探討與A型流行性感冒病毒PA蛋白質相互作用的細胞蛋白質hnRNP M

Abstract

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，找出八個可能與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。 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)確認兩者之間具有交互作用；經過RNase A的處理樣本之後，也證實兩者之間並非透過RNA而結合。最後，我們利用免疫螢光分析的技術，在在共軛焦顯微鏡(confocal microscop)下觀察到PA與hnRNP M同定位(co-localize)在細胞核中，再一次驗證兩者具有直接交互作用的可能性。 我們接著想了解此交互作用對病毒或宿主有甚麼生理意義。首先，使用溶斑分析法，發現hnRNP M具有抑制流感病毒複製的效果。而後以即時定量聚合酶鏈鎖反應與西方墨點法分析受病毒感染的細胞樣本，發現病毒感染能促使hnRNP M產生。而使用干擾素α處理細胞也能促使hnRNP M表現。另一方面，我們在受病毒感染的細胞樣本中發現干擾素β的產生。 以上結果顯示，PA與hnRNP M的交互作用可能會導致hnRNP M負向調控病毒複製功能，而這可能與細胞的免疫機制相關，但其中的詳細的活化路徑仍需要透過更多實驗來釐清。

Influenza A virus contains an RNA-dependent RNA polymerase (RdRp) which consists of viral proteins PA, PB1 and PB2. RdRp plays an important role in influenza A virus 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. To further investigate the role of PA protein in virus life cycle, 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 cellular proteins that interact with PA. To confirm the interaction between PA and hnRNP M, co-immunoprecipitation (co-IP) assay was performed. The results showed that PA did specifically interact with hnRNP M. The interaction between PA and hnRNP M (both are RNA-binding proteins) was not mediated by RNA because RNase A treatment did not abolish their interaction. The immunofluorescence assay (IFA) using confocal microscope also indicated that PA and hnRNP M co-localized in the nucleus, further confirming the interaction between these two proteins. Next, we performed plaque assay to investigate the influence of hnRNP M on influenza virus replication. The data showed that hnRNP M reduced the efficiency of virus replication. Through real-time RT-PCR and western blot, we showed that the expression level of hnRNP M was induced by influenza virus infection. We also found that hnRNP M could be induced by interferon α, but not by interferon β. Together, these data suggest that hnRNP M may be one of the cellular anti-viral proteins that are induced upon influenza A virus infection. Further investigation is required to clarify the anti-viral role of hnRNP M in influenza A virus infection.