細胞蛋白質hRrp43在A型流行性感冒病毒感染中所扮演的角色

The regulatory role of hRrp43 in Influenza A virus multiplication

蒲品靜(Pin-Ching Pu)

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(2015年)

https://doi.org/10.6342/NTU.2015.11141

摘要

PB2是流行性感冒的病毒蛋白，其功能是參與cap snatching，在cap snatching的過程中具有結合到宿主pre-mRNA的帽蓋上的功能。PB2與PB1及PA構成RNA-dependent RNA polymerase (RdRp)，參與流感病毒的轉錄及複製。
為了找出能與A型流行性感冒病毒PB2蛋白交互作用的細胞蛋白，本實驗室利用酵母菌雙雜合系統 (yeast two-hybrid system) 找出細胞蛋白質hRrp43 (exosome complex的組成蛋白之一) 會和流感病毒PB2進行交互作用，並利用免疫共沉澱法以及GST pull-down方法，證明細胞蛋白質hRrp43與流感病毒蛋白質PB2具有交互作用。而在本研究中，利用不同的hRrp43-specific shRNAs在H1299細胞株中knockdown細胞蛋白質hRrp43，證明了knockdown hRrp43會抑制A型流感病毒之複製，並且排除此現象是由shRNA所造成的off-target現象。更進一步證明感染不同病毒株 (WSN33及PR8病毒株) 或是在不同細胞株 (H1299及A549細胞株) 中，knockdown細胞蛋白hRrp43皆會抑制流感病毒的複製。此外，當knockdown exosome中的其他組成蛋白，亦能抑制流感病毒的複製，因此可以推定exosome complex會參與調控A型流感病毒之複製繁殖。
為了瞭解hRrp43是如何參與調控A型流感病毒之複製繁殖，利用GST pull-down方法，找出病毒蛋白質PB2主要利用271-522胺基酸序列與hRrp43進行交互作用，而胺基酸序列511-759則有較弱的交互作用，此兩區域皆有細胞核定位訊號 (nuclear localization signal, NLS)，因此利用免疫螢光染色觀察病毒入核情形，證明knockdown hRrp43會影響病毒蛋白質vRNP入核的情形。此外，利用qRT-PCR證實knockdown hRrp43會影響流感病毒mRNA、cRNA及vRNA之表現量。
另外，發現hRrp43-knockdown細胞可以釋放出某些物質去抑制流感病毒的複製，利用cDNA微陣列分析發現hRrp43-knockdown細胞中有21個基因的表現量會高於GFP-knockdown細胞兩倍。在這些表現量提高的基因中，很多被發現是interferon-stimulated genes。利用qRT-PCR再次確認後，證實knockdown hRrp43會使得IFN-β1表現量較高，導致下游ISGs包括IFIT1、IFIT2、IFIT3、IFITM2、IFITM3以及OASL的表現量均較控制組高，因此，由上述的結果中找到一個A型流行感冒病毒複製繁殖需要的host factor，並且提供一個控制流感病毒感染的新標靶。

關鍵字

A型流行性感冒病毒 ； PB2蛋白質 ； hRrp43 ； 病毒轉錄與複製 ； 干擾素刺激基因

並列摘要

Influenza A virus (IAV) PB2 protein, a cap binding protein, is a subunit of viral RNA-dependent RNA polymerase which transcribes and replicates RNA genome of influenza A virus.
Our previous studies demonstrated that hRrp43 could interact with PB2 by using GST pull-down and co-immunoprecipitation assays. In this study, we used different hRrp43-specific shRNAs to knockdown hRrp43 in H1299 cells. Our data showed that the viral protein production and viral propagation were dramatically decreased in H1299 cells transduced with different hRrp43 shRNAs, indicating that knockdown of hRrp43 indeed can lead to inhibition of influenza A viral propagation and these phenotype is not caused by off-target effect of shRNA. We also showed that the replication of different strains (including WSN33 and PR8 viruses) of IAV was inhibited in hRrp43-knockdown H1299 and A549 cells, indicating that the knockdown phenotype can be observed in different cells or using different viral strains. hRrp43 is a subunit of exosome, a large complex involved in RNA processing. To study whether an intact exosome is required for influenza A viral replication, we knocked down hRrp45, which is another subunit of exosome. We found that viral replication was also down-regulated in hRrp45-knockdown cells, indicating that an intact exosome is required for influenza A viral replication. The hRrp43-knockdown cells could secrete factors that can inhibit IAV replication. Finally we showed that 21 cellular genes were up-regulated more than 2 fold in hRrp43- knockdown H1299 cells compared to control cells by using cDNA microarray analysis. Among these up-regulated genes, many of them were found to be interferon-stimulated genes. By using qRT-PCR, we confirmed that IFN-β1 and the ISGs including IFIT1, IFIT2, IFIT3, IFITM2, IFITM3 and OASL were significantly up-regulated in H1299-shhRrp43 cells. Together, these results not only identify an intrinsic host factor that can facilitate IAV multiplication but also provide a new target for controlling IAV infection.

並列關鍵字

Influenza A virus ； PB2 ； hRrp43 ； transcription and replication ； interferon-stimulated gene

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