細胞蛋白hnRNP M在A型流感病毒感染下所辦演的角色

The role of cellular protein hnRNP M in Influenza A virus infection

黃子軒(Zih-Hsuan Huang)

指導教授 ： 王萬波

國立臺灣大學/醫學院/微生物學研究所/碩士(2014年)

https://doi.org/10.6342/NTU.2014.10226

摘要

流感病毒的PA、PB1及PB2蛋白質構成了病毒特有的RNA-dependent RNA polymerase (RdRp)，參與病毒複製轉錄的過程，在病毒生活史扮演重要的角色。本實驗室為了更了解病毒複製轉錄的機制，挑選PA進行更進一步的研究。首先，以PA為餌，利用酵母菌雙雜合系統(yeast two-hybrid system)篩選Hela細胞的cDNA資料庫，找出了幾個可能與PA有交互作用的細胞蛋白質，並對其中的hnRNP M進行更深入探討。
在之前的研究中，我們利用帶有tag的PA與hnRNP M蛋白質進行免疫共沉澱法(Co-IP)以及GST pull-down assay，確認兩者之間具有交互作用；在RNase A的作用下，也證實兩者之間並非透過RNA而結合。接著我們想了解此交互作用對病毒或宿主有甚麼生理意義，使用了溶斑分析法((plaque assay))和螢光酶報導系統(Luciferase reporter assay)之後，發現hnRNP M具有抑制流感病毒複製的效果。
為了進一步了解hnRNP M抑制流感病毒複製的機制，我們純化了帶有tag的PA與hnRNP M蛋白質，利用in vitro的方式，發現hnRNP M可以影響PA內切酶的活性。此外，我們也發現到當細胞受到流感病毒感染下，細胞體內的hnRNP M會大量表現。我們也進一步的發現到hnRNP M可以被干擾素α、β誘導而產生。另一方面，我們也發現細胞樣本在受到病毒感染的情況下，會有干擾素α、β的產生。
以上結果顯示，hnRNP M會藉由與PA的交互作用影響PA內切酶的活性，負向調控病毒複製轉錄的功能，而hnRNP M也會受到第一型干擾素的誘導，因此我們推論hnRNP M可能是細胞中抵抗病毒的重要成員之一。

關鍵字

流行性感冒病毒 ； PA蛋白質 ； hnRNP M ； 流感病毒轉錄與複製

並列摘要

Influenza A virus contains an RNA-dependent RNA polymerase(RdRp) which consists of viral proteins PA, PB1 and PB2. It plays an important role in influenza viral transcription and replication. PA had been shown to be involved in vRNA synthesis and to activate viral mRNA synthesis by regulating the ‘cap-snatching’ process. In previous studies, our lab used yeast two-hybrid system to identify cellular factors that may interact with PA protein. We found that hnRNP M, an mRNA splicing factor, is one of the candidates of PA-interacting cellular proteins.
To further confirm the interaction between PA and hnRNP M, co-immunoprecipitation (Co-IP) and GST pull-down assays were performed. The results demonstrated that PA did specifically interact with hnRNP M. Moreover, we performed plaque assay and Luciferase reporter assay to investigate the influence of hnRNP M on influenza virus replication. Both data showed that hnRNP M reduced the efficiency of virus replication.
In order to figure out the mechanism of how hnRNP M reduced the efficiency of virus replication, we purified hnRNP M protein and PA protein. With in vitro assay, we found out that hnRNP M can reduce the endonuclease activity of PA. Furthermore, through western blot, we showed that the expression level of hnRNP M was induced by influenza virus infection. We also found out that hnRNP M could be induced by interferon α、β. And we also showed that interferon α、β was induced by influenza virus infection.
Together, these data suggest that hnRNP M may be one of the cellular anti-viral proteins that are induced upon influenza A virus infection.

並列關鍵字

Influenza virus ； PA ； hnRNP M ； influenza viral transcription and replication

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