FIH negatively regulates type I IFN signaling (A) Immunoblot (IB) of overexpressed FIH in H1299 cells. (B) Quantitative real-time PCR analysis of IFNB1, ISG56, and CXCL10 mRNA in H1299 cells transfected with empty vector (Vec) or Myc-FIH (FIH) for 18–24 h and then left infected with SeV, VSV, EMCV, or HSV for 6 h. (C) IB of p65 in p65+/+ or p65−/− H1299 cells. (D) Quantitative real-time PCR analysis of IFNB1, ISG56, and CXCL10 mRNA in p65−/− H1299 cells transfected with Vec or Myc-FIH (FIH) for 18–24 h and then left infected with SeV, VSV, EMCV, or HSV for 6 h. (E) IB of IRF3 in IRF3+/+ or IRF3−/− H1299 cells. (F) Quantitative real-time PCR analysis of IFNB1, ISG56, and CXCL10 mRNA in IRF3−/− H1299 cells transfected with Vec or Myc-FIH (FIH) for 18–24 h and then left infected with SeV, VSV, EMCV, or HSV for 6 h. (G) IB of IRF3 dimerization and phosphorylation in FIH+/+ and FIH−/− THP1 cells infected with SeV or VSV for 0–6 h. (H) ELISA of IFNβ in the supernatants of FIH+/+ and FIH−/− THP1 cells infected with SeV, VSV, or HSV-1 for 12 h.

FIH negatively regulates type I IFN signaling by targeting IKKε (A) Luciferase activity of the ISRE reporter in Vec control and FIH-overexpressing (FIH) (left) or the control and FIH KO (right) HEK293T cells transfected with the indicated vectors. (B) IB of whole cell lysates (WCLs) and anti-hemagglutinin (HA) immunoprecipitation (IP) in HEK293T cells transfected with the indicated vectors for 24 h. (C) IB of WCLs and proteins immunoprecipitated with anti-IKKε antibody from THP1 cells infected with VSV for the indicated times and detected by anti-FIH antibody. (D) IP for the interaction of Myc-FIH with full-length or truncated IKKε in HEK293T cells. (E) IP for the interaction of FLAG-IKKε with full-length or truncated FIH in HEK293T cells. (F) Luciferase activity of the ISRE reporter in IKKε+/+ H1299 cells (left) and IKKε−/− H1299 cells (right) transfected with the indicated vectors. (G) Quantitative real-time PCR analysis of IFNB1, ISG56, and CXCL10 mRNA in IKKε+/+ and IKKε−/− H1299 cells transfected with Vec or HA-FIH and then left infected with VSV for 6 h. (H) Microscopy imaging of VSV-GFP replication in IKKε+/+ and IKKε−/− H1299 cells transfected with Vec or HA-FIH and then left infected with VSV-GFP (1 MOI) for 24 h. (I) Flow cytometry analysis of VSV-GFP replication in IKKε+/+ and IKKε−/− H1299 cells transfected with Vec or HA-FIH and then left infected with VSV-GFP (1 MOI) for 24 h. (J) IB for IKKε expression and the efficiency of VSV-GFP virus infection in IKKε+/+ and IKKε−/− H1299 cells transfected with Vec (−) or HA-FIH (+). (K) Quantitative real-time PCR analysis of VSV mRNA in IKKε+/+ and IKKε−/− H1299 cells transfected with Vec or HA-FIH and subsequently infected with VSV-GFP (1 MOI) for 24 h. (L and M) IB of IRF3 phosphorylation in IKKε+/+ H1299 cells (L) and IKKε−/− H1299 cells (M) transfected with Vec or HA-FIH (FIH) and then left infected with VSV (+) for 3–6 h. Quantitation of phosphorylated IRF3 versus total IRF3 is shown on the right.

FIH negatively regulates type I IFN signaling through its enzymatic activity (A) Luciferase activity of the ISRE reporter (left) and IFNβ promoter reporter (right) in HEK293T cells transfected with Vec, wild-type FIH, the enzymatically inactive mutant of FIH (H199A), or other mutants, including FIH (H233/234A), FIH (H279/280A), and FIH (313A), and then left infected with SeV for 6 h. (B) Quantitative real-time PCR analysis of IFNB1, ISG56, and CXCL10 mRNA in FIH−/− HEK293T cells transfected with Vec, HA-FIH, or HA-FIH (H199A) for 24 h and then left uninfected (mock) or infected with SeV for 6 h (top), transfected without (control)/with poly(I:C) for 6 h (bottom). (C) Flow cytometry analysis (top) and microscopy imaging (bottom) of VSV-GFP replication in FIH−/− HEK293T cells transfected with Vec, HA-FIH, or HA-FIH (H199A) for 24 h and then left infected with VSV-GFP (1 MOI) for 16 h. (D) IB for the efficiency of VSV-GFP virus infection in FIH−/− HEK293T cells transfected with Vec, HA-FIH, or HA-FIH (H199A) for 24 h and then left infected with VSV-GFP (1 MOI) for 16 h. (E and F) Quantitative real-time PCR analysis of VSV mRNA (E) and plaque assay of virus titer (F) in FIH−/− HEK293T cells transfected with Vec, HA-FIH, or HA-FIH (H199A) for 24 h and then left infected with VSV-GFP for 16 h. (G) Quantitative real-time PCR analysis of IFNB1, ISG56, and CXCL10 mRNA in FIH−/− THP1 cells reconstituted with Vec, FIH, or FIH (H199A) and then left uninfected (mock) or infected with VSV (top) or HSV-1 (bottom) for 6 h. (H) ELISA of IFNβ in the supernatants of FIH−/− THP1 cells reconstituted with Vec, FIH, or FIH (H199A) and then left uninfected (mock) or infected with VSV (top) or HSV (bottom) for 12 h. (I) IB of IRF3 dimerization and phosphorylation in FIH−/− THP-1 cells reconstituted with Vec, FIH, or FIH (H199A) and then left uninfected (−) or infected (+) with VSV for 6 h. Quantitation of dimerized or phosphorylated IRF3 versus total IRF3 is shown at the bottom.

FIH catalyzes the asparaginyl hydroxylation of IKKε at asparagines 254, 700, and 701 (A–C) The hydroxylated residues in IKKε identified by MS analysis. The hydroxylated peptides are shown on the left, and the non-hydroxylated peptides are shown on the right (A, N254; B, N700; C, N701). (D) Extracted ion chromatograms corresponding to unmodified and hydroxylated forms of the IKKϵ peptide RITTEKPAGAIAGAQRRENGPLEW. MS chromatography analysis illustrates the relation abundance of hydroxylation at Asn-254 (N254) following FIH intervention: FIH overexpression (left), endogenous levels of FIH (center, ∼3%), and KO of FIH (right, <1%). (E) Extracted ion chromatograms corresponding to unmodified and hydroxylated forms of the IKKϵ peptide ASDLLDNNRIIERL. MS chromatography analysis illustrates the relation abundance of hydroxylation at Asn-700 (N700) following FIH intervention: FIH overexpression (left), endogenous levels of FIH (center ∼5%), and KO of FIH (right, <1%). (F) Extracted ion chromatograms corresponding to unmodified and hydroxylated forms of the IKKε peptide DNNRIIERL. MS chromatography analysis illustrates the relation abundance of hydroxylation at Asn-701 (N701) following FIH intervention: FIH overexpression (left), endogenous levels of FIH (center, ∼3%), and KO of FIH (right, <1%). (G) Sequence alignment of partial IKKε (aas 244–264) and (aas 690–710) from human, mouse, rat, pig, dog, and cat. The red boxes indicate the conserved asparagine residues (N254, N700, and N701). (H) Luciferase activity of the ISRE reporter in HEK293T cells transfected with Vec, IKKε (N6A), IKKε, or IKKε (3N/A). (I) Quantitative real-time PCR analysis of IFNB1, ISG56, and CXCL10 mRNA in IKKε−/− H1299 cells reconstituted with IKKε or IKKε (3N/A) (in which asparagines 254, 700, and 701 are mutated to alanine), followed by non-infection (−) or infection (+) with VSV for 6 h. (J) IB of IRF3 phosphorylation in IKKε−/− H1299 cells reconstituted with IKKε or IKKε (3N/A), followed by infection with VSV for 0–6 h. (K) Microscopy imaging of VSV-GFP replication in IKKε−/− H1299 cells reconstituted with IKKε or IKKε (3N/A), followed by infection with VSV-GFP (1 MOI) for 16 h. (L) Flow cytometry analysis of VSV-GFP replication in IKKε−/− H1299 cells reconstituted with IKKε or IKKε (3N/A), followed by infection with VSV-GFP (1 MOI) for 16 h. (M) IB for the efficiency of VSV-GFP virus infection in IKKε−/− H1299 cells reconstituted with IKKε or IKKε (3N/A), followed by non-infection (−) or infection (+) with VSV-GFP (1 MOI) for 16 h.

FIH attenuates K63-linked polyubiquitination of IKKε at lysine 416 (A) IB for ubiquitination of IKKε in HEK293T cells transfected with HA-tagged wild-type ubiquitin (HA-Ub), IKKε, and an increased amount of FIH for 24 h. (B) IB for polyubiquitination of IKKε in HEK293T cells transfected with FLAG-IKKε and HA-Ub, HA-tagged K48-only Ub (K48O) (in which all lysine residues are mutated to arginine residues except lysine 48), HA-tagged K48R Ub (K48R) (in which only lysine 48 is mutated to arginine), HA-tagged K63-only Ub (K63O) (in which all lysine residues are mutated to arginine residues except lysine 63), or HA-tagged K63R Ub (K63R) (in which only lysine 63 is mutated to arginine), together with Vec or Myc-FIH. (C) IB for polyubiquitination of IKKε in FIH+/+ and FIH−/− HEK293T cells transfected with FLAG-IKKε and HA-Ub (wild type [WT]), or HA-Ub (K63O) for 24 h. (D) IB for K63-linked polyubiquitination of endogenous IKKε in THP1 cells stimulated with VSV for 0–8 h. (E) IB for polyubiquitination, K48-linked polyubiquitination, and K63-linked polyubiquitination of endogenous IKKε in FIH+/+ and FIH−/− THP1 cells stimulated without (−) or with (+) VSV for 8 h. Cell lysates were co-immunoprecipitated with anti-IKKε antibody (α-IKKε) and then detected by anti-Ub, anti-K48-linked Ub, or anti-K63-linked Ub antibodies, respectively. (F) IB for polyubiquitination of IKKε in HEK293T cells transfected with FLAG-IKKε, and HA-Ub, Myc-FIH, or Myc-FIH (H199A) for 24 h. (G) IB for polyubiquitination, K48-linked polyubiquitination, and K63-linked polyubiquitination of endogenous IKKε in FIH−/− THP1 cells reconstituted with Vec, Myc-FIH, or Myc-FIH (H199A) (H/A), followed by non-infection (−) or infection (+) with VSV for 8 h. (H) IB for polyubiquitination of WT IKKε and its mutants IKKε (N254A) (in which asparagine 254 is mutated to alanine), IKKε (2N/A) (in which asparagines 700 and 701 are simultaneously mutated to alanine residues), and IKKε (3N/A) (in which asparagines 254, 700, and 701 are simultaneously mutated to alanine residues) in HEK293T cells transfected with HA-Ub and FLAG-IKKε (WT), FLAG-IKKε (N254A), FLAG-IKKε (2N/A), or FLAG-IKKε(3N/A) in the absence (−)/presence (+) of Myc-FIH. (I) IB for polyubiquitination of IKKε mutants IKKε (K30R) (in which lysine 30 is mutated to arginine), IKKε (K401R) (in which lysine 401 is mutated to arginine), or IKKε(K416R) (in which lysine 416 is mutated to arginine) in HEK293T cells transfected with HA-Ub and FLAG-IKKε (K30R), FLAG-IKKε (K401R), or FLAG-IKKε (K416R) with (+) or without (−) overexpression of Myc-FIH. (J) IB for K63-linked polyubiquitination of IKKε mutants IKKε (K30R), IKKε (K401R), or IKKε (K416R) in HEK293T cells transfected with HA-Ub (K63O) and FLAG-IKKε (K30R), FLAG-IKKε (K401R), or FLAG-IKKε (K416R) with (+) or without (−) overexpression of Myc-FIH.

FIH attenuates K63-linked polyubiquitination of IKKε triggered by the cIAP1/cIAP2/TRAF2 E3 Ub ligase complex (A) Luciferase activity of the ISRE reporter in HEK293T cells transfected with the indicated vectors for 24 h. (B) Luciferase activity of the ISRE reporter in HEK293T cells transfected with the indicated vectors for 24 h and then left infected with SeV for 8 h. (C) IB for polyubiquitination of IKKε in HEK293T cells transfected with HA-Ub and FLAG-IKKε or Myc-cIAP1 in the absence (−)/presence (+) of FIH for 24 h. (D) IB for polyubiquitination of IKKε in HEK293T cells transfected with HA-Ub and FLAG-IKKε or Myc-cIAP2 in the absence (−)/presence (+) of FIH for 24 h. (E) IB for polyubiquitination of IKKε in HEK293T cells transfected with HA-Ub and FLAG-IKKε or Myc-TRAF2 in the absence (−)/presence (+) of FIH for 24 h. (F) CoIP for the interaction between FIH and IKKε, cIAP1, cIAP2, or TRAF2 in HEK293T cells transfected with the indicated vectors, including Myc-FIH, together with the FLAG Vec, FLAG-IKKε, FLAG-cIAP1, FLAG-cIAP2, or FLAG-TRAF2 for 24 h. (G) CoIP for the interaction between IKKε and cIAP1 in HEK293T cells with (FIH)/without (Vec) overexpression of FIH. HEK293T cells were transfected with the indicated vectors, including FLAG-IKKε and Myc-cIAP1 together with/without HA-FIH. (H) CoIP for the interaction between IKKε and cIAP2 in HEK293T cells with (FIH)/without (Vec) overexpression of FIH. HEK293T cells were transfected with the indicated vectors, including FLAG-IKKε and Myc-cIAP2 together with/without HA-FIH. (I) CoIP for the interaction between IKKε and cIAP1 in HEK293T cells with (FIH)/without (Vec) overexpression of FIH. HEK293T cells were transfected with the indicated vectors, including FLAG-IKKε and Myc-TRAF2 together with/without HA-FIH. (J) IB for polyubiquitination of IKKε (K416R) in HEK293T cells transfected with HA-Ub and FLAG-IKKε (K416R) in the absence (−)/presence (+) of Myc-cIAP1.

FIH-deficient mice are more resistant to viral infection (A) Survival (Kaplan-Meier curve) of Fih+/+ and Fih+/− mice (n = 6 or 7 per group) at different time points after intraperitoneal injection with a high dose of VSV (1 × 106 plaque-forming units [PFUs] per mouse) or EMCV (1 × 105 PFUs per mouse) and monitored for 8 days. (B) ELISA of Ifnβ in serum from Fih+/+ and Fih+/− mice (n = 7 per group) after intraperitoneal injection of VSV (1 × 106 PFUs per mouse) for 24 h. (C) Microscopy imaging of hematoxylin and eosin (H&E)-stained lung sections from Fih+/+ and Fih+/− mice treated with PBS or VSV (1 × 106) for 24 h. Quantitation of injury area in lung by ImageJ is shown on the right. Scale bar, 200 μm. (D and E) Quantitative real-time PCR analysis of Ifnb, Cxcl10, and VSV mRNA and plaque assay for VSV titers in the lungs (D) and spleen (E) from Fih+/+ and Fih+/− mice after intraperitoneal injection of VSV (1 × 106 PFUs per mouse) for 24 h. (F and G) Quantitative real-time PCR analysis of Ifnb, Cxcl10, and EMCV mRNA and plaque assay for EMCV titers in the lungs (F) and spleen (G) from Fih+/+ and Fih+/− mice after intraperitoneal injection of EMCV (1 × 105 PFUs per mouse) for 24 h.