Biochemical characterization of cell-associated and extracellular products of the Friend spleen focus-forming virus env gene.
The mature product of the env gene of Friend spleen focus-forming viruses (F-SFFV) is efficiently released from both leukemia cells and infected fibroblasts. Analyses of the kinetics of env protein synthesis and secretion in NRK cells infected with the Lilly-Steeves strain of SFFVp indicated that this product, gp65, was formed rapidly and remained stably associated with cells for up to 4 hr, at which point it was first detected in supernatant medium. By 12 hr after synthesis, greater than 95% of gp65 was found extracellularly. The release of this component was effectively blocked by 10 mM 1-deoxynojirimycin, an inhibitor of oligosaccharide processing, demonstrating a requirement for processing of high mannose precursor oligosaccharides in the secretion of gp65. Similar oligosaccharide substituents were found on cell-associated and extracellular forms of gp65. Enzymatic deglycosylation experiments demonstrated that in addition to the predicted four N-linked oligosaccharides, gp65 contains O-linked carbohydrates which are resistant to the action of peptide N-Glycanase F, but sensitive to neuraminidase and O-Glycanase. These structures may be related to O-linked oligosaccharides previously found on the env gene products of murine leukemia viruses. Comparison of the sizes of the deglycosylated forms of cell-associated and supernatant gp65 demonstrated that the extracellular molecules are approximately 3 kDa smaller than the cell-associated components. These data suggest the involvement of proteolysis at a C-terminal site in the release of gp65 from the plasma membrane.
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Laboratory of Retroviral Biology, Public Health Research Institute, New York, New York 10016.
Honnen
W J
WJ
D002462:Cell Membrane / Q000378:metabolism; D002999:Clone Cells; D005347:Fibroblasts; D015686:Gene Products, env / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D015752:Genes, env; D006026:Glycoside Hydrolases / Q000378:metabolism; D007700:Kinetics; D009052:Leukemia Virus, Murine / Q000235:genetics*; D009844:Oligosaccharides / Q000032:analysis; D011499:Protein Processing, Post-Translational; D015531:Radioimmunoprecipitation Assay; D013155:Spleen Focus-Forming Viruses / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure
D015686:Gene Products, env; D009844:Oligosaccharides; D006026:Glycoside Hydrolases