We analysed the terminal repeats of Epstein-Barr virus (EBV) in DNAs isolated from six lethal midline granuloma (LMG) biopsies. A single fused terminal fragment could be detected in each case, indicating that these angiocentric peripheral T cell lymphomas represent clonal proliferations of cells infected with EBV on a single occasion. Using reverse transcriptase-PCR, we detected EBV nuclear antigen (EBNA) 1 and latent membrane protein (LMP) 1, but not EBNA 2 messages in LMG biopsy RNAs. The splicing pattern of the EBNA 1 message was consistent with the usage of a promoter localized in the BamHI F fragment (F promoter). The BamHI W fragment repeats and LMPcoding sequences were highly methylated in all cases. In contrast, the LMP regulato~T¢ sequences were found to be hypomethylated or partially methylated, as in LMPexpressing nasopharyngeal carcinomas.

We have shown previously that the EBNA 1 and latent membrane protein encoding regions of the EpsteinBarr virus (EBV) genome are highly methylated at CCGG sequences in the Burkitt's lymphoma (BL)-derived cell line Rael, but are unmethylated in a lymphoblastoid cell line (LCL) harbouring the same virus. To examine whether this is a regular phenomenon, we compared the methylation patterns of selected regions (BamHl C, W, H, M, E, K and N fragments) of EBV DNA in representative EBVcarrying cell types of normal and neoplastic origin. Analysis of HpalI and MspI cleavage patterns showed that all probed regions were highly methylated in all six BL biopsy samples, but hypomethylated in the four LCLs immortalized by the virus. EBV DNA was also highly methylated in the nude mouse-passaged C15 nasopharyngeal carcinoma strain and partially methylated in the C18 strain. Eight BL lines propagated in vitro, ranging from a typical BL group I to a more LCLlike group III phenotype, showed heterogeneous levels of methylation. Rael, the only stable group I cell line, carried highly methylated viral genomes. The other cell lines, which have drifted to an LCL-like blastic phenotype to various degrees, showed more moderate or low viral DNA methylation. Two sublines of the BL cell line Jijoye, which could be classified as groups II and III, respectively, showed a corresponding difference in EBV DNA methylation. To assess the possible influence of hypomethylated linear EBV DNA molecules produced in lytically infected cells, the virus producer P3HR-1 and Jijoye M13 lines were compared for DNA methylation before and after treatment with phosphonoformic acid (PFA), an inhibitor of the viral DNA polymerase. PFA treatment resulted in a shift towards a more methylated pattern in both regions (BamHI W and E) assayed, but had no effect on virus non-producer lines (Rael, CB-M1-RaI-STO and Jijoye p79).

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The genome of Epstein-Barr virus (EBV) codes for two non-translated small RNA molecules, EBER 1 and 2. We found that both EBERs are expressed in the major EBV-carrying cell types, group I and III Burkitt's lymphoma (BL) cell lines, lymphoblastoid cell lines (LCLs) and in two nude mouse-passaged nasopharyngeal carcinoma (NPC) tumours. The relative amount of EBER 1 and EBER 2 varied in different host cells but did not correlate with the cellular phenotype. The EBER coding and flanking sequences were predominantly hypomethylated at HpalI sites not only in LCLs which usually carry hypomethylated EBV genomes but also in BL and NPC cell lines harbouring EBV episomes that are highly methylated in other regions. Thus, the EBER trancription units, actively transcribed by RNA polymerase III in the major EBV-carrying cell types, represent a methylation-free region in the EBV genome similarly to regulatory sequences of the latent membrane protein gene when the latter is transcribed by RNA polymerase II.

CpG-methylation blocks the activity of RNA polymerase II transcribed promoters in most cases. In contrast, the role of DNA methylation in the regulation of RNA polymerase III transcribed promoters is less clarified. There are two untranslated viral RNAs (EBER-1 and EBER-2) in most malignant cells carrying latent Epstein-Barr virus (EBV) genomes. We found that in vitro methylation blocked binding of the cellular proteins c-Myc and ATF to the 5 0 -region of the EBER-1 gene, and silenced the expression of the EBER-1 and EBER-2 genes, transcribed by RNA polymerase III, in transfected cells.