Fig. 4

Nuclear accumulation of YFP-Vpr upon co-incubation of viruses with target cells. a ASLVpp labeled with YFP-Vpr (green) and Gag-imCherry (red) were pre-bound to CV-1 cells expressing TVA950 in the cold (left panels), and virus entry was initiated by shifting to 37 °C. At 45 min post-initiation, 70 mM of NH₄Cl was added to block fusion and fully recover the YFP fluorescence in acidic endosomes (middle panels). Virus-cell incubation in the presence of fusion inhibitory R99 peptide (50 μg/ml) abrogates nuclear accumulation of YFP-Vpr, but not virus uptake (right panels). The top panels are three-color (Hoechst/YFP-Vpr/Gag-imCherry) images, while the bottom panels show only the YFP-Vpr and Gag-imCherry channels for clarity. The left and middle panels are the same image field at different time points, while the right panels are from a different experiment carried out in the presence of R99. White lines are drawn through the nuclei to generate respective intensity profiles shown in d. Inset in the lower middle panel shows the enlarged boxed area. b High-resolution confocal image of an optical slice through the middle of the CV-1 cell nucleus stained with Hoechst-33342 after incubation with MOI of ~0.05 of ASLVpp co-labeled with YFP-Vpr and Gag-imCherry. c Line histograms through nuclei corresponding to images in (a) depict the degree of spatial overlap of YFP-Vpr (green), mCherry (red) and Hoechst (blue) signals before raising the temperature and after 45 min at 37 °C in the absence or in the presence of R99 peptide. d Images of YFP-Vpr/Gag-imCherry labeled HXB2 pp particles pre-bound to CV-1-derived target cells before (left) and after (middle) incubation at 37 °C for 3 h. Parallel samples (right) were incubated for 3 h in the presence of 5 μM of HIV-1 fusion inhibitor C52L. Three-color images (upper panels) and two-color images (lower panels) are shown for the ease of identification of the YFP-Vpr signal within the Hoechst-stained nuclei (blue)