A, RT-PCR analysis of CXCR4 mRNA levels in the stably transfected U87. CD4 cells expressing the different CXCR4 variants. GAPDH was coamplified as an internal reference. The ratios of CXCR4 band intensity to the respective GAPDH band intensity were determined by densitometric analysis. B, flow cytometric analysis of the membrane expression of CXCR4 in the stably transfected cell lines. The black curves represent CXCR4-specific staining by the 12G5 mAb (left), reacting with an epitope located in ECL2, by the 173 mAb (middle), or by the 2B11 mAb (right), directed against the amino-terminal domain of CXCR4. The white histograms represent the background fluorescence caused by aspecific binding of the respective secondary antibodies.
Inhibition of SDF-1-induced intracellular calcium mobilization by AMD3100 in the different transfectants. After loading with the fluorescent calcium indicator Fluo-3, the cells were preincubated for 10 min withor without compound and were then stimulated with the chemokine at 2, 10, or 50 ng/ml depending on the cell line (seeMaterials and Methods). The transient increase in the intracellular calcium concentration was recorded by monitoring the change in fluorescence of the cells as a function of time using the Fluorometric Imaging Plate Reader (FLIPR). Top five graphs, SDF-1-induced calcium flux in the absence of AMD3100 (open symbols) or after preincubation of the cells with AMD3100 at 1 μg/ml (closed symbols). Each data point represents the mean fluorescence of quadruplicate microplate wells. The data of one experiment representative of four are shown. Bottom graph, concentration-dependence of the antagonistic activity of AMD3100. ○, CXCR4[WT]; ▪, CXCR4[D262N]; ▴, CXCR4[H281A]; ♦, CXCR4[D171N]; ●, CXCR4[D171N,D262N].
Inhibition of 12G5 mAb binding to the different CXCR4 mutants by AMD3100. The stably transfected U87.CD4.CXCR4 cells were treated for 15 min with AMD3100 at 1 μg/ml and were then stained with PE-conjugated 12G5 mAb and analyzed by flow cytometry. An isotype control mAb was used to correct for aspecific staining. The mean fluorescence intensity in the untreated positive control of each cell line was set equal to 100% (no inhibition of 12G5 binding). Antibody binding in the AMD3100-treated cell samples was expressed as a percentage of the corresponding positive control, and the percentage of inhibition was calculated. The bars represent the mean ± S.D. of three independent experiments.
PCR-based detection of virus entry into the U87. CD4 transfectants expressing the different CXCR4 mutants. Two hours after infection with HIV-1 NL4.3 or HIV-1 HE, total DNA was extracted from the cells and analyzed by semiquantitative PCR using HIV-1-specific primers from the LTR R/U5 region. DNA samples from mock-infected cells produced no signal (data not shown). DNA recovery was controlled by PCR with β-actin-specific primers. The results shown are from one representative experiment.
A, cytopathic effect (giant cell formation) at day 5 after infection of the different U87.CD4.CXCR4 cell variants with HIV-1 NL4.3. Note the manifest syncytium formation in the U87.CD4.CXCR4[WT], U87.CD4.CXCR4[D171N], and U87.CD4.CXCR4[H281A] cell cultures, which is significantly less pronounced in the U87.CD4.CXCR4[D262N] and totally absent in the U87.CD4.CXCR4[D171N,D262N] cell cultures. Mock-infected U87.CD4.CXCR4[WT] cells are shown as a negative control. Similar images were obtained in several repeat experiments. B, viral core p24 Ag production by HIV-1 NL4.3- and HIV-1 HE-infected U87.CD4.CXCR4[WT], U87.CD4.CXCR4[D171N], U87.CD4.CXCR4[D262N], U87.CD4.CXCR4[D171N,D262N], and U87.CD4.CXCR4- [H281A] cell cultures. The p24 Ag enzyme-linked immunosorbent assays were performed on cell culture supernatants collected at day 6 after infection with the NL4.3 strain and at day 4 after infection with the HE strain. The results represent the mean of at least two independent experiments.