A型流行性感冒病毒PB2蛋白質與細胞蛋白質PIAS1 之間的交互作用

Abstract

流感病毒的 PA、PB1及PB2 蛋白質為構成其病毒特有的RNA-dependent RNA polymerase (RdRp)，其中PB2蛋白質具有結合到宿主pre-mRNA的帽蓋上，對於病毒複製或是RNA 轉錄都是不可或缺的。本實驗室在找尋可能與PB2 進行交互作用的細胞蛋白質時，利用酵母菌雙雜合系統(Yeast two-hybrid system)，找出細胞蛋白質PIAS1會與PB2進行交互作用。 我們利用GST pull-down assay和co-immunoprecipitation確認PB2會與PIAS1結合，而為了測試PIAS1對於流感病毒的生長所造成之影響，利用流感病毒螢光酶報導系統(Luciferase Reporter Assay)，發現在大量表現PIAS1時會抑制病毒聚合酶功能的效果；反之，利用PIAS1 shRNA降低細胞中PIAS1的量，則會提升病毒聚合酶功能。這個結果顯示，PIAS1會抑制A型流感病毒的轉錄與複製。 而由於PIAS1在細胞中為SUMO E3 Ligase，因此想知道是否PIAS1與PB2交互作用而進一步將PB2 SUMOylation。我們首先必須先了解PB2是否會SUMOylation的現象。而在293T細胞中，進行in vivo SUMOylation，發現A型流感病毒WSN/33的PB2蛋白質的確有SUMOylation的現象。而在我們初步的結果中，也猜測PIAS1在PB2的SUMOylation扮演著角色。 為了進一步了解PB2的SUMOylation對流感病毒的影響，我們利用電腦分析尋找PB2上可能會有SUMOylation的位點。其中在lysine339與718附近的氨基酸序列符合SUMO consensus sequence。因此利用site-directed mutagenesis建構PB2 K339R, K718R, K339,718R。利用in vivo SUMOylation測試這些mutants，發現仍然可以進行SUMO1 modification，但是在流感病毒螢光酶報導系統比較正常與突變的PB2，發現突變的PB2能提升病毒聚合酶功能。關於PB2的SUMO1 modification的影響還需進一步的實驗證明。

Influenza A virus uses an RNA-dependent RNA polymerase (RdRp) consisting of PA, PB1 and PB2 proteins to transcribe and replicate its RNA genome. PB2, which is a cap binding protein, plays an important role in influenza A viral transcription and replication. In the process of searching for cellular proteins that interact with PB2, our lab previous used yeast two-hybrid system to identify PB2-interacting cellular proteins and found that PIAS1 is one of the cellular proteins that can interact with PB2. In this study, we confirmed the interaction between PB2 and PIAS1 by GST pull-down assays and co-immunoprecipitation assays. We used minigenome luciferase reporter assay to test whether PIAS1 could affect influenza A viral transcription and replication. We found that when PIAS1 was overexpressed, it could suppress the activity of viral RdRp. On the other hand, when PIAS1 was knocked down by expressing pias1shRNA, the activity of viral RdRp was significantly increased. These data indicate that PIAS1 plays an inhibitory role in influenza A virus transcription and replication. PIAS1 is a SUMO E3 ligase. It is possible that through interacting with PB2, PIAS1 plays a role in PB2 sumoylation. To investigate this, we first tested whether PB2 could be sumoylated in the cells. By using in vivo sumoylation assay, we showed that PB2 was sumoylated in 293T cells. Our preliminary data also suggest that PIAS1 plays a role in PB2 sumoylation. To understand the effect of the sumoylation on PB2 function, we used computer analysis to identify potential sumoylation sites on PB2. The amino acid sequences surrounding K339 and K718 match the SUMO consensus sequence. We then constructed K339R, K718R, and K339,718R PB2 mutants by site-directed mutagenesis. We used in vivo sumoylation assay to test these mutants, and found that those mutants still could be sumoylated. But in minigenome luciferase reporter assay, our data indicated that all three PB2 mutants had significantly higher activity than wild-type PB2. To identify the effect of PB2 SUMOylation awaits further investigation.