Computer-assisted analysis of the Epstein-Barr virus (EBV) open reading frame BILF2 (B95-8 nucleotides 150,525 to 149,782) predicts that it codes for a membrane-bound glycoprotein. [3H]glucosamine labeling of cells infected with vaccinia virus recombinants that expressed the BILF2 open reading frame revealed several diffuse species of glycoproteins of around 80,000 and 55,000 daltons. A monoclonal antibody derived from spleens of mice immunized with EBV immunoprecipitated the EBV-derived protein made by the vaccinia virus recombinants and also precipitated a late envelope glycoprotein with a mobility of 78,000 to 55,000 from EBV-producing cells. N-Glycanase treatment of the immunoprecipitated BILF2 product from EBV-producing cells resulted in a polypeptide of 28 kilodaltons, closely agreeing with the predicted molecular mass for the unmodified BILF2 gene product. Western (immuno-) blots using recombinant infected cells as a source of antigen showed that the majority of EBV-seropositive individuals have a serum antibody response to the BILF2-encoded gp78/55.
SUMMARYEpstein-Barr virus (EBV) membrane antigen glycoproteins gp340 and gp220 are encoded by a single gene. We have inserted this gene into a bovine papillomavirus (BPV) vector and expressed gp340/220 in mammalian cells under the control of the mouse metallothionein promoter. The proteins produced were of similar Mr, showed similar antigenic specificity and were transported to the same subcellular location as the authentic gp340/220. The inclusion of heavy metal ions in the medium had no effect on the levels of gp340/220, which were approximately the same as those found in standard EBV-transformed lymphoblastoid cell lines, e.g. B95-8. Cells that expressed gp340/220 were selected by several rounds of fluorescence-activated cell sorting, but on passage they rapidly lost the ability to express this glycoprotein. In contrast to this we found that BPV-transformed cells expressing a truncated version of gp340/220 still produced it at significant levels after extended passage.
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