An Inducible Cell-Cell Fusion System with Integrated Ability to Measure the Efficiency and Specificity of HIV-1 Entry Inhibitors
Figure 6
Design and testing of a dual reporter assay measuring inhibition efficiency and specificity.
A. A dual reporter system was designed by incorporating the R-Luc gene, under the control of a tTA-responsive promoter, into effector cells. When these cells are fused to target cells, two readouts are measured sequentially: F-Luc activity measures the extent of cell-cell fusion and R-Luc activity measures any off-target effect. Specific fusion inhibitors are expected to give low F-Luc and high R-Luc activities, whereas unrelated inhibition results in low activities of both luciferases. B. Various compounds were tested with the dual reporter system using H-AD8#15Ren effector cells and CEM-R5L1#21 target cells. Both F-Luc and R-Luc activities were measured and normalized to those activities seen in cells without any inhibitor. The final concentration of DMSO during the cell-cell fusion assay was 0.1% unless otherwise indicated. T20, T20 peptide (118 nM); Mar, Maraviroc (1.18 µM); CHX, cycloheximide (100 µg/ml); RPA, RPA-T4 (anti-CD4 antibody, 1 nM); OKT, OKT4 (anti-CD4 antibody, 1 nM); Con.Ab, Control antibody (1 nM); NBD, NBD-556 (9.1 µM); Dox, doxycycline (2 µg/ml); EF, efavirenz (an HIV-1 reverse transcriptase inhibitor, 312 nM).