Genome-Wide DNA Methylation as an Epigenetic Consequence of Epstein-Barr Virus Infection of Immortalized Keratinocytes

Abstract

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 involving DNA methylation 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 cellular DNA methylation 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 the DNA methylation changes were silent, 65 cellular genes that acquired CpG methylation showed altered transcript levels. Genes with increased transcript levels frequently acquired DNA methylation within the gene body while those with decreased transcript levels acquired DNA methylation near the transcription start site. Treatment with the DNA methyltransferase 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. IMPORTANCEHere, we show that EBV infection of oral keratinocytes led to CpG island hypermethylation as an epigenetic scar of prior EBV infection that was retained after loss of the virus. Such EBV-induced epigenetic modification recapitulated the hypermethylated CpG island methylator phenotype (CIMP) observed in EBV-associated carcinomas. These epigenetic alterations not only impacted gene expression but also resulted in delayed calcium and methylcellulose-induced keratinocyte differentiation. Importantly, these epigenetic changes occurred in cells that were not as genetically unstable as carcinoma cells, indicating that EBV infection induced an epigenetic mutator phenotype. The impact of this work is that we have provided a mechanistic framework for how a tumor virus using the epigenetic machinery can act in a "hit-and-run" fashion, with retention of epigenetic alterations after loss of the virus. Unlike genetic alterations, these virally induced epigenetic changes can be reversed pharmacologically, providing therapeutic interventions to EBV-associated malignancies. E pstein-Barr virus (EBV) is a prevalent gammaherpesvirus i...

Highly Efficient CRISPR/Cas9-Mediated Cloning and Functional Characterization of Gastric Cancer-Derived Epstein-Barr Virus Strains

“…Intact BAC clones were rescued from the pools of EBV-infected keratinocytes, indicating that the SNU719-BAC and YCCEL1-BAC viruses were episomally maintained (data not shown). This observation corresponds well with a previous report that telomerase-immortalized oral keratinocytes could be infected with Burkitt's lymphoma-derived EBV strain Akata, and EBV genomes were subsequently lost from the infected cells (30). In contrast, when HDK1-K4DT cells were infected with a derivative of EBV strain B95-8 [BART(ϩ) B95.8 virus (14)], the majority of hygromycin-resistant cells were GFP negative (Fig.…”

5-hydroxymethylation of the EBV genome regulates the latent to lytic switch

“…S1), although staining was visible in the normal surrounding cells. Interestingly, NOKs (telomerase-immortalized NOKs), which support an unusually low level of lytic EBV promoter methylation in comparison with other EBVinfected epithelial cell lines (16,17), had easily detectable 5hmC-modified DNA, even in the less differentiated basal cells (Fig. 1C).…”

“…Lytic viral gene expression following EBV infection of normal epithelial cells likely reflects the ability of the other EBV immediate-early (IE) protein, BRLF1 (R), to efficiently activate unmethylated lytic viral promoters. We recently showed that overexpression of R, but not Z, induces lytic EBV gene expression and replication in a latently infected telomerase-immortalized normal oral keratinocyte (NOKs) line, where the lytic viral promoters remain largely unmethylated (16,17). R activates lytic gene expression by binding to R-response elements (RREs) with a consensus sequence of GNCCN 9 GGNG (N 9 is a nine-nucleotide spacer region that can be any sequence) (18) or indirectly by interacting with cellular transcription factors (13).…”

Destabilization of the Human Epigenome: Consequences of Foreign DNA Insertions

“…The different transgenomic clones differ in their profiles. Viewed under the perspective of previously published investigations from our [5][6][7]16] and from other [11][12][13][14] laboratories, it is apparent that the insertion of foreign DNA into an established mammalian genome can lead to genome-wide destabilizations of cellular transcription and methylation patterns. We have not yet conducted a systematic investigation into the epigenetic trans effects of transgenome size, its From the sum total of all our previous investigations on this topic, we conclude that the integrations of Ad12, bacteriophage λ or plasmid DNA as well as the currently used pC1-5.6 plasmid have all led to alterations of methylation and transcriptional profiles in the recipient cells [5][6][7].…”

“…In Epstein-Barr virus (EBV)-transformed human lymphoblastoid cell lines, in EBV-infected cells [11,12] or in human macrophages carrying integrated HIV-1 proviral genomes [13], cellular DNA methylation and transcription patterns have been significantly altered. In induced pluripotent human stem cells with multiple integrated foreign genes, genome-wide alterations of CpG methylation profiles have been described [14].…”