鑑定並分析與A形流行性感冒病毒之PB2蛋白作用的細胞因子

Abstract

Influenza A virus的RNA聚合脢，是一個由三個蛋白質所組成的聚合體，分別是PB1,PB2以及PA。我們在這裡試著找出會和PB2作用的細胞內因子，並且研究它們互相做用的生理意義為何。 將病毒的PB2蛋白之C端蛋白質序列305-759作餌，利用酵母菌雙雜合系統 (Yeast two hybrid)，去篩選HeLa細胞的cDNA library，找出可能會和PB2作用的細胞內因子。篩選後得到8個基因，我們選擇其中幾個有興趣的候選人，往下做更深入的研究，其中包括HAX-1。 利用可表現帶有Flag的PB2蛋白質體，及融合其他tag的候選基因，共同轉染入細胞株293 FT大量表現，利用抗FLAG及其他的抗體，來進行免疫沉澱、GST pull-down，來更加的確認找到的細胞因子與PB2的作用是否真實。並且初步找出PB2與HAX-1結合的片段主要在中間第271-522的胺基酸序列，而C端的第636-759胺基酸序列有較弱的結合。在目前的結果中，初步證明細胞因子HAX-1會與PB2結合。 再往下做更近一步的分析，將細胞內的HAX-1利用RNAi knockdown後，病毒的複製有上升的情形，代表HAX-1可能會抑制病毒的生長。而在類病毒基因體的冷光報導蛋白系統中，大量表現HAX-1會使得冷光蛋白酶的表現下降，則暗示HAX-1也許是影響病毒RNA聚合酶的功能。因為HAX-1是已知的會對抗細胞凋亡 (apoptosis) 的蛋白，所以我們想看PB2的表現是否會影響HAX-1抗apoptosis的效果，但這方面的研究目前還沒有結論，必須要再多加改進。另外，在實驗的過程中，我們意外地發現共同表現HAX-1及PB2後，HAX-1會進入細胞核，而入核是否為PB2將HAX-1帶入核內或利用其它的機制以及HAX-1入核的功能為何，也有待釐清。

PB2 is a component of influenza A RNA polymerase complex and has been shown to play an important role in influenza A viral transcription and replication. To study the role of PB2 involved in influenza A viral replication, we set to fish out cellular factors that interact with PB2 by using yeast two-hybrid system. We found that eight cellular proteins could interact with PB2 C-terminal region. Among them, HAX-1 was confirmed to interact with PB2 by co-immunoprecipitation and GST pull-down assays. HAX-1, a multifunction protein, has been shown to have an anti-apoptotic function and play roles in regulating mRNA transport. To study the role of HAX-1 in influenza A viral replication, HAX-1 knock-down H1299 cells were established by using lentivirus expressing shRNA against HAX-1. We found that influenza A viral production was significantly increased in HAX-1 knock-down cells, indicating that HAX1 plays an inhibitory role in influenza A viral replication. To study the mechanism underlying HAX-1 inhibition of influenza A replication, we transfected PB2-Flag alone, HA-HAX-1 alone, or PB2-Flag + HA-HAX-1 into 293FT cells and detected the location of PB2-Flag and HA-HAX-1 by confocal microscope. HA-HAX-1 localized in the cytoplasm in the absence of PB2. This data is consistent with the previous reports that HAX-1 mainly localized in the mitochondria. Interestingly, when HA-HAX-1 was co-expressed with PB2-Flag, a protein known to localize in the nucleus, a significant proportion of HA-HAX-1 was found in the nucleus. These data indicate that PB2 may translocate HAX-1 into nucleus by interacting with the latter. The significance of this finding and how PB2 interaction with HAX-1 may facilitate influenza A viral production is discussed.