Single-molecule analysis of human immunodeficiency virus type 1 gp120-receptor interactions in living cells

Abstract

A quantitative description of the binding interactions between human immunodeficiency virus (HIV) type 1 envelope glycoproteins and their host cell surface receptors remains incomplete. Here, we introduce a single-molecule analysis that directly probes the binding interactions between an individual viral subunit gp120 and a single receptor CD4 and/or chemokine coreceptor CCR5 in living cells. This analysis differentiates single-molecule binding from multimolecule avidity and shows that, while the presence of CD4 is required for gp120 binding to CCR5, the force required to rupture a single gp120-coreceptor bond is significantly higher and its lifetime is much longer than those of a single gp120-receptor bond. The lifetimes of these bonds are themselves shorter than those of the P-selectin/PSGL-1 bond involved in leukocyte attachment to the endothelium bonds during an inflammation response. These results suggest an amended model of HIV entry in which, immediately after the association of gp120 to its receptor, gp120 seeks its coreceptor to rapidly form a new bond. This "bond transfer" occurs only if CCR5 is in close proximity to CD4 and CD4 is still attached to gp120. The analysis presented here may serve as a general framework to study mechanisms of receptor-mediated interactions between viral envelope proteins and host cell receptors at the single-molecule level in living cells.

FIG. 1.

Schematic of the MFP used to measure binding interactions between individual HIV-1 gp120 molecules and receptors in a living host cell. (A) Schematic of the molecular force probe used in the measurements of gp120-receptor binding interactions. (B) A low density of recombinant BaL gp120 molecules are cross-linked to a cantilever, which is gently brought into contact with a cell. The cell is engineered to express either CD4 alone, coreceptor/chemokine CCR5 alone, or both CD4 and CCR5. The time of contact between cell and cantilever is kept to a minimum (<1 ms) to ensure mostly single-molecule interactions. The cantilever is subsequently pulled with a controlled reproach velocity. The time-dependent force applied on the bond between a gp120 molecule and a single cell receptor and the time-dependent deformation of the cell membrane-bound proteins are recorded simultaneously. The gp120-functionalized cantilever is shown in black when a gp120-receptor bond is subjected to a pulling force before rupture and in light gray after bond rupture.

FIG. 2.

Force-distance spectra for gp120-CD4 interactions in living cells. Typical profiles of the time-dependent force acting on a gp120-CD4 bond under tension as a function of the time-dependent deformation of that bond are shown. From these force-distance spectra, the k_off rates of the bonds between HIV-1 envelope proteins and host cell receptors can be readily extracted. The probability of binding events between gp120 and its cell receptors depends on the contact time between cantilever and cell surface, the density of gp120 on the cantilever, and the approach force used to bring the cantilever in contact with the cell. These parameters are controlled to ensure mostly single-molecule interactions, as shown by single arrows in profiles 6 and 10 (numbered from the top) to indicate single-bond breakage. The two arrows in profile 1 show a rare case of double-bond breakage. Rupture force (in piconewtons) and loading rate (in piconewtons per second) are extracted from the height of the peak (in piconewtons) at the rupture point and from the product of the slope right before the peak (in piconewtons per micrometer) and the reproach velocity (in micrometers per second), respectively. Here, the reproach velocity was 15 μm/s. Experiments were conducted using CD4⁺ GHOST cells.

FIG. 3.

The interactions between gp120-coated cantilevers and cell receptors are specific. Typical force-distance spectra for the binding association between individual gp120 and CD4 molecules in the absence (first spectrum) and presence (second spectrum) of an anti-CD4 function-blocking monoclonal antibody (mAb), for a cantilever without cross-linked gp120 (third spectrum), and for CD4⁺ cells in the presence of sCD4 (fourth spectrum) are shown. Experiments were conducted using CD4⁺ GHOST cells.

FIG. 4.

Single-molecule analysis of gp120-CD4 binding interactions. (A) Rupture force (in piconewtons) required to break a single gp120-CD4 bond as a function of the loading rate (in piconewtons per second) to which the bond is subjected. The unstressed dissociation rate k⁰_off and the reactive compliance x_β of the bond are calculated from this curve through Bell's model analysis (see text). This curve features a break at a characteristic loading rate of ∼350 pN/s, which separates a regimen where the bond lifetime is long at low loading rates from a regimen where the bond lifetime is shorter at high loading rates (see text). (B) Distributions of rupture forces required to break a single gp120-CD4 bond at different reproach velocities. (C) Schematic of the intermolecular potential of the gp120-CD4 interaction qualitatively based on data shown in panel A. Red represents the inner barrier potential of the gp120-CD4 interaction at high loading rates; blue represents the outer barrier potential at low loading rates. The width of each potential well is taken as the reactive compliance at low and high loading rates. Experiments were conducted using CD4⁺ GHOST cells.

FIG. 5.

Single-molecule analysis of gp120-CCR5 and gp120-CCR5/CD4 bonds. (A) Typical force-distance spectra for the gp120-CCR5 bond in the presence of sCD4, gp120-CCR5/CD4, and gp120-CCR5 in the absence of sCD4. (B) Distributions of rupture forces to break a single bond between a single gp120 molecule and the protein complex CCR5/sCD4 at different reproach velocities. (C) Rupture force for single gp120-CCR5 bonds (circles) in the presence of sCD4 and single gp120-CCR5/CD4 bonds (squares) as a function of the loading rate applied to those bonds. Here again, characteristic ruptures occur in the force-loading rate curves, at threshold loading rates of ∼450 pN/s for the gp120-CCR5/sCD4 complex and ∼650 pN/s for the gp120-CCR5/CD4 complex. All experiments were conducted using CD4⁺ CCR5⁺ GHOST Hi-5 cells.