Identification of a novel protein encoded by the BamHI A region of the Epstein-Barr virus

An unusual set of 3' coterminal, spliced mRNAs transcribed through the BamHI A fragment have been previously identified in nasopharyngeal carcinoma (NPC) tissues. These RNAs have also been detected at low levels in Burkitt's lymphoma (BL) cell lines and Epstein-Barr virus (EBV)-transformed lymphocytes. Sequence analyses of clones from a cDNA library derived from the C15 NPC tumor indicated that the primary transcripts are differentially spliced, giving rise to a family of related transcripts, all of which encompass the BARF0 open reading frame (ORF) at the 3' end of the transcripts. One cDNA was identified that extended the BARF0 ORF at the 5' end, forming the RK-BARF0 ORF. In this study, a rabbit antiserum to a synthetic peptide representing an amino acid sequence encoded by the BARF0 ORF was prepared. This antiserum detected a glutathione S-transferase-BARF0 fusion protein and both BARF0 and RK-BARF0 proteins expressed from transfected constructs in H1299 cells. The serum also immunoprecipitated the 20-kDa BARF0 and 30-kDa RK-BARF0 in vitro-translated proteins. Immunoblot analyses identified a protein doublet of 30 and 35 kDa in all of the EBV-infected cell lines tested. Cellular fractionation studies revealed that the proteins were membrane associated. The sizes of the proteins detected in cell lines and their association with membranes suggests that they are likely encoded by the RK-BARF0 transcript, which is predicted to contain a membrane localization signal. The proteins were also detected in protein extracts prepared from NPC biopsies and a BL biopsy but not from hairy leukoplakia, a permissive EBV infection. These results reveal that the rightward RNA transcripts from the BamHI A region of EBV encode one or more proteins that are expressed in latently infected cells and in tumor tissue.