Epstein-Barr virus gene methylation and variation in latency

Epstein-Barr virus is a human herpesvirus and approximately 95 % of the adults carry the virus in a latent form. EBV has been implicated in the pathogenesis of human malignancies such as Burkitt's lymphoma and nasopharyngeal carcinoma. DNA methylation is one of the mechanisms that is involved in control of gene expression in viruses. We therefore investigated the methylation status of the EBV genome and determined the possible role of methylation in latent gene expression and activation. We have used two methods for analysing the methylation status of the EBV genome in cells with different forms of latency. One sequence-specific method based on restriction enzyme digestion was applied.

The second method used involved chemical alteration of cytosines followed by sequencing. We examined two control regions of EBV for methylation: the origin of latent replication (ori P) and the LMP 1 regulatory sequence (LRS). In all cell lines and EBV-carrying tumours tested ori P was always found to be unmethylated, whereas the LRS was unmethylated in LMP1-expressing cells and methylated in LMP1-negative cells. Furthermore, we demonstrated that demethylation of the viral genome is independent of DNA replication. We can conclude from these studies that methylation is likely to play a role in the control of EBV-gene expression.

A second aim of the thesis was to explore reliable and simple methods for detection of virus strain variation. We found a correlation between restriction fragment length polymorphisms (RFLPs) within exons coding for Epstein Barr virus nuclear antigen (EBNA) 1, EBNA 3, EBNA 6 and the size of the respective proteins. Based on the strong correlation between an n x 39 bp repeat in the EBNA 6-coding region and the size of the protein, we developed a polymerase chain reaction (PCR) assay over this repetitive sequence. We have shown that it is possible with our PCR-based method, to differentiate between types A and B and variants thereof in clinical samples. We have also shown by ELISA that a peptide derived from the n x 39 bp repeat is a major epitope for antibody reactivity.