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標題: 竹嵌紋病毒三重疊基因區第二轉譯蛋白拓蹼結構及功能特性之分析
Topological and Functional Analyses of the Triple-Gene-Block Protein 2 of Bamboo Mosaic Virus
作者: 許琇婷
Hsu, Hsiu-Ting
關鍵字: Bamboo Mosaic Virus
出版社: 生物化學研究所
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摘要: 竹嵌紋病毒(Bamboo mosaic virus,簡稱BaMV)三重疊基因區(triple gene block)之三個轉譯蛋白與該病毒於細胞間的轉移有關,因此稱之為移動蛋白。根據胺基酸序列分析,第二轉譯蛋白(簡稱TGBp2)被推測為一穿膜蛋白。本研究利用差異性離心的方法,進行植物細胞成分之劃分,發現TGBp2蛋白主要存在於感染竹嵌紋病毒的白蔾及菸草葉片細胞之膜狀成分中。接著,利用化學萃取法及trypsin處理感病植物膜狀成分中的membrane vesicles,證實TGBp2的確為一穿膜蛋白。為了進一步瞭解TGBp2蛋白的topological及biochemical properties,本研究利用liposome體外重組技術,構築含有TGBp2之proteoliposome,並以化學萃取法和trypsin digestion的方式,處理此proteoliposome樣品,發現體外重組之proteoliposome與感病植物膜狀成分中的membrane vesicles上的TGBp2蛋白相似都具有一種主要的membrane topology。由於經由trypsin剪切所獲得之穩定TGBp2分解產物,分子質量和完整TGBp2蛋白差異不大,推測TGBp2蛋白之N-端及C-端domains可能均暴露在所製備之membrane vesicle的外表。進一步sequential maleimide modification實驗,證實proteoliposome上TGBp2蛋白之C-端的確暴露在proteoliposome的外表。此外,TGBp2蛋白也具有自我組裝形成multimer的能力。 Potexvirus屬的病毒RNA會與三重疊基因區第一轉譯蛋白(簡稱TGBp1)以及鞘蛋白(簡稱CP)形成蛋白核酸複合體。一般猜測TGBp2蛋白可能會藉由與TGBp1或CP蛋白間的交互作用或者是其與病毒RNA結合,來協助病毒核酸在細胞間之移動。目前,TGBp2蛋白能否和TGBp1或CP蛋白交互作用,並未獲得正面結果。不過,藉由fluorescence spectroscopy和UV-crosslinking實驗,發現Triton X-100 micelle中的TGBp2蛋白,具有與單股RNA非專一性結合能力。推測TGBp2蛋白至少可能藉由與病毒RNA之非專一性結合,來協助病毒在細胞間之移動。
The triple gene block protein 2 (TGBp2) of Bamboo mosaic virus (BaMV) has been proposed to be a transmembrane protein; however, its features remain unclear. Here, we used biochemical approaches to determine its topological and biochemical properties. Our data reveal that TGBp2 is mainly associated with the endoplasmic reticulum membrane. The resistance of TGBp2 in proteoliposomes, prepared from both the BaMV-infected tissues and in vitro reconstitution system, to both chemical extraction and trypsin digestion confirmed that it is indeed an integral membrane protein. On the basis of the minor change in the size of the major stable TGBp2-derived tryptic fragment from the monomeric TGBp2, as well as the sensitivity of the cysteine residues at the C-terminal tail of TGBp2 to maleimide modification, we suggest that TGBp2 adopts a topology with both its short N- and C-terminal tails exposed to the outer surface of the endoplasmic reticulum. Moreover, TGBp2 is able to self-assemble as revealed by the ability to detect multimeric TGBp2 in the presence of crosslinker or oxidation agent. TGBp2 is thought to assist the movement of the viral ribonucleoprotein (RNP) complex by protein-protein or protein-RNA interactions. Using tyrosine fluorescence spectroscopy and UV-crosslinking assays, the TGBp2 solubilized with Triton X-100 was found to interact with viral RNA in a non-specific manner. These results raise the possibility that TGBp2 facilitates the intracellular delivery of viral RNA through a non-specific protein-RNA interaction mechanism.
其他識別: U0005-1507200911184300
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