Abstract
The roles of glucose and mannose trimming in the maturation of Sindbis virus in BHK cells have been investigated using inhibitors of glycoprotein oligosaccharide processing. In the presence of the glucosidase inhibitor N-methyl-1-deoxynojirimycin the viral glycoproteins were equipped with oligosaccharides of the composition Glc3Man8,9(GlcNAc)2 and the yield of virus in the extracellular medium was reduced as a result of a block in the proteolytic cleavage of the precursor (pE2) of the E2 viral envelope glycoprotein. The mannosidase I inhibitor 1-deoxymannojirimycin (dMM) also inhibited the appearance of virus in the medium and the oligosaccharides on the viral glycoproteins had the composition Man9(GlcNAc)2. However, pE2 was cleaved to E2 under these conditions, and it was found that when the yield of virus from the cells and medium together was considered, there was no difference between untreated and dMM-treated cultures, suggesting the presence of intracellular virus particles in the dMM-treated cultures. When examined by electron microscopy, the dMM-treated cultures were found to contain intracellular virus particles. In addition, nucleocapsids were found lining intracellular membranes. These observations taken together with the plaque test data intimate that Sindbis virus preferentially buds from internal membranes in BHK cells treated with dMM. The results confirm the essential role of glucose trimming in the Sindbis virus-BHK cell system and suggest that the initial stages of mannose removal may be important too.