Functional links between the fusion peptide-proximal polar segment and membrane-proximal region of human immunodeficiency virus gp41 in distinct phases of membrane fusion.
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.
Bellamy-McIntyre
Anna K
AK
Macfarlane Burnet Institute for Medical Research and Public Health, Prahran, Victoria 3004, Australia.
Lay
Chan-Sien
CS
Baär
Séverine
S
Maerz
Anne L
AL
Talbo
Gert H
GH
Drummer
Heidi E
HE
Poumbourios
Pantelis
P
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
D006023:Glycoproteins; D015699:HIV Envelope Protein gp120; D015700:HIV Envelope Protein gp41; D011993:Recombinant Fusion Proteins; D007930:Leucine; D000409:Alanine