Identification of an Internal Promoter of the Latent Membrane Protein 1 Gene of Epstein—Barr Virus

The latent membrane protein 1 (LMP 1) gene of an Epstein–Barr virus (EBV) variant derived from an nasopharyngeal carcinoma (NPC) biopsy in Taiwan was isolated and characterized (Chen et al., 1992). Transient expression of the genomic sequence containing this gene showed two proteins with molecular masses of 62 kD and 50 kD, respectively, recognized by LMP-specific antibody S12. To determine if these two proteins were derived from independent promoters, we generated a series of mutant plasmids from plasmid pT7(E) that contained the upstream and the coding regions of the LMP 1 gene. These mutants were introduced into a human epithelial cell line, C33A, and LMP 1 proteins were examined by Western blotting analysis with the S12 antibody. Data showed that plasmid with a fragment containing approximately 3 kb of upstream sequence of LMP 1 gene produced the 62-kD protein. Removal of 2.7 kb of the upstream sequence (plasmid Δ2710, deleted to nucleotide 169,571) resulted in the production of both 62-kD and 50-kD proteins. This suggested that the upstream sequence interfered with the production of the 50-kD protein. Plasmid DNA deleted to Acc I site (nucleotide 169,223) generated only the 50-kD protein, designated as tr-LMP. Further deletion to nucleotide 169,038 resulted in the expression of another smaller LMP1 (49 kD, named as str-LMP1). The region between nucleotides 168,789 and 169,038 was tested to see if it possessed a promoter activity by using the luciferase gene as a reporter. Data showed that this region contained promoter activity with a level compatible to the previously reported ED-L1A promoter. Furthermore, RNase protection assay identified a transcript initiated from this region. Therefore, our results suggested that the NPC-LMP 1 gene contains two internal promoters, ED-L1A and ED-L1B, that direct the expression of tr-LMP and str-LMP 1, respectively. The biological significance of using these promoters to express LMP1-related proteins requires further investigation.