Fig. 8

YFP-Vpr shedding is rate-limiting for nuclear entry and is not modulated by capsid stability. a Nuclear GFP-Vpr and Hoechst fluorescence before and after GFP photobleaching. b Fluorescence recovery after photobleaching. Circles are normalized means and SEM of 10 nuclei. Inset The GFP-Vpr signal recovery after photobleaching (circles), the line is a double-exponential fit to the data. c Kinetics of Vpr nuclear accumulation in CV-1/TVA950 and A549/TVA950 cells in the presence or absence of 50 μg/ml of R99 peptide. d Kinetics of single ASLVpp fusion and YFP-Vpr shedding in CV-1- or A549-derived cells measured as the time-point of mCherry disappearance from dual-labeled ASLVpp and complete loss of YFP-Vpr, respectively. Circles represent normalized cumulative plots for signal disappearance from ASLVpp. Lifetimes of post-fusion cores were measured as the difference in disappearance times of mCherry and YFP signals for the same particle. e Synchronized fusion of ASLV from endosomes. ASLVpp was allowed to enter CV-1-derived cells for 45 min at 37 °C in the presence of 70 mM NH₄Cl. Viral fusion was initiated by replacing NH₄Cl with imaging buffer, and the kinetics of fusion (release of mCherry) and loss of YFP-Vpr was measured (left axis). The corresponding appearance of YFP-Vpr in the nucleus in the same imaging field was determined as a fold-increase over that prior to initiation of synchronous fusion from endosomes (open circles, right axis). f CV-1/TVA950 cells inoculated with YFP-Vpr-labeled VSVpp containing either the wild-type (WT) HIV-1 capsid (R9 backbone) or one of the two capsid mutants, K203A (destabilizing) and 5Mut (stabilizing). The amount of cell-bound viruses was equalized based on cell-associated YFP-Vpr fluorescence and viruses were allowed to fuse for 2 h at 37 °C in the presence of 20 μΜ MG132. The nuclear YFP-Vpr signal was measured at indicated time intervals and normalized to the value at 2 h. Data-points are means and SEM from four image fields each