Mechanism of membrane perturbation by the HIV-1 gp41 membrane-proximal external region and its modulation by cholesterol

Abstract

Membrane-activity of the glycoprotein 41 membrane-proximal external region (MPER) is required for HIV-1 membrane fusion. Consequently, its inhibition results in viral neutralization by the antibody 4E10. Previous studies suggested that MPER might act during fusion by locally perturbing the viral membrane, i.e., following a mechanism similar to that proposed for certain antimicrobial peptides. Here, we explore the molecular mechanism of how MPER permeates lipid monolayers containing cholesterol, a main component of the viral envelope, using grazing incidence X-ray diffraction and X-ray reflectivity. Our studies reveal that helical MPER forms lytic pores under conditions not affecting the lateral packing order of lipids. Moreover, we observe an increment of the surface area occupied by MPER helices in cholesterol-enriched membranes, which correlates with an enhancement of the 4E10 epitope accessibility in lipid vesicles. Thus, our data support the view that curvature generation by MPER hydrophobic insertion into the viral membrane is functionally more relevant than lipid packing disruption.

Fig. 1.

HIV MPER designation and model for its association with membranes Panel A: sequence of MPER peptide used in this study. 4E10 epitope residues underlined. Numbering is based on the prototypic HXBc2 viral isolate. Panel B: model for the cognate ELDKWASLWNWFNITNWLWYIK peptide in association with a membrane monolayer. The structure adopted in detergent micelles was obtained from the Protein Data Bank (PDB ID: 2PV6) and rendered using Swiss-PDB-viewer. The insertion depths for the depicted residues L669, F673 and I675 are based on electro paramagnetic spectroscopy determinations [12].

Fig. 2.

Secondary structure and MPERp activity as a function of the Chol content in the bilayer. A) CD spectra of MPERp in association with PC vesicles containing different Chol mole ratios as indicated in the panels. The lipid and peptide concentrations were 1 mM and 30 μM, respectively. B) Effect of Chol on MPERp-induced ANTS leakage kinetics. The peptide was added to a vesicle suspension (100 μM lipid) at the time indicated by the arrow (t=50 s). The peptide-to-lipid ratio was 1:150. Chol mole fractions are indicated for each curve. The dotted traces follow peptide incorporation into the vesicles monitored through energy transfer from tryptophans to membrane-residing d-DHPE.

Fig. 3.

Grazing incidence X-ray diffraction data (symbols) and corresponding fits (lines): scattering intensity, integrated over q_Z range, against scattering vector q_XY of (A) DPPC/Chol (87:13, molar ratio) monolayer before (rhombs) and after MPERp (inverted triangles) injection; (B) DPPC/Chol (54:46, molar ratio) monolayer before (rhombs) and after MPERp (inverted triangles) injection.

Fig. 4.

X-ray reflectivity data (symbols) and corresponding fits (lines) normalized by Fresnel reflectivity plotted against scattering vector q_Z of (A) DPPC/Chol (87:13, molar ratio) monolayer before (rhombs) and after MPERp (inverted triangles) injection; (B) DPPC/Chol (54:46, molar ratio) monolayer before (rhombs) and after MPERp (inverted triangles) injection.

Fig. 5.

Inhibition of MPERp-induced vesicle contents leakage by 4E10. A) Effect of antibody addition to the ongoing leakage. POPC:Chol (2:1, molar ratio) vesicle samples (100 μM lipid) were treated with 1 μM peptide and, subsequently supplemented with 10 μg/ml of 4E10 (addition time indicated by the arrow). The dotted traces follow the leakage kinetics in the absence of antibody. B) 4E10-induced inhibition percentages plotted as a function of the Chol mole fraction. Rate reduction caused by antibody with respect to the leakage control without antibody was calculated by correcting 0% extent of leakage to the time point of antibody addition, and subsequently measuring increment of leakage after 20 s in both samples. C) Peptide mass percentage distribution between the monolayer slabs under the experimental conditions used for X-ray scattering assays (indicated by the arrows in the previous panel). Preferential location of the peptide into the HG slab correlates with better 4E10 epitope recognition-blocking.

Fig. 6.

Model to explain MPER-induced bilayer perturbation and its dependence on Chol. A) When Chol levels are low the peptide penetrates deeper into the monolayer, the accessibility to 4E10 epitope is hindered and the free peptides induce transient permeabilization of the bilayer [41]. B) Chol levels as those existing at the viral envelope lead to shallower MPER insertion and increased accessibility to the 4E10 epitope. Moreover, the surface occupied by each peptide increases and the monolayer thickness decreases. We surmise that the elastic stress generated in bilayers by the expansion of one monolayer can be relaxed in two ways: 1) free peptides may generate toroidal, stable aqueous pores [41,43]; and 2) in the context of the viral gp41, transmembrane domains lock MPER sequences into a ring-like configuration at the membrane interface. Experimental evidence for involvement of 5–7 trimers at the fusion site has been obtained by electron tomography [49]. Elastic stress is released in this case through the formation of the protruding bulges (see reference [26] for a discussion on membrane fusion driven by curvature generation).