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. The envelope glycoprotein complex (Env) 4 of human immunodeficiency virus type 1 (HIV-1) comprises a trimer of receptor binding gp120 subunits in non-covalent association with a trimer of transmembrane gp41 subunits on the surface of infected cells and virions. Viral entry is initiated when gp120 binds to cell-surface CD4 molecules, inducing structural changes within the gp120 core domain, and leading to the formation of the binding site for the chemokine co-receptors, CCR5 and/or CXCR4 (1-4). gp120 comprises 5 variable loops (V1-V5) that exist outside the gp120 core; V3, and to a lesser degree V1 and V2, contribute to chemokine receptor binding specificity (5-7). The sequential binding of gp120 to CD4 and chemokine receptor triggers the refolding of gp41 into a trimer of hairpins, which mediates membrane fusion (8, 9). gp41 is a class I fusion glycoprotein, being structurally homologous to the fusion glycoproteins of other retroviruses, orthomyxoviruses, paramyxoviruses, filoviruses, and coronaviruses. The gp41 ectodomain is comprised of an N-terminal fusion peptide, connected through a flexible polar segment to a coiled coil-forming amphipathic ␣-helix (N-helix), a centrally located disulfide-bonded loop, a C-terminal amphipathic ␣-helix (C-helix), and a membrane-proximal tryptophan-rich region (MPR) (10 -16). The ectodomain is anchored to the viral envelope by a C-terminally located transmembrane domain (TMD), which precedes an ϳ150-residue cytoplasmic domain.The majority of the gp41 ectodomain appears to be buried by the gp120 trimer. This model for gp41 in the context of prefusion Env is based on the findings that the epitopes of monoclonal antibodies (mAbs) encompassing the fusion peptide and polar segment (residues 521-538), disulfide bonded region (579 -613), and C-helix (644 -663), are largely occluded in pre- * This work was supported by the National Health and Medical ResearchCouncil of Australia Grants 296200 and 345413, American Foundation for AIDS Research Grant 106610-36-RGNT, and Sidaction. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 4 The abbreviations used are: Env, envelope glycoprotein; HIV-1, human immunodeficiency virus type 1; N-helix, N-terminal coiled coil forming ␣-helix of gp41; C-helix, C-terminal ␣-helix of gp41; MPR, membrane proximal region; TMD, transmembrane domain; mAb, monoclonal antibody; sCD4, soluble CD4; DiO, 3,3Ј-dioctadecyloxacarbocyanine perchlorate; DiI, 1,1Ј-dioctadecyl-3,3,3Ј,3Ј-tetramethylindocarbocyanine perchlorate; PBS, phosphate-buffered saline; MBP, maltose-binding protein; MALDI, matrixassisted la...