探討A型流行性感冒病毒蛋白質PA與細胞蛋白質Siva-1之交互作用

Interaction between Influenza A virus PA protein and cellular protein Siva-1

蔡紹宇(Shao-Yu Tsai)

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(2010年)

https://doi.org/10.6342/NTU.2010.02782

摘要

流感病毒具有一組特別的RNA-dependent RNA polymerase(RdRp)，其構成為病毒蛋白質PA, PB1 及PB2；其中PA已知參與了病毒vRNA的合成以及mRNA的轉錄，而為了更加清楚PA在病毒的生活史中所扮演的角色，本實驗室以全長的PA做為餌，利用酵母菌雙雜合系統(yeast two-hybrid system)篩選Hela細胞的cDNA資料庫，找出可能會與PA有交互作用的細胞蛋白質。經過篩選後得到八個候選基因，其中挑出了Siva-1進一步確認其與PA的交互作用與機制。
首先將全長的PA與Siva-1再次以酵母菌雙雜和實驗確認其交互作用。之後利用帶有tag的PA與Siva-1蛋白質進行共同免疫沉澱法(co-IP)以及GST pull-down，確認了兩者在in vivo及in vitro的情況下的確會結合。為了更進一步確認兩者結合的部位，將Siva-1分成N端、中間段以及C端後，發現只有中間含有death domain homology region(DDHR)的部分(a.a 48-124)才與PA有較強的結合能力。
接著為了瞭解此交互作用對病毒或宿主有何生理意義，以流感病毒感染Siva-1
-knockdown的細胞株後，發現產生的病毒比對照組多了約兩倍。但是在能夠測試病毒基因複製與轉錄的螢光酶報導系統的實驗中，發現Siva-1-knockdown的細胞株與對照組間並無差異。以上結果顯示Siva-1可能會負向的調控病毒的複製，但其機制並非透過影響病毒基因的轉錄或複製。
最後，報導指出Siva-1為一種促細胞凋亡蛋白質，我們以流式細胞儀分析後證實Siva-1的確可以引發細胞凋亡，並且將PA與Siva-1共同表現時，可發現凋亡的細胞數目會降低。由於病毒常會藉由調控細胞的凋亡機制以增進本身的複製，而我們的實驗結果即指出A型流感病毒可能會利用PA與Siva-1間的交互作用阻止Siva-1進行細胞凋亡以促進病毒複製，但其詳細機制則需要更多實驗才能證實。

關鍵字

A型流感病毒 ； 細胞凋亡

並列摘要

Influenza A virus contains an RNA-dependent RNA polymerase that composed of viral proteins PA, PB1 and PB2. Among them, PA has been shown to play important roles in vRNA synthesis and to activate viral mRNA synthesis by promoting PB1’s endonuclease activity. To further investigate the role of PA in virus life cycle, our lab set to identify cellular factors that may interact with PA by using yeast two-hybrid system. We found that Siva-1, a protein that has an apoptotic activity, is one of the potential PA-interacting cellular proteins.
To further confirm the interaction between PA and Siva-1, full-length Siva-1 was fused with Gal4 activation domain to generate pGAD-GH -Siva-1. This plasmid was then co-transformed with pGBDU-C1-PA into yeast to verify the interaction between PA and Siva-1 in a two-hybrid assay. We found that PA indeed can interact with Siva-1. We also demonstrated that PA can interact with Siva-1 in vivo and in vitro by co-immunoprecipitation and GST pull-down assays and that PA may interact with the death domain homology region (DDHR) of Siva-1.
To study the role of Siva-1 in influenza A virus replication, influenza A virus was used to infect Siva-1-knockdown NPC-TW04 and H1299 cells. By using plaque assays, we found that the production of influenza A virus was significantly increased in the Siva-1 knockdown cells, suggesting that Siva-1 plays an inhibitory role in influenza A virus multiplication. To further study whether Siva-1 could affect the replication of influenza A viral genome, a replication-reporter system that can be used to measure the replication of influenza A viral genome was used. We found that the replication rate of influenza viral genome is similar in Siva-1 knockdown cells and control cells, indicating that Siva-1 has no effect on the replication of influenza viral genome. Taken together, these data suggest that Siva-1 may affect the multiplication of influenza A virus at stages other than the replication of viral genome.
Siva-1 has been reported to have an apoptotic activity. To study the functional outcome of PA-Siva-1 interaction, we tested whether PA could affect the apoptotic activity of Siva-1. We found that Siva-1, as reported, could induce apoptosis and that PA could more or less block apoptosis induced by Siva-1. Viruses are well known to encode anti-apoptotic proteins to facilitate their own multiplication. Whether the interaction between PA and Siva-1 could prevent apoptosis and facilitate virus multiplication during influenza A virus infection remains to be determined. Our data, though not prove, point to this possibility.

並列關鍵字

Influenza A virus ； PA ； Siva-1 ； apoptosis

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