Fig 2: HSV-2 ICP27 blocks IRF3 activation by physical interaction. (A) HSV-2 ICP27 interferes with the nuclear translocation of IRF3. HeLa cells were seeded in 35 mm glass-bottom dishes and transfected with empty vector pcDNA3.1(+), plasmid expressing HSV-2 ICP27-HA or influenza virus NS1-HA. At 24 h post-transfection, cells were stimulated with or without 100 HAU ml−1 SeV for 16 h and prepared for immunofluorescence assay. IRF3 was detected under 647 nm wavelength, ICP27-HA or NS1-HA under 488 nm wavelength, and nuclei under 405 nm wavelength. (B) HSV-2 ICP27 inhibits IRF3 phosphorylation. HEK 293T cells were seeded in 6-well plates and transfected with pcDNA3.1(+), plasmid expressing HSV-2 ICP27 or influenza virus NS1. At 24 h post-transfection, cells were stimulated with or without 100 HAU ml−1 SeV for 16 h. Nuclear and cytoplasmic proteins were isolated, and phosphorylated IRF3 was detected with a p-IRF3 mAb. (C) HSV-2 ICP27 interacts with endogenous IRF3. HEK 293T cells were seeded in 6-well plates and transfected with pcDNA3.1(+) or HSV-2 ICP27-Flag. At 24 h post-transfection, cells were stimulated with or without 100 HAU ml−1 SeV for 16 h and lysed for immunoprecipitation assay. One representative experiment out of three is shown (A–C). (D) Kinetics of ICP27-IRF3 binding. The kinetics of binding was performed on a Forte-Bio Octet Red System. Purified IRF3 was conjugated with biotin at a molecular weight ratio of 1:3. Ten microgram/milliliter biotinylated IRF3-Flag was coupled to Biosenors and immersed in different concentration of ICP27-Flag (50, 200, 500, or 800 nM) for association and disassociation. The response in nm shift was recorded as a function of time. KD (M) = 2.03E-08.

Fig 3: Intraperitoneal administration of nitroalkene compound CP-36 dampens STING-dependent inflammationin vivo.(A–C) Mice were administrated with CP-36 i.p. (10 or 30 mg/kg) 2 h before activating STING with i.p. injection of cAIM(PS)2 Difluor (Rp/Sp) (0.2 mg/kg) for 4 h. Immunoblot analysis of homogenized and lysed (A) lung and (B) liver tissue to evaluate the levels of pTBK1, TBK1, pSTAT1, STAT1 and Viperin with (C) densitometry quantification of protein levels. The plots include data from individual mice represented in immunoblots. Bars indicate mean ± SEM and are normalized to the cAIM(PS)2 Difluor (Rp/Sp) group, expressed as a percentage. (D–F) WT or STING-KO BMDMs were pre-treated with CP-36 (5 and 10 μM) or reduced CP-36 (R-CP-36, 5 and 10 μM) for 2 h, followed by cGAMP stimulation (4 μg/mL) for either 3 h or 6 h. (D) Immunoblot analysis of whole-cell lysate (representative blot) to evaluate levels of pSTING, STING, pIRF3 and IRF3 and (E) densitometry quantification of pSTING/STING protein levels after 3 h cGAMP stimulation. Three independent experiments are plotted. (F) qPCR analysis of Ifnb1 and Cxcl-10 expression was quantified relative to 18S mRNA levels after 6 h of cGAMP stimulation. BMDM cultures obtained from 6 individual mice (3 male and 3 female mice) are plotted. Bars indicate mean ± SEM and are normalized to cGAMP levels, expressed as a percentage. Statistical analyses were performed using Welch's t-test, yielding the following p values: For tissues: pTBK1: p = 0.0293, TBK1: p = 0.0283, Viperin: p = 0.0311, STAT1: p = 0.0159. For BMDMs: pSTING/STING: p = 0.0184, Ifnb1: p < 0.0001, Cxcl-10: *, p = 0.0367 and **, p = 0.0076. * Indicates p < 0.05, ** indicates p < 0.01, and **** indicates p < 0.0001.

Fig 4: The cGAS-STING pathway activation and antitumor immunity after treatments.G1:Saline, G2:Saline+L, G3: MHMS, G4: MnCO+TSSI + L, G5: THMS + L, G6: MTHMS + L. a Schematic illustration of gas nanoadjuvant-mediated cGAS-STING promotion. b Western blotting assay of STING, TBK1, IRF-3, and phosphorylation of proteins in tumor. Samples derived from the same experiment and gels/blots were processed in parallel. Experiment was repeated twice independently with similar results. c Flow cytometric assay of mature DC (CD11c+CD80+CD86+) in TDLNs (n = 4 mice). The p values of G6 to G5 and G6 to G4 are 0.0003 and <0.0001. d Flow cytometric assay of tumor-infiltrating CD8+ in CD3+ T cells (n = 4 mice). The p values of G6 to G5 and G6 to G4 are 0.0001 and <0.0001. e Flow cytometric assay of tumor-infiltrating CD4+Foxp3+ Tregs (n = 4 mice). The p values of G6 to G5 and G6 to G4 are both <0.0001. Flow cytometric assay of tumor-infiltrating f M2-like macrophages (CD206hiCD11b+F4/80+) and g M1-like macrophages (CD80hiCD11b+F4/80+) (n = 4 mice). f p values of G6 to G5 and G6 to G4 are 0.0006 and 0.0001. g p values of G6 to G5 and G6 to G4 are 0.0002 and <0.0001. h Flow cytometric assay of CD3+CD8+CD62LlowCD44hi TEM in spleen (n = 4 mice).The p values of G6 to G5 and G6 to G4 are 0.0001 and <0.0001. Data represent mean ± SD. Data were compared through one-way ANOVA using Tukey post-hoc test. Source data underlying b–h are provided as a Source Data file.

Fig 5: Summary figure.SARS-CoV-2 infection in epithelial cells leads to an early inflammatory response that correlates with virus RNA replication followed by a late interferon response that can be amplified by activated non-infected bystander cells. Virus infection and replication causes cellular stress that can lead to mitochondrial stress/damage, DNA damage, and cell death. Each of these responses can result in the activation of the cGAS-STING axis via cellular DNA leading either to direct activation of NF-κB or to TBK1-mediated activation of IRF3 and NF-κB. In SARS-CoV-2 infected cells, the TBK1 and IRF3 pathways are blocked by the action of several viral proteins. Therefore, STING activation leads to a predominant NF-κB response causing production and release or pro-inflammatory cytokines. Created with BioRender.com.