FIG 4.

P2X1R antagonists prevent functional engagement of CXCR4 and CCR5 by HIV-1 Env. HXB2pp or BaL26pp were prebound to TZM-bl cells in the cold, and fusion was initiated by shifting the temperature to 37°C for 90 min and measured by a BlaM assay. (A) Abrogation of HIV-1 fusion with TZM-bl cells with high concentrations of fusion inhibitors and NF279. The CD4 binding-site-targeting compound BMS-806 (10 μM); the CXCR4 and CCR5 binding inhibitors AMD3100 and TAK-779, respectively (both at 7 μM); the gp41 6-helix bundle inhibitor C52L (7 μM); and NF279 (10 μM for HXB2pp and 50 μM for BaL26pp) were added immediately prior to virus-cell coincubation at 37°C. (B) Schematic protocol for creating a temperature-arrested stage (TAS) of HIV-1 fusion prior to the addition of fusion inhibitors or NF279. CoR, coreceptor. (C) A TAS of fusion between HXB2pp or BaL26pp and TZM-bl cells was created as depicted in panel B, and fully inhibitory concentrations of fusion inhibitors or NF279 (at the concentrations described above for panel A) were added before raising the temperature to induce fusion. (D) Illustration of a TAS protocol similar to the one depicted in panel B, but incubation at a subthreshold temperature was carried out in the presence of a fully inhibitory concentration of NF279, after which time the antagonist was washed out and viruses/cells were incubated at 37°C in the absence or in the presence of high concentrations of fusion inhibitors. (E) Inhibition of HXB2pp and BaL26pp fusion with TZM-bl cells after preincubation at a subthreshold temperature in the presence of NF279, as depicted in panel D. Fusion inhibitors were added at the concentrations indicated above for panel A, immediately prior to shifting the temperature to 37°C. Data in panels A, C, and E are the normalized mean BlaM activities and standard deviations from three experiments carried out in triplicate. **, P < 0.01; ***, P < 0.001; NS, not significant; ND, not determined.