Mechanisms of activation of interferon regulator factor 3: the role of C-terminal domain phosphorylation in IRF-3 dimerization and DNA binding

Abstract

The interferon regulatory transcription factor (IRF-3) is activated by phosphorylation of Ser/Thr residues clustered in its C-terminal domain. Phosphorylation of these residues, which increases the negative charge of IRF-3, results in its dimerization and association with DNA, despite the increase in repulsive electrostatic interactions. To investigate this surprising effect, the dimerization of IRF-3 and two phosphomimetic mutants, 2D (S396D, S398D) and 5D (S396D, S398D, S402D, T404D and S405D), and their binding to single-site PRDI and double-site PRDIII–PRDI DNA sequences from the IFN-β enhancer have been studied. It was found that: (a) the mutations in the C-terminal domain do not affect the state of the DNA-binding N-terminal domain or its ability to bind target DNA; (b) in the 5D-mutant, the local increase of negative charge in the C-terminal domain induces restructuring, resulting in the formation of a stable dimer; (c) dimerization of IRF-3 is the basis of its strong binding to PRDIII–PRDI sites since binding of 5D to the single PRDI site is similar to that of inactivated IRF-3. Analysis of the binding characteristics leads to the conclusion that binding of dimeric IRF-3 to the DNA with two tandem-binding sites, which are twisted by ∼100° relative to each other, requires considerable work to untwist and/or bend the DNA.

Functional Characterization of Murine Interferon Regulatory Factor 5 (IRF-5) and Its Role in the Innate Antiviral Response

“…Activated MuIRF-5 Stimulates Transcription of IFNA and -B Promoters-As we have shown previously, HuIRF-5 v4, stimulates transcriptional activity of IFNA gene promoters in reporter assays and induces several IFNA genes in NDV-infected 2fTGH cells (which lack IRF-7) (23,41). We therefore examined whether MuIRF-5 is also capable of stimulating type I IFN promoters in infected cells by employing the luciferase reporter assay.…”

Human Bocavirus NP1 Inhibits IFN-β Production by Blocking Association of IFN Regulatory Factor 3 with IFNB Promoter

“…In some cases, cells were mock infected or infected with SeV (100 HA U ml 21 ) at 24 h posttransfection for 16 h. Cells were lysed in 300 ml lysis buffer and clarified by centrifugation at 16,000 3 g for 5 min at 4˚C. Co-IP assays were performed using a Dynabeads Protein G Immunoprecipitation Kit (Invitrogen).…”

“…At 24 h posttransfection, cells were mock infected or infected with SeV (100 HA U ml 21 ) for 8 h, fixed in 4% paraformaldehyde for 10 min at room temperature (RT), and permeabilized with 0.2% Triton X-100 for 15 min at RT. After three rinses with PBS, cells were incubated in blocking solution (PBS supplemented with 3% BSA and 5% normal goat serum) overnight at 4˚C and then incubated with mouse anti-HA Ab (Sigma-Aldrich) and rabbit anti-IRF-3 (PROTEINTECH group) for 1 h at RT.…”

“…Recognition of PAMPs by PRRs, such as retinoic acid-inducible protein I (RIG-I), signals through adaptor protein mitochondrial antiviral signaling protein (MAVS). MAVS activation leads to the activation of IkB ε (IKK-ε) and TANK-binding kinase 1 (TBK-1), which phosphorylate IRF-3 to induce its dimerization and translocation into the nucleus (21,22). Activated IRF-3 and other transcription factors assemble on the IFN-a/b promoter in a cooperative manner to initiate IFN-b transcription (23,24).…”

MIP-T3 Is a Negative Regulator of Innate Type I IFN Response

“…Phosphorylation of IRF3 is a critical event in IFN production (54). Phosphorylated IRF3 would dimerize and translocate into the nucleus to activate the IFN-b promoter (55). To shed light on the mechanism by which MIP-T3 inhibits IFN production, we next explored whether MIP-T3 might affect RIG-I-induced and TBK1-induced phosphorylation of IRF3.…”