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標題: A 型流行性感冒病毒非結構蛋白 NS1 與其宿 主因子 TAP 之作用區域分析
The study on interaction domain between nonstructural protein 1 (NS1) of influenza A virus and host factor TAP
作者: 蔡咏良
Tsai, Yung-Liang
關鍵字: 流感病毒;NS1;非結構蛋白;TAP
出版社: 生物化學研究所
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流行性感冒病毒曾在中世紀引起了大流行,而今每10~20 年都會有一波的流
感疫情,其病毒變異性及造成的影響都是很強烈的。A 型流行性感冒病毒擁有8
段單股的RNA 基因,其中第八個片段為nonstructural(NS)gene,可轉譯出兩
個非結構性蛋白,NS1A 與NS2A。病毒非結構蛋白NS1A 為約26KDa 大小的蛋
白,具有和RNA 及蛋白質結合的性質,能和許多宿主蛋白結合,例如RIG-1、
RHA、TAP、CPSF30 等等,為病毒對抗宿主免疫系統上的關鍵,與病毒的複製
和抑制宿主干擾素反應有關。本實驗中主要探討TAP、RHA 和CPSF 因子之間
對NS1A 的結合交互關係。NS1A 蛋白結合蛋白Tip-associated protein (TAP)
和mRNA 運輸出細胞核有關,NS1A 可能會藉由和其形成複合體,影響宿主蛋
白的核輸出,並將病毒的mRNA 輸出細胞核。將TAP 與CPSF 的NS1A 的結合
片段同時和NS1A 作用,發現NS1A 只能偵測到一種的結核蛋白訊號。另外,
RHA 蛋白同時為NS1A 及TAP 的結合因子,本實驗除了証明TAP 及RHA 不以
RNA 作為對NS1A 的中間結合因子,為直接性蛋白對蛋白結合關係,也將其與
TAP 同時對NS1A 進行競爭結合。實驗結果發現在增加RHA 後,TAP 對於NS1A
的結合能力有顯著的下降,兩者可能是對NS1A 相同區域具有交互作用。接著我
想找尋TAP 主要對NS1A 進行結合的最小區域,所以利用基因重組選殖TAP 的
突變刪除株,再對NS1A 進行GST pull-down,最終找尋到TAP61-104 區域為和
NS1A 結合的主要區域。以二級結構分析軟體預測,發現其多數為random coli
的部分,少數可能為extended strands 的結構。

Influenza A virus is a worldwide pathogen, which caused about 40 million people
die in 1918. The nonstructural protein 1 (NS1A), which was encoded by the eighth
RNA genome of Influenza A virus, plays an important role against host antiviral
response during virus infection. The NS1A has multifunction: i) its N-terminal
domain RNA-binding can bind to itself dsRNA to protect them from host immune
system detect. ii) Its C-terminal effector domain can bind to host factor to affect their
functions, such as RIG-1, PKR and CPSF30. We focus on several nuclear factors that
NS1A interact with, such as RHA, TAP, and CPSF30. RHA and TAP are involved in
mRNA export, whereas CPSF30 is required for 3' poly-A addition of mRHA. First,
we mixed the GST-fusion NS1A with 35S label RHA, TAP, CPSF30 to analyze their
interactions. Second, we performed NS1A competitive binding experiment between
CPSF30 and TAP as well as between TAP and RHA. Finally, we performed maping
analysis in order to identify the minimal domain of TAP for NS1A binding. Our
results indicated that NS1A can't interact with the two associated factor at the same
time. In addition, TAP amino acid residues 61-104 fragment is the minimal domain
required for NS1A association. According to the secondary structural prediction, the
fragment is composed of extended strands and random coli.
其他識別: U0005-1208200913344500
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