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Analyses of the RNA-Binding Properties of the Triple Gene Block Protein 1 and Coat Protein of Bamboo Mosaic Potexvirus
|摘要:||竹嵌紋病毒（bamboo mosaic potexvirus，簡稱 BaMV）的三重疊基因區（triple gene block ; TGB）所轉譯生成的三個蛋白，參與病毒在植物中的移動，故稱為移動蛋白。以往利用immunogold標示，配合電子顯微鏡的觀察，發現感病植物細胞中第一移動蛋白（TGBp1）常以聚結體的方式存在，而在其附近則有絲狀病毒顆粒的存在。另外，在TGBp1蛋白和病毒核酸結合的研究中發現，不論是由感染BaMV的植物細胞內純化或是由大腸桿菌大量表現純化所得之可溶性部分TGBp1蛋白，均只有在低離子強度下（50 mM NaCl）才明顯具有和病毒核酸較佳的結合能力，但事實上植物細胞中之離子強度卻高於此數值許多。最近一些研究報告顯示potexvirus之病毒核酸、三重疊基因區的第一個轉譯蛋白（TGBp1）及鞘蛋白（coat protein，CP）會形成 ribonucleoprotein 複合物，於植物細胞中進行移動。基於以上事實，推測在植物細胞生理環境下，可能有某些因子會協助TGBp1與病毒核酸結合，而病毒鞘蛋白或許就是其中之一因子。為證明鞘蛋白是否能協助TGBp1與病毒核酸結合，本實驗首先利用大腸桿菌T7大量表現系統，大量生產及純化TGBp1及鞘蛋白。此兩蛋白經變性和重新摺疊處理後，取可溶性部分蛋白和以[a-32P]CTP標定的病毒核酸，進行UV cross-linking，分析此兩蛋白與核酸的結合能力。結果顯示，不論在 pH8.0或7.4，單獨的TGBp1及鞘蛋白均可與病毒核酸結合。進一步的實驗發現，在適當的蛋白與病毒RNA莫耳比條件下，TGBp1蛋白與鞘蛋白皆具有協助對方增加與病毒核酸結合的能力。|
Bamboo mosaic potexvirus (BaMV), which primarily infects members of the Bambusoideae, has a single-stranded positive-sense RNA genome that composes five major open reading frames (ORFs). Proteins encoded by ORFs 2, 3 and 4 of BaMV share similar features with the products of the corresponding ‘triple-gene-block' (TGB) of other potexviruses. All of them are required for the cell-to-cell movement of viral RNA. With the aid of immunogold labeling and electron microscopy, it has been demonstrated that TGBp1 is associated with electron dense crystalline bodies (EDCBs) observed in cytoplasm, nuclei and nucleoli of infected leaf tissues, with coat protein (CP) and virus particle being detected in the neighbourhood of EDCBs. It has been also reported that the soluble form of TGBp1 possesses nonspecific RNA-binding activity at an ionic strength (50 mM NaCl) much lower than that expected for plant cells. Recently, it was reported that the viral RNA, TGBp1 and CP form ribonucleoprotein complex, which is able to move from cell to cell in the plant tissues. On the basis of the above-mentioned fact, it was assumed that there are certain factors, which are able to assist the binding of TGBp1 to viral RNA under physiological condition. And CP may be one of the factors. To prove this idea, the TGBP1 and CP proteins were first overproduced and purified. After denaturation and refolding, the soluble forms of these two proteins (TGBp1 and CP) were assayed for their RNA-binding activity through the use of a UV-cross linking technique. The present data revealed that TGBp1 or CP was able to bind RNA by itself at both pH8.0 and 7.4. Moreover, both TGBp1 and CP were able to enhance the RNA-binding activity of each other at certain molar ratios of protein to viral RNA.
|Appears in Collections:||生物化學研究所|
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