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Biosynthesis, structure, and biological activities of envelope protein gp65 of murine coronavirus
K Yokomori1, N La Monica, S Makino
1Department of Microbiology, University of Southern California, School of Medicine, Los Angeles 90033.
Abstract:
We have previously shown that gp65 (E3) is a virion structural protein which varies widely in quantity among different strains of mouse hepatitis virus (MHV). In this study, the biosynthetic pathway and possible biological activities of this protein were examined. The glycosylation of gp65 in virus-infected cells was inhibited by tunicamycin but not by monensin, suggesting that it contains an N-glycosidic linkage. Glycosylation is cotranslational and appears to be complete before the glycoprotein reaches the Golgi complex. Pulse-chase experiments showed that this protein decreased in size after 30 min of chase, suggesting that the carbohydrate chains of gp65 undergo trimming during its transport across the Golgi. This interpretation is supported by the endoglycosidase treatment of gp65, which showed that the peptide backbone of gp65 did not decrease in size after pulse-chase periods. This maturation pathway is distinct from that of the E1 or E2 glycoproteins. Partial endoglycosidase treatment indicated that gp65 contains 9 to 10 carbohydrate side chains; thus, almost all of the potential glycosylation sites of gp65 were glycosylated. In vitro translation studies coupled with protease digestion suggest that gp65 is an integral membrane protein. The presence of gp65 in the virion is correlated with the presence of an acetylesterase activity. No hemagglutinin activity was detected.
Insights
Mouse hepatitis virus (MHV) structural protein gp65 (E3) undergoes N-linked glycosylation and carbohydrate trimming in the Golgi apparatus. Its presence correlates with acetylesterase activity, but not hemagglutinin activity.
Area of Science:
- Virology
- Molecular Biology
- Glycobiology
Background:
- Mouse hepatitis virus (MHV) is a significant pathogen in mice.
- gp65 (E3) is a virion structural protein with variable expression levels across MHV strains.
Purpose of the Study:
- To elucidate the biosynthetic pathway of the MHV gp65 (E3) protein.
- To investigate the biological activities associated with gp65 (E3).
Main Methods:
- Inhibition of glycosylation using tunicamycin and monensin.
- Pulse-chase experiments to track protein maturation.
- Endoglycosidase treatment to analyze carbohydrate chains.
- In vitro translation coupled with protease digestion.
- Correlation analysis between gp65 presence and enzyme activity.
Main Results:
- Glycosylation of gp65 is N-linked, cotranslational, and completed before Golgi entry.
- Carbohydrate chains of gp65 are trimmed in the Golgi, distinct from E1/E2 glycoproteins.
- gp65 contains 9-10 N-linked glycosylation sites, with most being utilized.
- gp65 is an integral membrane protein.
- gp65 presence correlates with acetylesterase activity; hemagglutinin activity was absent.
Conclusions:
- The maturation pathway of gp65 (E3) involves N-linked glycosylation and subsequent carbohydrate trimming.
- gp65 (E3) is an integral membrane protein associated with MHV acetylesterase activity.