IDENTIFICATION OF A PROMOTER FOR THE LATENT MEMBRANE-PROTEIN-1 GENE OF EPSTEIN-BARR-VIRUS THAT IS SPECIFICALLY ACTIVATED IN HUMAN EPITHELIAL-CELLS

Latent membrane protein 1 (LMP 1) is one of two Epstein-Barr virus (EB V)-encoded proteins that expressed in nasopharyngeal carcinoma (NPC) c ells, Previous studies showed that a 3,5-kb transcript of the LMP 1 ge ne, in addition to the 2,8-kb transcript, was detected in a B95-8-EBV- containing, nude mice-passaged NPC tumor, C15, This indicated that a t ranscript was initiated from a region 5' to the putative promoter, ED- L1. We have isolated an EBV variant from a NPC tissue, and this virus strain contained a more pathogenic LMP 1 gene. DNA sequence analysis o f the 5'-upstream region showed distinct variations as compared to tha t of B95-8 strain, To test if the LMP 1 gene of the NPC strain also co ntained an upstream promoter, we generated a series of deletion plasmi ds encompassing positions -1,030 to +20 of the LMP 1 promoter and test ed for their abilities to drive the expression of the reporter gene in human epithelial cell lines, C-33A and NPC-TW076, We found that the r egion between -643 and -496 contained a promoter activity that was app roximately five-fold higher than the putative promoter, ED-LI, This re gion between -643 and -496 was designated as ED-L1E, C-33A cells conta ining the genomic clone pT7(E) or the clone that had deleted a 94-bp E D-L1 sequence (Delta 94) was used to determine the transcription initi ation sites by RNase protection assay, Results showed that a transcrip tion initiation site was located at nucleotide 170,099 (''A'') of EBV genome. The transcript was expressed in NPC biopsies and in human prim ary normal epithelial cells transfected with pT7(E) and Delta 94, resp ectively, as examined by reverse transcriptase-polymerase chain reacti on (RT-PCR) analysis, Furthermore, the ED-LIE was not regulated by the EBV-encoded nuclear antigen 1-mediated transcriptional enhancer famil y of repeats (FR) in C-33A cells, Our results suggested that the ED-L1 E was specifically activated in epithelial cells, The biological signi ficance of the selective usage of the ED-L1E promoter was discussed.