Fig. 4: HIV-1 VRCs recruit CPSF6 to NSs.

a–d Timecourse of CPSF6 accumulation at NS-localized HIV-1 complexes. TZM-bl cells stably expressing SNAP-Lamin nuclear envelope marker and MDMs (untreated) were infected at MOI of 5 and fixed at indicated time points. Where indicated, PF74 (25 μM) was added to samples 30 min before fixation (denoted 6 h + PF74). Cells were immunostained for NSs (SC35) and endogenous CPSF6. Uninfected cells were used as controls. Association of nuclear INmNG-labeled VRCs with SC35+ compartments in MDMs (a) and TZM-bl cells (b). White dashed circles and contours show NS-associated IN puncta, yellow arrows point to IN puncta that recruited CPSF6, white arrows in 6 h + PF74 treatment point to the loss of CPSF6 signal from IN puncta. Analysis of the ratio of CPSF6/INmNG fluorescence in nuclear IN puncta residing inside (SC35(+)) and outside (SC35(−)) NSs in MDMs (c) and TZM-bl cells (d). Note: raw INmNG and CPSF6 fluorescent intensities are shown in Supplementary Fig. 5a–d. Scale bar is 5 μm in (a, b). Data in (c, d) are presented as median values (yellow lines) ± SEM at 95% CI. Obtained by analysis of >80 nuclei in each cell type for each time point, from three independent experiments/donors. The number of MDM IN complexes analyzed in (c) was 31, 79, 353, and 80 for 2, 4, 6 h, and 6 h + PF74, respectively. In (d), >200 IN puncta in TZM-bl cells were analyzed for all time points. Statistical analysis was performed using nonparametric Mann–Whitney rank-sum test (nsp > 0.05; ***p < 0.001). In (c, d), analyses for the same sample inside and outside NSs are shown in blue, and the p values for the effect of PF74 treatment at 6 hpi are shown in red. All nuclear IN spots were analyzed without an exception. Source data are provided as a Source Data file.