Detection and characterization of a glycoprotein encoded by the mouse mammary tumor virus long terminal repeat gene

Mouse mammary tumor virus (MMTV) is a retrovirus that causes mammary tumors in susceptible mice. MMTV contains a unique open reading frame (ORF) in the unique 3' region of the proviral long terminal repeat (LTR) with the potential to encode a 36-kDa protein. However, the ORF gene product has not been detected in murine mammary tissues or cell lines. We utilized the baculovirus expression vector system to generate large amounts of the ORF protein. Putative ORF gene products of 36 and 45 kDa were detected as unique proteins in extracts of insect cells infected with recombinant baculovirus (LTR-ORF BV), and the identities of these proteins as viral gene products were confirmed immunologically. Antipeptide antisera were generated in rabbits against peptides chosen from computer-predicted hydrophilic regions of the ORF coding sequence. These antisera reacted specifically by immunoprecipitation and by immunoblot with the proteins expressed in LTR-ORF BV-infected insect cells, as well as with MMTV LTR ORF in vitro translation products. Polyclonal antisera were raised against two putative ORF protein species partially purified from insect cells. These sera specifically immunoprecipitated viral protein products translated in vitro. In vitro translation of MMTV LTR ORF transcripts in the presence of canine pancreatic microsomal membranes generated a higher-molecular-weight ORF gene product, indicating that the ORF protein is modified by N-linked glycosylation. This glycosylated ORF product comigrated with the larger ORF protein species produced in infected insect cells. The gp45 product was metabolically labeled with [3H] mannose, [3H] galactose, and [3H] N-acetyl-D-glucosamine in insect cells, whereas this incorporation was inhibited in the presence of tunicamycin. Digestion of gp45 with endoglycosidase H yielded the lower-molecular-weight ORF protein p36. These observations suggest that the ORF glycoprotein contains hybrid N-linked oligosaccharides. Demonstration of the modified nature of the ORF gene product will facilitate characterization of ORF protein expression in murine tissues.