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Brefeldin A and monensin arrest cell surface expression of membrane glycoproteins and release of rubella virus
1Department of Pathology, University of British Columbia, Research Centre, Vancouver, Canada.
Abstract:
The maturation of rubella virus (RV) glycoproteins E2 and E1 was examined by using brefeldin A (BFA) and monensin. BFA, which induces the rapid redistribution of Golgi enzymes residing in the Golgi complex into the endoplasmic reticulum (ER), was used to locate the intracellular site for the modification of carbohydrate side-chains on RV E1 and E2 proteins. The monovalent ionophore monensin, which inhibits intracellular transport of proteins through the ER-Golgi complex, was used to block the transport of E1 and E2 glycoproteins through the Golgi complex. BFA and monensin effectively blocked the cell surface expression of RV E2 and E1 proteins, secretion of an anchor-free form of E2 and budding of RV from the plasma membrane. For O-linked glycosylation, addition of N-acetylgalactosamine and galactose to E2 protein was found to take place in the medial to the trans Golgi. A dramatic change in the intracellular distribution of RV structural proteins was observed when transfected COS cells were treated with BFA or monensin, although the proteolytic processing of RV structural protein precursor was not affected. In the presence of BFA or monensin, virus release from infected Vero cells was only 0.1% of the intracellular virus, and the intracellular virus titre decreased as well. Our results suggest that O-linked glycosylation on the E2 protein occurred in the post-ER region and the transport of RV structural proteins to the Golgi complex and post-Golgi compartment may be a rate-limiting step in RV assembly and budding.
Insights
Rubella virus (RV) glycoprotein maturation involves O-linked glycosylation in the Golgi complex. Transport to the Golgi is crucial for RV assembly and budding, as blocking it significantly reduces virus release.
Area of Science:
- Virology
- Cell Biology
- Glycobiology
Background:
- Rubella virus (RV) is an important human pathogen.
- Understanding the maturation of RV glycoproteins is essential for developing antiviral strategies.
- The roles of the endoplasmic reticulum (ER) and Golgi complex in RV glycoprotein processing are not fully understood.
Purpose of the Study:
- To investigate the intracellular sites of rubella virus (RV) glycoprotein E2 and E1 maturation.
- To determine the impact of brefeldin A (BFA) and monensin on RV glycoprotein processing and virus assembly.
- To elucidate the role of O-linked glycosylation in RV pathogenesis.
Main Methods:
- Treatment of infected cells with brefeldin A (BFA) to redistribute Golgi enzymes to the ER.
- Treatment of infected cells with monensin to inhibit ER-Golgi transport.
- Analysis of RV glycoprotein E1 and E2 maturation and cell surface expression.
- Quantification of RV release and intracellular virus titers.
Main Results:
- BFA and monensin treatment blocked cell surface expression of RV E1 and E2 glycoproteins.
- O-linked glycosylation of E2 protein occurred in the medial to trans Golgi.
- Virus release was significantly inhibited (0.1% of intracellular virus) in the presence of BFA or monensin.
- Proteolytic processing of RV structural protein precursor was unaffected by BFA or monensin.
Conclusions:
- O-linked glycosylation of rubella virus E2 protein occurs in the post-ER region.
- Transport of RV structural proteins through the Golgi complex and post-Golgi compartments is a rate-limiting step for RV assembly and budding.
- Inhibition of ER-Golgi transport severely impairs rubella virus release.