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. 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...

Induction of Antibodies Specific for Gp41 of HIV-1 by Gene Gun DNA Vaccination

“…Other authors had shown an interaction between MPER and FPPR during infection [18][19][20][21][22]. These findings suggested that the interaction between the MPER and the FPPR during infection may generate a conformation able to induce 2F5/4E10-like neutralising antibodies.…”

“…There may be several reasons for the failure of many of the attempts to induce 2F5/4E10-like neutralising antibodies including our new approach: First, although it has been shown, that peptides derived from the FPPR increase the binding of mAb 2F5 to its epitope in the MPER/ E2 domain [14] and there is an interaction between MPER and FFPPR [18][19][20][21][22], it is still unknown whether this interaction is also required for the induction of 2F5/4E10-like broadly neutralising antibodies. Second, it remains unclear, whether the DNA immunisation generated the correct membrane associated context.…”

“…Interaction of both antibodies with lipids, their long hydrophobic CDR3 loop and cross-reactivity with cardiolipin have been discussed controversially as possible reasons for the failure of all previous immunisation attempts [15][16][17]. Since there is evidence for intramolecular interactions between the MPER and the FPPR during infection and neutralisation [18][19][20][21][22] and a peptide corresponding to the sequence of the FPPR has been shown to increase binding of mAb 2F5 to its epitope [14], it was suggested that the FPPR may be required to generate a conformation which will be able to induce broadly neutralising antibodies of the 2F5 and 4E10.…”

The Membrane-Proximal External Region of the Human Immunodeficiency Virus Type 1 Envelope: Dominant Site of Antibody Neutralization and Target for Vaccine Design

“…The role of the MPER in fusion is further supported by the ability of three bNt MAbs (2F5, 4E10, and Z13) to recognize epitopes in this region; thus binding to the MPER by these MAbs blocks infection, presumably by interfering with one of the critical steps required for viral entry. A recent study evaluated the crucial role of the FP-proximal PR and the MPER during HIV-1 infection using two different modes of viral transmission: cell-to-cell and virus-to-cell fusion (13). Interestingly, Ala substitution of Trp 666 , Trp 672 , Phe 673 , and Ile 675 in the MPER reduced viral entry potential by ϳ120-fold without affecting cell-to-cell fusion.…”

Immunising with the transmembrane envelope proteins of different retroviruses including HIV-1

“…[41][42][43][44][45] In addition we had shown that the binding of 2F5 is enhanced when in addition to the peptide containing its epitope a peptide corresponding to the FPPR is present, suggesting that the interaction between both peptides induces a conformation allowing a better binding of 2F5. 46 Whether this conformation is also required to induce broadly neutralizing antibodies remains unclear.…”