Bovine interleukins 2 and 4 expressed in recombinant bovine herpesvirus 1 are biologically active secreted glycoproteins

The open reading frames encoding bovine interleukin 2 (bolL-2) and bovine interleukin 4 (bolL-4) were integrated into the unique short segment of the genome of bovine herpesvirus 1 (BHV-1) and expressed under control of the murine cytomegalovirus (MCMV) immediate-early 1 (iel) enhancer-promoter element or the MCMV early 1 (el) promoter, Madin-Darby bovine kidney cells infected with the recombinant viruses secreted bolL-2 or bolL-4 into the culture medium. Secretion was inhibited by the presence of brefeldin A during the infection, indicating that export from the cells was dependent on a functional Golgi apparatus, Treatment of the secreted interleukins with hl-glycosidase F reduced the apparent molecular mass of recombinant BHV-1-expressed bolt-2 from 22 kDa to 16 kDa and that of bolL-4 from 20 kDa to 13 kDa, which demonstrated that both cytokines contain N-linked oligosaccharides, Digestion with neuraminidase and O-glycosidase had no detectable effect on the apparent molecular masses, suggesting that BHV-1-expressed bolt-2 and bolL-4 are not, or only slightly, O-glycosylated, In vitro experiments demonstrated the biological activity of recombinant BHV-1-expressed bolt-2 and bolL-4 by their ability to maintain the proliferation of bovine 4325 T cells and activated bovine B cells, respectively, In conclusion, we show that bolt-2 and bolL-4 are secreted from recombinant BHV-1-infected cells as biologically active glycoproteins.