Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/24006
標題: A型流感病毒H1N1之NS1蛋白其二聚體化對其功能之影響
The effect of dimeric structure of NS1 of influenza A virus (H1N1) on its functions
作者: 鄭雅心
Cheng, Ya-Hsin
關鍵字: influenza A virus;A型流感病毒;NS1;dimeric structure;NS1;二聚體化
出版社: 生物化學研究所
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摘要: 
A型流行性感冒病毒之非結構蛋白 (nonstructural protein,NS1) 是一個多功能蛋白,同時也是病毒的毒性因子,然而其各種功能與毒性機轉間之關係仍不十分清楚。NS1可區分為兩個區域,分別為N端雙股核醣核酸結合區域 (double-stranded RNA binding domain,RBD) 及C端因子結合區域 (effector domain) ,NS1蛋白雙股核醣核酸結合區域與雙股核醣核酸結合可抑制RNaseL系統;此外,NS1蛋白還會與一些宿主細胞因子結合,認為能抵抗宿主的抗病毒反應。例如,NS1蛋白與CPSF30結合會影響pre-mRNA processing,因此抑制細胞mRNA 出核。由於NS1蛋白會形成二聚體結構,我們想探討二聚體與NS1的功能之相關性。因此本實驗室利用定點突變構築數種NS1突變株試著破壞NS1的二聚體以研究二聚體對其功能性之影響。首先把不同NS1質體DNA轉染入人類胚胎腎細胞進行 luciferase 分析,以分析突變後對干擾素分泌的影響。之後再利用GST pulldown與bacterial two hybrid方式雙重檢驗不同突變對NS1形成二聚體的影響。除了檢驗突變點對二聚體的影響,我們也利用 EMSA 去檢查突變點對NS1與雙股核醣核酸結合之能力。此外本實驗室亦利用GST pulldown檢驗NS1蛋白上不同突變點對於NS1與宿主因子 (僅針對CPSF30) 的結合是否有顯著的影響。從已有實驗結果來看,S103F/I106M/R35A/R46A突變株抑制干擾素的分泌強於S103F/I106M突變株,不僅如此,S103F/I106M/R35A/R46A亦喪失二聚體化能力以及和雙股核醣核酸結合之能力;而且對與CPSF30結合能力明顯大於其他突變株。此結果暗示了,當我們試著破壞 NS1 的二聚體結構,希望能降低對細胞的影響,有可能得到適得其反的結果。

The influenza A virus non-structural protein 1 (NS1) is a multifunctional protein and a virulence factor. It is considered to antagonize host innate immune responses. However, there is no clear correlation between the function of NS1 and its virulence. NS1 structure can be divided into two distinct functional domains. An N-terminal double-stranded RNA binding domain (RBD), which binds double-strand RNA and inhibit the activity of RNase L system and a C-terminal effector domain. NS1 has been shown to associate with several cellular proteins via the effector domain, including CPSF 30, a cellular factor required for 3´ end processing of cellular pre-mRNA, thereby inhibiting the production of all cellular mRNAs. As NS1 can dimerize and is coupled with dsRNA binding activity, we want to understand the correlation between its dimeric structure and functions by constructing several NS1 mutants disrupting the NS1 dimeric interface. We then examine the dimerization activity of NS1 mutants via GST pulldown assay and bacteria two hybrid assay, we use EMSA technique to analyze the dsRNA binding activity. Unexpectedly, we found the S103F/I106M/R35A/R46A mutant represses interferon-beta expression stronger than the S103F/I106M mutant, although the dimerization and dsRNA binding activity of S103F/I106M/R35A/R46A are impaired severely. Therefore, it should be cautious in the attempt of disrupting NS1 dimeric interface to decrease its virulence toward host cell.
URI: http://hdl.handle.net/11455/24006
其他識別: U0005-1208200915475600
Appears in Collections:生物化學研究所

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