Epstein-Barr virus gene methylation
and variation in latency
Kerstin 1. Falk
Microbiology and Tumor Biology Center (MTC)
Karolinska Institute, Box 280, S- 171 77 Stockholm, Sweden
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. Further
more, we demonstrated that demethylation of the viral genome is indepen
dent 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 dinical
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.
K~y wor~: EBV, Methylation, PCR, EBNA 6, ori P, ori Lyt
ISBN 91-628-2374-