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標題: 竹嵌紋病毒三重疊基因區第一及第二轉譯蛋白功能特性之研究
The functional properties of the triple gene block protein 1 and 2 of bamboo mosaic potexvirus.
作者: 許琇婷
Hsu, Hsiu-Ting
關鍵字: bamboo mosaic potexvirus
出版社: 生物化學研究所
摘要: 竹嵌紋病毒(bamboo mosaic virus,簡稱BaMV)三重疊基因區(triple gene block)之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。其中第一轉譯蛋白(簡稱TGBp1)於感染竹嵌紋病毒之植物葉細胞中會形成不溶性的結晶性蛋白聚結體,而第二轉譯蛋白(簡稱TGBp2),則推測為一膜蛋白。本研究旨在探討此TGBp1結晶性蛋白聚結體如何發揮其功能,以及其功能發揮是否和TGBp2有關。 先前的研究結果顯示,感病植物TGBp1結晶性蛋白聚結體具有NTP-binding及NTPase兩種活性,但卻缺乏RNA-binding的能力,不過TGBp1結晶性蛋白聚結體經變性及重新折疊後所得到的可溶性TGBp1蛋白,則同時具有上述三種活性。因此推測在感病植物細胞中,TGBp1結晶性蛋白聚結體在某種狀態下應該會釋放出同時具有上述三種功能之TGBp1蛋白,藉此協助病毒在細胞間轉移。本研究結果顯示,病毒RNA和鞘蛋白之存在,有助於TGBp1結晶性蛋白聚結體釋放出具有NTP-binding和NTPase活性的TGBp1蛋白。由於病毒RNA的存在會影響此釋放出的TGBp1蛋白和NTP-binding的能力,因此我認為此釋放出的TGBp1蛋白也具有和RNA結合的能力。 另外,本研究利用差異性離心進行植物細胞成分之劃分,證實TGBp2蛋白主要存在於感染竹嵌紋病毒的白蔾葉片細胞之膜狀成分中。進一步以化學方法處理膜狀成分,則證實TGBp2為一嵌入型膜蛋白。將感病植物葉片細胞之膜狀成分樣品以Ficoll gradient分離,結果發現鞘蛋白、TGBp1及TGBp2蛋白與Phospholipid在ficoll gradient中同分布,這種現象說明鞘蛋白、TGBp1蛋白可能會以蛋白-核酸複合體(ribonucleoprotein complex)的形式,與存在於膜上的TGBp2蛋白有相互作用。另外,利用liposome之體外重組技術,我也構築了含有TGBp2之proteoliposome,可以進一步進行TGBp2與TGBp1相互作用關係的研究。
The TGBp1 and TGBp2 proteins of bamboo mosaic potexvirus are encoded by the first and second overlapping genes of the triple-gene- -block, of which the products are thought to play roles in virus movement between cells. The TGBp1 was found to form inclusion bodies associated with virus particles in the BaMV-infected tissues. The TGBp2 was suggested to be a membrane protein. The TGBp1 inclusion from the BaMV-infected tissues possessed the NTP-binding and NTPase activities, but was unable to bind RNA. However, the soluble TGBp1 protein, which was obtained from denaturation and refolding of both the E. coli overexpressed and endogenous TGBp1 inclusions, possessed the above three activities. We proposed that the endogenous TGBp1 inclusion is an active source of the soluble TGBp1 protein that is able to release functional TGBp1. In this experiment, we found that the dissociation of functional TGBp1 from the inclusions is enhanced by the presence of viral RNA and capsid protein. The TGBp1 protein, which was released from the inclusions, possessed the NTP-binding, NTPase, as well as RNA-binding activities. The TGBp2 protein has been able to be detected reproducibly mainly in the membrane and cell wall fractions of the BaMV-infected Chenopodium quinoa leaves. The timing of expression of TGBp2 was approximately parallel with those found for TGBp1 and capsid proteins. Since TGBp2 remained associated with membrane even after extraction with a solution containing 1 M Na2CO3, 4 M urea, or 1 M KCl, it was believed that TGBp2 is an integral membrane protein. After ficoll gradient centrifugation of the membrane fraction of the BaMV-infected tissues, TGBp1, TGBp2, capsid protein, and phosholipid were sedimented at about same positions of the gradient. In vitro TGBp2 refolding and TGBp2-containing proteoliposome reconstitution have been performed successfully. The proteoliposome thus obtained can be of use in our future biochemical study of TGBp2 as well as the mutual interaction among the three movement proteins of bamboo mosaic potexvirus.
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