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 ca rcinoma (NPC) tissues. These RNAs have also been detected at low level s in Burkitt's lymphoma (BL) cell lines and Epstein-Barr virus (EBV)-t ransformed lymphocytes. Sequence analyses of clones from a cDNA librar y derived from the C15 NPC tumor indicated that the primary transcript s are differentially spliced, giving rise to a family of related trans cripts, all of which encompass the BARF0 open reading frame (ORF) at t he 3' end of the transcripts. One cDNA was identified that extended th e 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 seq uence 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. T he 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 membr ane associated. The sizes of the proteins detected in cell lines and t heir association with membranes suggests that they are likely encoded by the RK-BARF0 transcript, which is predicted to contain a membrane l ocalization signal. The proteins were also detected in protein extract s prepared from NPC biopsies and a BL biopsy but not from hairy leukop lakia, a permissive EBV infection. These results reveal that the right ward RNA transcripts from the BamHI A region of EBV encode one or more proteins that are expressed in latently infected cells and in tumor t issue.