Membrane-proximal external HIV-1 gp41 motif adapted for destabilizing the highly rigid viral envelope

Abstract

Electron microscopy structural determinations suggest that the membrane-proximal external region (MPER) of glycoprotein 41 (gp41) may associate with the HIV-1 membrane interface. It is further proposed that MPER-induced disruption and/or deformation of the lipid bilayer ensue during viral fusion. However, it is predicted that the cholesterol content of this membrane (∼45 mol %) will act against MPER binding and restructuring activity, in agreement with alternative structural models proposing that the MPER constitutes a gp41 ectodomain component that does not insert into the viral membrane. Here, using MPER-based peptides, we test the hypothesis that cholesterol impedes the membrane association and destabilizing activities of this gp41 domain. To that end, partitioning and leakage assays carried out in lipid vesicles were combined with x-ray reflectivity and grazing-incidence diffraction studies of monolayers. CpreTM, a peptide combining the carboxyterminal MPER sequence with aminoterminal residues of the transmembrane domain, bound and destabilized effectively cholesterol-enriched membranes. Accordingly, virion incubation with this peptide inhibited cell infection potently but nonspecifically. Thus, CpreTM seems to mimic the envelope-perturbing function of the MPER domain and displays antiviral activity. As such, we infer that CpreTM bound to cholesterol-enriched membranes would represent a relevant target for anti-HIV-1 immunogen and inhibitor development.

Figure 1

Models for gp41 MPER-TMD region organization at the HIV membrane and designation of the CpreTM and MPERp sequences used in this study. (A) Proposed models for the location of gp41 MPER-TMD at the Chol-enriched viral membrane. The cryoET density map contours of envelope spikes reported by Zhu et al. (5) and Zanetti et al. (12) are compared (left and right gray areas, respectively). Sequences depicted roughly parallel to the membrane plane (left) span amphipathic-at-interface and interfacial MPER subdomains (7). Cylinders inserted into the membrane represent the TMD anchors. (B) Sequences of the peptides used in this study. The diagram also shows positions and sequences for the potential cholesterol recognition/interaction amino acid consensus (outlined residue sequence) and 4E10 epitope (underlined residues). Sequence and numbering are according to the prototypic HXBc2 isolate.

Figure 2

Effect of Chol on the lytic activities and binding capacities of MPER peptides. (A, left) Effect of Chol on CpreTM-induced leakage kinetics. The peptide was added at the time indicated by the arrow (t = 50 s) at a peptide/lipid ratio of 1:500. Lipid concentration was 100 μM. Chol mole fractions are indicated for each curve. (A, right) Partitioning curves estimated from the fractional change in Trp fluorescence with increasing POPC or POPC/Chol (1:1) concentrations (squares and circles, respectively). Peptide concentration was 0.5 μM. Lines correspond to the best fit of the experimental values to a hyperbolic function (see Materials and Methods in the Supporting Material). (B) MPERp peptide was used at a peptide/lipid ratio of 1:150 in the leakage assays (left). Otherwise, conditions are as in A.

Figure 3

Binding to vesicles and ANTS leakage induced by CpreTM (upper) and MPERp (lower) as a function of the Chol mole fraction. Plotted values correspond to leakage (extents measured 60 min after peptide addition) and binding to membranes relative to those measured for vesicles devoid of Chol (solid and open circles, respectively). Dotted and dashed curves indicate values of the area compressibility moduli as measured by Needham and Nunn (33) and Henriksen et al. (34), respectively. Otherwise, experimental conditions are as in Fig. 2.

Figure 4

Inhibition of viral infectivity induced by CpreTM and MPERp. (A) Dose dependency of pseudovirus infection inhibition. Viruses were pseudotyped with HIV-1 Env (HXB2) or VSV-G (solid and dotted lines, respectively). The infection of TZM-bl target cells was monitored in both cases by flow cytometry as the green fluorescent protein signal in infected cells. (B) Specificity of the inhibitory effect. HIV-1 and VSV pseudovirions (solid and open columns, respectively) were incubated with 5 μM of CpreTM or MPERp. T-20 fusion inhibitor (0.1 μM) was included as a positive control for HIV-1 specificity. The asterisk denotes p < 0.005 by Student's test for unpaired data. Graphs in both panels display the mean ± SD of four measurements in two independent experiments.

Figure 5

Dose dependency and mechanism of CpreTM-induced content leakage from POPC (circles and solid lines), POPC/Chol (4:1) (squares and dotted lines), and POPC/Chol (2:1) (triangles and dashed lines) LUVs. (A) Partitioning curves estimated from the fractional change in Trp fluorescence with increasing lipid concentration. The fitting curves are superimposed. (B) ANTS leakage (extent measured 60 min after peptide addition) measured as a function of the peptide/lipid mole ratio. The lines link the experimental values for better appreciation of the differences. (C) Fluorescence requenching analysis to establish the mechanism of membrane permeabilization. Internal quenching (Q_in) was measured as a function of the ANTS released (f_out) after incubation with peptide (see Supporting Material).