Extended Data Fig. 4: Vpx inhibits NF-κB signaling induced by cGAS-STING from a lentiviral expression vector or virus infection. | Nature Microbiology

Extended Data Fig. 4: Vpx inhibits NF-κB signaling induced by cGAS-STING from a lentiviral expression vector or virus infection.

From: HIV-2/SIV Vpx targets a novel functional domain of STING to selectively inhibit cGAS–STING-mediated NF-κB signalling

Extended Data Fig. 4

A,B, Vpx expressed from a lentiviral expression vector inhibited NF-κB signaling induced by cGAS-STING. HEK293T cells were co-transfected with the NF-κB promoter luciferase vector, pRL-TK Renilla, Myc-cGAS, STING-Flag, and pLVX-HIV-2 ROD Vpx as indicated. After 24h, NF-κB promoter activity was analyzed (A), and the cell lysates were analyzed by immunoblotting with anti-Flag or anti-HA antibody (B) (n = 3 independent biological experiments). Means and standard deviations are presented. The statistical significance analyses were performed using a two-sided unpaired t-test. C,D, Vpx expressed from an SIVmac infectious clone inhibited NF-κB signaling induced by cGAS-STING. HEK293T cells were transfected with SIVmac wild-type or SIVmacΔVpx for 12h. Subsequently the NF-κB promoter, pRL-TK Renilla, Myc-cGAS, and STING-Flag vectors were transfected as indicated for 24h. NF-κB promoter activity was analyzed by luciferase reporter assays (C), and the cell lysates were analyzed by immunoblotting with antibody targeting Pr55Gag or Flag (D) (n = 3 independent biological experiments). Means and standard deviations are presented. The statistical significance analyses were performed using a two-sided unpaired t-test. E,F, Vpx expressed during viral infection inhibited NF-κB signaling induced by cGAS-STING. EA.hy926 cells were infected with an equal amount of SIVmac or SIVmacΔVpx virus for 12h. Subsequently, the cells were transfected with ISD (2 μg/mL) for another 12h. Cell lysates were analyzed by immunoblotting with anti-SAMHD1 and anti-CAp27 antibodies (E). (A representative immunoblotting result out of n = 3 independent biological experiments is shown.) Total RNA was prepared and analyzed for the transcriptional level of the indicated genes by RT-qPCR (F) (n = 3 independent biological experiments). Means and standard deviations are presented. The statistical significance analyses were performed using a two-sided unpaired t-test. G, Schematic description of the experimental procedure. H, HEK293T cells were infected with an equal amount of SIVmac or SIVmacΔVpx virus for 12h. Subsequently, the cells were transfected with the NF-κB promoter luciferase vector, pRL-TK Renilla, Myc-cGAS, and STING-Flag as indicated. NF-κB promoter activity was measured 24h after transfection, and the cell lysates were analyzed by immunoblotting with the indicated antibodies (n = 3 independent biological experiments). Means and standard deviations are shown. The statistical significance analyses were performed using a two-sided unpaired t-test. I, BMDCs were infected with SIVmac or SIVmacΔVpx. After 4 h, the medium was changed, and the cells were cultured with or without STING agonist (20 μM CDA) for another 36h. Then BMDCs were harvested, and CD86 protein expression was tested by flow cytometry (n = 3 independent biological experiments). The gating strategy is shown. Numbers indicate the percentage in the gate. The bar graph shows the increased numbers of CD86 positive cells after STING agonist treatment (number of CD86 positive cells treated with STING agonist subtracts number of CD86 positive cells treated without STING agonist). Bar graph data are from n = 3 independent biological experiments. Means and standard deviations are shown. The statistical significance analyses were performed using a two- sided unpaired t-test.

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