Epstein–Barr virus‐induced epigenetic alterations following transient infection

Abstract

Epstein-Barr virus (EBV) is a known tumor virus associated with an increasing array of malignancies; however, the association of the virus with certain malignancies is often erratic. To determine EBV’s contributions to tumorigenesis in a setting of incomplete association, a transient model of infection was established where a clonal CCL185 carcinoma cell line infected with recombinant EBV was allowed to lose viral genomes by withdrawal of selection pressure. Global gene expression comparing EBV-negative, transiently infected clones to uninfected controls identified expression changes in over 1000 genes. Among downregulated genes, several genes known to be DNA methylated in cancer were identified including E-cadherin and PYCARD. A cadherin switch, increased motility and enhanced cellular invasiveness present in EBV-positive cells were retained following viral loss indicating an epigenetic effect. Repression of PYCARD expression was due to increased promoter CpG methylation, whereas loss of E-cadherin expression after transient EBV infection did not correlate with increased DNA methylation of the E-cadherin promoter. Rather, repression of E-cadherin was consistent with formation of a repressive chromatin state. Decreased histone 3 or 4 acetylation at the promoter and 5’ end of the E-cadherin gene was observed in an EBV-negative, transiently infected clone relative to the uninfected controls. These results suggest that EBV can stably alter gene expression in a heritable fashion in formerly infected cells, while its own contribution to the oncogenic process is masked.

Succumbing to the laws of attraction

“…For example, alterations in DNA methylation and changes to gene expression in the host genome are known to be induced by Epstein Barr infection. 84,85 This is consistent with evidence supporting an infectious etiology in at least some forms of CFS 86 and the relative success of recent trials involving depletion of the B cell population, a reservoir for EBV infection. 87 Even though care was taken to involve the principal regulatory interactions no model is complete.…”

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

“…Total cellular RNA was harvested in RNA STAT60 (Tel-Test, Friendswood, TX) and purified per the manufacturer's instructions. Ten micrograms of total RNA was used to make cDNA as previously described (30). One hundred nanograms of cDNA was amplified using Go Taq DNA Polymerase (1 unit/reaction; Promega), 5ϫ Go Taq buffer (Promega), 500 nM specific primer (see Table S1 in the supplemental material), and 200 nM each deoxynucleoside triphosphate (dNTP) (Illustra dNTP Polymerization Mix; GE Healthcare).…”

“…RNA was harvested 3 days after seeding using STAT60 (Tel-Test) according to the recommended protocol. Transcriptional profiles from duplicate seedings of cells were determined as previously described (30). Fifteen micrograms of fragmented, biotin-labeled cRNA was hybridized to Affymetrix U133 Plus 2.0 GeneChips.…”

“…EBV infection of germinal center B cells not only was shown to alter DNMT expression, resulting in changed DNA methylation patterns at particular regions, but also induced the histone 3 lysine 27 demethylase, KDM6B, potentially regulating genes differentially expressed in Hodgkin's lymphoma (28,29). However, in carcinoma cells, we previously demonstrated that EBV infection led to epigenetic silencing at the PYCARD and E-cadherin gene loci involving DNA methylation and repressive chromatin modifications, respectively (30). Importantly, these epigenetic changes were maintained following loss of the virus and indicated that their heritable nature was not dependent on continued viral gene expression (30).…”

“…However, in carcinoma cells, we previously demonstrated that EBV infection led to epigenetic silencing at the PYCARD and E-cadherin gene loci involving DNA methylation and repressive chromatin modifications, respectively (30). Importantly, these epigenetic changes were maintained following loss of the virus and indicated that their heritable nature was not dependent on continued viral gene expression (30). A limitation of studies using cancer cells is the propensity for genomic instability that results in complex genetic and epigenetic changes that may confound epigenetic events induced by EBV infection.…”