Time-Resolved Imaging of Single HIV-1 Uncoating In Vitro and in Living Cells
Fig 5
Analyses of spontaneous and CsA-induced release of CypA-DsRed from post-fusion cores.
(A) CsA-induces loss of CypA-DsRed from the IN-sfGFP labeled particle 1 in a TZM-bl cell at 4 h.p.i. Particle 2 retained CypA-DsRed. The cell nucleus is colored blue. The lower image panels show blow up views of the two particles at indicated times after CsA addition. Scale bar 10 μm. (B) Kinetics of spontaneous, apparently complete loss of CypA-DsRed from IN-sfGFP labeled cores in TZM-bl cells within 80 min of imaging, starting at 2, 4 or 6 h after infection. CsA (10 μM) was added at 80 min after beginning of image acquisition. (C-G) Single particle tracking analysis of CypA-DsRed loss from IN-sfGFP puncta in TZM-bl cells at 6 h post-infection. Cells were imaged for 80 min, at which point 10 μM CsA was added and imaging continued for 40 min. Examples of a steady level of the CypA marker (C), as well as partial (D) and nearly complete (E) loss of CypADsRed are shown. (F) Typical stable CypA-DsRed and IN-sfGFP signals in the presence of Nevirapine (10 μM). Dashed lines in panels C-F mark the time of CsA addition. (G) Analysis of the single particle tracking results exemplified in panels C-F. The changes in CypA-DsRed intensity within the 80 min imaging window were measured for 142 randomly chosen post-fusion cores in control samples and 122 cores in Nevirapine treated samples. The CypA-DsRed loss was categorized as no release (less than 40% loss of the initial signal), slow (40–70% of the signal) and quick (>70% loss) during the 80 min imaging interval. The fraction of particles that did not release CypA-DsRed within 80 min of imaging increased from 43.0% in control to 65.6% in Nevirapine-treated cells, whereas the slow and quick release events dropped from 42.4% to 32.8% and from 14.6% to 1.6%, respectively.