Selective interactions of polyanions with basic surfaces on human immunodeficiency virus type 1 gp120 - PubMed (original) (raw)

Selective interactions of polyanions with basic surfaces on human immunodeficiency virus type 1 gp120

M Moulard et al. J Virol. 2000 Feb.

Abstract

It is well established that the gp120 V3 loop of T-cell-line-adapted human immunodeficiency virus type 1 (HIV-1) binds both cell-associated and soluble polyanions. Virus infectivity is increased by interactions between HIV-1 and heparan sulfate proteoglycans on some cell types, and soluble polyanions such as heparin and dextran sulfate neutralize HIV-1 in vitro. However, the analysis of gp120-polyanion interactions has been limited to T-cell-line-adapted, CXCR4-using virus and virus-derived gp120, and the polyanion binding ability of gp120 regions other than the V3 loop has not been addressed. Here we demonstrate by monoclonal-antibody inhibition, labeled heparin binding, and surface plasmon resonance studies that a second site, most probably corresponding to the newly defined, highly conserved coreceptor binding region on gp120, forms part of the polyanion binding surface. Consistent with the binding of polyanions to the coreceptor binding surface, dextran sulfate interfered with the gp120-CXCR4 association while having no detectable effect on the gp120-CD4 interaction. The interaction between polyanions and X4 or R5X4 gp120 was readily detectable, whereas weak or undetectable binding was observed with R5 gp120. Analysis of mutated forms of X4 gp120 demonstrated that the V3 loop is the major determinant for polyanion binding whereas other regions, including the V1/V2 loop structure and the NH(2) and COOH termini, exert a more subtle influence. A molecular model of the electrostatic potential of the conserved coreceptor binding region confirmed that it is basic but that the overall charge on this surface is dominated by the V3 loop. These results demonstrate a selective interaction of gp120 with polyanions and suggest that the conserved coreceptor binding surface may present a novel and conserved target for therapeutic intervention.

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Figures

FIG. 1

FIG. 1

DexS inhibition of MAb binding to the conserved coreceptor binding site. Monomeric recombinant sgp120 derived from the MN or JRFL strain of HIV-1 was captured on the solid phase by an antibody to the conserved COOH terminus, then preincubated without (−) or with (+) 12.3 μM DexS prior to addition of sequential dilutions of gp120 antibodies specific for the CD4bs (IgG1b12) (A), the V3 loop (447-52D) (B), and the CD4i epitopes 17b (C) and 48d (D). Detection of bound MAb was carried out by ELISA, and the results are presented as optical densities at 450 nm. Each datum point is the mean of values obtained from triplicate samples, and each experiment was repeated on at least three separate occasions.

FIG. 2

FIG. 2

Inhibition of MAb binding to oligomeric Env expressed on the surface of HIV-1MN-infected cells. Cells were preincubated with various concentrations of DexS (A) or ATA (B) before being washed, and gp120-specific MAbs were added. Bound MAb was detected by indirect immunofluorescence and flow cytometry, and the results are expressed as percent inhibition of MAb binding relative to the positive (no inhibitor) and negative (no MAb) controls. Each datum point is the mean of values for triplicate samples.

FIG. 3

FIG. 3

DexS inhibition of gp120-CXCR4 interactions. (A) A3.01 (CD4+ CXCR4+) T cells were preincubated with various concentrations of DexS prior to the addition of HIV-1MN gp120. The CXCR4-specific MAb 12G5 or the CD4-specific MAb Q4120 was subsequently added. (B) A3.01 cells were preincubated with DexS prior to addition of MAb 12G5 or Q4120. Bound MAb was detected by indirect immunofluorescence and flow cytometry, and the results are expressed as percent inhibition of MAb binding relative to the positive (no inhibitor) and negative (no MAb) controls. Each datum point is the mean of values obtained from triplicate samples.

FIG. 4

FIG. 4

Inhibition of HIV-1 infectivity by DexS. HIV-1HXBc2 and HIV-1JRCSF were preincubated for 1 h at 37°C with the concentrations of DexS shown, then added to activated PBL for a further 1 h. After being washed, the cells were cultured for 7 days, and then the production of virus was measured by detection of cell-free viral p24 in the supernatant. The results, expressed as picograms of p24 per milliliter, were calculated from the optical densities at 492 nm. Each bar represents the mean of values for three replicate wells, and the error bars indicate ± 1 standard deviation.

FIG. 5

FIG. 5

Direct binding of [35S]heparin to immobilized gp120. gp120 and mutated forms thereof were incubated with [35S]heparin before being blotted onto a nitrocellulose membrane. The radioactivity in bound material was determined with a scintillation counter, and results are expressed as counts per minute. (A) Binding of [35S]heparin to 400 nM gp120HXBc2, gp120JRFL, and V3 loop deletion mutants thereof. (B) Binding of [35S]heparin to 170 nM gp120HXBc2, and mutants thereof, precomplexed (or not) with a molar excess of MAb 48d. The background value for [35S]heparin represents binding to the filter in the absence of protein. Each bar represent the mean of values for triplicate samples, and the error bars represent ± 1 standard deviation.

FIG. 6

FIG. 6

SPR analysis of X4-derived gp120–heparin interactions: overlay of sensorgrams showing binding of WT and mutated forms of gp120HXBc2 to immobilized heparin. sgp120 (used at concentrations of [from top to bottom] 45, 30, 22.5, 15, and 11.5 nM) was injected for 6 min over a heparin-activated surface at a flow rate of 15 μl/min to analyze the association phase, after which running buffer alone was injected to analyze the dissociation phase. (A) Binding curves for wild-type gp120HXBc2. (B) Scatchard plot of the equilibrium binding data directly measured on the sensorgrams after a 25-min period of interaction. (C) Binding curves for gp120HXBc2, alone or preincubated with a 10-fold molar excess of MAb 50-23, 48d, or both (from top to bottom). (D) Maximum responses measured at the end of the association phases shown in panel C. (E and F) Binding curves for gp120HXBc2Δ82ΔC5ΔV1V2 (E) and gp120HXBc2Δ82ΔC5ΔV1V2V3 (F) injected at (from top to bottom) 90, 60, 45, and 30 nM over the heparin surface.

FIG. 7

FIG. 7

SPR analysis of R5X4- and R5-derived gp120–heparin interactions: overlay of sensorgrams showing binding of gp120 to immobilized heparin. (A and B) Binding curves for R5X4 gp12089.6 (A) and R5X4 gp120W61D (B) injected at (from top to bottom) 60, 45, 30, 22.5, and 15 nM. (C and D) Binding curves for wild-type R5 gp120JRFL (C) and V3 loop-deleted gp120JRFL (D) injected at (from top to bottom) 90, 60, 45, and 30 nM over the heparin surface. Binding conditions were similar to those described in the legend to Fig. 6. S, seconds.

FIG. 8

FIG. 8

Modeling of the electrostatic potential for the coreceptor binding surface of the gp120 trimer. The molecular models of the gp120 trimer shown here are orientated with the trimer axis perpendicular to the page, showing the conserved surfaces of the HIV-1 clones HXBc2, MN, 89.6, and JRFL. The top panels show the gp120 core, the middle panels show the V3 loop, and the bottom panels show the V3 loop integrated into the gp120 core. These models are depicted in a Cα worm representation (left column) and an electrostatic surface representation (other four columns). The Cα worm representations for the different strains are essentially indistinguishable: the one corresponding to the HXBc2 sequence is shown with the core colored rust brown and the V3 loop colored green. The electrostatic potentials were calculated with the program Delphi and are depicted at the solvent-accessible surface, which is colored according to the local electrostatic potential, ranging from dark blue (most positive, corresponding to 10 kT/e) to red (most negative). The figure was prepared with the program GRASP (56).

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