EB 病毒極早期蛋白 Rta 在上皮細胞中造成病毒再活化之分子機制

Molecular mechanisms of Epstein-Barr virus immediate-early protein Rta-mediated viral reactivation in epithelial cells

陳嬿如(Yen-Ju Chen)

指導教授 ： 陳振陽

共同指導教授 ： 林素芳(Su-Fang Lin)

國立臺灣大學/醫學院/微生物學研究所/博士(2014年)

https://doi.org/10.6342/NTU.2014.02695

摘要

許多研究指出EB病毒(Epstein-Barr virus)進入溶裂期複製是造成鼻咽癌的成因之一。EB病毒極早期蛋白Rta是一個轉錄活化子，其表現可以活化EB病毒進入溶裂期複製。先前的研究發現，單純表現Rta蛋白於上皮細胞中可以使細胞週期停滯於G1期，最後導致細胞老化。在誘導型Rta表現HEK293細胞株(293TetER)所建立EB病毒潛伏感染系統EREV8中加入doxycycline誘導Rta蛋白表現後，細胞中的EB病毒開始進行溶裂期複製並且產生出具感染性的病毒顆粒。另外，我們也發現在這幾個Rta表現細胞中都有細胞生長速率降低和細胞代謝活性減少的共同特徵。我們猜測Rta蛋白在上皮細胞造成的細胞週期停滯可能是引起EB病毒再活化的重要因素。基因微陣列晶片分析與相對蛋白質表現動力學分析結果顯示，Rta蛋白可以促使細胞週期抑制子p21和SFN表現量增加，並同時降低一系列細胞週期正向調控子的表現，包括MYC，以限制細胞週期進行。
基因的轉錄作用與基因體的拓撲結構息息相關。例如基因體拓撲結構的組織者CCCTC-binding factor (CTCF)可控制活化態或是抑制態染色質的空間結構並且調控基因的表現。之前的報導指出CTCF結合於MYC基因的啟動子區域可以幫助MYC的轉錄作用。利用電腦分析，Rta與CTCF都辨認著富含GC的DNA序列，且此兩種DNA序列具有高度的相似性。而在Rta可能的DNA結合位置附近常常帶有CTCF的結合位點，因此我們推測Rta蛋白可能會藉由干擾CTCF結合於細胞與病毒基因體以同時控制細胞週期與病毒生活史的進行。結合染色質免疫沉澱法與DNA甲基化分析，我們發現Rta蛋白結合於MYC、CCND1、JUN和CDK6基因調控區域後，該區域的DNA甲基化作用也隨之增加。重要的是，在這些增加甲基化修飾的DNA片段上，CTCF的結合量隨之減少。
同樣的，在EB病毒或卡波西氏瘤疱疹病毒潛伏感染的細胞中，CTCF蛋白也可以分別牽引病毒基因體形成特殊結構，使潛伏期基因能夠被表現出來。而當Rta表現時可藉由提升CpG的甲基化進而干擾CTCF蛋白結合在病毒基因體上的潛伏/再活化控制區與溶裂期DNA複製起始點。因此，Rta藉由干擾CTCF蛋白結合在細胞基因體的能力，使某些基因表現靜默。另一方面，在病毒基因體中Rta藉由移除CTCF而活化病毒溶裂期複製之進行。
本論文的研究結果提供了在上皮細胞中Rta蛋白造成細胞週期停滯與EB病毒再活化、進入溶裂期複製的分子機制。

關鍵字

EB 病毒 ； Rta ； 細胞週期停滯 ； 病毒的再活化 ； CTCF ； DNA 甲基化

並列摘要

Accumulating evidence indicated that the lytic cycle replication of Epstein-Barr virus (EBV) also played roles in the development of nasopharyngeal carcinoma (NPC). Rta, one of the immediate–early proteins of EBV, is a transcriptional activator that reactivates viral lytic cycle replication. In previous studies, Rta expression in the epithelial cells efficiently arrests cell cycle at G1 phase followed by cellular senescence. Therefore, this thesis aimed to investigate (i) the molecular mechanism of Rta-mediated cell cycle arrest; (ii) the correlation between cell cycle arrest and viral reactivations. First, EBV genome-harboring system was established in a doxycycline-inducible Rta expression cell line in HEK293 cell (293TetER), designated as EREV8. Upon doxycycline induction, Rta expression triggered the latent EBV genome from latent to lytic cycle replication and finally produced infectious viral particles. Next, we observed that the common features of these cell lines were with reduced cell growth rate and decreased cellular metabolic activities upon Rta expression. Further, the results of microarray analysis and comparative protein expression kinetics analysis, Rta upregulated the expression levels of cell cycle inhibitors p21 and SFN, and downregulated a number of positive cell cycle regulators to restrict cell cycle progression, including MYC.
The gene transcriptional regulation is highly correlated with the genome topology. CCCTC-binding factor (CTCF), the principal genome organizer, participates in regulating spatial compartments of active and repressive chromatins and modulating gene expression. It was reported that CTCF bound on the promoter region to support the transcription of MYC. Through in silico analysis, we found that the binding sites of CTCF and Rta are both GC rich and share similarities. Many potential Rta binding sites are adjacent to strong CTCF binding peaks. Therefore, we hypothesized that Rta interferes with CTCF binding in the host and viral genomes, which in turn modulate cell cycle and viral lytic cycle progressions simultaneously. Combining chromatin immunoprecipitation assays and DNA methylation analysis, we observed that the CpG methylation levels of the regulatory regions of MYC、CCND1、JUN、and CDK6 were increased upon Rta binding. Importantly, increased CpG methylation of these regions was accompanied by decreased CTCF occupancy.
CTCF loops viral genomes that are essential for EBV and KSHV latency. Similarly, Rta elevated the CpG methylation and interfered with CTCF binding on the latency/reactivation control regions and the oriLyt regions of EBV and KSHV genomes. Together, our results indicated that, via interference with CTCF binding, in the host genome Rta may function as a transcriptional repressor in silencing gene expression, while in the viral genome Rta acts as an activator for lytic gene loci by removing a topological constraint initiated by CTCF.
Taken together, the results in this thesis provide the molecular mechanism of Rta-mediated cell cycle arrest and viral reactivations in EBV infected epithelial cells.

並列關鍵字

EBV ； Rta ； cell cycle arrest ； viral reactivation ； CTCF ； DNA methylation

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