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1 Department of Tumor Biology, Karolinska Institute, Box 60 400, 10401 Stockholm, Sweden
and2 Laboratoire d'Immunobiologie Cellulaire, U.A. 1156 CNRS, Institut Gustave Roussy, 94805 Villejuif Cedex, France
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 EBNA 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 the NPC tumour, but were demethylated in the derived LCLs. In the LMP-expressing coding exons were methylated. The EBNA 1 coding exon was methylated in the Rael line and in NPC, in spite of expression. In contrast, CpG pairs in ori P 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 (ori P and 5' flanking sequences) also suggest a specific role for methylation in the regulation of EBNA and LMP expression.
Keywords: Epstein-Barr virus, methylation, latent membrane protein
Received 8 March 1989;
accepted 19 July 1989.
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