Inhibitory effects of SCH-C and AMD3100 on replication of 8 different HIV-1 clinical isolates in U87.CD4.CCR5.CXCR4 cells. Viruses were added after 10 minutes of preincubation with SCH-C and AMD3100 at 1000 ng/ml or as a mixture of both at a 1:1 ratio each at 1000 ng/ml. Viral p24 core Ag production (pg/ml) was measured by ELISA 5 days after infection of cells. The coreceptor use of each isolate is shown between brackets. The data of one representative experiment out of two are presented.

Flow cytometric analysis of the membrane expression of CD4, CCR5 and CXCR4 in the U87.CD4.CCR5.CXCR4 double-transfected cell line (3 panels, left column) and in the single-transfected U87.CD4.CCR5 cell line (3 panels, central column) and U87.CD4.CXCR4 cell line (3 panels, right column). The gray curves represent staining by the specific CD4 (clone SK3), CXCR4 (clone 12G5) and CCR5 (clone 2D7) monoclonal antibodies in, respectively, the upper, central and lower row. The mean fluorescence intensity (MFI) of the CD4, CXCR4 and/or CCR5 positive cells is indicated in the right hand corner of each histogram. Aspecific background fluorescence is indicated by the white peaks.

Microscopic view of cytopathic effect (giant cell formation) at day 5 after infection of U87.CD4.CCR5.CXCR4 cells with the clinical isolates CI #10, CI #14 and CI #17, which each have a different coreceptor use as shown between brackets. Uninfected cells are shown as the negative control (upper row). Note the enormous giant cell formation in the untreated virus-infected cell cultures whereas such cytopathic effect is totally absent in infected cells exposed to a 1:1 combination of SCH-C and AMD3100 at 1000 ng/ml. Independently, SCH-C and AMD3100 prevented giant cell formation in cell cultures inoculated with R5 HIV-1 and X4 HIV-1, respectively, but not in cell cultures infected with the dual tropic R5/X4 HIV-1 isolate.

A. Concentration-dependent inhibition of LD78β- and SDF-1-induced intracellular calcium mobilization by SCH-C and AMD3100 in CCR5- and CXCR4-transfected U87.CD4 cells and in the double-transfected U87.CD4.CCR5.CXCR4 cells. The Fluo-3 loaded cells were preincubated for 10 minutes with SCH-C at 200, 40, 8 and 1.6 ng/ml or AMD3100 at 1000, 200, 40 and 8 ng/ml. Then U87.CD4.CCR5 cells were stimulated with LD78β at 100 ng/ml (upper left graph) and the U87.CD4.CXCR4 cells were stimulated with SDF-1 at 20 ng/ml (upper right graph). U87.CD4.CCR5.CXCR4 cells were stimulated with either LD78β at 100 ng/ml (lower left graph) or SDF-1 at 20 ng/ml (lower right graph). The transient increase in intracellular calcium concentration was recorded by monitoring the change in green fluorescence intensity of the cells (y-axis) as function of time (x-axis) using the Fluorometric Imaging Plate Reader (FLIPR). Each data point represents the average value of the fluorescence measured in quadruplicate. The data of one representative experiment of four are shown. B. LD78β- and SDF-1-induced intracellular calcium mobilization and blocking by SCH-C and AMD3100. Fluo-3 loaded cells were preincubated with a 1:1 combination of SCH-C and AMD3100 at 1000 ng/ml for 10 minutes, after which LD78β (100 ng/ml) and SDF-1 (20 ng/ml) were added sequentially at timepoints 20 seconds and 222 seconds respectively (arrows). The transient increase in intracellular calcium concentration was recorded by monitoring the change in green fluorescence intensity of the cells (y-axis) as function of time (x-axis) using the FLIPR. Each data point represents the average value of the fluorescence measured in quadruplicate. The data of one representative experiment out of four are shown.