Extended Data Fig. 2: Characterization of stable cell lines (HEK293T and Expi293F) expressing wild-type human CCR5.

a, Chemokine receptor assay. HEK293T and HEK293T-CCR5 (stable) cells were treated with different concentrations of CCL5. F_t/F₀ is a fluorescence-signal ratio proportional to that of intracellular cAMP concentration at 40 min after CCL5 activation and at time 0. The dose–response curves were plotted for both HEK293T (black) and HEK293T-CCR5 (red) cells. The experiment was carried out in quadruplicate, and repeated at least three times with similar results. Error bars indicate the standard deviation calculated by the STDEV function in Excel. b, Flow cytometry histograms of HIV-1 gp120 binding to CCR5 expressed on the cell surfaces in the absence (orange) or presence (red) of soluble CD4. HEK293T cells (black), CCR5-expressing cells only (grey) and CCR5-expressing cells with soluble CD4 only (blue) were negative controls. The experiment was repeated independently at least twice with similar results. c, HIV-1 Env-mediated cell–cell fusion. HEK293T cells stably transfected with CCR5 were mixed with HIV-1 Env (gp160)-expressing cells in the absence or presence of soluble CD4. The CCR5 cells fuse with CD4-triggered Env cells very efficiently, and form large syncytia that cover almost the entire well. The experiment was repeated independently twice with similar results. d, Chemokine receptor assay by various ligands. As in a, Expi293F and Expi293F-CCR5 (stable) cells were treated with CCL5, gp120, CD4 or the complex of gp120 and CD4. The dose–response curves were plotted for both Expi293F as a control (left) and Expi293F-CCR5 (right) cells, with different ligands as indicated. The experiment was carried out in quadruplicate and repeated at least three times with similar results. Error bars indicate the standard deviation calculated by the STDEV function in Excel. e, Left, kinetic curves of 5 representative wells of HEK293T-CCR5 cells treated with 5 different ligands as indicated. ATP activates the endogenous G_q-coupled G-protein-coupled receptor (P2Y receptor), as a positive control. The ratio represents fluorescence intensity divided by baseline intensity. Right, dose–response curve of each ligand. The y axis is a background-subtracted ratio (peak fluorescent intensity ratio − 1). We conclude that our gp120 and gp120–CD4 do not activate G-protein-mediated calcium flux at the concentrations tested here. The experiment was carried out in quadruplicate and repeated twice with similar results. Error bars indicate the standard deviation calculated by the STDEV function in Excel.