| Summary: | 博士 === 國立臺灣大學 === 微生物學研究所 === 102 === 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.
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