A型流行性感冒病毒PB2蛋白質與細胞蛋白質HNRPM之交互作用

Abstract

中文摘要 流感病毒的PB2蛋白質為構成其病毒特有的RNA-dependent RNA polymerase (RdRp) 的重要一員，對於病毒複製或是RNA轉錄都是不可或缺的。在實驗室找尋可能與PB2進行交互作用的細胞蛋白質時，利用共免疫沈澱法(co-IP) 發現細胞中的HNRPM可能與PB2結合，而在前人的研究中也證實HNRPM可能會結合上PA-PB2- PB1-TAP tag複合體。為了更進一步確認PB2能與HNRPM結合，將PB2分為許多片段刪除組，再使用GST pull-down assay觀察到PB2可能透過其中間片段 (胺基酸序列281-511) 之間與HNRPM結合。由於HNRPM與PB2都可以與RNA結合，為了避免HNRPM與PB2之間的交互作用是透過RNA，在進行GST pull-down assay之前，細胞使用RNase A去除RNA，GST-PB2仍然可以與HNRPM結合，顯示PB2與HNRPM之間的結合不是透過RNA。此外，使用共軛聚焦顯微鏡也觀察到GFP-PB2與Flag-HNRPM都可以共同位在細胞核中。 測試HNRPM對於influenza virus的複製所造成之影響，利用shHNRPM分別在HEK293 與NPC-TW04細胞株內篩選出HNRPM knock-down之穩定細胞株。而流感病毒在HNRPM knock-down之穩定細胞株內的複製效率下降，顯示HNRPM可能影響到流感病毒的複製機轉。在luciferase reporter assay中觀察到HNRPM可能可以影響流感病毒的轉錄或複製。 觀察流感病毒PB2對於HNRPM本身alternative splicing activity之影響，將FGFR2 minigene質體送入293T細胞中，利用semi-quantitative PCR (semi-qPCR) 來觀察splicing產物，結果發現少量的PB2 可以些微刺激HNRPM的活性，大量表現PB2則會抑制其活性。更進一步探討HNRPM是否參與病毒內M mRNA splicing之調控，使用流感病毒感染HNRPM-knockdown之NPC-TW04混合穩定細胞株，並使用semi-qPCR來偵測M1 mRNA、M2 mRNA及mRNA3。經過定量後發現在HNRPM-knockdown穩定細胞株中M2 mRNA的比例較低，推測HNRPM可能影響了流感病毒M mRNA splicing過程。而HNRPM如何影響M mRNA splicing過程以及流感病毒轉錄與複製過程仍須更加細部探討。

PB2, a component of influenza A RNA polymerase complex, plays an important role in influenza A viral transcription and replication. In the process of searching for cellular proteins that interact with PB2, we accidentally found that PB2 could interact with cellular HNRPM protein, an mRNA splicing factor, by using co-immunoprecipitation assay. This finding is consistent with the previous report that HNRPM can be pull-down by PA-PB2-PB1-TAP tag complex. To further confirm that PB2 can interact with HNRPM, GST pull-down assays were performed. We found that PB2 indeed can interact with HNRPM and this interaction was mediated through medial region (amino acids 281-511) of PB2. Since both PB2 and HNRPM can bind RNA, it is important to rule out the possibility that the interaction is mediated through RNA. We thus treated the cell extract with RNase A before performing GST pull-down assays. We found that HNRPM could still be pull-down by GST-PB2, indicating that PB2 can interact with HNRPM even in the absence of RNA. We also found that the HNRPM and PB2 were co-localized in the nucleus in the immunofluorescence assays. To test the effect of HNRPM on influenza A viral replication, we generated HNRPM knock-down cell line by using lentivirus expressing shRNA against HNRPM. We found that influenza A viral replication was slightly decreased in HNRPM knock-down cell line, indicating that HNRPM is involved in influenza A viral replication. This conclusion was further supported by the finding that the viral RNA-dependent RNA replication rate was decreased in the viral replication reporter assay. To test the effect of PB2 on the alternative splicing activity of HNRPM, we transfected the FGFR2 minigene, HNRPM , and PB2 plasmids into 293T cells. Through semi-quantitative PCR, we found that low level of PB2 could slightly stimulate HNRPM activity whereas high level of PB2 could inhibit HNRPM activity. To test whether HNRPM was involved in the regulation of influenza A M mRNA splicing, we infected HNRPM-knockdown NPC-TW04 cells and the parental cells with influenza A virus and quantitated the level of M1 mRNA, M2 mRNA, and mRNA3 in the infected cells. We found that the relative proportion of M2 mRNA was decreased in HNRPM knock-down cells, indicating that HNRPM is involved in regulating M mRNA splicing. How HNRPM affects M mRNA splicing and what role HNRPM may play in the influenza A viral transcription and replication require further investigation.