Extended Data Fig. 6: The DCAF1 binding- defective Vpx mutant Q76R also impairs cGAS- STING-triggered NF-κB activation and silencing DCAF1 does not affect Vpx-mediated cGAS- STING inhibition.

A,B, HEK293T cells were co-transfected with the NF-κB promoter luciferase vector, pRL-TK Renilla, Myc-cGAS, STING- Flag, and increasing amounts of SIVmac HA-Vpx (wild-type or Q76R) as indicated. At 24 h after transfection, the cell lysates were detected by immunoblotting with anti-SAMHD1, anti- Flag, or anti-HA antibody (A). The NF-κB promoter activity was analyzed by luciferase reporter assays (B). C,D, HEK293T cells were co-transfected with the NF-κB promoter luciferase vector, pRL-TK Renilla, Myc-cGAS, STING-Flag, and SIVmac HA- Vpx (wild-type or Q76R) as indicated. The cell lysates were detected by immunoblotting with anti-SAMHD1, anti-Flag, or anti-HA antibody 24h after transfection (C). The mRNA levels of NFKBIA, IER3, GADD45B, and CXCL8 were analyzed by RT- qPCR (D). E,F, HEK293T cells were co-transfected with the NF- κB promoter luciferase vector, pRL-TK Renilla, Myc-cGAS, STING-Flag, and increasing amounts of HIV-2 ROD HA-Vpx (wild-type or Q76R) as indicated. After 24h, the cell lysates were detected by immunoblotting with anti-SAMHD1, anti- Flag, or anti-HA antibody (E). The NF-κB promoter activity was analyzed by luciferase reporter assays (F). G,H, HEK293T cells were co-transfected with the NF-κB promoter luciferase vector, pRL-TK Renilla, Myc-cGAS, STING-Flag, and HIV-2 ROD HA-Vpx (wild-type or Q76R) as indicated. The cell lysates were detected by immunoblotting with anti-SAMHD1, anti-Flag, or anti-HA antibody 24 h after transfection (G). The mRNA levels of NFKBIA, IER3, GADD45B, CXCL10, and CXCL8 were analyzed by RT-qPCR (H). I, HEK293T cells transduced with shNT or shDCAF1 were generated. Transduced HEK293T cells were co- transfected with the NF-κB promoter luciferase vector, pRL-TK Renilla, Myc-cGAS, STING-Flag, and HA-Vpx (SIVmac, HIV-2 ROD, or HIV-2 7312A) as indicated. The cell lysates were detected by immunoblotting with anti-DCAF1, anti-Flag, or anti-HA antibody 24h after transfection. J, NF-κB promoter activity was analyzed by luciferase reporter assay using parallel samples prepared as described above. (One representative immunoblotting result out of n = 3 independent biological experiments is shown (A, C, E, G, I)). Data are from n = 3 independent biological experiments. Means and standard deviations (B, D, F, H, J) are shown. The statistical significance analyses were performed using a two-sided unpaired t-test.