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Seven virus-encoded proteins are regularly expressed in Epstein-Barr virus (EBV)-transformed lymphoblastoid (LCL) cell lines: the EBV nuclear antigens EBNA 1 to 6 and the latent membrane protein (LMP). In nasopharyngeal carcinoma (NPC), only EBNA 1 is regularly expressed; LMP is detected in about 50% of the tumours. In Burkitt's lymphoma (BL) tumours, only EBNA 1 is expressed. Also, in BL-derived cell lines that maintain the phenotypic markers characteristic of the in vivo tumour (group I), only EBNA 1 is expressed. EBV was rescued by induction or cocultivation from one BL cell line with a restricted group I pattern, and from one NPC tumour, into normal B cells. In the resulting LCLs EBNA 1 to 6 and LMP were expressed. We assessed the level of methylation in the genes encoding ENBNA 2 and LMP by restriction fragment analysis using the methylation-sensitive enzymes SmaI and HpaII. These genes were extensively methylated in the group I BL line Rael and the nude mouse-passaged C15 NPC tumour, but were demethylated in the derived LCLs. In the LMP-expressing NPC C15 tumour the 5' flanking region of the gene was hypomethylated, whereas the coding exons were methylated [corrected]. The EBNA 1 coding exon was methylated in the Rael line and in NPC, in spite of expression. In contrast, CpG pairs in oriP were originally hypomethylated and remained so after their transfer to LCLs. The cell phenotype-dependent pattern of EBV gene methylation correlated with the phenotype-dependent pattern of EBNA and LMP expression. The specific patterns of methylation localized to controlling regions (oriP and 5' flanking sequences) also suggest a specific role for methylation in the regulation of EBNA and LMP expression.

journal of general virology

The Role of Methylation in the Phenotype-dependent Modulation of Epstein-Barr Nuclear Antigen 2 and Latent Membrane Protein Genes in Cells Latently Infected with Epstein-Barr Virus