HIV-1 hemifuson with the plasma membrane can be distinguished from dynasore-mediated quenching of the viral membrane marker. (A) TZM-bl cells were pretreated with 60 μM dynasore (Santa Cruz) in HBSS++ and centrifuged with HXB2 Gag-mKO/DiD virus. Cells were washed with HBSS++ and imaged in the same buffer (upper panel), in 60 μM dynasore (lower panel), or in 60 μM dynasore + 1 μM of C52L (not shown) for 30 min at 37°C. mKO is pseudocolored green and DiD is pseudocolored red. Scale bar is 20 μm. (B) The changes in DiD intensity induced by dynasore were evaluated based on the mean fluorescence of at least 100 virions positive for both Gag-mKO and DiD. To compensate for the focal drift and imaging artifacts related to the cell movement, the ratio of mean DiD/mKO fluorescence signals is plotted over time. MLV Gag-mKO/DiD or Gag-GFP/DiD particles pseudotyped with HXB2 Env (C and E) or JRFL Env (D and F) were bound to TZM-bl cells pretreated with 60 μM dynasore in HBSS++. Individual viruses were tracked and analyzed for changes in fluorescence intensity. Single particle tracking in the presence of dynasore demonstrated either the fast DiD decay (C andD) or slow decay (E and F) phenotypes in the presence of dynasore. (G) The fraction of co-labeled HXB2 pseudoviruses undergoing fast lipid transfer or endosomal fusion (content release) in control experiments or in the presence of 60 μM dynasore with or without 1 μM C52L.