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1 Department of Pathology and Laboratory Medicine, University of Pennsylvania
and The2 Wistar Institute, Philadelphia, Pennsylvania 19104, U.S.A.
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.
Present address: Department of Laboratory Medicine, Washington University, St Louis, Missouri, 63110, U.S.A.
> Present address: National Heart Lung and Blood Institute, Bethesda, Maryland 20892, U.S.A.
Received 16 August 1990;
accepted 6 November 1990.
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