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標題: 參與竹嵌紋病毒於植物細胞中移動 之宿主蛋白搜尋
The search for host protein factor(s) involved in the movement of Bamboo mosaic virus in plant cells
作者: 黃依婷
Yi-Ting Huang
關鍵字: 無;no
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竹嵌紋病毒(Bamboo mosaic virus, BaMV)利用其三重疊基因區(triple gene block,TGB)所轉譯出的三個移動蛋白,TGBp1、TGBp2 和 TGBp3,協助病毒在植物宿主中移動 本實驗室曾提出兩種移動蛋白協助病毒在宿主細胞內運輸的。模式。在第一種模式中,TGBp2、TGBp3 會和病毒顆粒結合,再招募 TGBp1,形成 TGBp1-TGBp2-TGBp3-virion 的移動複合體,沿著內質網絡向原生質絲(plasmodesmata, PD)移動。第二種模式則是 TGBp2 會誘導形成含有 TGBp2 及TGBp3 的內質網囊泡,並攜帶著病毒顆粒,沿著細胞骨架 actin 移動至 PD,在此移動過程中 TGBp1 會加入,參與協助病毒通過原生質絲。但是,除了上述病毒移動蛋白外 是否有植物宿主蛋白也存在此病毒移動複合體中 協助病毒移動,,,卻仍不清楚?本研究乃希望搜尋可能和病毒移動複合體結合,並協助病毒移動的宿主蛋白。由於病毒在細胞間轉移,必定要通過 PD,因此我首先嘗試從感病植物的 PD 中萃取病毒移動複合體,並利用 anti-HA 進行免疫沉澱,純化出含有TGBp3:HA 的可能病毒移動複合體。不過,存在 PD 內含有 TGBp3:HA 的病毒移動複合體含量過少,不利於我進行宿主蛋白的搜尋。因此,我將搜索範圍擴大到整個感病葉組織,亦即從全葉組織中萃取含有 TGBp3:HA 病毒移動複合體的樣品 並以 anti-HA 進行免疫沉澱 純化含有 TGBp3:HA 的病毒移動複合體可惜,,,。
銀染的結果顯示,此複合體中並沒有任何宿主蛋白的存在。為了突破搜尋不到宿主蛋白的困境,我改從感病葉純化病毒顆粒,希望能在純化的病毒顆粒中,找到可能協助病毒移動的宿主蛋白。LC-MS-MS 的分析結果顯示,病毒顆粒上存在著宿主的 RuBP carboxylase。進一步以免疫沉澱分析 RuBP carboxylase 與病毒移動複合體中的那一個元件具有交互作用,發現其與 TGBp1 與 CP 具有交互作用的能力。但是,這樣的交互作用對 BaMV 病毒在細胞中的移動是否具有重要性,則有待進一步的探討。

Bamboo mosaic virus (BaMV) encodes three functionally coordinated movement proteins, TGBp1, TGBp2 and TGBp3, from the triple gene block (TGB) of virus genome to facilitate its cell-to-cell movement in host plant. Our lab has proposed two modes to explain how movement proteins assistant in virus movement. In first mode,
TGBp2 and TGBp3 associate stably with virions, and recruit TGBp1 to form TGBp1-TGBp2-TGBp3-virion movement complex. This complex then moved alongside the ER network toward the plasmodesmata (PD). In second mode, the virion associates with the TGBp2 induced ER-derived TGBp2- and TGBp3-containing membrane vesicles. The virion-associated vesicles are then transported to the PD through actin filament. The TGBp1 would be recruited to the virion-associated vesicles during the process of trafficking. However, in addition to the movement protein, TGBp1,TGBp2 and TGBp3, whether host factors also are included in the virus movement complex to assist virus movement remains unknown.
This study is aimed to search for host factors that would associate with movement complex of virus and assist in virus movement. Because plant viruses need to pass through PD during intercellular movement, I first tried to extract movement complex of virus from the PD of infected plant tissues, and purify TGBp3:HA containing
movement complex by immunoprecipitation with anti-HA. However, the amount of TGBp3:HA containing movement complex in the PD was too low for me to looking for host factors. Thus, I expanded the search to entire leaf tissues, that is, I extracted virus movement complex from the entire leaf tissues of infected plants and purify
TGBp3:HA containing movement complex by immunoprecipitation with anti-HA.
Unfortunately, no host protein factor was observed after SDS-PAGE and silver staining. In order to break the plight for not finding host factors, I turned to purify
virions from infected leaves and hope to search for host factors that assist in virus movement. The results of LC-MS-MS showed that the virions contained host factor,
named RuBP carboxylase. I further utilized immunoprecipitation to analysis the interaction partner of RuBP carboxylase movement complex of virus. The results showed that TGBp1 and CP could interact with RuBP carboxylase. However, how are those interactions important to viral movement remain unknown.
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