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: S R Burger, A T Remaley, J M Danley, J Moore, R J Muschel, W H Wunner, S L Spitalnik
• Journal: The Journal of general virology (J Gen Virol) Vol. 72 ( Pt 2) Pg. 359-67 (Feb 1991) ISSN: 0022-1317 [Print] England
• PMID: 1993876 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Antigens, Viral
• Glycoproteins
• Lectins
• Viral Envelope Proteins
• Tunicamycin

Topics

• Animals
• Antigens, Viral (immunology)
• Biological Transport
• Blotting, Southern
• Carbohydrate Sequence
• Cell Line
• Cricetinae
• Fluorescent Antibody Technique
• Gene Expression
• Glycoproteins (genetics, immunology, metabolism)
• Glycosylation
• Lectins
• Molecular Sequence Data
• Mutation
• Precipitin Tests
• Rabies virus (genetics)
• Transfection
• Tunicamycin (pharmacology)
• Viral Envelope Proteins (genetics, immunology, metabolism)