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  • 學位論文

鑑定並分析與A型流行性感冒病毒之PA蛋白作用的細胞因子

Identification and characterization of cellular factors interacting with influenza A virus PA protein

指導教授 : 王萬波

摘要


流行性感冒病毒屬於正黏液科(Orthomyxoviridae)的RNA病毒,大小約為80~120nm,具有八段負股的RNA,分別是-PB2、PB1、PA、HA、NP、NA、M、NS,其中PB2、PB1、PA、NP則是參與流感病毒複製的主要蛋白質。而流感病毒不同於其他RNA病毒的地方,在於流感病毒感染宿主細胞後會進入細胞核中進行複製,而一般RNA病毒則是在細胞質中複製。 本文的研究著重於在流行性感冒病毒在細胞內的複製機轉,以及宿主細胞因子對流感病毒的複製是否會造成影響。因為已經有許多研究指出,宿主細胞蛋白質會跟病毒蛋白質結合並影響病毒的複製過程。由於PA的功能目前尚未被完整定義,為了找出可以跟PA有交互作用的細胞蛋白質,我們初步的實驗是利用酵母菌雙雜交的方式,發現參與在流感病毒複製的PA subunit會跟HAX1、HNRPM、hCLE、SIVA、FTH1、PRDX1、MMAB等七個基因所encode的蛋白質進行交互作用。為了觀察HAX1與HNRPM是否真正參與在病毒的複製過程中,我們利用lenitivirus包裹shRNA 感染目標細胞,藉此抑制細胞中HAX1或HNRPM基因mRNA的表現。在HAX1或HNRPM基因的表現即使被抑制卻不影響細胞生長的前提下,接著以流感病毒WSN33病毒株感染基因表現被抑制的細胞株,觀察是否會影響A型流感病毒在細胞內的複製能力。 在H1299_shHAX1細胞中,可以發現HAX1基因表現被抑制之後,流感病毒vRNA的複製有稍微受到影響,但不影響病毒mRNA的合成。而在NPCTW04_HNRPM細胞中,則發現抑制HNRPM之後,病毒的vRNA與mRNA合成都降低,表示病毒的複製及轉錄能力均受到影響。因此推測,這兩個基因有可能藉著和PA之間的交互作用,來影響病毒的複製過程。但PA在人類細胞中是否真的會與HAX1或HNRPM結合,以及抑制的機轉為何都還需要再研究。

並列摘要


Influenza virus is a member of Orthomyxoviridae RNA virus which size is about 80~120nm. Influenza A virus encode eight negative-strand RNA segment which are PB2, PB1, PA, HA, NP, NA, M, NS. Different to other RNA virus, influenza A virus replicates in the nucleus. The four influenza A viral proteins, PB1, PB2, PA, and NP, comprise viral RNA replication complex, and are absolutely required for viral RNA replication and transcription. Among them, the function of PA in influenza A viral RNA replication is least known. Several cellular proteins have been shown to interact with the subunits of influenza A RNA polymerase complex. The interaction between some cellular factors and viral replication complex has been shown to be essential for viral replication and transcription. In this study, we aimed to identify cellular proteins that can interact with the PA protein of influenza A virus by using yeast two-hybrid screening. We found that seven cellular proteins, including HAX1, HNRPM, SIVA, hCLE, FTH1, PRDX1, MMAB, might interact with PA protein. Among them, hCLE was reported previously to interact with PA. To study whether PA-interacting proteins, HAX1 and HNRPM, were involved in influenza A viral replication and transcription, we knocked down HAX1 or HNRPM expression in the cells by lentivirus expressing HAX1 or HNRPM siRNA. We found that knockdown of HAX1 and HNRPM did not affect cell growth. Influenza A vRNA, but not viral mRNA, synthesis was reduced in HAX1-knockdown cells, suggesting HAX1 is involved in influenza A viral RNA synthesis. As regard to HNRPM, we found that both vRNA and viral mRNA synthesis is reduced in HNRPM-knockdown cells, suggesting that HNRPM is involved in influenza A viral replication and transcription. How HAX1 and HNRPM affect influenza A viral replication and transcription and whether PA indeed interacts with HAX1 and HNRPM in the cells remains to investigated. The elucidation of these issues may shed light on the mechanism of influenza A replication.

並列關鍵字

influenza RNA virus PA cellular factor RNA-dependent RNA polymerase HAX1 HNRPM

參考文獻


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