FIG. 5.

CCR5-binding ability of soluble recombinant envelope glycoproteins. Equivalent amounts of recombinant soluble gp120 envelope glycoproteins labelled with [³⁵S]cysteine and [³⁵S]methionine were preincubated with 0.5 μg of sCD4 per ml for 1 h at room temperature. Cf2Th-CCR5 cells (10⁶ cells/well in six-well plates), which stably express human CCR5 (17), were overlayed with the gp120-sCD4 mixture and were incubated at 37°C for 2 h. The cells were subsequently washed with phosphate-buffered saline plus 2% fetal calf serum and were lysed with NP-40 buffer. The cell lysates were then immunoprecipitated with serum from an HIV-1-infected individual, and the amount of precipitated gp120 was determined by densitometry of sodium dodecyl sulfate-polyacrylamide gels. As a control, the binding assay was also performed with the 89.6 gp120 glycoprotein in the absence of sCD4 (Mock). Two independent clones of each recombinant envelope glycoprotein were used in the analysis, and the experiments were carried out at least twice.