Figure S6.

HIV-1 infectivity and nuclear entry upon CPSF6 knockdown in SupT1-R5 cells, related to Figure 5

(A–C) CPSF6 knock-down in SupT1-R5 cells. Cells were transduced with AAVs expressing non-silencing (NS) or CPSF6 shRNA. Mock-transduced cells were used as additional control. 72 h after transduction, cell viability and CPSF6 knock-down efficiency were analyzed. (A) The number of viable cells was determined by trypan blue exclusion. The graph shows mean values and SD from three independent experiments. (B) CPSF6 knock-down efficiency. Cells were fixed, immunostained with anti-CPSF6 antibody, followed by staining with secondary antibody conjugated with AlexaFluor 488. The proportion of CPSF6-positive cells was determined by flow cytometry. Data are representative of three independent experiments. Statistical significance was calculated using an unpaired two-tailed t test, ^∗∗∗p < 0.0001. (C) Representative histogram of CPSF6 signal intensity for cell populations analyzed by FACS. For the experiment shown, 12,332 (mock), 15,195 (NS shRNA) and 13,454 (CPSF6 shRNA) events were analyzed. (D–F) HIV-1 infectivity and nuclear import in SupT1-R5 cells upon CPSF6 knock-down. (D) Effect of CPSF6 knock-down on HIV-1 infectivity. AAV-transduced cells were infected with wild-type HIV-1_NL4-3. At 48 h p.i., cells were fixed, immunostained for intracellular HIV-1 CA and infection was scored by flow cytometry. The graph shows mean values and SEM from three independent experiments performed in triplicates. Statistical significance was assessed by unpaired two-tailed Student’s t test. n.s., not significant. (E, F) Effect of CPSF6 knock-down on virus nuclear entry. AAV-transduced cells were infected with IN.mScarlet carrying NNHIV particles for 90 min at 16°C and then shifted to 37°C to initiate virus entry. Cells were fixed 15 h after temperature shift and immunostained for CPSF6 (green) and lamin B (cyan). Arrowheads indicate IN.mScarlet-positive complexes (red) in the nucleus (white arrowheads) of cell expressing non-silencing (NS) control shRNA, and IN.mScarlet signals associated with nuclear envelope (empty arrowheads) in cell expressing CPSF6 shRNA. Scale bars: 2.5 μm. (F) Box-and-whisker plot shows the numbers of IN.mScarlet positive complexes in nuclei of cells expressing NS or CPSF6 shRNA. Outliers were identified by Tukey’s test. Statistical significance was assessed by unpaired two-tailed Student’s t test, ^∗∗∗p < 0.0001.