Induction of INKIT by Viral Infection Negatively Regulates Antiviral Responses through Inhibiting Phosphorylation of p65 and IRF3

Abstract

The transcription factors p65 and IRF3 play key roles in the induction of cellular antiviral responses. Phosphorylation of p65 and IRF3 is required for their activity and constitutes a key checkpoint. Here we report that viral infection induced upregulation of INKIT, an inhibitor for NF-κB and IRF3 that restricted innate antiviral responses by blocking phosphorylation of p65 and IRF3. INKIT overexpression inhibited virus-induced phosphorylation of p65 and IRF3 and expression of downstream genes. In contrast, knockdown or knockout of INKIT had the opposite effect: Inkit mice produced elevated levels of type I interferons and proinflammatory cytokines and were more resistant to lethal viral infection compared to wild-type. INKIT interacted with IKKα/β and TBK1/IKKɛ, impairing the recruitment and phosphorylation of p65 and IRF3. Viral infection induced IKK-mediated phosphorylation of INKIT at Ser58, resulting in its dissociation from the IKKs. Our findings thus uncover INKIT as a regulator of innate antiviral responses.

Human Cytomegalovirus DNA Polymerase Subunit UL44 Antagonizes Antiviral Immune Responses by Suppressing IRF3- and NF-κB-Mediated Transcription

“…The transcription factors IRF3 and NF-B are critically involved in virus-triggered induction of downstream genes (47). The induction of downstream genes by IRF3 and NF-B promotes an antiviral state that limits viral replication.…”

Induction of OTUD4 by viral infection promotes antiviral responses through deubiquitinating and stabilizing MAVS

“…Reagents, antibodies, and constructs Poly(I:C) was used as described previously. 56 Cycloheximide (CHX), MG132 and bafilomycin A1 were purchased from Sigma. 44 Immuno-reagents were obtained as follows: Mouse control IgG (Santa Cruz Biotechnology, sc-2025); rabbit control IgG (Millipore, 12-370); HRP-conjugated goat-anti mouse or rabbit IgG (Thermo Scientific, PA1-86717 and SA1-9510); HRP-conjugated mouse anti-FLAG (Sigma, A8592); HRP-conjugated goat anti-mouse IgG, F(ab′) 2 fragment specific (Jackson Immuno Research,115-035-006); HRPconjugated goat anti-rabbit IgG, F(ab′)2 fragment specific (Jackson Immuno Research,111-035-006); mouse anti-FLAG (Sungene, KM8002); anti-GFP (Sungene, KM8009); anti-β-Actin (KM9001); anti-GAPDH (KM9002); anti-Myc (KM8003); anti-Tubulin (KM9003); anti-HA (COVANCE, MMS-101R); anti-Ubiquitin (sc-8017); anti-pIκBα (Cell Singling Technologies, 9246L); anti-mouse MAVS (sc-365333) and anti-IRF3 (sc-33641); Rabbit anti-ubiquitin K48specific linkage (Millipore, 05-1307); anti-ubiquitin K63-specific linkage (Millipore, 05-1308); anti-TBK1 (Abcam, 96328-11); anti-p-TBK1 (Abcam, 109272), anti-IRF3 (sc-9082); anti-p-IRF3 (Cell Singling Technologies, 4947S); anti-IκBα (sc-371); anti-mouse MAVS (CST 4983s) and anti-human MAVS (BETHYL, A300-782A).…”

“…The DUB expression library, ISRE and IFN-β promoter luciferase reporter constructs, mammalian expression plasmids for MAVS, MAVS truncations, p65, MAVS, TBK1, IRF3, IRF7, RIG-I, ubiquitin and anti-VSV were kindly provided by Dr. Hong-Bing Shu (Wuhan University) and have been previously described. 44,45,56 Mammalian expression plasmids for OTUD4 and OTUD4 mutants and truncations were constructed by standard molecular biology techniques.…”

“…The experiments were performed as previously described. 44,45,56,58,59 In brief, cells were lysed in Nonidet P-40 lysis buffer containing 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, and 1% protease and phosphatase inhibitor cocktail (Biotool). Cell lysates were subjected to SDS-PAGE and immunoblot analysis was performed with the appropriate antibodies.…”

“…These experiments were performed as previously described. 56,60 Cells were fixed with 1% formaldehyde and quenched by glycine and then washed three times with PBS and then harvested in ChIP lysis buffer (50 mM Tris·HCl pH 8.0, 1% SDS, 5 mM EDTA) followed by sonication to give DNA fragments of 400-600 bp. The lysate was centrifuged at 4°C for 15 min and ChIP dilution buffer (20 mM Tris·HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100) was added to the supernatant (4:1 volume).…”