Salamon D, Takacs M, Myöhänen S, Marcsek Z, Berencsi G, Minarovits J
2nd Department of Pathology, Semmelweis University of Medicine, Budapest, Hungary.
Biol Chem. 2000 Feb;381(2):95-105. doi: 10.1515/BC.2000.014.
Latent episomal genomes of Epstein-Barr virus, a human gammaherpesvirus, represent a suitable model system for studying replication and methylation of chromosomal DNA in mammals. We analyzed the methylation patterns of CpG dinucleotides in the latent origin of DNA replication of Epstein-Barr virus using automated fluorescent genomic sequencing of bisulfite-modified DNA samples. We observed that the minimal origin of DNA replication was unmethylated in 8 well-characterized human cell lines or clones carrying latent Epstein-Barr virus genomes as well as in a prototype virus producer marmoset cell line. This observation suggests that unmethylated DNA domains can function as initiation sites or zones of DNA replication in human cells. Furthermore, 5' from this unmethylated region we observed focal points of de novo DNA methylation in nonrandom positions in the majority of Burkitt's lymphoma cell lines and clones studied while the corresponding CpG dinucleotides in viral genomes carried by lymphoblastoid cell lines and marmoset cells were completely unmethylated. Clustering of highly methylated CpG dinucleotides suggests that de novo methylation of unmethylated double-stranded episomal viral genomes starts at discrete founder sites in vivo. This is the first comparative high-resolution methylation analysis of a latent viral origin of DNA replication in human cells.
人类γ疱疹病毒——爱泼斯坦-巴尔病毒（Epstein-Barr virus，EBV）的潜伏游离型基因组，是研究哺乳动物染色体DNA复制和甲基化的合适模型系统。我们使用亚硫酸氢盐修饰的DNA样本进行自动荧光基因组测序，分析了EBV DNA复制潜伏起始位点处CpG二核苷酸的甲基化模式。我们观察到，在8个特征明确的携带潜伏EBV基因组的人类细胞系或克隆中，以及在一个原型病毒产生型狨猴细胞系中，DNA复制的最小起始位点均未甲基化。这一观察结果表明，未甲基化的DNA结构域可作为人类细胞中DNA复制的起始位点或区域。此外，在该未甲基化区域的5'端，我们在大多数所研究的伯基特淋巴瘤细胞系和克隆中的非随机位置观察到了从头DNA甲基化的焦点，而淋巴母细胞系和狨猴细胞携带的病毒基因组中的相应CpG二核苷酸则完全未甲基化。高度甲基化的CpG二核苷酸的聚集表明，未甲基化的双链游离型病毒基因组的从头甲基化在体内从离散的起始位点开始。这是首次对人类细胞中DNA复制的潜伏病毒起始位点进行的比较高分辨率甲基化分析。
