Search

We reported earlier that variable expression of the Epstein-Barr virus (EBV) encoded membrane protein LMP 1 in nasopharyngeal carcinoma and the host-cell-phenotype-dependent activity of the BCR2 promoter (one of the possible initiator sites for transcripts of Epstein-Barr nuclear antigens) in Burkitt's lymphoma (BL) lines can be related to the methylation status of the 5'-flanking regulatory regions of the BNLF 1 and BCR2 promoter, respectively. Here we report that clones of the BL line Mutu that differ in expression of LMP 1 also show a differential methylation pattern of the LMP 1 regulatory sequences: this region is hypomethylated in an LMP 1 expressing (group III) clone but methylated in a group I clone that does not express LMP 1. We introduced in vitro methylated reporter plasmids carrying BNLF 1 and BCR2 enhancer-promoter sequences into the BL line Raji and found that overall methylation of 5'-CG-3' sequences by the Spiroplasma methylase Sssl significantly reduced their activity compared to unmethylated or mock-methylated controls. Methylation of 5-CCGG-3' sequences by Hpall methyltransferase gave similar results. On the contrary, methylation of 5'GCGC-3' sequences by Hhall methyltransferase gave similar results. On the contrary, methylation of 5'-GCGC-3' sequences by Hpal methyltransferase resulted only in a moderate reduction of BNLF 1 enhancer-promoter activity. These data support the notion that methylation at discrete sites within control regions of latent, growth-transformation associated EBV genes may contribute to silencing their expression.

Epstein-Barr virus (EBV) reveals two types of interactions with human B lymphocytes. In immortalized lymphoblastoid cell lines (LCL), nine virus-coded proteins are expressed: six nuclear (EBNA1-6) and three membrane proteins (LMP1, TP 1,2). In Burkitt's lymphoma (BL) tumors and representative cell lines (type I) only EBNA-1 is expressed. The viral genomes are carried by the cells in an extrachromosal, episomal form. We have earlier described an overall difference in EBV DNA methylation between LCLs of normal B-cell origin and type I BL lines. EBV genomes are hypomethylated in the former, methylated in the latter. The origin of replication (oriP) for the episomal DNA is exempted from this overall methylation. This unmethylated region was analyzed in the BL derived type I cell line Rael. It starts just upstream of the coding exons for the small untranslated RNAs EBER I/II. It extends throughout oriP and includes several control elements between oriP and the first promoter for EBNA transcription (BCR2). The viral DNA was methylated around and downstream of that promoter, which is not used for EBNA-1 expression in Rael.