Effects of glucosamine, 2-deoxyglucose, and tunicamycin on glycosylation, sulfation, and assembly of influenza viral proteins

Abstract

The effects of glucosamine, 2-deoxy-D-glucose (2-DG), and tunicamycin (TM) on influenza viral glycoproteins was investigated. The electrophoretic mobility of the hemagglutinin (HA) polypeptide progressively increased with increasing concentration of glucosamine, accompanied by a decrease in the incorporation of [ 3H]fucose into the glycoprotein. However, a significant amount of the label was detected in association with HA even when cells were treated with glucosamine concentrations as high as 40 m M In this case, the peak of [ 3H]fucose label associated with HA was distinct from HA 0 (HA in which glycosylation is maximally inhibited by a given inhibitor) 2 detected with labeled amino acid precursors; the former showed slower electrophoretic mobility than the latter, indicating that HA glycoproteins produced in the presence of glucosamine are heterogeneous with respect to the extent of glycosylation. Analysis of the smooth cytoplasmic membrane fraction showed that a significant amount of 35SO 4 −2 was also incorporated into HA polypeptides synthesized in cells treated with 40 m M glucosamine; the peak of 35S label coincided with that of [ 3H]fucose label rather than HA 0. When cells were treated with increasing concentrations of 2-DG, the incorporation of 35SO 4 −2 2 into HA decreased in parallel with that of [ 3H]glucosamine, and no incorporation of either label was detected in cells treated with 10 m M 2-DG. TM completely inhibited the incorporation of [ 3H]glucosamine into viral glycoproteins at a concentration as low as 0.29 μ M. Although HA 0 was not resolved in whole cells treated with TM, it was clearly resolved in isolated smooth membrane fractions. No 35S0 4 −2 label was detected in the HA 0 protein. The presence of HA 0 in smooth membranes of TM-treated cells suggests that neither glycosylation nor sulfation is essential for membrane association or migration of HA from rough to smooth membranes. It also appears that such modifications of HA are not required for integration of the protein into plasma membranes since HA 0 was detected in plasma membranes isolated from both 2-DG and TM-treated cells. Formation of virus particles was observed in the presence of 2-DG, although the yield was markedly reduced. Such particles were found to possess aberrant glycoproteins, and their hemagglutinating activity was significantly lower than that of control virions. Particles without hemagglutinating activity were also produced by TM-treated cells. Analysis of polypeptides of these particles showed that they contain unglycosylated proteins but lack any detectable glycoproteins.