Extended Data Fig. 5: Vpx suppressed cGAS- STING function is SAMHD1 independent and Inhibition of cGAS- STING-mediated NF-κB signaling is a conserved function of Vpx proteins from diverse HIV-2 and SIV viruses.

A, Aliquots of the samples used were analyzed by RT-qPCR. RNA was isolated, followed by detection of NFKBIA, IER3, and CXCL8 mRNA levels by RT-qPCR (n = 3 independent biological experiments). Means and standard deviations are shown. The statistical significance analyses were performed using a two- sided unpaired t-test. B, The graph represents the location of siRNA-1, siRNA-2, and shRNA targeting in the SAMHD1 coding region. C, HEK293T cells were transfected with siNT, siSAMHD1-1, or siSAMHD1-2 for 24 h and then transfected NF- κB-Luc or pRL-TK Renilla, together with STING-Flag and Myc- cGAS, empty vector or SIVmac Vpx. Promoter transactivation was analyzed by luciferase reporter assay, and the protein expression levels were analyzed by immunoblotting with antibody targeting SAMHD1 or GAPDH (n = 3 independent biological experiments). One representative immunoblotting result out of n = 3 independent experiments is shown. Means and standard deviations are presented. The statistical significance analyses were performed using a two-sided unpaired t-test. D, HEK293T cells were co-transfected with NF- κB promoter luciferase vector, pRL-TK Renilla, Myc-cGAS, STING-Flag, and HIV/SIV HA-Vpx as indicated. The cell lysates were detected by immunoblotting with anti-SAMHD1, anti- Flag, or anti-HA antibody. (One representative immunoblotting result out of n = 3 independent biological experiments is shown.) E, The mRNA levels of NFKBIA, CXCL8, and CXCL10 were analyzed by RT-qPCR (n = 3 independent biological experiments). Means and standard deviations are shown. The statistical significance analyses were performed using a two-sided unpaired t-test.