Fig. 9: Interplay between LEN, CPSF6 and HIV-1.
From: Prion-like low complexity regions enable avid virus-host interactions during HIV-1 infection
a Antiviral activities after LEN addition to HEK293T cells at indicated time points post-infection. The normalized and averaged data (+/− SD) from three independent experiments are shown. Source data are provided as a Source Data file. b Representative immunoblotting to show the expression levels of CPSF6 in parental, CPSF6 KO (CKO) and CPSF6 overexpressing HEK293T cells. The experiment was repeated 3 times independently with similar results. c Antiviral activities of LEN in indicated cells with varying levels of CPSF6. VSV-G pseudotyped HIV-scarlet viruses (MOI = 0.5) were used to infect indicated cells. The infection levels in different cell lines without LEN treatment were 43.9 ± 2% (WT), 38.5 ± 0.9% (CKO) and 38.4 ± 1.6% (WT + CPSF6). The normalized and averaged data (+/− SD) from three independent experiments are shown in c. Source data are provided as a Source Data file. d TZM-bl cells were infected with INmNG-labeled fluorescent HIV-1 pseudoviruses for 8 h to allow virus nuclear import40. At 8 hpi, cells were treated with DMSO or indicated concentrations of LEN for 30 min, fixed with PFA and immuno-stained for endogenous CPSF6. Single Z-slice images show nuclear IN-puncta (green) and endogenous CPSF6 (red). Yellow dashed circles and white arrows point to nuclear IN-puncta colocalized or not-colocalized with CPSF6, respectively. Insignificant nuclear import occurs within 30 min of LEN treatment. Scale bar is 5 μm. e Analysis of background subtracted CPSF6 fluorescence associated with nuclear IN puncta in d. All data points (n = 553 for DMSO; n = 740 for 0.5 nM LEN; n = 389 for 5 nM LEN; n = 746 for 50 nM LEN; n = 309 for 500 nM LEN; n = 412 for 5000 nM LEN) obtained from a total of three independent experiments are overlayed. Statistical significance of comparison of indicated concentration of LEN versus DMSO control was determined by non-parametric Mann-Whitney two-tailed rank sum test. P-values for 0.5, 5, 50, 500 and 5000 nM LEN were 0.21, 0.66, 1.3E-24, 1.7E-22, 2.5E-28, respectively. ***p < 0.0001. ns: not significant. Source data are provided as a Source Data file. f Isolated HIV-1 cores were incubated with 2 μM GST-CPSF6261-358 for 20 min. DMSO control or indicated concentrations of LEN were added to the mixture and incubated for 20 min. The HIV-1 cores pulled-down by GST-CPSF6261-358 through glutathione sepharose beads were detected by ELISA. The averaged data (+/− SD) from three independent experiments are shown. Source data are provided as a Source Data file. LEN lenacapavir, a.u. arbitrary units.
