Bovine interleukins 2 and 4 expressed in recombinant bovine herpesvirus 1 are biologically active secreted glycoproteins.
The open reading frames encoding bovine interleukin 2 (boIL-2) and bovine interleukin 4 (boIL-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 (ie1) enhancer-promoter element or the MCMV early 1 (e1) promoter. Madin-Darby bovine kidney cells infected with the recombinant viruses secreted boIL-2 or boIL-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 N-glycosidase F reduced the apparent molecular mass of recombinant BHV-1-expressed boIL-2 from 22 kDa to 16 kDa and that of boIL-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 boIL-2 and boIL-4 are not, or only slightly, O-glycosylated. In vitro experiments demonstrated the biological activity of recombinant BHV-1-expressed boIL-2 and boIL-4 by their ability to maintain the proliferation of bovine 4325 T cells and activated bovine B cells, respectively. In conclusion, we show that boIL-2 and boIL-4 are secreted from recombinant BHV-1-infected cells as biologically active glycoproteins.
Kühnle
G
G
Institute of Molecular and Cellular Virology, Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, Insel Riems, Germany.
Collins
R A
RA
Scott
J E
JE
Keil
G M
GM
D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D005822:Genetic Vectors; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D007242:Herpesvirus 1, Bovine / Q000235:genetics*; D007376:Interleukin-2 / Q000235:genetics* / Q000378:metabolism; D015847:Interleukin-4 / Q000235:genetics* / Q000378:metabolism; D016366:Open Reading Frames; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D002241:Carbohydrates; D006023:Glycoproteins; D007376:Interleukin-2; D011993:Recombinant Fusion Proteins; D015847:Interleukin-4