Aging, Environment, & DiseaseDNA Methylation and Hydroxymethylation

Lasting impressions: an epigenetic imprint of past viral infection

Christine E. Birdwell and Rona S. Scott

Department of Microbiology and Immunology, Center for Molecular and Tumor Virology, Louisiana State University Health Sciences Center-Shreveport, Shreveport, LA 71130


Lasting impressions: an epigenetic imprint of past viral infection Epstein-Barr virus (EBV) is a prevalent human herpesvirus affecting over 90% of the adult population worldwide. Most individuals harbor EBV asymptomatically as lifelong infections. However, in rare cases, EBV is associated with epithelial and B cell malignancies. The viral life cycle is dependent on host epigenetic machinery to regulate viral gene expression programs that ensure viral persistence and spread. Infection with EBV can lead to epigenetic changes in the host cell as well. Several EBV proteins interact with host epigenetic machinery, such as the latent membrane proteins ability to induce the activity of the DNA methyltransferases (DMNT) [1-3]. The consequences of which can best be seen in EBV-associated malignancies with the CpG island methylator phenotype (CIMP) [4, 5]. CIMP is where CpG islands upstream of tumor suppressors are hypermethylated and this phenotype has a strong correlation with EBV. Whether EBV directs the DNA methylation changes, or if the epigenetic changes are due to the carcinogenic process are still being defined.

To observe EBV-dependent epigenetic changes in epithelial cells, our lab infected immortalized normal oral keratinocytes (NOKs) with EBV for several passages and then allowed infected cells to naturally lose the virus (transient infection). If EBV induced epigenetic changes, these epigenetic alterations would be retained in clones that lost EBV. EBV infected NOKs showed a delay in differentiation in response to calcium and methylcellulose, two well established methods of inducing keratinocyte differentiation. Importantly, this phenotype was maintained in EBV-negative transiently infected cells, suggestive of epigenetic alterations. Given that EBV can modulate the DNMTs, we used reduced representation bisulfite sequencing to map the methylation status of CpG residues genome wide. The methylation state of 7 to 8 million unique CpG residues was mapped. Further analysis revealed over 13,000 differentially methylated CpGs that were common in cells exposed to EBV compared to uninfected parental controls. Of particular interest was the increase in methylation at CpG islands associated with 27 genes in cells exposed to EBV compared to uninfected controls, a phenomenon reminiscent of the CIMP phenotype seen in vivo in EBV-associated carcinomas.

To correlate DNA methylation to functional changes in gene expression, the transcriptional profiles following transient EBV infection were analyzed compared to uninfected parental controls. Notably, 65/260 differentially expressed genes had evidence of gains in DNA methylation in cells exposed to EBV. The location of DNA methylation correlated with the change in expression. Downregulated genes acquired DNA methylation typically near the transcription start site (TSS) while upregulated genes acquired DNA methylation much farther from the TSS. In fact, 31/33 downregulated genes had evidence of increased DNA methylation in CpG islands/shores in EBV infected and transiently EBV infected clones compared to uninfected cells. Treatment with a DNMT inhibitor, decitabine, increased RNA levels in a set of downregulated genes similar to RNA levels in uninfected controls, suggesting that the increased DNA methylation was responsible for the altered regulation [6].

In conclusion, our observations suggest that EBV infection of epithelial cells results in epigenetic changes to the host genome that have long lasting phenotypic consequences even after loss of the virus, and define a potential mechanism for viral “hit-and-run” oncogenesis.

 

1. Hino R, Uozaki H, Murakami N, Ushiku T, Shinozaki A, Ishikawa S, Morikawa T, Nakaya T, Sakatani T, Takada K, & Fukayama M (2009). Activation of DNA methyltransferase 1 by EBV latent membrane protein 2A leads to promoter hypermethylation of PTEN gene in gastric carcinoma. Cancer research, 69 (7), 2766-74 PMID: 19339266

2. Tsai CL, Li HP, Lu YJ, Hsueh C, Liang Y, Chen CL, Tsao SW, Tse KP, Yu JS, & Chang YS (2006). Activation of DNA methyltransferase 1 by EBV LMP1 Involves c-Jun NH(2)-terminal kinase signaling. Cancer research, 66 (24), 11668-76 PMID: 17178861

3. Tsai CN, Tsai CL, Tse KP, Chang HY, & Chang YS (2002). The Epstein-Barr virus oncogene product, latent membrane protein 1, induces the downregulation of E-cadherin gene expression via activation of DNA methyltransferases. Proceedings of the National Academy of Sciences of the United States of America, 99 (15), 10084-9 PMID: 12110730

4. Chong JM, Sakuma K, Sudo M, Ushiku T, Uozaki H, Shibahara J, Nagai H, Funata N, Taniguchi H, Aburatani H, & Fukayama M (2003). Global and non-random CpG-island methylation in gastric carcinoma associated with Epstein-Barr virus. Cancer science, 94 (1), 76-80 PMID: 12708478

5. Hutajulu SH, Indrasari SR, Indrawati LP, Harijadi A, Duin S, Haryana SM, Steenbergen RD, Greijer AE, & Middeldorp JM (2011). Epigenetic markers for early detection of nasopharyngeal carcinoma in a high risk population. Molecular cancer, 10 PMID: 21535891

6. Birdwell CE, Queen KJ, Kilgore P, Rollyson P, Trutschl M, Cvek U, & Scott RS (2014). Genome-wide DNA methylation as an epigenetic consequence of Epstein-Barr virus infection of immortalized keratinocytes. Journal of virology PMID: 25056883

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Rona Scott

Rona Scott

  • mousa

    It was a very exciting article. it can be used for future treatment to both viral infections and tumor derived from them.