Fig 1.
Distinct gp120s show discordant binding levels to CCR5 but not to CD4 or the E51 mAb.
A Saturation binding curves of 35S-gp120s to HEK-R5 membranes in the presence of 400 nM sCD4. Specific binding of gp120s was deduced by subtracting from total binding the non-specific binding (NSB). NSB was measured using 10 μM of the CCR5 antagonist maraviroc (MVC) and was observed to increase linearly with the concentration of 35S-gp120, as expected in the case of low affinity binding. Similar results were obtained when measuring NSB on parental HEK 293T cells. Relative to total binding, mean NSB values at a concentration of 10 nM gp120 represented 59.3%, 27.3%, 12%, 24.3%, 20.1%, 17% and 54.3% for gp120 #10, 25, 34, 38, 48, 50 and 58, respectively. These differences proved mainly due to differences in the extents of specific binding, and not to differences in NSB between the distinct gp120s. One representative experiment out of at least three independent experiments performed in duplicate is shown. On the whole, three to four independent batches of purified gp120s were used for these experiments, with similar results. Data points were described by a one-site (gp120 #25, 34, 38, 48, 50) or a two-site binding model (gp120 #10) or linear regression (gp120 #58). B The Bmax values (means ± SD) for the gp120s were deduced from the saturation curves using the Prism Software. n.d.: not determined. The binding levels of JR-FL gp120 and gp120 #34 to CCR5 were compared using both proteins at their KD (i.e. 4.35 nM for JR-FL gp120 [45] and 7 nM for gp120 #34 (S1 Table)). Binding of JR-CSF gp120 was also determined at 4.35 nM, although the KD value for this protein is not known. C, D The Scatchard transformations of the binding data were deduced using Prism. For the sake of clarity, the linear Scatchard plots for gp120 Bx08, #38, #48 and #59 are not represented. E Specific binding of 10 nM 35S-gp120 to intact HEK-R5 cells (1.106), with 200 nM sCD4 (n = 2). Relative to total binding, mean NSB values (determined as in A) represented 19%, 27.9%, 6.9%, 3.4%, 10% and 39.8% for gp120 #1, 10, 25, 34, 50 and 58, respectively. F Specific binding to HEK-CD4 cell membranes of 35S-gp120s used at a concentration equal to their KI for CD4 (means ± SEM of 3 experiments). KI were deduced from competition binding experiments of the anti-CD4 Q4120 mAb by unlabeled gp120 (inset). NSB was determined using parental HEK cells and represented less than 10% of total binding. ns: P > 0.05 using unpaired two-tailed Student t test. G SPR measurement of the binding of 100 nM gp120 to the mAb E51 immobilized on a CM4 sensorchip, alone or with 200 nM sCD4. Results are means ± SEM (n = 2). Inset: Representative sensorgrams for gp120 #34.
Fig 2.
Different HIV-1 gp120s show different modes of binding to CCR5.
The panels show competition binding experiments of 10 nM 35S-gp120 #34 to HEK-R5 membranes by increasing concentrations of unlabeled gp120 #34 (A), #58 (A), #50 (B) and #10 (C). In all experiments, unlabeled gp120 #25 was used as an internal control (A, B and C). Representative experiments out of 3 independent experiments performed in duplicate are shown. Results (means ± SEM) were normalized for non-specific binding (0%) and specific binding in the absence of competitor (100%, B0). In the panels, data points are fitted according to a one-site (gp120 #25, 34 and 58) or a two-site (gp120 #50, F value = 43.9; p < 0.0001) competitive binding model or a sigmoidal dose-response model with a variable slope (gp120 #10). The Hill slope (nH) values were deduced from fitting of the competition curves according to a sigmoidal dose-response model with a variable slope.
Fig 3.
Distinct HIV-1 gp120s are differentially sensitive to CCR5 dimerization.
A Membranes from HEK 293 cells transfected with FLAG/SNAP-tagged WT-CCR5 or L196K-CCR5 cDNA show similar receptor expression levels. Fluorescence intensity (at 620 nm) of the SNAP substrate BG-Lumi4-Tb bound to receptors increases linearly with membrane quantity, in contrast to non specific binding (NSB) to parental membranes. B FRET efficacy was measured between SNAP- and CLIP-tagged receptors labeled with lumi4-Tb and d2, respectively, and expressed as the ratio of the fluorescence intensities at 665 nm (d2) and 620 nm (Lumi4-Tb) (λex = 320 nm). HEK cells were transfected in such a way that WT-CCR5, L196K-CCR5 and L208K-CCR5 have similar cell surface expression levels and that the FRET acceptor(CLIP)/donor(SNAP) ratio is non-saturating and kept constant in all experiments. Mutation of Leu-196 reduces CCR5 dimerization, in contrast to Leu-208 that resides outside the dimerization interface [52] (n = 3). C, Saturation binding curves of the CCR5 chemokine 125I-CCL3 to FLAG/SNAP-tagged WT-CCR5- or L196K-CCR5-expressing membranes. Results indicate that L196K-CCR5 has a 36%-fold lower Bmax value than WT-CCR5 (2.5 vs 3.9 pmole/mg of protein, respectively). In the range of the 125I-CCL3 concentrations tested, however, similar affinity constant values were deduced from the curves for WT- and L196K-CCR5 (KD = 0.36±0.18 and 0.47±0.13 nM, respectively). D Saturation binding curves of 35S-gp120 #25 or #34 to FLAG/SNAP-tagged WT-CCR5- or L196K-CCR5-expressing membranes. E Specific binding of 10nM 35S-gp120/sCD4 to WT-CCR5-, L196K-CCR5- or L196C-CCR5-expressing membranes. * P<0.05; ** P <0.01; *** P <0.001, vs binding to WT-CCR5 in unpaired two-tailed Student t test. Results (means ± SEM, n = 3–6) are expressed as % specific binding relative to 35S-gp120 #34 binding to WT-CCR5. F Binding of mAbs 2D7, 45531 or anti-Flag M2 to FLAG/SNAP-tagged WT- or L196K-CCR5-expressing A3.01 or HEK cells was revealed by AlexaFluor 647-conjugated secondary Ab and flow cytometry analysis (n = 3).
Fig 4.
Model for the role of CCR5 dimerization in gp120 binding.
CCR5 exists in at least two distinct dimer conformations (D1 and D2), which coexist with receptor monomers (M). The orientations of TM5 and ECL2 are different in D1 and D2. Depending on the nature of the tip of the V3 loop (V3) and perhaps its distance/position relative to the bridging sheet (BS), distinct gp120s differ in the way they engage D1 and D2 while binding similarly receptor monomers. Some gp120s (e.g. gp120 #34, brown colored) bind D1 and D2 in 2:2 and 1:2 stoichiometries, respectively, and conversely for other gp120s (e.g. gp120 #25, blue colored). A In WT-CCR5-expressing cells, D1 is predominant over D2 and monomers explaining why the number of gp120 #34 molecules bound to these cells is higher than that of gp120 #25 molecules (Figs 1 and 3 and S2 Fig). B L196K-CCR5 preferentially adopts the D2 conformation, in which the conformation of ECL2 differs, compared to that in D1. D2 binds gp120 #25 and #34 in 2:2 and 1:2 stoichiometries, respectively. This explains why the number of gp120 #25 molecules bound to L196K-CCR5-expressing cells is higher than that to WT-CCR5-expresing cells, and conversely for gp120 #34 (Fig 3D and 3E). Equilibrium arrows in A and B show that the proportion of dimers is decreased in the case of L196K-CCR5, as compared to WT-CCR5.
Fig 5.
Role of CCR5 dimerization in HIV-1 entry into T-cells.
A Fusion kinetics of BlaM-vpr-containing virus #25 or 34 with CD4+ A3.01 T-cells expressing FLAG/SNAP-tagged WT-CCR5 or L196K-CCR5. Data points represent means ± SEM of 2 independent determinations (out of 5). B Gating strategy of A3.01 cells expressing WT-CCR5 (red) or L196K-CCR5 (blue) at a low (GMFI = 111 and 123 for WT-CCR5 and L196K-CCR5, respectively), intermediate (599 and 533) or high level (2903 and 2382). Shown are representative flow cytometry plots of cell side scatter vs receptor expression level revealed with the AlexaFluor 647-conjugated anti-FLAG mAb M2. On the whole cell populations, both receptors were expressed at similar expression levels (GMFI = 362 and 312 for WT-CCR5 and L196K-CCR5, respectively). Comparable results were obtained using the unconjugated M2 revealed by an AlexaFluor 647-conjugated goat anti-mouse IgG (GAM) (GMFI = 5871 and 3342) C, D and E The virus-cell fusion experiments shown in A were analyzed with A3.01 T-cells expressing low (C), intermediate (D) or high (E) cell surface level of either WT- or L196K-CCR5. F Levels of fusion at 3 h for the indicated viruses were measured with A3.01 T-cells expressing low (L) or high (H) level of either WT- (red bars) or L196K-CCR5 (blue bars). Results are means ± SEM of 3 independent determinations (except for the non-M and M-tropic viruses JR-CSF and JR-FL where n = 1). In panels A, C, D, E and F, results are expressed as percents of fusion relative to the maximum extent of fusion (Fmax) of virus #34 with L196K-CCR5-expressing cells. This Fmax value, expressed as the percentage of cells containing BlaM-vpr, ranged between 40–60% and did not vary with the receptor expression level.
Fig 6.
Blood-derived HIV-1 isolates depend on CCR5 plasticity for infection of MDMs.
A MDMs from 6 different healthy donors were infected by virus clones pseudotyped with gp160 #1, #10, #25, #34, #50 or #58, the M-tropic or the non-M-tropic HIV-1 strain JR-FL or JR-CSF, respectively, or recombinant viral populations generated from plasma (P) or cerebrospinal fluid (CSF) of two patients with HIV-1-associated encephalitis (P#B and P#J). Virus quantities that produced comparable levels of infectivity in T-cells isolated from the same donors (i.e. 105 RLU) were used. Of note, similar results were obtained after normalizing the virus quantities to the p24 content. Results represent infectivities of the viruses in MDMs, determined in duplicate, and normalized to infectivity of JR-FL (arbitrarily set at 100%). B Sensitivity of viruses to sCD4. Some of the previous viruses were tested for their sensitivity to inhibition by increasing concentrations of sCD4 in infection experiments of PHA/IL-2- activated CD4 T-cells. The virus amounts were selected in such a way that infectivities in the absence of sCD4 were similar (i.e. 105 RLU of luciferase activity). The data points shown are from one representative experiment performed in duplicate, out of three experiments performed independently. The independent experiments were carried out with the lymphocytes from different healthy donors. Infectivity of the viruses was expressed as percentage of that measured in the absence of sCD4 (100%). Inhibition curves were fitted according to a sigmoidal dose-response model with a variable slope. C The panel shows sCD4 IC50 values that were deduced from inhibition curves with GraphPad Prism 6. Results are means ± SD of 3 independent determinations. D Dose-response inhibitions of infection of activated CD4 T-cells by increasing concentrations (ranging between 10−12 and 3x10-8 nM) of the anti-CD4 mAb Q4120 were carried out with the indicated viruses. Then, IC50 values for inhibition of infection by Q4120 were calculated with GraphPad Prism 6. The data shown here represent means ± SEM of two independent experiments performed in duplicate. E Infection assays of Affinofile cells expressing CCR5 at a high level (≈ 105 receptors/cell, stimulated by 2 μM ponasterone A) and increasing amounts of CD4 stimulated by 7 different concentrations of minocycline ranging between 0.08 and 2.5 ng/ml (see “Materials and methods” for further details). Data points represent infectivities of viruses that were normalized to infectivity in Affinofile cells expressing the highest levels of CCR5 and CD4 (CD4high/CCR5high Affinofile cells), arbitrarily taken as 100%. One representative experiment out of three independent experiments performed in duplicate is shown. In those experiments, the quantities of the different viruses were adjusted in such a way that they generated similar RLU in CD4high/CCR5high Affinofile cells (105). However, similar results were also obtained when normalizing the quantities of viruses to the p24 content. F Infectivities of the indicated pseudotyped viruses in CD4low/CCR5high affinofile cells (cells that have not been stimulated by minocycline, and where the CD4 expression level is minimal, i.e. in the range of 4000 receptors/cell), expressed as percentage of the infectivity in CD4high/CCR5high Affinofile cells). G Receptor expression levels at the surface of untransduced (grey line) or transduced MDMs with pTRIP ΔU3-expressing FLAG-tagged WT-CCR5 (blue line) or L196K-CCR5 (magenta line). Results were obtained using the anti-Flag mAb M2 (left panel) or the anti-CCR5 mAb 2D7 (right panel) revealed by an AlexaFluor 647-conjugated goat anti-mouse IgG (GAM) and flow cytometry analysis. With the mAb M2, GMFI values equal to 7836 and 2010 were found for WT-CCR5 and L196K-CCR5. Background signal of untransduced MDMs labeled with GAM alone is represented as red lines. H Levels of fusion of BlaM-vpr-containing virus #25, virus #34, JR-CSF or JR-FL to untransduced or transduced MDMs. Fusion was measured by flow cytometry analysis 3 h post-inoculation, as described in the “Materials and Methods” section. Each data point represents the level of fusion of the indicated virus clone with MDMs from one donor, expressed as a percentage of MDMs where CCF2 is cleaved by BlaM. On the whole, four independent experiments with MDMs from four healthy individuals were done. The panel displays the means +/- SD of the results obtained from these 4 experiments.