Extended Data Fig. 2: Selective inhibition of cGAS-STING- but not RIG-I card- or IRF3-5D-triggered immune activation by SIVmac.

A,Vpx has no effect on cGAS or STING expression. Expression vectors for Myc-cGAS and STING-Flag were co-transfected with increasing amounts of SIVmac Vpx in HEK293T cells. After 24h, cell lysates were detected by immunoblotting with Myc, HA, and Flag antibodies (A representative immunoblotting result out of n = 3 independent biological experiments is shown.). B,C,D, HEK293T cells were transfected with NF-κB- Luc (B,C) or IRF3-Luc (D) with increasing amounts of SIVmac HA-Vpx, in the presence of RIG-IN-Flag (B), Myc-cGAS, and STING-Flag expression vectors (C), or IRF5-5D-Flag vector (D), respectively. pRL-TK Renilla was used as the internal control. Transactivation of the luciferase reporter was determined 24h after transfection, and the protein expression levels were analyzed by immunoblotting with anti-Flag or anti-HA antibody (n = 3 independent biological experiments). GAPDH served as a loading control. Means and standard deviations are presented; a two-sided paired t-test was performed. E, CD4+ T cells were infected with equal amounts of SIVmacΔVpx, SIVmacΔVpx+Vpx, or WT virus for 4h, the medium was changed, and SeV (20 HA/mL) was added to the medium and incubated for another 8 h. Total RNA was prepared from the harvested cells and analyzed for the transcriptional level of the indicated genes by RT-qPCR. Different shapes represent different donors, with three independent experiments for each donor. A single mean value, depicted as a single horizontal line, is shown and marked in red. Standard deviations are presented. (n = 3 independent biological experiments.) F, cGAS-STING- stimulated genes and the influence of Vpx. Heat map of the percentages of genes stimulated by cGAS+STING (FC>2) and regulated by Vpx .G, The numbers in the pie charts indicate the percentage (3.2%) of NF-κB-regulated genes (29) among the total cGAS-STING-stimulated genes (906) (FC>1.5) (left) and the percentage (90%) of NF-κB-regulated genes (29) suppressed by Vpx (26) (right) from microarray data. NF-κB- regulated genes are shown in purple, and others in dark yellow; NF-κB-regulated genes suppressed by Vpx are in red, and others in blue.