Proposal model for the direct role of IRF-3 in both constitutive and induced RIG-I expression.

<p>Upon viral infection, a low level of constitutively expressed RIG-I recognizes viral RNA, inducing the cells to reach an antiviral state. In the antiviral state, IRF-3 is phosphorylated in response to RLR signaling and translocates to the nucleus to induce type I IFNs. Our findings indicate that activated IRF-3 is also able to directly enhance the expression of RIG-I to enhance antiviral signaling. Infected cells produce IFNs, which subsequently activate STAT1, leading to the robust expression of RIG-I in neighboring cells.</p

IRF-3 binds to IRF-E in the RIG-I promoter.

<p>(A) EMSA was performed using a DIG-labelled RIG-I IRF-E wild-type (WT) probe. Nuclear extracts were prepared from poly I:C-transfected HeLa cells. For the supershift assay, rabbit anti-IRF-3 antibody was pre-incubated with the reaction mixture. (B) The labeled RIG-I IRF-E-WT probe was combined with 500 nM (r)GST or (r)GST-IRF-3(5D) fusion protein at various concentrations (500 nM, 125 nM, and 50 nM; wedges). Arrows indicate the IRF-3-oligonucleotide probe complex.</p

IRF-3 binding to the RIG-I promoter <i>in vivo</i>.

<p>HeLa cells were transfected with poly I:C (100 ng) for 4 h and fixed; the DNA was then fragmented. Chromatin immunoprecipitation was performed using an anti-IRF-3 antibody or control IgG. The means (±SD) of three experiments are shown. †<i>P</i> < 0.05</p

Critical Role of IRF-3 in the Direct Regulation of dsRNA-Induced Retinoic Acid-Inducible Gene-I (RIG-I) Expression

<div><p>The cytoplasmic viral sensor retinoic acid-inducible gene-I (RIG-I), which is also known as an IFN-stimulated gene (ISG), senses viral RNA to activate antiviral signaling. It is therefore thought that RIG-I is regulated in a STAT1-dependent manner. Although RIG-I-mediated antiviral signaling is indispensable for the induction of an appropriate adaptive immune response, the mechanism underlying the regulation of RIG-I expression remains elusive. Here, we examined the direct regulation of RIG-I expression by interferon regulatory factor 3 (IRF-3), which is an essential molecule for antiviral innate immunity. We initially found that RIG-I can be induced by dsRNA in both IFN-independent and IRF-3-dependent manners. A sequence analysis revealed that the RIG-I gene has putative IRF-3-binding sites in its promoter region. Using a combination of cellular, molecular biological, and mutational approaches, we first showed that IRF-3 can directly regulate the expression of RIG-I via a single IRF-element (IRF-E) site in the proximal promoter region of the RIG-I gene in response to dsRNA. IRF-3 is considered a master regulator in antiviral signaling for the generation of type I interferons (IFNs). Thus, our findings demonstrate that RIG-I expression induced by the IRF-3-mediated pathway may serve as a crucial antiviral factor for reinforcing a surveillance system against viral invasion through the regulation of the cytoplasmic viral sensor RIG-I.</p></div

Characterization of RIG-I-IRF-E for IRF-3 binding.

<p>(A) Description of oligonucleotide probes of wild-type (WT) (-22 to +8) and mutant (MT) RIG-I IRF-E are shown. Putative IRF-3-binding sites are indicated in lowercase and underlined. (B) The RIG-I IRF-E-WT and RIG-I IRF-E-MT probes were mixed with the nuclear extract as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163520#pone.0163520.g006" target="_blank">Fig 6</a>, and an EMSA was then performed. A competition assay was performed based on the addition of a 50-fold molar excess of unlabeled WT or MT probes. (C) The labeled WT or MT probes were combined with recombinant (r)GST or (r)GST-IRF-3(5D) fusion protein at various concentrations (500 nM). Arrows indicate the IRF-3-oligonucleotide probe complex. N.S.: non-specific signal.</p

IRF-E on the RIG-I promoter regulates the transcriptional activity of RIG-I.

<p>(A) Putative consensus sequences of ISRE, GAS and IRF-E within the proximal region of the RIG-I promoter are shown. (B) A series of single (double or triple) deletion constructs on the RIG-I promoter is shown. (C) U3A and U5A cells were co-transfected with the RIG-I deletion constructs and a renilla luciferase expression vector as shown in (B); †<i>P</i> < 0.05, *<i>P</i> < 0.01.</p

Promoter analysis of the human RIG-I gene.

<p>(A) Putative consensus sequences of STAT1, ISRE, c-Rel and IRF-E on the RIG-I promoter are shown. (B) and (C) HeLa cells were co-transfected with pGL4.11 (empty) or serial human RIG-I luciferase reporter constructs and Renilla luciferase expression vector (pGL4.74) for 24 h. The cells were further transfected with poly I:C (100 ng) for 4 h. The reporter activities are shown as relative values, specifically ratios of the firefly luciferase activities driven by the RIG-I promoters to the Renilla luciferase activities. The means (±SD) of three experiments are shown; †<i>P</i> < 0.05, *<i>P</i> < 0.01.</p

STAT1- and type I IFN-independent RIG-I expression requires IRF3.

<p>HeLa (A), and 2fTGH, U3A, and U5A cells (B) were transfected with siRNA against IRF-1 or IRF-3 or control siRNA for 48 h and then transfected with poly I:C (100 ng) for 4 h. The expression levels of RIG-I were determined by quantitative RT-PCR. The means (±SD) of three experiments are shown; †<i>P</i> < 0.05, *<i>P</i> < 0.01.</p

Intracellular localization of IRF-1 and IRF-3.

<p>(A) HeLa cells were subjected to the following treatments: control (a-c, j-l), transfected with poly I:C (100 ng, for 4 h) (d-f, m-o), or treated with IFN-γ (2 ng/mL, for 4 h) (g-i, p-r). The cells were then fixed with 4% paraformaldehyde and co-stained for IRF-1 (red, left panels) or IRF-3 (red, right panels). Nuclei were counterstained with DAPI (blue). A representative result for five random fields is shown. (B) HeLa cells were transfected with poly I:C (100 ng, for 4 h) or treated with IFN-γ (2 ng/mL, for 4 h). The cells were then harvested in lysis buffer and fractionated. The cell extracts were subsequently subjected to SDS-PAGE, blotted, and probed with anti-IRF-1, anti-IRF-3, anti-HSP90, or anti-histone H1 antibodies. The results are representative of three independent experiments.</p

Non-Canonical Role of IKKα in the Regulation of STAT1 Phosphorylation in Antiviral Signaling

<div><p>Non-self RNA is recognized by retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), inducing type I interferons (IFNs). Type I IFN promotes the expression of IFN-stimulated genes (ISGs), which requires the activation of signal transducer and activator of transcription-1 (STAT1). We previously reported that dsRNA induced STAT1 phosphorylation via a type I IFN-independent pathway in addition to the well-known type I IFN-dependent pathway. IκB kinase α (IKKα) is involved in antiviral signaling induced by dsRNA; however, its role is incompletely understood. Here, we explored the function of IKKα in RLR-mediated STAT1 phosphorylation. Silencing of IKKα markedly decreased the level of IFN-β and STAT1 phosphorylation inHeH response to dsRNA. However, the inhibition of IKKα did not alter the RLR signaling-mediated dimerization of interferon responsive factor 3 (IRF3) or the nuclear translocation of nuclear factor-κB (NFκB). These results suggest a non-canonical role of IKKα in RLR signaling. Furthermore, phosphorylation of STAT1 was suppressed by IKKα knockdown in cells treated with a specific neutralizing antibody for the type I IFN receptor (IFNAR) and in IFNAR-deficient cells. Collectively, the dual regulation of STAT1 by IKKα in antiviral signaling suggests a role for IKKα in the fine-tuning of antiviral signaling in response to non-self RNA.</p></div