Fig. 6: NS localization stabilizes VRCs in the nucleus.

TZM-bl cells were infected with VSV-G pseudotyped HIVeGFP labeled with INmNG for 4 h (half-time of nuclear import), at which time DMSO, Nevi (10 μM), or the indicated concentrations of PF74 was added and cells were fixed after 30 min or after 2 h of PF74 application to quantify nuclear IN puncta (a, b) and cytosolic CA-positive IN puncta (c) or incubated for additional 20 h to measure infection (d). a, b A high dose of PF74 results in the displacement of IN puncta from NSs and disappearance of IN puncta. a Single Z-stack images showing the presence of CA/p24 immuno-labeled (red) IN puncta (green) in the nucleus (blue) of 25 µM PF74 treated cells after 30 min (top panel) or 2 h (bottom panel). Analysis from 30 to 40 cells in each of the three independent experiments showing the average number of IN puncta per nucleus (b) or CA-positive IN puncta in the cytoplasm (c) is shown. d The fraction of eGFP-expressing TZM-bl cells treated with indicated drugs at 4 hpi was determined at 24 h. Dashed lines represent baseline nuclear import upon drug addition (b) and 50% of DMSO infection (d), respectively. Scale bar in (a) is 5 μm and inset in (a) is 1 μm. Error bars in panels (b–d) are mean values ± SEM. Data in (b, c) are from >80 nuclei/cells containing >1500 IN complexes and three independent experiments (a–d). The statistical significance in (b–d) was determined by two-tailed Student’s t test (ns, p > 0.05; ***p < 0.001). Results of statistical analyses in (d) vs. DMSO control is shown in blue vs. Nevi that is shown in red. Statistical analysis in (b) with respect to a 30 min treatment with DMSO is shown in blue. Source data are provided as a Source Data file.