Jansson A, Masucci M, Rymo L
Department of Clinical Chemistry, Gothenburg University, Sahlgren's Hospital, Sweden.
J Virol. 1992 Jan;66(1):62-9. doi: 10.1128/JVI.66.1.62-69.1992.
Eight of the nine viral antigens known to be expressed in in vitro Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines are downregulated in EBV-carrying Burkitt lymphomas (BL). Only EBNA1 can be detected in BL biopsies and BL-derived cell lines that maintain the representative phenotype during culture in vitro (group I BL lines). This restricted pattern of viral gene expression is accompanied by extensive EBV DNA methylation and can be reversed by treatment with the demethylating agent 5-azacytidine. Transcription of the genes encoding the six transformation-associated EBNAs can be initiated from one of two promoters located in the BamHI C and W regions, respectively, of the virus genome. We show that discrete sites within the BamHI W enhancer region are methylated in the group I BL lines Rael, Cheptage, and Elijah and become unmethylated after 5-azacytidine treatment that induces the expression of EBNA2. Demethylation correlates with activation of transcription from the BamHI W promoter as determined by S1 protection analysis. Reporter plasmids in which the W enhancer sequences were linked to the chloramphenicol acetyltransferase gene were active in untreated Rael, Cheptage, and Elijah cells, demonstrating that all of the required transcription factors are present in group I BL cells. Conversely, in vitro methylation of the enhancer sequences abolished their activity. The results suggest that methylation of control regions in the EBV genome may play a critical role for the regulation of viral gene expression in tumor cells.
已知在体外爱泼斯坦-巴尔病毒（EBV）转化的B淋巴母细胞系中表达的9种病毒抗原中的8种，在携带EBV的伯基特淋巴瘤（BL）中表达下调。在体外培养过程中保持典型表型的BL活检组织和BL来源的细胞系（I组BL细胞系）中，仅能检测到EBNA1。这种病毒基因表达的受限模式伴随着广泛的EBV DNA甲基化，并且可以通过用去甲基化剂5-氮杂胞苷处理来逆转。编码6种与转化相关的EBNAs的基因转录可以分别从位于病毒基因组BamHI C区和W区的两个启动子之一起始。我们发现，在I组BL细胞系Rael、Cheptage和Elijah中，BamHI W增强子区域内的离散位点发生了甲基化，而在诱导EBNA2表达的5-氮杂胞苷处理后这些位点变得去甲基化。如通过S1保护分析所确定的，去甲基化与BamHI W启动子的转录激活相关。其中W增强子序列与氯霉素乙酰转移酶基因相连的报告质粒在未处理的Rael、Cheptage和Elijah细胞中具有活性，这表明I组BL细胞中存在所有必需的转录因子。相反，增强子序列的体外甲基化消除了它们的活性。结果表明，EBV基因组中调控区域的甲基化可能在肿瘤细胞中病毒基因表达的调控中起关键作用。
