Cholesterol enhances influenza binding avidity by controlling nanoscale receptor clustering

Abstract

Influenza virus infects cells by binding to sialylated glycans on the cell surface. While the chemical structure of these glycans determines hemagglutinin-glycan binding affinity, bimolecular affinities are weak, so binding is avidity-dominated and driven by multivalent interactions. Here, we show that membrane spatial organization can control viral binding. Using single-virus fluorescence microscopy, we demonstrate that the sterol composition of the target membrane enhances viral binding avidity in a dose-dependent manner. Binding shows a cooperative dependence on concentration of receptors for influenza virus, as would be expected for a multivalent interaction. Surprisingly, the ability of sterols to promote viral binding is independent of their ability to support liquid-liquid phase separation in model systems. We develop a molecular explanation for this observation via molecular dynamics simulations, where we find that cholesterol promotes small-scale clusters of glycosphingolipid receptors. We propose a model whereby cholesterol orders the monomeric state of glycosphingolipid receptors, reducing the entropic penalty of receptor association and thus favoring multimeric complexes without phase separation. This model explains how cholesterol and other sterols control the spatial organization of membrane receptors for influenza and increase viral binding avidity. A natural consequence of this finding is that local cholesterol concentration in the plasma membrane of cells may alter the binding avidity of influenza virions. Furthermore, our results demonstrate a form of cholesterol-dependent membrane organization that does not involve lipid rafts, suggesting that cholesterol's effect on cell membrane heterogeneity is likely the interplay of several different factors.

Fig. 1. Sterols enhance influenza binding avidity to target membranes. Relative influenza binding avidity to planar bilayers containing 10 mol% of the indicated sterol was measured using a single-virus binding assay (ESI Fig. 1†); GD1A is shown as entirely in the upper leaflet in supported bilayers as suggested for G_M1. Cholesterol-containing membranes bound significantly more influenza particles than membranes lacking sterol (p < 0.005). Of the six cholesterol analogues tested, only cholestenone increased binding significantly compared to cholesterol (p < 0.0005). In addition to sterol, target bilayers contained 1 mol% GD1a, 20 mol% DOPE, 0.5 mol% OG-DHPE and remaining mol% POPC.

Fig. 2. Viral binding is dose-dependent on sterol concentration and cooperative in receptor concentration. (A) Dose-dependence of viral binding as a function of mol% sterol. Both cholesterol and cholestenone enhanced membrane binding avidity in a dose-dependent manner but cholestenone demonstrated a more marked effect at low concentrations. Above 10 mol%, both sterols showed a roughly linear dose dependence. Membranes containing 40 mol% cholestenone did not form homogenous supported bilayers and could not be measured. All values are normalized to 10 mol% cholesterol. (B) Cooperativity analyzed by fitting binding versus GD1a receptor concentration using the Hill equation. Hill coefficients of 2.36 ± 0.85 and 2.83 ± 0.61 for cholesterol and cholestenone respectively were not significantly different (p = 0.15).

Fig. 3. Cholesterol slows GD1a–GD1a dissociation rates in molecular dynamics simulations. Simulation snapshots for membranes containing 20 mol% cholesterol and (A) 4 mol% GD1a or (B) 10 mol% GD1a. GD1a molecules showed reversible clustering in simulations containing 0, 10 or 20 mol% cholesterol. Cholesterol is rendered in purple, GD1a in red, and phospholipids in gray. (C and D) Probability density functions for GD1a–GD1a dissociation rate constants estimated from simulations. These compare rates over the range 0–20 mol% cholesterol at either 4 mol% or 10 mol% GD1a. Cholesterol significantly reduced k_off compared to sterol-free membranes under all of these conditions (p < 10^–6).

Fig. 4. Thermodynamic model for cholesterol-mediated increase in GD1a nanoclusters based on simulations. (A) Thermodynamic model for GD1a multimerization and association with cholesterol. Multimerization has a favorable enthalpy but unfavorable entropy. We postulate that cholesterol has a differential ordering effect on GD1a in the monomeric reference state as compared to the multimer. This decreases the entropic penalty of multimerization, increasing the overall fraction of multimers at equilibrium and thus viral binding avidity. Snapshots are rendered of monomers and multimers in simulated membranes containing 4 mol% GD1a. The monomer–dimer equilibrium is shown for simplicity, but the same model applies to more complex multimers. (B) Orientational order parameters for GD1a lipid tails in simulated membranes containing 1 mol% GD1a. Increasing concentrations of cholesterol progressively order GD1a tails, suggestive of an overall decrease in entropy.