X4-gp120 modulates CXCR4 dynamics and nanoclustering.

Single-particle tracking analysis of JKCD4+X4- cells transiently transfected with CXCR4-AcGFP on fibronectin (FN)-, FN + CXCL12-, or FN + X4-gp120-coated coverslips (828 particles in 96 cells on FN; 2,997 in 95 cells on FN + CXCL12 and 1,547 in 91 cells on FN + X4- gp120) n = 3. A) Percentage of mobile and immobile CXCR4-AcGFP particles at the membrane of cells treated as indicated. B) Diffusion coefficients (D1–4) of mobile particles at the membrane of cells treated as indicated with the median value of each experiment (black circles) and the median of all trajectories (dotted black lines). (****p ≤ 0.0001). C) Frequency of CXCR4-AcGFP particles containing monomers and dimers (≤2) or nanoclusters (≥3), mean ± SD calculated from mean spot intensity (MSI) values of each particle as compared with the value of monomeric CD86-AcGFP (980 ± 86 a.u., **p ≤ 0.01, ***p ≤ 0.001). D) Intensity distribution of individual CXCR4-AcGFP trajectories on unstimulated and CXCL12 or X4-gp120-stimulated cells. Graph shows the distribution of all trajectories, with the mean value of each experiment (black circles) and the median of all trajectories (dotted black lines) (n = 3; ****p ≤ 0.0001). Statistical significance was determined by two-way ANOVA in panels A and C and by non- parametric Kruskal-Wallis tests followed by Dunn’s test for panels B and D.

gp120-VLPs are mature particles that express a low number of Env trimers.

A) Representative images of clarified VLPs visualized by STED microscopy. Upper panels show images of the indicated VLPs stained for Gag p24 (blue) and gp120 (red). Lower panels show 10× magnification of equivalent images. White arrows indicate mature VLPs (p24 condensation). (B) Percentage of mature VLPs, analyzed from the images in A) using TrackAnalyzer in ImageJ, based on p24 intensity and aggregation level (mean ± SD; n = 2; ****p ≤ 0.0001; the significance indicated on immature VLPs bar shows the difference with all other conditions). C) Percentage of VLPs expressing gp120 on their surface, as analyzed in ImageJ (mean ± SD; n = 2; ***p ≤ 0.001). D) Distribution of gp120 mean fluorescence intensity. Each spot corresponds to the mean fluorescence intensity for each analyzed VLP in a.u. The black line represents the mean of all values (****p ≤ 0.0001). E) Frequency of gp120 intensity/particle. Statistical significance was determined by one-way-ANOVA followed by Tukey’s multiple comparisons test in panels B and C and by Mann-Whitney analysis for panel D.

gp120 VLPs modulate CXCR4 dynamics and nanoclustering.

Single- particle tracking analysis of JKCD4+X4- cells transiently transfected with CXCR4- AcGFP, on fibronectin (FN)-, FN + VLPs-, or FN + gp120 VLPs-coated coverslips (1,087 particles in 159 cells on FN; 1,400 in 153 cells on FN +VLPs and 1,061 in 160 cells on FN + gp120 VLPs) n = 6. A) Diffusion coefficients (D1–4) of mobile particles at the membrane of cells treated as indicated. Figure shows the mean value of each experiment (black circles) and the median of all trajectories (dotted black lines) (n = 6; ****p ≤ 0.0001). B) Frequency of CXCR4-AcGFP particles containing monomers and dimers (≤2) or nanoclusters (≥3) in cells treated as indicated. Mean ± SD calculated from mean spot intensity (MSI) values of each particle as compared with the value of monomeric CD86-AcGFP (980 ± 86 a.u., **p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001). C) Intensity distribution (arbitrary units, a.u.) from individual CXCR4-AcGFP trajectories on cells treated as indicated. Graph shows the distribution of all trajectories, with the mean value of each experiment (black circles) and the median of all trajectories (dotted black lines) (n = 6; ****p ≤ 0.0001). Statistical significance was determined by non-parametric Kruskal-Wallis tests followed by Dunn’s test for panels A and C, and by two-way ANOVA in panel B.

gp120 VLPs modulate CXCR4R334X dynamics and nanoclustering.

Single- particle tracking analysis of JKCD4+X4- cells transiently transfected with CXCR4R334X- AcGFP, on fibronectin (FN)-, FN + VLPs-, or FN + gp120 VLPs-coated coverslips (341 particles in 63 cells on FN; 610 in 54 cells on FN + VLPs and 707 in 63 cells on FN + gp120 VLPs) n = 2. A) Intensity distribution (arbitrary units, a.u.) from individual CXCR4R334X-AcGFP trajectories on cells treated as indicated. Graph shows the distribution of all trajectories, with the mean value of each experiment (black circles) and the median of all trajectories ± SD (dotted black lines) (n = 2; ****p ≤ 0.0001). B) Diffusion coefficients (D1–4) of mobile single particle trajectories at the membrane of cells treated as indicated. Figure shows the mean value of each experiment (black circles) and the median of all trajectories (dotted black lines) (n = 2; n.s. not significant, *p ≤ 0.05, ****p ≤ 0.0001). C) Frequency of CXCR4R334X-AcGFP particles containing monomers plus dimers (≤2) or nanoclusters (≥3), ± SD calculated from mean spot intensity values of each particle as compared with the value of monomeric CD86-AcGFP (*p ≤ 0.05, ***p ≤ 0.001). Statistical significance was determined by non-parametric Kruskal-Wallis tests followed by Dunn’s test for panels A and C, and by two-way ANOVA in panel B.

CD4 forms heterodimers with CXCR4 and CXCR4R334X.

FRET saturation curves generated using HEK-293T cells transiently transfected with a constant amount of CD4-CFP DNA (2 μg) and increasing amounts of A) CXCR4-YFP (0.5–8.0 μg), B) CXCR4R334X-YFP (0.5–8.0 μg) or C) 5HT2B DNA (0.5–12 μg). KD and FRETmax values were calculated using a nonlinear regression equation for a single binding-site model (n = 2). D) FRET efficiency in HEK-293 cells transiently transfected with CXCR4-YFP/ CD4-CFP (ratio 15:9), in the absence or presence of gp120 VLPs or Env(-) VLPs. Data shows FRET efficiency (arbitrary units, a.u.) (mean ± SD; n = 3; n.s. not significant, *p ≤ 0.05, ***p ≤ 0.001). E) FRET efficiency in HEK-293 cells transiently transfected with CXCR4R334X-YFP/CD4-CFP (ratio 15:9), in the absence or presence of gp120-VLPs or Env(-) VLPs. Data shows FRET efficiency (a.u.) (mean ± SD; n = 3; n.s. not significant, *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001). Statistical significance was determined by unpaired t-test in panels D and E.

X4-gp120 promote similar internalization patterns of CXCR4 and CXCR4R334X receptors.

A) Surface receptor expression of CXCR4 (white dots) or CXCR4R334X (black dots), after stimulation with CXCL12 (blue lines) or X4-gp120 (red lines). Results show mean ± SD of the percentage of receptor expression at the cell surface (n = 3). B) Surface receptor expression of CD4 in JK CD4+ CXCR4+ (white dots) or JK CD4+ CXCR4R334X (black dots), after stimulation with CXCL12 (blue lines) or X4-gp120 (red lines). Results show mean ± SD of the percentage of receptor expression at the cell surface (n = 3). Statistical significance was determined by one-way-ANOVA of AUC (*p ≤ 0.05, **p ≤ 0.01).

CD4/CXCR4 and CD4/CXCR4R334X complexes support similar HIV-1 infection.

The presence of CXCR4R334X on JKCD4+ cells does not alter gp120 binding and increases fusion events with target cells expressing HIV pHXB2 envelope. A) Binding of X4-gp120 to target cells expressing CD4 and CXCR4 or CD4 and CXCR4R334X analyzed by flow cytometry. Cells were incubated with 0.3 μg/mL of X4- gp120 at 37°C for 30 minutes. Data show MFI (arbitrary units, a.u.) mean ± SD; (n = 2). Statistical significance was determined using Student’s t-test (n.s.= not significant). B) Cell-cell fusion between JKHXBc2-expressing HIV-1 envelope and different target cells (JKCD4+CXCR4+, JKCD4+CXCR4- and JKCD4+CXCR4R334X). Prior to co-culture, each cell type was loaded with the corresponding cell-tracker. Data show the percentage of fusion events ± SD (n = 6). We used as reference the fusions events detected in JKCD4+CXCR4+ cells (100%). Statistical significance was determined by one-way- ANOVA (*p < 0.05, ****p ≤ 0.0001). C) Representative biparametric histograms from cells in B showing CMAC versus orange fluorophores. D) Human PBMCs isolated from a WHIM patient (WHIM) and three healthy donors (HD1-3) in two independent experiments were infected with X4-pseudotyped HIV-1NL4-3 (MOI: 0.001). At 2 hours post infection (p.i.), supernatant samples were obtained at different time points (days post-infection) and p24 levels (pg/mL) in each sample were determined using a commercial ELISA. Results show mean ± SD (n = 2).