Binding of the 2F5 Monoclonal Antibody to Native and Fusion-Intermediate Forms of Human Immunodeficiency Virus Type 1 gp41: Implications for Fusion-Inducing Conformational Changes

Abstract

We investigated how the broadly neutralizing monoclonal antibody 2F5 affects the human immunodeficiency virus type 1 envelope glycoprotein as it undergoes receptor-induced conformational changes and show that 2F5 binds both native and fusion-intermediate conformations, suggesting inhibition of a late step in virus entry. We also demonstrate conformational changes in the C heptad of gp41.The envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) consists of a surface (gp120) subunit that attaches virus to target cells and a noncovalently associated transmembrane protein (gp41) (Fig. 1A) that mediates fusion between virus and target cell membranes. These subunits are synthesized as a fusion-inactive precursor (gp160) that is cleaved in the synthetic pathway to generate mature Env. gp120 binding to CD4 and chemokine receptors on the host cell triggers fusion-inducing conformational changes in gp41, leading to entry of the viral nucleocapsid into the host cell cytoplasm.The mechanism of gp41-mediated membrane fusion is not fully understood. A widely accepted model of HIV entry postulates that gp41 undergoes major refolding steps as it mediates membrane fusion (Fig. 1B, reviewed in reference 5). In this model, gp41 transitions from its native, metastable conformation as it exists on the surface of virus or infected cells to a final fusion-active conformation consisting of a thermostable six-helix bundle structure (Fig. 1C). This structure forms when two heptad repeat regions in the gp41 ectodomain self assemble into a trimer of hairpins, where the N-and C-terminal heptad repeats align in an antiparallel manner in each hairpin monomer (4,35,37). The N heptads form a triple-stranded coiled coil in the internal layer of the six-helix bundle, and the C-heptad repeat helices form the external layer. It is believed that gp120 binding to receptors loosens the association of gp120 with gp41, which in turn releases the fusion peptide at the N terminus of gp41 to insert into the target membrane. Subsequent folding of this fusion-intermediate conformation into the six-helix bundle structure probably facilitates fusion by bringing membranes together as Env adopts a more thermodynamically stable conformation.We investigated gp41 conformational changes by analyzing how two gp41 monoclonal antibodies (MAbs) with epitopes in the C heptad of gp41 bind Env under various conditions (Fig. 1A). The first antibody, 2F5, well known for being one of the few broadly neutralizing and protective HIV antibodies (20, 28), binds to a core epitope containing the residues ELDKWA at the C terminus of the C heptad (24, 27). The second antibody, D50, also binds a linear peptide from the C heptad but is not neutralizing (7). D50 was generated in mice immunized with a secreted, uncleaved, oligomeric form of Env (8). In enzyme-linked immunosorbent assay experiments (data not shown), we confirmed that both MAbs bind the same C-heptad gp41 peptide (DP-178/T20, residues 638 to 673 of the HXB2 Env) but not an overlapping C-heptad gp41 pe...

Oligomer-Specific Conformations of the Human Immunodeficiency Virus (HIV-1) gp41 Envelope Glycoprotein Ectodomain Recognized by Human Monoclonal Antibodies

“…The MAb NC-1 was generated by immunizing a mouse with a model gp41 polypeptide from the HIV-1 HXB2 strain, and was shown to bind to the gp41 six-helical bundle of several CXCR4-tropic HIV-1 strains. 48,50 This MAb may be less useful for studying CCR5-tropic HIV-1 envelope glycoproteins, as we found that MAb NC-1 did not detectably interact with the CCR5-using envelope glycoproteins examined in this study.…”

A fusion-intermediate state of HIV-1 gp41 targeted by broadly neutralizing antibodies

“…Several reports have provided evidence that 2F5 may target the fusion-intermediate conformation of gp41 (28,29), but previous attempts to mimic the intermediate state have been limited largely to constructs containing only the HR1 and HR2 fragments (9), which could not be used to resolve the issue. In contrast, our gp41-inter constructs contain nearly the full-length gp41 ectodomain, including the full epitopes for 2F5 and 4E10.…”

Immunogenicity of recombinant human immunodeficiency virus type 1-like particles expressing gp41 derivatives in a pre-fusion state

“…Given that mAb 2F5 favorably binds to its epitope in native gp160 and/or transient fusion intermediate state of gp41 [48][49][50], we have constructed three different gp41 variants in a prefusion state and presented these target epitopes in the MPER on the surface of HIV VLP. We report here an immunogenicity study of the VLP/ gp41 variants and the attendant complexities of epitope display and immunodominance observed.…”