Fig. 5

Nuclear accumulation of YFP-Vpr correlates with virus input. a, b Varying dilutions of YFP-Vpr/Gag-imCherry labeled ASLVpp were pre-bound to CV-1/TVA950 (a) or A549/TVA950 (b) cells in the cold followed by incubation at 37 °C for 2 h in the presence of 20 μΜ MG132. The nuclear YFP-Vpr was measured in fixed samples in the absence (filled circles) or in the presence (open circles) of 50 μg/ml R99 peptide. Data are means and SEM from four image fields. Linear regression lines are shown. c ASLVpp fusion-mediated nuclear accumulation of YFP-Vpr in CV-1/TVA950 cells in the absence (filled circles) or in the presence (gray circles) of 20 μΜ MG132. Data represent the ratio of nuclear YFP-Vpr to the signal prior to initiation of fusion. d The fusion efficiency of ASLVpp/YFP-Vpr/Gag-imCherry with CV-1/TVA950 or A549/TVA950 cells, as determined by single particle tracking. Bars are means and SEM from three experiments each, with total number of dual-labeled viral particles indicated. e Correlation between the fraction of viral YFP-Vpr accumulated in the nucleus after 2 h at 37 °C (see the legend to a) and the slope of viral fusion (BlaM signal) vs the viral p24 input (see Additional file 3: Figure S6). Every point represents a distinct ASLVpp or VSVpp preparation. f ASLVpp (lanes 1, 2) and VSVpp (lanes 3, 4) were produced using the wild-type HIV-1 R9ΔEnv backbone and co-labeled with YFP-Vpr (and BlaM-Vpr to enable measurements of virus-cell fusion shown in Fig. 5a). The viruses either contained (odd lanes) or lacked (even lanes) Gag-imCherry. Equal amounts of p24 from virus preparations were subjected to SDS-PAGE and blotted for p24 (HIV IG antibody) or YFP-Vpr (GFP antibody). The loading order: 1 ASLVpp/YFP-Vpr, 2 ASLVpp/YFP-Vpr/Gag-imCherry, 3 VSVpp/YFP-Vpr and 4 VSVpp/YFP-Vpr/Gag-imCherry. The numbers are the respective fractions (%) of viral YFP-Vpr that entered the nucleus in live cell experiments within 2 h