Regulated expression of Sindbis and vesicular stomatitis virus glycoproteins in Saccharomyces cerevisiae.

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RESUMO

cDNAs encoding either the structural proteins (capsid and glycoproteins E1 and E2) of Sindbis virus or the glycoprotein of vesicular stomatitis virus (VSV) were fused to the Saccharomyces cerevisiae galactokinase gene (GAL1) promoter and inserted into a yeast shuttle vector. After addition of galactose to yeast transformed with this vector, 2.5-3% of total yeast protein synthesis was detected as virus proteins by specific anti-virus protein antibodies. In cells containing the Sindbis virus structural genes, the virus capsid protein was effectively released from the nascent polypeptide and two endoglycosidase H-sensitive glycoproteins were produced. One of these was identical in its gel mobility to E1 and the other appeared to be p62, a precursor to E2. A low level of E1 protein was detected on the cell's surface membranes. A single molecular weight species of glycosylated VSV glycoprotein was produced and half of the total protein could be detected at the surface membranes of yeast. Addition of long mannose chains and acylation of the virus proteins with fatty acids were not observed. Formation of virus proteins was also examined in yeast secretory mutants; one of these (sec53) failed to glycosylate the virus proteins.

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