Incomplete complex oligosaccharides in semliki forest virus envelope proteins arrested within the cell in the presence of monensin

Abstract

Semliki Forest virus-infected chicken embryo fibroblasts were treated with 10 μ m-monensin at 3.5 hours after infection. The release of infectious virus ceased within 30 minutes after the addition of the drug. Accumulation of virus envelope proteins on the plasma membrane was arrested also within 30 minutes, as revealed by a radioimmune assay. In the presence of monensin, the envelope protein precursor p62, labeled with [ 35S]methionine for 15 minutes starting at four hours post-infection, was efficiently cleaved to E2 and E3 during a chase period of 105 minutes. Electron micrographs taken six hours post-infection revealed intracellular maturation of virions into dilated vesicles in the area of the Golgi complex. Numerous nucleocapsids were attached to the cytoplasmic side of the vesicle membranes. Envelope protein E3 labeled for 30 minutes at 4 hours post-infection with [ 3H]mannose in the presence of monensin was isolated after a 90-minute chase period and digested extensively with Pronase. The glycopeptides were studied using successive digestions with specific exoglycosidases and endoglycosidases. The products were analyzed by gel filtration, lectin affinity and paper chromatography. In monensin-treated cells, the intracellular E3 protein, which in the virion has only complex glycans, had three alternate categories of glycans: (1) complex glycans with fewer sialic acid residues than in the glycans of virion E3; (2) high mannose type glycans; and (3) a mixture of intermediate glycans with novel structures.