Monitoring early fusion dynamics of human immunodeficiency virus type 1 at single-molecule resolution

Abstract

The fusion of human immunodeficiency virus type 1 (HIV-1) to host cells is a dynamic process governed by the interaction between glycoproteins on the viral envelope and the major receptor, CD4, and coreceptor on the surface of the cell. How these receptors organize at the virion-cell interface to promote a fusion-competent site is not well understood. Using single-molecule force spectroscopy, we map the tensile strengths, lifetimes, and energy barriers of individual intermolecular bonds between CCR5-tropic HIV-1 gp120 and its receptors CD4 and CCR5 or CXCR4 as a function of the interaction time with the cell. According to the Bell model, at short times of contact between cell and virion, the gp120-CD4 bond is able to withstand forces up to 35 pN and has an initial lifetime of 0.27 s and an intermolecular length of interaction of 0.34 nm. The initial bond also has an energy barrier of 6.7 k(B)T (where k(B) is Boltzmann's constant and T is absolute temperature). However, within 0.3 s, individual gp120-CD4 bonds undergo rapid destabilization accompanied by a shortened lifetime and a lowered tensile strength. This destabilization is significantly enhanced by the coreceptor CCR5, not by CXCR4 or fusion inhibitors, which suggests that it is directly related to a conformational change in the gp120-CD4 bond. These measurements highlight the instability and low tensile strength of gp120-receptor bonds, uncover a synergistic role for CCR5 in the progression of the gp120-CD4 bond, and suggest that the cell-virus adhesion complex is functionally arranged about a long-lived gp120-coreceptor bond.

FIG. 1.

Schematic of the instrument used to measure the micromechanics and kinetics properties of single molecular bonds between an infectious HIV-1 virion and individual cell receptors on a live host cell. (A) Schematic of the detection components of the MFP and the flexible cantilever placed just above a host cell. (B) Pseudovirus particles are cross-linked to a triangular cantilever, which is delicately brought into contact with a cell displaying either major receptor CD4, coreceptor CCR5 (or CXCR4), or both on its surface. (C) Typical force deflection traces recorded during the retraction of the cantilever. Ruptures of virion-cell bonds are marked by arrows. (D) Probability of formation of bonds between a virion and a host cell. The distribution displays Poisson characteristics (see text). (Inset graph) Probability of formation of bonds when only force deflection traces displaying at least one bond adhesion are analyzed. The time of contact between cell and virion was ∼1 μs.

FIG. 2.

Test of binding specificity and characterization of virion-receptor interactions at single-molecule resolution. (A) Test of specificity of MFP measurements and frequency of binding interactions between Env glycoproteins and CD4⁻ CCR5⁺ living cells in the presence of sCD4, in the absence of sCD4, in the presence of a function-blocking antibody against CCR5 (CCR5 mAb), or in the absence of virions attached to the cantilever (No virus), respectively. (B) Test of specificity of MFP measurements and frequency of binding interactions between Env glycoproteins and CD4⁻ CCR5⁺ living cells in the absence of added molecules, in the presence of a function-blocking antibody against CD4 (CD4 mAb; B4), in the presence of sCD4, or in the absence of virions (No virus), respectively. (C) Comparison of CD4⁺ CCR5⁺ cells infected to express GFP with pseudotyped virus with and without LC-SMCC treatment. (D) Mean adhesion force of the gp120-CD4 bond as a function of loading rate (pN/s) for CD4⁺ CCR5⁻ parental cells. Fit of this curve using Bell's model yielded a bond dissociation constant, k_off⁰, of 3.73 s⁻¹ and a bond reactive compliance, x_β, of 0.34 nm. (E) Distribution of adhesion bond forces obtained experimentally (triangles) or computed using a Monte Carlo simulation (line) based on Bell model's kinetic parameters (see text for details). The retraction velocity of the cantilever was maintained at 10 μm s⁻¹.

FIG. 3.

Histograms of adhesion forces of single intermolecular bonds for increasing contact time between pseudovirus and host cell. Time-dependent histograms of adhesion forces and mean adhesion forces of intermolecular bonds between Env glycoproteins and receptors on CD4⁺ CCR5⁻ GHOST parental cells (A and B), CD4⁺ CCR5⁺ GHOST Hi-5 cells (C and D), and CD4⁻ CCR5⁺ HOS.R5 cells (E and F). Mean adhesion forces at times >0 s were considered statistically significant different from the values at no contact time. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data were collected with a retraction velocity of 10 μm/s.

FIG. 4.

Energy barriers, lifetimes, and molecular elastic constant of bonds between Env glycoprotein and cellular receptors. (A and B) Time-dependent minimum value of the free energy (ΔG^‡) describing the binding adhesion interactions between Env glycoproteins and receptors on CD4⁺ CCR5⁻, CD4⁺ CCR5⁺, CD4⁻ CCR5⁺, and CD4⁺ CXCR4⁺ cells in the absence (A) and the presence (B) of small-molecule inhibitors. (C and D) Time-dependent molecular spring constants, κ_m, of gp120-CD4 and gp120(sCD4)-CCR5 bonds in the absence (C) and the presence (D) of small-molecule inhibitors. (E and F) Time-dependent distance from the free energy minimum to the point of bond rupture, x^‡, of gp120-CD4 and gp120(sCD4)-CCR5 bonds in the absence (E) and presence (F) of small-molecule inhibitors. Data were collected with a retraction velocity of 10 μm/s.

FIG. 5.

Dissociation rates calculated from adhesion distributions and normalized by the initial (i) dissociation rate of the gp120-CD4 bond. (A) Dissociation rates k₀ for virion gp120 adhesion with CD4⁺ CCR5⁻, CD4⁺ CCR5⁺, CD4⁻ CCR5⁺, and CD4⁺ CXCR4⁺ cell lines normalized by the no-contact time k₀ for CD4⁺ CCR5⁻ cells. (B) k₀ values (normalized by the initial control CD4⁺ CCR5⁻ value as in A) for CD4⁺ CCR5⁺ and CD4⁻ CCR5⁺ cell lines in the presence of small-molecule inhibitors. (C) Example fit of the theoretical probabilistic equation to experimental data obtained from CD4⁺ CCR5⁺ cells with no contact time. (D) Effect of small-molecule inhibitors (w/inhibitor) on k₀ from CD4⁺ CCR5⁺ and CD4⁻ CCR5⁺ cell lines normalized by the corresponding values without (w/o) small-molecule inhibitors. Data were collected with a retraction velocity of 10 μm/s.

FIG. 6.

Histograms of adhesion forces of single intermolecular bonds between pseudovirus and host cell in the presence of small-molecule inhibitors. Time-dependent histograms of adhesion forces and mean adhesion forces of single intermolecular bonds formed between HIV-1 and CD4⁺ CCR5⁺ GHOST Hi-5 cells in the presence and absence (control) of BMS-806 (A and B), CD4⁻ CCR5⁺ HOS.R5 cells in the presence of sCD4 and in the presence and absence of BMS-806 (C and D), and CD4⁻ CCR5⁺ HOS.R5 cells in the presence of sCD4 and in the presence and absence of T20 (E and F). Data were collected with a retraction velocity of 10 μm/s.

FIG. 7.

Schematic of the kinetic and mechanical parameters describing early fusion dynamics of HIV-1. Mean adhesion force (f), dissociation rate (k₀), change in free energy (ΔG^‡), and distance from the free energy minimum (x^‡) of the bonds involved in early HIV-1 fusion dynamics. The values above and below the arrows compare the mean adhesion forces and dissociation rates of the bonds corresponding to the binding and conformational states linked by the arrows. The initial binding of CD4 to gp120, the conformation change of gp120, and finally gp120 binding to CCR5 are illustrated here above their corresponding energy potentials. Data were collected with a retraction velocity of 10 μm/s.