Extended Data Fig. 10: Influence of SIVmac Vpx on STING-mediated NF- κB signaling and viral infection. | Nature Microbiology

Extended Data Fig. 10: Influence of SIVmac Vpx on STING-mediated NF- κB signaling and viral 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. 10

A,B, Jurkat cells were infected with CAp27-normalized SIVmac or SIVmacΔVpx virus for 48h. The productive viral infection was analyzed by flow cytometry (n = 3 independent biological experiments) using antibody against CAp27 (A). Means and standard deviations are presented. The statistical significance analyses were performed using a two-sided unpaired t-test. Intracellular Pr55Gag was analyzed by immunoblotting with the anti-CA antibody. GAPDH was used as a loading control (B) (Immunoblotting is a representative of n = 3 independent biological experiments.) C, Influence of SIVmac Vpx on STING- mediated p50 nuclear translocation. Jurkat cells were infected with equal amounts of SIVmacΔVpx or SIVmacΔVpx+HA-Vpx, SIVmacΔVpx+Vpx ET16/17AA, or SIVmacΔVpx+Vpx RS51/52AA virus and treated with STING agonist as in Fig. 5. Cells were harvested, and the nuclear and cytoplasmic fractions were separated 12h after infection. Proteins were analyzed by immunoblotting using anti-p50, anti-HA, anti-IRF3, anti- GAPDH, or anti-histone antibody. Immunoblotting is representative of n = 2 independent biological experiments. D,E, STING is critical for Vpx-mediated suppression of the STING agonist-triggered anti-viral effect. STING-silenced Jurkat (shSTING) or control (shNT) cells were monitored by immunoblotting using anti-STING and anti-GAPDH antibody (D) (A representative immunoblotting result out of n = 2 independent biological experiments is shown.) STING-silenced Jurkat (shSTING) or control (shNT) cells were infected with equal amounts of SIVmac or SIVmacΔVpx virus. The medium was changed 4h later and replaced with fresh medium containing DMSO or STING agonist. Supernatants from the Jurkat cells were collected at 48h and 72h after infection and analyzed for viral infectivity using the TZM-bl luciferase reporter assay (E) (n = 3 independent biological experiments). Means and standard deviations are presented. The statistical significance analyses were performed using a two-sided unpaired t-test. F, Jurkat cells were infected with equal amounts of SIVmac or SIVmacΔVpx virus; 4h later, the medium was changed, and SeV (20 HA/mL) was added to the medium. Cells and total mRNA were prepared, and mRNA was analyzed by RT-qPCR to determine the transcription levels of the indicated genes (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, Jurkat cells were infected with SIVmacΔVpx or SIVmacΔVpx+HA-Vpx virus for 12h, and then infected with HIV-1 NL4-3ΔEnvGFP virus for 48h. Infected cells were harvested, and GFP-positive cells were tested by flow cytometry (n = 3 independent biological experiments). Means and standard deviations are presented. The statistical significance analyses were performed using a two-sided unpaired t-test. H, CD4+ T cells were infected with equal amounts of SIVmacΔVpx incorporated with Vpx WT, ET16/17AA, or RS51/52AA mutant virus and treated with STING agonist or not. Total cells were harvested, and the protein expression levels were analyzed by immunoblotting with anti-IRF3-p, anti-IFIT3, anti-ISG15, anti-STING, anti-cGAS, anti-HA, or anti-GAPDH antibody. A representative immunoblotting result out of n = 2 independent biological experiments is shown.

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