Related Experiment Videos
Structure and function of rabies virus glycoprotein
Abstract:
Of the three major proteins associated with the rabies virus membrane, only the glycoprotein (G-protein) was found to be located on the external surface of the viral membrane. A minor glycoprotein (gp 50) detected by SDS-polyacrylamide gel electrophoresis (PAGE) of rabies virus appeared to be a breakdown product of the G-protein. Glycoprotein prepared by treatment of rabies virus with Triton X100 and purified by isoelectric focusing was found to be homogeneous with respect to size and isoelectric point. The apparent molecular weight of this component isolated under nondenaturing conditions is approximately 400,000 daltons. The same material analyzed by PAGE was found to be comprised solely of polypeptide chains of the G-protein (MW 80,000 daltons). Therefore, the Triton X100-released material represents homopolymers of the G-protein. The purified rabies virus glycoprotein (G) is only structural protein of the virus that induces the formation of virus-neutralizing antibodies and which confers immunity to animals. The total protective activity of the virus was recovered in the purified G protein preparation. The protective activity of G protein increased with purification : 9 ng of G protein was required to protect 50% of the mice as compared to 1.63 microgram of the virus. The number of oligosaccharide side chains on rabies virus glycoprotein was investigated. Analysis of glycopeptides obtained by protease digestion of desialated glycoprotein revealed three discrete glycopeptides. Comparison of the protease digestion products from desialated and from untreated glycoprotein indicated a heterogeneity among the glycopeptides in the sialic acid content. Two major tryptic glycopeptides were isolated from desialated rabies virus glycoprotein and were analyzed after protease digestion; one contained two oligosaccharide side chains and the other contained a single oligosaccharide side chain.
Insights
The rabies virus glycoprotein (G-protein) is the sole external viral protein and induces virus-neutralizing antibodies. Purified G-protein confers immunity, with its protective activity increasing significantly upon purification.
Area of Science:
- Virology
- Immunology
- Structural Biology
Background:
- The rabies virus membrane contains three major proteins, with only the glycoprotein (G-protein) exposed externally.
- A minor glycoprotein (gp 50) is likely a degradation product of the G-protein.
Purpose of the Study:
- To characterize the rabies virus G-protein.
- To investigate its role in inducing virus-neutralizing antibodies and conferring immunity.
Main Methods:
- Rabies virus treatment with Triton X100 and purification by isoelectric focusing.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for molecular weight analysis.
- Protease digestion and glycopeptide analysis to study oligosaccharide side chains.
Main Results:
- Purified G-protein exists as homopolymers (approx. 400,000 daltons) composed of 80,000 dalton polypeptide chains.
- The G-protein is the only structural protein responsible for inducing virus-neutralizing antibodies and conferring immunity.
- Protective activity was concentrated in the purified G-protein, requiring significantly less G-protein than whole virus for protection.
- Analysis revealed three distinct glycopeptides, with heterogeneity in sialic acid content and isolation of two major tryptic glycopeptides with one or two oligosaccharide side chains.
Conclusions:
- The rabies virus G-protein is a key immunogenic component responsible for protective immunity.
- Its purification enhances its protective efficacy, highlighting its potential as a vaccine candidate.
- Structural analysis of the G-protein's glycosylation provides insights into its function.