Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36078
標題: 探討竹嵌紋病毒核酸複製&;#37238;之類解旋酵素活性區與外鞘蛋白質交互作用之生物意義
Investigating the Biological Significance of the Interaction between the Helicase-like Domain of Replicase and the Coat Protein of Bamboo Mosaic Virus
作者: 顏玉婷
Yen, Yu-Ting
關鍵字: Bamboo Mosaic Virus;竹嵌紋病毒;Helicase-like Domain;Coat Protein;類解旋酵素活性區;外鞘蛋白質
出版社: 生物科技學研究所
摘要: 
竹嵌紋病毒 (Bamboo mosaic virus, BaMV) 為單股正意核醣核酸病毒,其基因體包含五個轉譯架構區(open reading frames, ORFs)。ORF1 可轉譯出大小約 155 kDa 的複製酵素,從N端到C端分別具有戴帽酵素活性區、類解旋酵素活性區及核醣核酸聚合酵素活性區。ORF5 由約 1 kb 的次基因體所轉譯,產物為病毒的外鞘蛋白質,參與了病毒的組裝且與病毒在植物體內的移動有關。先前研究利用酵母菌雙雜交篩選系統,發現類解旋酵素活性區與外鞘蛋白質有交互作用的現象。利用易錯聚合鏈反應的方法,對外鞘蛋白質的核&;#33527;酸序列進行隨機突變,再藉由細菌雙雜交系統分析,找到會影響與類解旋酵素之間的交互作用突變的氨基酸殘基 I130M/D170N/A209G/N218K 以及 N210S。其中單一突變胺基酸N210S便能影響外鞘蛋白質與類解旋酵素之間的交互作用。因此本實驗將繼續探討外鞘蛋白質與類解旋酵素交互作用的生理意義。首先,將 A209G 與 N210S 共同以及分別構築於竹嵌紋病毒感染載體 (pCBG) 之外鞘蛋白質片段上,接種於菸草及白藜。發現 A209G、N210S以及 A209G/N210S 等突變皆可抑制病毒在菸草中的系統性移動。在接種白藜的實驗中,N210S的突變亦減少了病毒在細胞與細胞之間的移動,而 A209G和 A209G/N210S 的突變則完全破壞病毒在細胞與細胞間的移動。藉由菸草原生質體轉染試驗顯示,外鞘蛋白質的累積量並不會因突變而有所減少。進一步抽取總核醣核酸,進行北方墨點法分析,發現病毒基因體的累積量亦不受影響。在穿透式電子顯微鏡的分析中,於感染的原生質體中可發現突變的病毒顆粒的長度較野生型的病毒顆粒長,顯示外鞘蛋白質突變的病毒株仍然可以形成病毒顆粒,推估突變的外鞘蛋白質與核醣核酸的結合並不受到影響。

Bamboo mosaic virus (BaMV) has a single-strand RNA genome consisting of five open reading frames (ORFs). ORF1 encodes a 155-kDa replicase containing a capping enzyme domain, a helicase-like domain (HLD), and a RNA-dependent RNA polymerase domain, while ORF5 encodes a 25-kDa viral coat protein (CP) which plays roles in encapsidation as well as the viral movement. By yeast-two hybrid screening, we discovered that HLD can bind CP. Previous study showed that two CP mutants, I130M/D170N/A209G/N218K and N210S, had weaker interaction with HLD. In this study, the biological meaning of the interaction between HLD and CP of BaMV has been investigated. First, we introduced A209G, N210S and A209G/N210S mutations on the ORF5 of the BaMV infectious clone (pCBG), and inoculated the engineered clones onto the leaves of Nicotiana benthamiana and Chenopodium quinoa. We found that A209G, N210S and A209G/N210S mutations abolished the viral systemic movement in N. benthamiana. It also suggested that viral cell-to-cell movement was restricted by N210S and abolished by A209G and A209G/N210S in C. quinoa. These mutations had no influence on accumulation of CP and genomic RNA inside protoplasts. In TEM analysis, the length of the mutant viral particles were longer than that of the wild type in inoculated protoplasts suggesting that the mutant CPs still be able to bind the viral RNA for virion formation.
URI: http://hdl.handle.net/11455/36078
Appears in Collections:生物科技學研究所

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