title: Stable expression of rabies virus glycoprotein in Chinese hamster ovary cells.
abstract: The rabies virus glycoprotein (G protein) has several important functions and is a major antigenic stimulus of the host immune system following rabies virus infection or vaccination. We developed a model system for studying the role of N-linked glycosylation in the intracellular transport and antigenicity of this molecule. The full-length cDNA of the G protein of the ERA strain of rabies virus was inserted into the eukaryotic shuttle vector pSG5 and then stably transfected into wild-type Chinese hamster ovary (CHO) cells and mutant CHO cell lines defective in glycosylation. Transfected wild-type CHO cells expressed the G protein (detected by immunofluorescence) on the cell surface in a manner similar to rabies virus-infected cells. The transfected wild-type CHO cells were shown by immunoprecipitation to produce a protein of 67K that comigrated with the fully glycosylated G protein isolated from virus-infected cells or purified virions. Treatment of the transfected cell lines with tunicamycin completely blocked surface expression and resulted in the intracellular accumulation of the G protein, suggesting that the presence of N-linked oligosaccharides is important for transport of this glycoprotein to the plasma membrane. The G protein cDNA was also expressed in the lectin-resistant CHO cell lines Lec 1, Lec 2 and Lec 8. In these cells initial N-linked glycosylation does occur, but later steps in processing of the oligosaccharides are blocked. In each case, the G protein was expressed on the surface of lectin-resistant CHO cells in a similar manner to expression on wild-type CHO cells. This suggests that various different N-linked oligosaccharide structures support intracellular transport of this glycoprotein. Thus, stably transfected CHO cell lines will provide a useful model system for further studies of the role of N-linked glycosylation in trafficking and antigenicity of the rabies virus G protein.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia.
authors.forename: S R
authors.initials: SR
authors.lastname: Burger
authors.forename: A T
authors.initials: AT
authors.lastname: Remaley
authors.forename: J M
authors.initials: JM
authors.lastname: Danley
authors.forename: J
authors.initials: J
authors.lastname: Moore
authors.forename: R J
authors.initials: RJ
authors.lastname: Muschel
authors.forename: W H
authors.initials: WH
authors.lastname: Wunner
authors.forename: S L
authors.initials: SL
authors.lastname: Spitalnik
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000276:immunology; D001692:Biological Transport; D015139:Blotting, Southern; D002240:Carbohydrate Sequence; D002460:Cell Line; D006224:Cricetinae; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006023:Glycoproteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D006031:Glycosylation; D037102:Lectins; D008969:Molecular Sequence Data; D009154:Mutation; D011233:Precipitin Tests; D011820:Rabies virus / Q000235:genetics*; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
chemical_list: D000956:Antigens, Viral; D006023:Glycoproteins; D037102:Lectins; D014759:Viral Envelope Proteins; D014415:Tunicamycin