Genome-wide DNA methylation as an epigenetic consequence of Epstein-Barr virus infection of immortalized keratinocytes

The oral cavity is a persistent reservoir for Epstein-Barr virus (EBV) with lifelong infection of resident epithelial and B cells. Infection of these cell types results in distinct EBV gene expression patterns regulated by epigenetic modifications involvingDNAmethylation and chromatin structure. Regulation of EBV gene expression relies on viral manipulation of the host epigenetic machinery that may result in long-lasting host epigenetic reprogramming. To identify epigenetic events following EBV infection, a transient infection model was established to map epigenetic changes in telomerase-immortalized oral keratinocytes. EBV-infected oral keratinocytes exhibited a predominantly latent viral gene expression program with some lytic or abortive replication. Calcium and methylcelluloseinduced differentiation was delayed in EBV-positive clones and in clones that lost EBV compared to uninfected controls, indicating a functional consequence of EBV epigenetic modifications. Analysis of global cellularDNAmethylation identified over 13,000 differentially methylated CpG residues in cells exposed to EBV compared to uninfected controls, with CpG island hypermethylation observed at several cellular genes. Although the vast majority of theDNAmethylation changes were silent, 65 cellular genes that acquired CpG methylation showed altered transcript levels. Genes with increased transcript levels frequently acquiredDNAmethylation within the gene body while those with decreased transcript levels acquiredDNAmethylation near the transcription start site. Treatment with the DNAmethyltransferase inhibitor, decitabine, restored expression of some hypermethylated genes in EBV-infected and EBV-negative transiently infected clones. Overall, these observations suggested that EBV infection of keratinocytes leaves a lasting epigenetic imprint that can enhance the tumorigenic phenotype of infected cells.