A型流行性感冒病毒PA蛋白質與細胞蛋白質hnRNP M之交互作用

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HNRPM進行更深入探討。 我們利用帶有tag的PA與HNRPM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，我們將PA及HNRPM分別分成多個片段，再以免疫共沉澱法找尋彼此間交互作用的區域。實驗結果顯示，PA與HNRPM皆是用本身的N端與對方進行交互作用。最後，我們利用免疫螢光分析的技術，在螢光顯微鏡下看到PA與HNRPM共同定位在細胞核中，再一次驗證兩者具有交互作用的可能性。 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FGFR2 minigene系統，將其送到293T細胞中，再利用semi-quantitative PCR (semi-qPCR)來觀察splicing的產物。結果發現PA的多寡並不會影響HNRPM調控alternative splicing的功能。而在能夠測試病毒聚合酶複製及轉錄功能的螢光酶報導系統實驗中，發現在細胞中過量表現HNRPM會有抑制病毒聚合酶功能的效果。以上結果顯示，PA與HNRPM的交互作用可能會引導HNRPM負向調控病毒複製轉錄的功能，其中的詳細機制需要透過更多的實驗才能證實。

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. 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 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. The interaction between PA and hnRNP M (both are RNA-binding proteins) was not mediated through RNA because RNase A treatment did not abolish their interaction. The immunofluorescence assay (IFA) also indicated that PA and hnRNP M co-localized in the nucleus, further confirming the interaction between these two proteins. Next, we used PA and hnRNP M deletion mutants to map the interaction regions on each protein. Our data indicated that both PA and hnRNP M use their N-terminal regions to interact with each other. Knowing the interaction between PA and hnRNP M, we then tested whether PA would affect hnRNP M’s alternative splicing activity. hnRNP M is known to promote alternative splicing of the FGFR2 minigene. By quantifying the splicing products of the FGFR2 minigene in the presence or absence of PA, we found that PA did not affect hnRNP M’s activity on alternative splicing. We then tested whether hnRNP M would affect influenza A viral replication. An influenza replication-reporter system was used to measure the transcription and replication activity of viral RdRp. It turned out that overexpression of hnRNP M reduced the activity of RdRp. Further investigation is required to clarify the role of hnRNP M in viral transcription and replication.