title: Effects of mutations in glycosylation sites and disulphide bonds on processing, CD4-binding and fusion activity of human immunodeficiency virus envelope glycoproteins.
abstract: Site-directed mutagenesis was used to study the biological significance of a disulphide bridge and two N-linked oligosaccharides in the CD4-binding region of the envelope glycoproteins of human immunodeficiency virus type 1. Mutagenesis was performed in a phage M13 system at sites corresponding to the cysteine residue (amino acid 402) and the asparagine residues (390 and 447) of the env gene. The mutated env gene was inserted into a recombinant vaccinia virus under the control of the vaccinia virus 7.5K promoter and the expression of mutated env proteins was analysed by SDS-PAGE, a conventional indirect immunofluorescence assay and by a fluorescence-activated cell sorter. Cysteine 402 was found to be essential for the specific cleavage of gp160 into gp120 and gp41, and for intracellular transport of the protein to the cell surface. CD4-binding and syncytium formation assays demonstrated that the disulphide bridge of cysteine 402 stabilized a conformation essential for receptor binding as well as syncytium formation by CD4+ cells. No altered biological activity compared to that of the wild-type proteins could be detected for the mutant proteins lacking the N-glycosylation sites. These data show that the two conserved glycans attached to asparagine residues 390 and 447 do not play any active role in the formation of the disulphide bridge involving cysteine 402 or in the maintenance of an active conformation of the protein, despite their location within the functionally important CD4-binding region.
authors.affiliation: Department of Clinical Virology, University of Göteborg, Sweden.
authors.forename: A
authors.initials: A
authors.lastname: Bolmstedt
authors.forename: A
authors.initials: A
authors.lastname: Hemming
authors.forename: P
authors.initials: P
authors.lastname: Flodby
authors.forename: P
authors.initials: P
authors.lastname: Berntsson
authors.forename: B
authors.initials: B
authors.lastname: Travis
authors.forename: J P
authors.initials: JP
authors.lastname: Lin
authors.forename: J
authors.initials: J
authors.lastname: Ledbetter
authors.forename: T
authors.initials: T
authors.lastname: Tsu
authors.forename: H
authors.initials: H
authors.lastname: Wigzell
authors.forename: S L
authors.initials: SL
authors.lastname: Hu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015704:CD4 Antigens / Q000502:physiology*; D002460:Cell Line; D003545:Cysteine / Q000032:analysis; D004220:Disulfides; D005814:Genes, Viral; D006031:Glycosylation; D015497:HIV-1 / Q000235:genetics* / Q000276:immunology; D006367:HeLa Cells / Q000166:cytology / Q000276:immunology / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
chemical_list: D015704:CD4 Antigens; D004220:Disulfides; D014759:Viral Envelope Proteins; D003545:Cysteine