EBV infection alters DNA methylation in primary human colon cells: a path to inflammation and carcinogenesis?

EBV is associated to several types of human cancers and changes in DNA methylation are reported to contribute to viral-driven carcinogenesis, particularly in cancers of epithelial origin. In previous study, we have shown that EBV infects human primary colonic cells (HCoEpC) and replicates in these cells, inducing pro-inflammatory and pro-tumorigenic effects, mostly prevented by inhibiting viral replication by PAA. Interestingly, the EBV-induced effects correlated with DNMT1 upregulation and were counteracted by pretreating cells by 5-AZA, suggesting a role for DNA hypermethylation. Based on this background, in this study, we investigated the methylation changes induced by EBV infection in HCoEpC, in the presence or in the absence of PAA or ERK1/2 and STAT3 inhibitors, pathways known to be activated by EBV and involved in dysregulating methylation in tumor cells. The genome wide-methylation analysis performed in this study allowed us to identify several biological processes and genes affected by this epigenetic change, as a possible underlying mechanism leading to the pathologic effects induced by EBV previously observed. In particular, we found that the virus induced intense methylation changes, that hypermethylation was prevalent over hypomethylation, and that several genes involved in embryogenesis and carcinogenesis and inflammation were affected. Primary human colonic epithelial cells (HCoEpC) were infected with EBV virions isolated by B95–8 cells (V), pretreated with PD0325901 and AG490 before infection (PD-AG-V), pretreated with phosphonoacetic acid (PAA-V), and treated with these compound in the absence of infection (PD-AG and PAA) or left untreated and uninfected as negative control (CT). Genomic DNA was purified using the PureLink genomic DNA mini kit and sheared to ~6 kb fragment size using Covaris G tubes. Nanopore sequencing libraries were prepared using the native library prep kit SQK-LSK110 and sequenced with R9.4.1 MinION flow cells (FLO-MIN106).