A型流行性感冒病毒PB2蛋白質與細胞蛋白質PIAS1之交互作用

Abstract

PB2是流行性感冒的病毒蛋白，其功能是參與cap snatching，與PB1、PA、NP共同構成RNA-dependent RNA polymerase (RdRp)，可進行virus transcription及replication。PB2蛋白在cap snatching的過程中具有結合到宿主pre-mRNA的帽蓋上的功能。 為了更深入研究流感病毒複製的機轉，及在流感病毒複製過程中可能參與的細胞內蛋白有哪些、它們參與或影響了流感病毒複製過程中的哪個部分，本實驗室利用酵母菌雙雜合系統(Yeast two-hybrid system)，分別找出了胞內會與各流感病毒蛋白有交互作用的蛋白質。 PIAS1是其中一種會和流感病毒蛋白PB2有交互作用的細胞蛋白質，它的功能是SUMO E3 ligase，可將其他蛋白質做SUMOylation的修飾。我們想藉由對PB2和PIAS1之間交互作用的探討和研究，希望能更深入了解流感病毒轉錄和複製的機制。 SUMO是small ubiquitin like modifier的簡稱，它是一種後轉譯修飾，會影響及調控許多細胞內的生理功能。 先前實驗室已利用酵母菌雙雜合系統(Yeast two-hybrid system)及GST pull down assay再次確認PB2和PIAS1之間的交互作用關係。我們再利用co-immunoprecipitation確認PB2和PIAS1在細胞內確實有交互作用。 而由於PIAS1在細胞中為SUMO E3 Ligase，因此我們想知道PIAS1與PB2的交互作用的意義是否包括PIAS1會將PB2 SUMOylation。因此首先我們想要知道PB2是否會被SUMOylation修飾。在先前的研究中已發現PB2在in vitro有被SUMOyation的情形。我們使用293T細胞進行in vivo SUMOylation，並使用immunoprecipitation實驗分析。發現A型流感病毒WSN/33的PB2蛋白質的確有in vivo SUMOylation的現象。 確定了PB2有被SUMOylation的情形後，接著我們想要尋找PB2的SUMOylation sites。先前實驗室利用電腦分析尋找PB2上可能會有SUMOylation的位點，發現在lysine339與718附近的氨基酸序列符合SUMO consensus sequence，因此實驗室先前已利用site-directed mutagenesis建構了PB2 K339R, K718R, K339,718R mutants。我們利用in vivo SUMOylation測試這些mutants，發現lysin 339有可能是PB2的SUMOylation sites 之一。 接著，我們想知道PIAS1是否會在PB2的SUMOylation扮演角色。我們改變293T細胞中的PIAS1 protein 表現量，並分析PB2的 SUMOylation情形。實驗結果發現WSN/33 PB2蛋白的SUMOylation的情形在knockdown PIAS1時會下降，overexpress PIAS1時上升。 因此我們懷疑PB2可能是PIAS1的SUMOylation target，是PIAS1將PB2做了SUMO這個修飾。 為了釐清PIAS1及SUMO對PB2的影響，我們利用免疫螢光染色觀察大量表現PIAS1時PB2的入核情形。實驗發現PB2在正常的情況下是會入核的，但在大量表現PIAS1的情況下，PB2入核的情形會受到影響。另外，PB2 K339R,K718R, K339,718R mutants的入核情形在正常情況下和wild type PB2是相似的，但在大量表現PIAS1的情況下，mutants的入核情形，相較於wild type PB2其受PIAS1大量表現的影響較小。 另外，為了更進一步測試PIAS1對流感病毒複製的影響，我們分別使用knockdown PIAS1及正常細胞感染流感病毒並測試其病毒蛋白表現量。實驗發現，若將PIAS1 knockdown，流感病毒蛋白的表現情形會上升。反之如果大量表現PIAS1，使用流感病毒溶斑分析，可發現流感病毒的複製在PIAS1大量表現的細胞，有明顯的減少情形。依這些實驗的結果推斷，PIAS1的存在，對流感病毒的複製是抑制的效果。 總結這些研究結果，顯示PIAS1可能藉由將PB2 SUMOylation的方式，影響PB2的入核等等，進而影響influenza virus的複製。

Influenza A viral PB2 protein, a cap binding protein which functions together with PB1, PA and NP in the process of cap snatching, composes the influenza A viral RNA-dependent RNA polymerase (RdRp). It plays the role of binding to the cap of pre-mRNA while undergoing cap snatching process. RdRp, which contains the PB2 protein, can transcribe and replicate RNA genome of Influenza A virus. Our previous research team has make study on influenza A virus and its life cycle, which it used yeast two-hybrid system to identify PB2-interacting cellular proteins, and found that PIAS1, a SUMO E3 ligase, is one of the cellular proteins that can interact with PB2. By investigating the interaction between viral PB2 and cellular protein PIAS1, We would like to further explore the mechanism of influenza A viral transcription and replication. As we understand the interaction betweem PB2 and PIAS1 in vitro and in vivo, and PIAS1 is a SUMO E3 ligase, it is possible that through interacting with PB2, PIAS1 plays a role in PB2 SUMOylation. To begin with, we would like to discuss whether PB2 could be SUMOylated in cells. By using in vivo SUMOylation assay and immunoprepicitation, we found that PB2 was SUMOylated in 293T cells. Later on, more detailed studies confirmed that K339 might be one of the SUMOylation sites of PB2. Also, we would like to understand if PIAS1 can influence the SUMO status of PB2. The data shows that expression level of cellular PIAS1 protein can influence the amounts of SUMOylated PB2. According to the above research data, we deduced that PB2 might be a substrate of PIAS1. Furthermore, to investigate the influence of PIAS1 to PB2, we observed the PB2 nuclear import under normal or experimental condition by immunofluorescence staining. The data showed that PB2 will be imported into the nucleus under normal condition but this situation will be affected when PIAS1 was overexpressed. Besides, PB2 K339R,K718R, K339,718R mutants will be imported into nucleus, similar to the wild type ones under normal condiction. However, in the condition of overexpressing PIAS1, the intense of affection of nuclear import was less obvious compared to the wild type ones. Finally, to understand the effects of PIAS1 to influenza A viral transcription and replication, we tested the viral protein expression of infected normal and PIAS1 knocked down cells. The result showed that knocking down of PIAS1 enhanced the expression level of viral proteins. On the other hand, , plaque assay data also showed that overexpression of PIAS1 reduced the replication of influenza A virus. Research data indicates that PIAS1 plays an inhibitory role in influenza A virus transcription and replication. In a nutshell, we assumed that PIAS1 may SUMOylated PB2, and SUMOylation of PB2 by PIAS1 may influence the nuclear import of PB2, thereby affects the transcription and replication of influenza A virus.