Time-Resolved Imaging of Single HIV-1 Uncoating In Vitro and in Living Cells
Fig 4
Tight association of CypA-DsRed with post fusion HIV-1 cores.
(A) Approximately 100,000 TZM-bl cells were inoculated with ~1 ng (MOI 0.4) of IN-sfGFP (green) and CypA-DsRed (red) labeled pseudoviruses at 37°C, fixed at 4 h post-infection and immunostained for CA/p24. Cell nucleus stained with Hoechst-33342 is colored gray. Scale bar 5 μm. (B) The fraction of double-labeled IN-sfGFP/CypA-DsRed and IN-sfGFP/p24 puncta in TZM-bl cells infected as in panel A after varied times post-infection normalized to the control experiments in which viral fusion was prevented by 50 mM NH4Cl. Data are means and SEM from 4 fields of view. (C) TZM-bl cells were infected with pseudoviruses for 4 h, as in panel A, treated with 10 μM CsA or DMSO for 30 min at 37°C, fixed and imaged. Cell nuclei are colored blue. Scale bar 5 μm. (D) Quantification of CsA-induced CypA-DsRed displacement from post-fusion cores shown in panel C. The mean fluorescence intensity of CypA-DsRed and CA/p24 within IN-sfGFP spots was calculated for control and CsA-treated samples. Data are means and SD from 4 fields of view. (E) The number of intact post-fusion cores at indicated times after infection carried out in the presence of Nevirapine (Nevir, 10 μM) or DMSO. The number of post-fusion cores per cell was calculated as the difference between the number of colocalized IN-CypA spots per cell in control and CsA-treated samples for each time point. Data are normalized to NH4Cl control. Results are means and SEM from 4 fields of view. (F) TZM-bl cells were infected with ~1.5 ng p24 of viruses bearing WT CA, and the E45A and K203A mutants for 2 h. The number of post-fusion cores for each sample was determined from 5 independent experiments, as described in panel E, and normalized to number of WT CA.