HIV-1 infection is blocked after CXCR4 conformational change. (A) U87-CD4-CXCR4 and U87-CD4-CCR5 cells were incubated with NSC23766 and CXCR4 (detected by mAb 44717 or 4G10) and CCR5 (detected by mAb 2D7) surface signals were measured. Bars show the mean fluorescence intensity determined by flow cytometry and expressed as percentage ± SEM compared with untreated conditions (n = 3). (B) U87-CD4-CXCR4, and U87-CD4-CCR5 cells were inoculated, in the presence or absence of NSC23766, with the single round luciferase reporter HIV-1 pseudotyped with the X4-using envelope HxB2 or the R5-using envelope BaL-26, respectively. Forty-eight hours later, the infectivity of the virus was analyzed by the luciferase assay. Bars show the luciferase activity measured by a microplate luminometer and expressed as percentage ± SEM compared with untreated conditions (n = 3). (C) U87-CD4-CXCR4 cells were inoculated with the X4-virus NL4-3 in the presence or absence of NSC23766. After 48 hours, DNA samples were isolated and the early reverse transcriptase product R/U5 was quantified by qPCR. Bars show the number of proviral DNA copies corrected for differences in DNA input (β-actin) and expressed as percentage ± SEM compared with untreated conditions (n = 3). (D) PHA-stimulated PBMCs were inoculated with the X4-virus NL4-3 in the presence or absence of NSC23766. After 48 hours, DNA samples were isolated and R/U5 and pol proviral DNA were quantified by qPCR. Bars show the number of proviral DNA copies corrected for differences in DNA input (β-actin) and expressed as percentage ± SEM compared with untreated conditions (n = 4; *P < .05, **P < .01).