Nonkwelo C B, Long W K
Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Virology. 1993 Nov;197(1):205-15. doi: 10.1006/viro.1993.1581.
The regulatory mechanisms which control latency and reactivation of the Epstein-Barr virus (EBV) are not fully understood. To determine whether DNA methylation is involved, we examined the BamHI-H divergent promoter, which also encompasses the origin for lytic replication (oriLyt) of EBV. The divergent promoter was highly methylated in the stringently latent HH514cl16 cell line and largely unmethylated in the semipermissive FF41-1 cell line. Expression vectors in which the divergent promoter directed transcription of the chloramphenicol acetyltransferase gene were made. Using in vitro methylation and transient transfections, we found an inverse correlation between the number of sites methylated and level of gene expression. Similar patterns of inhibition were observed when the methylated promoter was activated by BZLF1 or BRLF1 and in lymphoid or epithelial cells. The role of two CpG dinucleotides in the BRLF1 binding sites of the divergent promoter was determined by site-directed mutagenesis. The results indicated that site-specific methylation of these CpGs was not solely responsible for inhibition of expression by methylation. DNA methylation also reduced DNA replication mediated by oriLyt. These results suggest that hypermethylation of the divergent promoter and oriLyt may suppress transcription and lytic replication of EBV.
控制爱泼斯坦-巴尔病毒（EBV）潜伏期和再激活的调控机制尚未完全明确。为了确定DNA甲基化是否参与其中，我们检测了BamHI-H分歧启动子，该启动子也包含EBV裂解复制起始位点（oriLyt）。在严格潜伏的HH514cl16细胞系中，分歧启动子高度甲基化，而在半允许性的FF41-1细胞系中则基本未甲基化。构建了由分歧启动子指导氯霉素乙酰转移酶基因转录的表达载体。通过体外甲基化和瞬时转染，我们发现甲基化位点的数量与基因表达水平呈负相关。当甲基化启动子被BZLF1或BRLF1激活时，以及在淋巴细胞或上皮细胞中，均观察到类似的抑制模式。通过定点诱变确定了分歧启动子BRLF1结合位点中两个CpG二核苷酸的作用。结果表明，这些CpG的位点特异性甲基化并非甲基化抑制表达的唯一原因。DNA甲基化还减少了oriLyt介导的DNA复制。这些结果表明，分歧启动子和oriLyt的高甲基化可能会抑制EBV的转录和裂解复制。
