title: Mutation-directed chemical cross-linking of human immunodeficiency virus type 1 gp41 oligomers.
abstract: The human immunodeficiency virus type 1 transmembrane protein gp41 oligomer anchors the attachment protein, gp120, to the viral envelope and mediates viral envelope-cell membrane fusion following gp120-CD4 receptor-chemokine coreceptor binding. We have used mutation-directed chemical cross-linking with bis(sulfosuccinimidyl)suberate (BS3) to investigate the architecture of the gp41 oligomer. Treatment of gp41 with BS3 generates a ladder of four bands on sodium dodecyl sulfate-polyacrylamide gels, corresponding to monomers, dimers, trimers, and tetramers. By systematically replacing gp41 lysines with arginine and determining the mutant gp41 cross-linking pattern, we observed that gp41 N termini are cross-linked. Lysine 678, which is close to the transmembrane sequence, was readily cross-linked to Lys-678 on other monomers within the oligomeric structure. This arrangement appears to be facilitated by the close packing of membrane-anchoring sequences, since the efficiency of assembly of heterooligomers between wild-type and mutant Env proteins is improved more than twofold if the mutant contains the membrane-anchoring sequence. We also detected close contacts between Lys-596 and Lys-612 in the disulfide-bonded loop/glycan cluster of one monomer and lysines in the N-terminal amphipathic alpha-helical oligomerization domain (Lys-569 and Lys-583) and C-terminal alpha-helical sequence (Lys-650 and Lys-660) of adjacent monomers. Precursor-processing efficiency, gp120-gp41 association, soluble recombinant CD4-induced shedding of gp120 from cell surface gp41, and acquisition of gp41 ectodomain conformational antibody epitopes were unaffected by the substitutions. However, the syncytium-forming function was most dependent on the conserved Lys-569 in the N-terminal alpha-helix. These results indicate that gp160-derived gp41 expressed in mammalian cells is a tetramer and provide information about the juxtaposition of gp41 structural elements within the oligomer.
authors.affiliation: St. Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia.
authors.forename: T L
authors.initials: TL
authors.lastname: McInerney
authors.forename: W
authors.initials: W
authors.lastname: El Ahmar
authors.forename: B E
authors.initials: BE
authors.lastname: Kemp
authors.forename: P
authors.initials: P
authors.lastname: Poumbourios
mesh_terms: D001665:Binding Sites / Q000235:genetics; D003432:Cross-Linking Reagents; D015699:HIV Envelope Protein gp120 / Q000235:genetics / Q000378:metabolism; D015700:HIV Envelope Protein gp41 / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D009154:Mutation
chemical_list: D003432:Cross-Linking Reagents; D015699:HIV Envelope Protein gp120; D015700:HIV Envelope Protein gp41