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Latent episomal genomes of EpsteinBarr 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 EpsteinBarr virus using automated fluorescent genomic sequencing of bisulfitemodified DNA samples. We observed that the minimal origin of DNA replication was unmethylated in 8 wellcharacterized human cell lines or clones carrying latent EpsteinBarr 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 Burkitts 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 highresolution methylation analysis of a latent viral origin of DNA replication in human cells.

De Novo DNA Methylation at Nonrandom Founder Sites 5' from an Unmethylated Minimal Origin of DNA Replication in Latent Epstein-Barr Virus Genomes