Synchronizing the HIV-1 fusion by pre-forming Env-CD4-coreceptor complexes does not redirect fusion to the plasma membrane. JRFL (A) or HXB2 (B) pseudoviruses carrying BlaM-Vpr were pre-bound to TZM-bl cells in the cold and further incubated for 2.5 h at 20.5°C (JRFL), 22-24°C (HXB2) to create a temperature-arrested stage (TAS) or at 4°C (control). Fusion was induced by raising the temperature to 37°C in DMEM/10% FBS. The extent of the functional CD4 and coreceptor engagement by the virus was evaluated by adding the respective fusion inhibitors BMS-806 (10 μM, CD4 binding), AD101 (10 μM, CCR5 binding) or AMD3100 (10 μM, CXCR4 binding) and measuring the BlaM activity. Alternatively, the time course of HIV-1 escape from C52L (1 μM) was measured by adding the peptide at indicated time points. (C) Similar to panel B, but, in addition to the time course of HXB2 escape from C52L, the kinetics of acquisition of resistance to the TB was also determined. (D) HXB2 pseudotyped viruses labeled with the MLV Gag-GFP and DiD were bound to TZM-bl cells in the cold and either immediately used for imaging experiments (blue circles) or further incubated for 2.5 h either at 4°C or, to create TAS, at 24°C. Fusion was trigged by raising the temperature to 37°C at the onset of imaging. Single viruses were tracked and individual fusion events quantified as described in Materials and Methods. The differences in the kinetics of the virus escape from C52L in panels B and C reflect the experimental variability due to differences in the viral stock and the cell passage numbers.