FIG. 1.

(A) Binding of gp120 to chemokine receptors. Radioiodinated Env proteins were tested for binding to 2 × 10⁵ to 5 × 10⁵ 293T cells transiently transfected with plasmids expressing full-length CD4, CCR5, or CXCR4 or with the parental vector pcDNA3. Binding was performed at RT for 1 h in the presence or absence of 100 nM sCD4 as indicated. Raw values of representative experiments repeated at least three times are shown without background subtraction. The strains used were M-tropic HIV-1 strains JRFL and CM235, T-tropic HIV-1 strains MN and HXB2, and SIVmac239. (B and C) Optimization of JRFL gp120 binding to CCR5. The binding assay was optimized by varying conditions such as cell number (B) or the amount of radiolabeled gp120 added (C). Results are from representative experiments, repeated at least twice, without any background subtraction. Optimization of sCD4 concentrations are shown in Fig. 8B. Optimal specific binding was achieved with 5% BSA, but signal/noise values of greater than 5:1 could also be achieved with 0.1% BSA (data not shown), enabling further modification of this assay for applications such as high-throughput screening. Unless otherwise indicated, all assays were performed with 2 × 10⁵ cells, 100 nM sCD4, and 5% BSA in HEPES⁺⁺ binding buffer, for 1 h at RT, and with wash buffer containing 500 mM NaCl.