title: Structure and cell surface maturation of the attachment glycoprotein of human respiratory syncytial virus in a cell line deficient in O glycosylation.
abstract: The synthesis of the extensively O-glycosylated attachment protein, G, of human respiratory syncytial virus and its expression on the cell surface were examined in a mutant Chinese hamster ovary (CHO) cell line, ldlD, which has a defect in protein O glycosylation. These cells, used in conjunction with an inhibitor of N-linked oligosaccharide synthesis, can be used to establish conditions in which no carbohydrate addition occurs or in which either N-linked or O-linked carbohydrate addition occurs exclusively. A recombinant vaccinia virus expression vector for the G protein was constructed which, as well as containing the human respiratory syncytial virus G gene, contained a portion of the cowpox virus genome that circumvents the normal host range restriction of vaccinia virus in CHO cells. The recombinant vector expressed high levels of G protein in both mutant ldlD and wild-type CHO cells. Several immature forms of the G protein were identified that contained exclusively N-linked or O-linked oligosaccharide side chains. Metabolic pulse-chase studies indicated that the pathway of maturation for the G protein proceeds from synthesis of the 32-kilodalton (kDa) polypeptide accompanied by cotranslational attachment of high-mannose N-linked sugars to form an intermediate with an apparent mass of 45 kDa. This step is followed by the Golgi-associated conversion of the N-linked sugars to the complex type and the completion of the O-linked oligosaccharides to achieve the mature 90-kDa form of G. Maturation from the 45-kDa N-linked form to the mature 90-kDa form occurred only in the presence of O-linked sugar addition, confirming that O-linked oligosaccharides constitute a significant proportion of the mass of the mature G protein. In the absence of O glycosylation, forms of G bearing galactose-deficient truncated N-linked and fully mature N-linked oligosaccharides were observed. The effects of N- and O-linked sugar addition on the transport of G to the cell surface were measured. Indirect immunofluorescence and flow cytometry showed that G protein could be expressed on the cell surface in the absence of either O glycosylation or N glycosylation. However, cell surface expression of G lacking both N- and O-linked oligosaccharides was severely depressed.
authors.affiliation: Department of Microbiology, University of Alabama Medical School, Birmingham 35294.
authors.forename: G W
authors.initials: GW
authors.lastname: Wertz
authors.forename: M
authors.initials: M
authors.lastname: Krieger
authors.forename: L A
authors.initials: LA
authors.lastname: Ball
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000096:biosynthesis* / Q000235:genetics; D002240:Carbohydrate Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D015707:HN Protein; D009154:Mutation; D011499:Protein Processing, Post-Translational; D012136:Respiratory Syncytial Viruses / Q000235:genetics / Q000502:physiology*; D014759:Viral Envelope Proteins; D014764:Viral Proteins
chemical_list: D000956:Antigens, Viral; D015707:HN Protein; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C116204:attachment protein G