Fig 2: PARP9 recruits and activates downstream PI3K p85.a Immunoblot analysis of endogenous proteins PARP9, mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K) regulatory subunit p85 precipitated with anti-PARP9, or immunoglobulin G (IgG) from whole-cell lysates of wild-type BMDC left infected (Mock) or infected with VSV at MOI of 1 for 2 h or 4 h. b Immunoblot analysis of purified Myc-tagged p85 with anti-Myc antibody (bottom blot), and immunoblot analysis of purified HA-tagged full-length PARP9 and serial truncation of PARP9 with deletion (?) of various domains alone with anti-HA antibody (top blot) or after incubation with Myc-tagged p85 and immunoprecipitation with anti-Myc antibody (middle blot). c Schematic diagram showing full-length p85 (Full) and serial truncations of p85 with deletion (?) of various domain (left margin); numbers at ends indicate amino acid positions (top). SH3, the Src-homology 3 domain; RhoGAP, Rho GTPase activating protein domain; SH2, the Src-homology 2 domain. d Immunoblot analysis of purified Myc-tagged PARP9 with anti-Myc antibody (bottom blot), and immunoblot analysis of purified HA-tagged full-length p85 and serial truncations of p85 with deletion of various domains alone with anti-HA antibody (top blot) or after incubation with Myc-tagged PARP9 and immunoprecipitation with anti-Myc antibody (middle blot). e Confocal microscopy of primary peritoneal macrophages from WT mice left infected (Mock) or infected with VSV at MOI of 1 for 2 h. p85 or TBK1 was stained with mouse anti-PI3 kinase p85 alpha monoclonal antibody (Cat: ab86714, Abcam) or mouse anti-TBK1 monoclonal antibody (Cat: NB100-56705, Novus Biologicals), followed by Alexa Fluor 488 goat anti-mouse secondary antibody (green), while PARP9 was stained with rabbit anti-PARP9 polyclonal antibody (Cat: LS-B9440, LifeSpan BioSciences), followed by Alexa Fluor 594 goat anti-rabbit secondary antibody (red). DAPI (4',6-diamidino-2-phenylindole) served as the nuclei marker (blue). Scale bars represent 10 µm. f Quantification of colocalization between PARP9 and p85 or TBK1 using ImageJ software (n = 3 per group). g Immunoblot analysis of endogenous proteins PARP9 and PI3K regulatory subunit p85 precipitated with anti-PARP9, or immunoglobulin G (IgG) from whole-cell lysates of human MDDC left transfected (Mock) or transfected with reovirus dsRNA (Reo-RNA) for 6 h. h Immunoblot analysis of endogenous proteins p85 and phosphorylated p85 (p-p85) precipitated with anti-p85 from whole-cell lysates of wild-type (WT) or PARP9 KO BMDC left infected (0 h) or infected with VSV or reovirus (Reo) at MOI of 1 for 0.5 or 1 h. i The relative amounts of PIP3 in the plasma membrane were normalized to the amount of immunoprecipitated p85 in WT and PARP9 KO BMDC after VSV infection (n = 3 per group). Error bars indicate standard error of the mean for results in f, i. NS, not significant (p > 0.05), ***p < 0.001, and p value was calculated by unpaired two-tailed Student’s t test. Data are representative of three independent experiments. Exact p values (f, p = 0.00007, p = 0.08; i, p = 0.0003, p = 0.00024).

Fig 3: TRAF3IP3 targets TRAF3 and TBK1.a–d Coimmunoprecipitation and immunoblotting using lysates from HEK293T cells transfected with the indicated expression vectors. e Immunoblotting of endogenous proteins with the indicated antibodies using lysates of THP-1 cells infected for various times with VSV (MOI = 1) followed by immunoprecipitation with anti-TRAF3IP3. f, g Coimmunoprecipitation and immunoblotting using lysates from HEK293T cells transfected with the indicated expression vectors. Data are one representative of at least three independent experiments. Source data are provided as a Source Data file.

Fig 4: DHX16 enhances activation of the IFN-I production pathway(A–D) A549s treated with siNTC or siDHX16 followed by IAV infection for 24 h (PR8 MOI = 0.1). (A) Phosphorylation of TBK1 and IRF3 (immunoblot). (B) IFN-ß and IFIT2 expression (qRT-PCR). (C) IFN-ß protein in supernatants (ELISA). (D) IAV viral titers (plaque assay).(E–G) A549s transfected with empty vector or DHX16 followed by IAV infection for 24 h (PR8 MOI = 0.1). (E) Phosphorylation of TBK1 and IRF3 (immunoblot). (F) IFN-ß and IFITM1 expression (qRT-PCR). (G) IAV viral titers (plaque assay).(H–M) PPMO-mediated in vivo silencing of DHX16 in BALB/c mice followed by IAV infection (PR8, 1000 PFU) (n = 3–4/group). (H) Schematics of experiment. (I) Validation of PPMO-mediated silencing in murine lung tissue (immunoblot). (J) IAV viral titers (plaque assay). (K) mIFN-ß and mIFITM1 expression (qRT-PCR). (L) IFN-ß protein in lung tissue (ELISA) (n = 5–8/group). (M) Weight loss from NTC or DHX16 PPMO-treated mice infected with IAV (PR8, 100 PFU) (n = 5/group).(N–Q) IFN-ß expression from siNTC or siDHX16 treated A549s infected with IAV (PR8 WT MOI = 0.1) (N) or ZIKV (FSS MOI = 0.1) (O), Calu-3s infected with SARS-CoV-2 (icSARS-CoV-2-mNG MOI = 1) (P) or A549s transfected with 3p-hpRNA (100 ng/mL) (Q) (qRT-PCR).Data are expressed as means (n = 3) ± SD or ± SEM as in (M). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (Student's t test or one-way ANOVA with Tukey's multiple comparisons). Data are representative of 2–3 independent experiments.See also Figures S2 and S3.

Fig 5: TRAF3IP3 promotes K48-dependent ubiquitination of TBK1.a Immunoblotting (top) and RT-PCR (bottom) analyses using HEK293T transfected with HA-TBK1 and Flag-IRF3 and increasing doses of Myc-TRAF3IP3. b–e Coimmunoprecipitation and immunoblotting using HEK293T transfected with indicated vectors. Data are one representative of at least two independent experiments. Source data are provided as a Source Data file.