Cholesterol-dependent membrane fusion induced by the gp41 membrane-proximal external region-transmembrane domain connection suggests a mechanism for broad HIV-1 neutralization

Abstract

The HIV-1 glycoprotein 41 promotes fusion of the viral membrane with that of the target cell. Structural, biochemical, and biophysical studies suggest that its membrane-proximal external region (MPER) may interact with the HIV-1 membrane and induce its disruption and/or deformation during the process. However, the high cholesterol content of the envelope (ca. 40 to 50 mol%) imparts high rigidity, thereby acting against lipid bilayer restructuring. Here, based on the outcome of vesicle stability assays, all-atom molecular dynamics simulations, and atomic force microscopy observations, we propose that the conserved sequence connecting the MPER with the N-terminal residues of the transmembrane domain (TMD) is involved in HIV-1 fusion. This junction would function by inducing phospholipid protrusion and acyl-chain splay in the cholesterol-enriched rigid envelope. Supporting the functional relevance of such a mechanism, membrane fusion was inhibited by the broadly neutralizing 4E10 antibody but not by a nonneutralizing variant with the CDR-H3 loop deleted. We conclude that the MPER-TMD junction embodies an envelope-disrupting C-terminal fusion peptide that can be targeted by broadly neutralizing antibodies.

Importance: Fusion of the cholesterol-enriched viral envelope with the cell membrane marks the beginning of the infectious HIV-1 replicative cycle. Consequently, the Env glycoprotein-mediated fusion function constitutes an important clinical target for inhibitors and preventive vaccines. Antibodies 4E10 and 10E8 bind to one Env vulnerability site located at the gp41 membrane-proximal external region (MPER)-transmembrane domain (TMD) junction and block infection. These antibodies display broad viral neutralization, which underscores the conservation and functionality of the MPER-TMD region. In this work, we combined biochemical assays with molecular dynamics simulations and microscopy observations to characterize the unprecedented fusogenic activity of the MPER-TMD junction. The fact that such activity is dependent on cholesterol and inhibited by the broadly neutralizing 4E10 antibody emphasizes its physiological relevance. Discovery of this functional element adds to our understanding of the mechanisms underlying HIV-1 infection and its blocking by antibodies.

FIG 1

Proposed model for HIV-1 Env-induced membrane fusion (A) and designation of the gp41 MPER-TMD region (B). The highlighted gp41 elements are as follows: FP, fusion peptide; NHR and CHR, amino- and carboxy-terminal helical regions, respectively; MPER, membrane-proximal external region; TMD transmembrane domain; 6-HB: 6-helix bundle. In panel B, MPER-TMD sequence variability within HIV-1 clade B is displayed as a WebLogo representation (75). Nonpolar amino acids are in blue. The green box above indicates the position of the 4E10 epitope. The tick marks indicate residues facing the paratope with helical periodicity. The diagram under the sequence delimits the helical subdomains and locates positions for nonhelical junctions. Bars below the helices span the sequences covered by the overlapping peptides used in this study.

FIG 2

Fusion activity of MPER-TMD peptides as a function of membrane rigidity. Levels of fusion (lipid-mixing assay) measured after a 10-min incubation with NpreTM, CpreTM, or TMDp were plotted against the Chol mole fraction. The peptide-to-lipid molar ratio was 1:25 in all cases. Plotted values are means ± standard deviations (SD) from three experiments. Membrane order (dotted line and squares) ranged from GP values of 0.05 (most fluid) to 0.6 (most rigid), as measured in GUVs. Arrows on top mark Chol contents in the plasma membrane of virus-producing H9 and MDM cells (26) and virions (24).

FIG 3

Fusion of POPC-Chol (1:1 molar ratio) vesicles induced by NpreTM, CpreTM, and TMDp peptides. (Top) Kinetics of fusion (lipid-mixing assay). Peptides were added to vesicle suspensions at the indicated peptide-to-lipid ratios. The time of addition was 50 s (arrow). The lipid concentration was 100 μM. (Bottom) Final extents of fusion. The percentage of lipid-mixing measured after a 10 min incubation of peptides with vesicles has been plotted as a function of the peptide concentration. Values are means ± SD from three different experiments.

FIG 4

MDS of CpreTM sequence interacting with POPC-Chol (1:1 molar ratio) lipid bilayers. (A) The simulation considered 4 peptides. The snapshot was taken at 112 ns. Peptides are displayed in stick-and-ribbon format, and phospholipids and Chol are shown in a space-filling representation. Close views in the right panels illustrate polar-head group engagement (top) and acyl-chain splaying (bottom). (B) The simulation considered 12 peptides. (Left) Snapshot of CpreTM-induced disruption of POPC-Chol (1:1) bilayers (taken at 360 ns). (Right) Close view illustrating the phospholipid extraction phenomenon.

FIG 5

Structural alterations of lipid bilayers. (A) MDS of membrane surface alteration by CpreTM. (Left) Snapshot of CpreTM interacting with POPC-Chol (1:1) lipid bilayers. The peptides are displayed in space-filling representation (gray), and phospholipids and Chol are shown in semitransparent molecular surface-and-stick representation (blue and red, respectively). (Right) Phospholipid head group protrusions (1) and acyl-chain exposure (2) when the peptide is omitted. (B) AFM height images of POPC-Chol (1:1) SPBs. An untreated control sample (left) is compared with SPBs that were treated with 0.01 and 0.1 μM CpreTM (center and right, respectively). Images of CpreTM-containing samples were obtained 30 min after peptide addition. Sizes of visual fields are 4.5 by 4.5 μm. Plots below the images display the height profiles for the trajectories indicated by the white lines. (C) Interactions of NpreTM (top) and CpreTM (bottom) with POPC-Chol (1:1) and POPC-Chol (4:1) membranes, respectively. (Left) MDS snapshots taken at 100 ns. Peptides (gray) and lipids (POPC, green; Chol, red) are displayed in space-filling representation. (Right) AFM height images (conditions were as described for panel B).

FIG 6

Fusion inhibition by 4E10 MAb. (A) (Left) Accessibility of the 4E10/10E8 epitope region (in green) on the membrane surface according to MDS of CpreTM interacting with POPC-Chol (1:1) bilayers. Side chain of Lys-683 is displayed in orange. Phospholipids (stick representation) and Chol (space-filling representation) are in blue and red, respectively. The snapshot was taken at 100 ns. (Right) Docking of the 4E10 paratope into the previous structure. To create the figure, the peptide bound to Fab4E10 in the crystal structure with PDB code 2FX7 was fitted into the simulated CpreTM peptide. The CDR-H3 loop and the side chains of Trp residues within are highlighted in yellow. (B) (Left) Vesicles were primed for fusion with CpreTM (a), and after 60 s (b), they were treated with 1 (red), 2 (blue), 5 (green), or 10 (orange) μg/ml of MAb4E10. The black trace corresponds to the control in the absence of antibody. Finally, the mixture was supplemented with fluorescently labeled vesicles, and the remaining fusion activity was monitored over time (c). (Right) Vesicles were primed with Cala peptide, and the MAb effect was assessed under the same conditions.

FIG 7

Comparison of the 4E10 Fab (left) and its derived ΔLoop mutant (right). (A) Circular dichroism spectra of Fab4E10-WT and Fab Fab4E10-ΔLoop mutant. Negative absorption at 217 nm observed in both cases was consistent with adoption of a main β-structure. (B) Binding to the soluble peptide epitope. Competitive enzyme-linked immunosorbent assays (ELISAs) were performed using plates coated with CpreTM (1.4 μM). Prior to being added to the plates, Fabs were preincubated for 30 min with serial dilutions of soluble peptide-epitope (NWFDITNWLWYIK-KKK). Percentages of binding inhibition were determined in duplicate and adjusted to saturation curves. (C) Cell entry inhibition assay. Pseudoviruses were preincubated with Fab, and single cell entry events were monitored by fluorescence-activated cell sorting (FACS) after incubation with TZM-bl target cells. Means ± SD of 4 measurements from 2 independent experiments are displayed. (D) Fusion inhibition. (Left) Vesicles were primed for fusion with CpreTM (a), and after 60 s (b) they were treated with 1, 2, 5, or 10 μg/ml of Fab4E10-WT, as indicated. The thicker trace on top corresponds to the control in the absence of antibody. (Right) At the time indicated by the arrow (b), vesicles were supplemented with 5 or 20 μg/ml of Fab4E10-ΔLoop, as indicated. Conditions are otherwise as described for Fig. 6.

FIG 8

Proposed activity for the gp41 sequence covered by CpreTM peptide during HIV membrane fusion. (A) MDS snapshot displaying phospholipid extraction from the interface and Chol stacking in the opposing monolayer. (B) (Left) Cartoon representation of lipids displayed in panel A. (Right) Chol molecules have been omitted to highlight their possible effects on the phospholipid matrix. Chol may help promote phospholipid extraction by stabilizing negative curvature of the monolayer (1) and/or by filling interlamellar voids (2). (C) Functioning of the section covered by the CpreTM sequence in the context of Env-mediated fusion. (Left) In the prefusion state (I in Fig. 1A), the CpreTM region may be concealed at the base of the ectodomain and inserted parallel to the membrane plane. (Center) Possible role in a prehairpin configuration (state II in Fig. 1A). Orienting conserved aromatic residues at the MPER-TMD junction perpendicular to the membrane plane may promote phospholipid extraction. The model supports the possibility of 4E10 binding to lipids concomitantly to the protein epitope. (Right) Closure of the hairpin might couple disruption of the viral membrane to fusion. Phospholipid molecules whose acyl chains are splayed may establish a lipid connection between contacting bilayers. The ectodomain is proposed to bend again at the ⁶⁷¹NWFD⁶⁷⁴ elbow in this state.