Hiv-1 exploits ccr5 conformational heterogeneity to escape inhibition by chemokines



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Fig. 1. Chemokine- and gp120-binding CCR5 represent different receptor populations. Binding of 0.1 nM 125I-CCL3 (A) or 10 nM 35S-gp120Bx08 (in the presence of 30 nM soluble CD4) (B, C and D) was displaced by increasing amounts (A, C and D) or a 100 nM concentration (B) of unlabeled chemokines. Results were normalized for non-specific binding (0%) and specific binding in the absence of competitors (B0, 100%) and fitted according to a one-site (panel A, CCL7, 6P4, 2P3 and 5P12 in panels C and D) or a two-site (16.2 < F value < 93.3 with p < 0.0001 for CCL3 in C and CCL4 and PSC in D) competitive binding model. In panel B, data points are means ± SEM of 5 independent determinations. Panels A, C and D show representative experiments out of 3-5 performed independently.

Fig. 2. Steric inhibition of gp120 binding to CCR5 does not contribute to the anti-HIV-1 activity of PSC-RANTES. HeLa P4C5 cells were incubated with MVC, 5P12-or PSC-RANTES at 37 or 4 °C before being infected by Bx08Ren viruses and treated as indicated in the text. Results represent the luciferase activity in the cell lysates, expressed as relative light units (RLU). A representative experiment out of 5 independent determinations is shown. Uninfected cells (NI) served as negative controls in those experiments.

Fig. 3. CCR5 coupling to NFG-proteins differentially influences native agonist chemokine and gp120 binding. (A) Total binding of 0.2 nM 125I-CCL3 to 5.105 A3.01-R5 cells (left panel) or membranes from CD4+ T-cells (15 g of proteins) (right panel) was measured in the presence or absence (control) of GTPS or Gpp(NH)p or after treatment of cells with PTX. Non specific binding was determined using the antagonist TAK779 or MVC. *, p < 0.05; **, p < 0.01 as compared to controls in unpaired two-tailed Student’s t test. Panels (B) and (C) are saturation experiments of 125I-CCL3 and 125I-CCL5 binding to HEK-R5 cell membranes, respectively. Specific binding was measured in the presence or in the absence of Gpp(NH)p. Total binding of 0.1 nM 125I-CCL3 (D) or 10 nM 35S-gp120 from the HIV-1 strains 25, 34 or Bx08 in complex with sCD4 (E) to membranes from HEK 293T cells expressing WT-CCR5 or R126N-CCR5 (R/N) was measured in the presence or absence (control) of Gpp(NH)p and/or MVC (nonspecific binding). Equal amounts of WT-CCR5 and R126N-CCR5 at the cell surface were confirmed by flow cytometry. Saturation binding experiments of 35S-gp120/sCD4 complexes revealed KD values (in nM) of 7.5, 8.3 and 9.9 for gp12025, gp12034 and gp120Bx08, respectively. (F) Displacement of 35S-gp120Bx08 binding by CCL3 was measured in the absence or presence of Gpp(NH)p. Data were fitted according to a two-site competitive binding model (F = 42 with p < 0.0001 and F = 5.5 with p = 0.0062 for data in the absence and in the presence of Gpp(NH)p, respectively). Panels show representative experiments out of at least 3 performed independently.

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