title: Functional links between the fusion peptide-proximal polar segment and membrane-proximal region of human immunodeficiency virus gp41 in distinct phases of membrane fusion.
abstract: The binding of CD4 and chemokine receptors to the gp120 attachment glycoprotein of human immunodeficiency virus triggers refolding of the associated gp41 fusion glycoprotein into a trimer of hairpins with a 6-helix bundle (6HB) core. These events lead to membrane fusion and viral entry. Here, we examined the functions of the fusion peptide-proximal polar segment and membrane-proximal Trp-rich region (MPR), which are exterior to the 6HB. Alanine substitution of Trp(666), Trp(672), Phe(673), and Ile(675) in the MPR reduced entry by up to 120-fold without affecting gp120-gp41 association or cell-cell fusion. The L537A polar segment mutation led to the loss of gp120 from the gp120-gp41 complex, reduced entry by approximately 10-fold, but did not affect cell-cell fusion. Simultaneous Ala substitution of Leu(537) with Trp(666), Trp(672), Phe(673), or Ile(675) abolished entry with 50-80% reductions in cell-cell fusion. gp120-gp41 complexes of fusion-defective double mutants were resistant to soluble CD4-induced shedding of gp120, suggesting that their ability to undergo receptor-induced conformational changes was compromised. Consistent with this idea, a representative mutation, L537A/W666A, led to an approximately 80% reduction in lipophilic fluorescent dye transfer between gp120-gp41-expressing cells and receptor-expressing targets, indicating a block prior to the lipid-mixing phase. The L537A/W666A double mutation increased the chymotrypsin sensitivity of the polar segment in a trimer of hairpins model, comprising the 6HB core, the polar segment, and MPR linked N-terminally to maltose-binding protein. The data indicate that the polar segment and MPR of gp41 act synergistically in forming a fusion-competent gp120-gp41 complex and in stabilizing the membrane-interactive end of the trimer of hairpins.
authors.affiliation: Macfarlane Burnet Institute for Medical Research and Public Health, Prahran, Victoria 3004, Australia.
authors.forename: Anna K
authors.initials: AK
authors.lastname: Bellamy-McIntyre
authors.forename: Chan-Sien
authors.initials: CS
authors.lastname: Lay
authors.forename: Séverine
authors.initials: S
authors.lastname: Baär
authors.forename: Anne L
authors.initials: AL
authors.lastname: Maerz
authors.forename: Gert H
authors.initials: GH
authors.lastname: Talbo
authors.forename: Heidi E
authors.initials: HE
authors.lastname: Drummer
authors.forename: Pantelis
authors.initials: P
authors.lastname: Poumbourios
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D015496:CD4-Positive T-Lymphocytes / Q000821:virology; D002460:Cell Line; D019281:Dimerization; D005822:Genetic Vectors; D006023:Glycoproteins / Q000737:chemistry; D015699:HIV Envelope Protein gp120 / Q000737:chemistry; D015700:HIV Envelope Protein gp41 / Q000737:chemistry*; D006801:Humans; D007930:Leucine / Q000737:chemistry; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
chemical_list: D006023:Glycoproteins; D015699:HIV Envelope Protein gp120; D015700:HIV Envelope Protein gp41; D011993:Recombinant Fusion Proteins; D007930:Leucine; D000409:Alanine