Imaging the HIV-1 fusion with lymphoid cells. HIV-1 JRFL Env pseudotyped MLV particles co-labeled with Gag-GFP and DiD were bound to CEM.NKR-CCR5.Luc cells by spinoculation at 4°C. Virus uptake was triggered by raising the temperature to 37°C just prior to imaging. (A) Hemifusion events at the cell surface were characterized by the loss of the DiD signal while the GFP-tagged viral content was retained. (B) Hemifusion with the plasma membrane is associated with restricted particle motility. (C) Upon endosomal fusion, the membrane marker, DiD, remained within the recipient endosome while the Gag-GFP signal was rapidly lost. (D) The virus shown in (C) underwent more extensive movement. (E) Kinetics of the individual hemifusion (red line) or fusion (green line) events plotted as cumulative distributions as a function of time. Arrows in A and C indicate the tracked co-labeled virus.