Fig. 4. Chemokine-mediated inhibition of HIV-1 infection, but not chemotaxis, is independent of G-proteins. PTX-treatment impaired PSC-RANTES-mediated chemotaxis of A3.01-R5 cells (A) but not the ability of the chemokine analog to inhibit infection of these cells by the Bx08Ren viruses (B). Panels show representative experiments out of at least three independent experiments.
Fig. 5. Native chemokines and RANTES analogs with agonist activity induce CCR5 downregulation in a G-protein independent manner. Native chemokines have a low potency in internalizing CCR5. (A-D) Cell surface expression of FLAG-tagged receptors stably expressed in HEK 293 cells was detected by flow cytometry as indicated in SI Materials and Methods. (A) CCR5 cell surface expression level (in Mean Fluorescence Intensity) was measured after stimulation or not by 300 nM PSC-RANTES (90 min, 37 °C) of cells treated or not by PTX. In panels (B) and (C), CCR5 downregulation is expressed as percent of maximum effect induced by PSC- or 6P4-RANTES, which showed equal potency (Table 2) and efficacy (panel (A)) in the assay. In panel (B), cells were stimulated by chemokines for 90 min at 37 °C. Data were fitted to a sigmoidal dose-response model with a variable slope, with bottom and top values constrained to equal 0 % and 100 %, respectively. Panel (C) shows time-dependent downregulation of CCR5 induced by 100 nM PSC-RANTES or CCL4. Data were analyzed using a one-phase exponential association function. In panel (D), the expression level of either WT- or 349-CCR5 at the surface of cells stimulated by CCL4 or PSC-RANTES (90 min, 37 °C) is expressed as percent of receptor expression level at the surface of untreated cells (100 %). Results are representative of two (C) or three (A, B and D) independent experiments.
Supporting information Colin et al. SI Materials and Methods
Ligands, Cells and Viruses. Radioactive chemokineswere from PerkinElmer Life Sciences. Dr F. Baleux (Institut Pasteur, Paris) provided CCL4. CCL5 and RANTES analogs were produced as described in ref. (22). CCL3 and CCL7 were purchased from R&D systems (Minneapolis, MN). Recombinant soluble human CD4 (sCD4) was from Protein Sciences Corp., Meriden, CT. Soluble, monomeric HIV-1 gp120 were produced using a Semliki forest virus (SFV) expression system and metabolically labeled with 35S-Met/Cys as in ref. (19), purified by affinity chromatography on Strep-Tactin columns (IBA, Goettingen, Germany) using the One-STrEP-tag fused to the gp120 C-terminus as a bait, and quantified by Coomassie blue staining using BSA as a standard. Gp120Bx08 was previously described (19). The sequences coding for gp12025, gp12034, gp12050 and gp12058, cloned into the SFV-derived expression vector pSFV2 or the pNL4-3Ren plasmid (v.i.), were isolated from biological virus clones (clones 341.14 5C6, 341.75 6C4, 1031.20 8C1 and 1031.65 9D8, respectively) obtained from PBMCs of two individuals (patients # 341 and 1031) of the Amsterdam Cohort studies on HIV-1 and AIDS (a gift from Dr H. Schuitemaker, University of Amsterdam, The Netherlands). PBMCs were collected 30 (gp12025), 128 (gp12034), 22 (gp12050) and 91 (gp12058) months after seroconversion at CD4+ T-cell counts of 630, 90, 520 and 50 cells/mL, respectively.
A3.01.R5 cells and HEK cells stably expressing WT-CCR5 (HEK-R5 cells), R126N-CCR5, FLAG-tagged WT- or 349-CCR5 were described previously (19, 27, 28, 37). CD4- and CCR5-expressing HeLa P4C5 cells were cultured at 37 °C under CO2 in DMEM supplemented with 10 % FCS, 100 U/mL penicillin, 100 mg/mL streptomycin, 1 mg/mL G418 and 300 mg/mL hygromycin B. Human CD4+ T-lymphocytes were purified from PBMCs of healthy blood donors (Etablissement Français du sang, The French Official Blood Bank) by Ficoll centrifugation (PAA) followed by immunomagnetic selection (Miltenyi Biotec) and maintained in phytohemagglutinin (1 mg/mL)- and interleukin 2 (300 IU/mL)-containing RPMI-1640 medium at 37 °C under CO2 before use.
The pBx08Ren and pJRRen plasmids were described previously (19, 38). The plasmids p25Ren, p34Ren, p50Ren and p58Ren were generated from the pNL4-3Ren plasmid previously described (38). Using the technique of overlapping PCR, the fragment from residue 34 to 480 in the NL4-3 gp120 was replaced by the corresponding region from gp12025, gp12034, gp12050 or gp12058. The sequences of primers used are available under request. Protocols for virus production in HEK 293T cells and quantification (p24) are described elsewhere (19).
In this work, guanosine 5’-0-(g-thio)triphosphate (GTPgS, from Sigma, St Louis, MO)) and Guanosine 5′-[β,γ-imido]triphosphate (Gpp(NH)p) were used at 200 and 100 M, respectively. Treatments of cells with the toxin from Bordetella pertussis (PTX) (Sigma) were carried out overnight at a 100 ng/mL concentration. The M2 anti-Flag monoclonal antibody is from Sigma (St Louis, MO).
Radioligand Binding Assays.Protocols for membrane preparations and 125I-CCL3 binding studies to cell membrane preparations or intact cells were described previously (19). Bmax determinations for 125I-CCL3 and 35S-gp120 in intact HEK-R5 cells and membrane preparations from these cells were previously carried out (19). For saturation binding of 125I-CCL5, CCR5-expressing membranes (1 mg of proteins/well) and the radioligand were incubated in 96-well basic Flashplates (PerkinElmer Life Sciences) for 90 min at room temperature, in the presence or in the absence of Gpp(NH)p and/or 10 mM MVC (from the AIDS Research and Reference Reagent Program) in a 0.1 mL final volume of binding buffer (19). Bound and unbound 125I-CCL5 were separated by centrifugation (800 x g, 10 min) at 4 °C and removal of supernatants. Displacement of 35S-gp120 in the presence of 30 nM sCD4 was performed as in ref. (19) except that incubations were done in Eppendorf tubes. To remove unbound 35S-gp120, membranes were pelleted at 4 °C (16.000 x g, 5 min) and then washed once with washing buffer (50 mM Hepes, pH 7.4, 5 mM MgCl2, 1 mM CaCl2 and 500 mM NaCl). Pellets were resuspended in Optiphase Supermix scintillation liquid and radioactivity was counted in a Wallac 1450 MicroBeta Trilux® (PerkinElmer Life Sciences).
Analysis of the binding data was made using the Prism software (GraphPad Software Inc., San Diego). The inhibition constants Kiof the competing chemokines were calculated according to the Cheng and Prusoff equation Ki = [IC50/(1+L/KD)], where L is the concentration of the radioligand; KDis the dissociation constant of the radioligand-CCR5 complex, and IC50 is the concentration of competing ligand displacing 50% of the specific binding of the radioligand (39). In competition experiments, an F test was used to determine whether the experimental data fitted better to a one-site or a two-site competitive binding model.
Chemotaxis assays. A3.01.R5 cells (1.5x105) in 0.08 mL of assay medium (RPMI-1640 supplemented with 20 mM HEPES and 1 % human serum AB) prewarmed at 37 °C were added to the upper chambers of HTS-Transwell-96 Well Permeable Supports with polycarbonate membrane of 5 mm pore size (Corning Inc.), and 0.235 mL of the same medium with or without (spontaneous migration) PSC-RANTES was added to the lower chambers. Chemotaxis proceeded for 6 h at 37 °C in humidified air with 5 % CO2. The number of cells migrating across the polycarbonate membrane was assessed by flow cytometry with a FACS Canto (BD Biosciences). Specific migration was calculated by subtracting spontaneous migration from the number of cells that migrated toward the chemokine.
Receptor downregulation.HEK cells stably expressing either WT-CCR5 or 349-CCR5 fused to a FLAG epitope at their N-terminal end, pretreated or not overnight with 100 ng/mL PTX, were detached from culture plates in EDTA-containing PBS, and then incubated at 37 °C in conical-bottom 96-well plates (5x105 cells/well) for the indicated periods of time in 0.25 mL final volume of assay medium (DMEM supplemented with 20 mM HEPES and 1 % BSA), in the presence or in the absence of the indicated concentrations of chemokines. Cells were then placed on ice, centrifuged (200 x g, 4 °C) and washed once in cold PBS supplemented with 1 % BSA and 0.1 % sodium azide. Cells were then incubated for 1 h at 4 °C in 0.2 mL of PBS/BSA buffer containing the anti-FLAG M2 monoclonal antibody (Sigma) at 2 mg/mL, washed once, and then further incubated with a horse anti-mouse secondary antibody conjugated to phycoerythrin (5 mg/mL) (Vector). We controlled that chemokines and M2 did not compete for binding to Flag-tagged CCR5. The amount of receptors remaining at the cell surface was assessed by flow cytometry using a FACS Canto (BD Biosciences) and expressed as indicated in the legend of Fig. 5.
Infection inhibition assays. HeLa P4C5 cells were cultured in flat-bottom 96-well plates (1.5x104 cells/well) for 24 h and then treated, or not, overnight with PTX (100 ng/mL). Unless otherwise mentioned, cells were then infected with 7 ng of p24 of each virus in the presence or in the absence of chemokines and/or PTX at 10 ng/mL. Regarding activated CD4+ T-cells and A3.01.R5 cells, 2x105 cells treated, or not, overnight with PTX (100 ng/mL) were dispensed in round or conical-bottom 96-well plates and then incubated with the viruses (2 ng or 7 ng of p24 for A3.01.R5 cells and CD4+ T-cells, respectively) in the presence or in the absence of the indicated concentration of chemokines and/or PTX at 10 ng/mL. Incubation of CD4+ T-cells was carried out in the presence of IL-2 at 300 IU/mL. At 48 h post-infection, cells were lysed, and viral replication was assessed by measuring luciferase activity (PROMEGA, Madison, WI), using the 96-well plate lumi/fluorimeter Mithras LB940 (Berthold).
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Table S1. Half-maximal effective concentrations (EC50) for chemokine-induced downregulation of WT- or 349-CCR5 in HEK 293 cells treated or not by PTX