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National Chung Hsing University Institutional Repository - NCHUIR > 生命科學院 > 生物化學研究所 > 依資料類型分類 > 碩博士論文 >  竹嵌紋病毒顆粒與含有三重疊基因區第三轉譯蛋白之病毒移動複合體之穩定結合

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/152317

標題: 竹嵌紋病毒顆粒與含有三重疊基因區第三轉譯蛋白之病毒移動複合體之穩定結合
The stable association of virion with the triple-gene-block protein 3-based movement complex of Bamboo mosaic virus
作者: 周遠霖
Chou, Yuan-Lin
Contributors: 張邦彥
Ban-Yang Chang
生物化學研究所
關鍵字: 竹嵌紋病毒;三重疊基因區;移動蛋白;免疫金標定
Bamboo mosaic virus;BaMV;TGB;IGL
日期: 2013
Issue Date: 2013-11-07 13:20:27 (UTC+8)
Publisher: 生物化學研究所
摘要: 竹嵌紋病毒(Bamboo mosaic virus, BaMV)三重疊基因區(triple gene block, TGB)所轉譯出的三個移動蛋白:TGBp1、TGBp2和TGBp3,參與病毒在宿主中的轉移。不過,它們協助病毒轉移的詳細機轉,卻仍待釐清。其中,TGBp2和TGBp3均屬源自內質網的膜蛋白。本研究除了希望瞭解TGBp3在膜上的拓樸特性外,亦希望藉由分析含有TGBp3的膜蛋白複合體,來闡釋TGBp3如何參與病毒在宿主中的移動。第一章TGBp3膜拓樸學的研究,我以能表現C端帶有HA tag之TGBp3的BaMV 病毒質體(pCB-P3HA)感染植物,並利用anti-HA抗體,偵測TGBp3在植物組織中之分佈。結果顯示,TGBp3主要存在富含內質網膜囊泡(ER-derived membrane vesicle)的P30樣品中。同時,我取在體外成功重組、含TGBp3的蛋白脂質體,以及由感病植物所製備、含有TGBp3的膜囊泡,以化學萃取、蛋白酶水解或化學修飾的方法進行分析,證實TGBp3是一C端暴露在膜外表的嵌入型膜蛋白。另外,若以交聯(crosslink)方式,處理含有TGBp3的膜囊泡,則可發現TGBp3以多聚體形式出現在膜上。在第二章論文研究中,我成功地自感病植物中萃取,並純化出含有TGBp3的膜蛋白大型複合體。經一系列的電泳分離和質譜鑑定,發現此複合體中含有TGBp1、TGBp2、TGBp3、外鞘蛋白、複製酶和病毒RNA等。將此含有TGBp3的病毒複合體先以分子篩管柱分離、再以免疫沈澱純化,並以穿透式電子顯微鏡觀察,發現此大型病毒複合體的核心架構,是由TGBp2、TGBp3和病毒顆粒(virion)所組成。進一步以免疫金標定分析純化的病毒顆粒,更證實上述觀點的正確性,亦確認TGBp1也可結合在此病毒複合體上。最後,雷射共軛焦螢光顯微鏡分析結果顯示,TGBp1需要TGBp2和TGBp3的協助,才能精準有效的座落在原生質絲(plasmodesmata, PD)中。綜合上述研究結果及參考文獻所記載的病毒移動模型,我提出了一個更為精細、且以TGBp2、TGBp3和病毒顆粒為核心架構的胞內病毒運輸模型。
Bamboo mosaic virus (BaMV) encodes three functionally coordinated movement proteins, TGBp1, TGBp2 and TGBp3 from triple gene block (TGB) of virus genome to facilitate its cell-to-cell movement in plant host. Among the three TGB proteins, TGBp2 and TGBp3 are known to associate with the endoplasmic reticulum (ER) membrane and the ER-derived vesicles. The ER- or vesicle-associated TGBp2 and TGBp3 presumably form a membrane complex to deliver the viruses. However, the identity of the “viral RNA cargo” and whether the cargo is able to associate with the TGBp2- and TGBp3-containing membrane complex during intracellular transport remain elusive. This study is aimed to elucidate the detail mechanism of virus movement by analyzing the topological properties of TGBp3 and dissecting the TGBp3-containing virus transport complex.
The first chapter of this dissertation is about the studies of the membrane topology of TGBp3. Taking advantage of an infectious plasmid clone of BaMV expressing the HA-tagged TGBp3 and an E. coli plasmid construct expressing His-tagged TGBp3, I have analyzed the membrane topology of TGBp3. The results of chemical extraction and partial proteolysis of TGBp3 in membrane vesicles prepared from both the BaMV-infected tissues and in vitro reconstituted proteoliposomes revealed that TGBp3 is an integral membrane protein with its C-terminal tail exposed on the outer surface of membrane vesicles. The exposure of TGBp3 C-terminal tail on the outer surface of membrane vesicles can be further confirmed by sequential maleimide modification of the conserved cysteine residues in the C-terminal tail of TGBp3. In addition, TGBp3 is able to self-interact as evidenced by its ability to form oligomers in the presence of chemical crosslinkers or oxidation agent.
In the second chapter, the Sarkosyl-extracted TGBp3-containing membrane protein complexes are isolated and analyzed by western blot, LC-MS/MS and RNA-dependent RNA polymerase assay. These results indicate that the TGBp3-containing membrane protein complexes may be composed of TGBp1, TGBp2, TGBp3, coat protein, replicase and viral RNA. The results of immunoprecipitation of the Sarkosy-extracted TGBp3-containing membrane protein complexes after a gel-filtration process revealed the existence of a stable TGBp2-TGBp3-virion complex in the extract, which can be substantiated by the results of transmission electron microscopy (TEM) of the purified virion sample. Moreover, I have clarified that TGBp3 plays a key role in virus cell-to-cell movement by enhancing the TGBp2- and TGBp3-dependent PD localization of TGBp1. Finally, a refined model based on our results and previously reported informations is proposed in the last paragraph of the 2nd chapter.
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