi)含體的酸性環(huán)境進(jìn)行，結(jié)果發(fā)現(xiàn)RDV基因組S2編碼的蛋白P2具有膜融合蛋白功能，對(duì)P2蛋白的功能單位分析發(fā)現(xiàn)，該蛋白膜融合必須的功能單位如N端的融合肽，以及兩個(gè)HR區(qū)，這些功能區(qū)對(duì)P2的膜融合功能是必須的。日本Omura教授在培養(yǎng)的感病葉蟬細(xì)胞系中證實(shí)上述結(jié)果。P8很早就被發(fā)現(xiàn)具有依賴于低pH的切割機(jī)制，在其N端和C端分別具有一個(gè)切割位點(diǎn)，天然病毒粒子中只存在C端的斷裂，而大腸桿菌表達(dá)的蛋白兩種切割同時(shí)存在。我們用MgCl2處理提純的病毒粒子使病毒解殼繼而獲得純化的P8蛋白。Western blotting結(jié)果表明N端和C端都發(fā)生了斷裂，這說明P8在天然病毒粒子中不發(fā)生N端的斷裂是由于病毒粒子的結(jié)構(gòu)特點(diǎn)引起的。氨基酸序列分析表明P8的C端斷裂產(chǎn)物P8N的C末端存在兩性螺旋結(jié)構(gòu)域。在細(xì)胞膜上表達(dá)這個(gè)結(jié)構(gòu)域會(huì)破壞細(xì)胞膜的穩(wěn)定性，導(dǎo)致細(xì)胞膜通透性增強(qiáng)，胞質(zhì)大量流失；當(dāng)把純化的P8和P8N蛋白與培養(yǎng)的昆蟲細(xì)胞共同孵育時(shí)，只有P8N能夠進(jìn)入細(xì)胞。這些結(jié)果說明P8在其C端的斷裂有助于其改變細(xì)胞膜通透性，幫助病毒粒子進(jìn)入宿主細(xì)胞。采用酵母雙雜交技術(shù)，以P8蛋白為誘餌，從水稻的cDNA文庫中篩選得到了與P8相互作用的宿主蛋白乙醇酸氧化酶（Glycolate oxidase, GOX）。免疫共沉淀實(shí)驗(yàn)證明P8與水稻和昆蟲的GOX都有相互作用；對(duì)表達(dá)P8和GOX的昆蟲細(xì)胞進(jìn)行免疫熒光結(jié)果也表明，P8在昆蟲細(xì)胞中的定位受到GOX的影響而向過氧化物酶體聚集。我們推測這種相互作用可能也影響了RDV在昆蟲宿主細(xì)胞內(nèi)的定位。綜上所述，該結(jié)果對(duì)闡明RDV以及同屬病毒進(jìn)入和侵染宿主機(jī)制具有重要的推動(dòng)作用，并提供了一個(gè)研究模型。這也是關(guān)于植物病毒蛋白介導(dǎo)宿主細(xì)胞膜融合的首次報(bào)道。關(guān)鍵詞： 水稻矮縮病毒，外殼蛋白，膜融合，膜通透性，乙醇酸氧化酶，七肽重復(fù)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