Interferon regulatory factor 1 (IRF1) and anti-pathogen innate immune responses

Abstract

The eponymous member of the interferon regulatory factor (IRF) family, IRF1, was originally identified as a nuclear factor that binds and activates the promoters of type I interferon genes. However, subsequent studies using genetic knockouts or RNAi-mediated depletion of IRF1 provide a much broader view, linking IRF1 to a wide range of functions in protection against invading pathogens. Conserved throughout vertebrate evolution, IRF1 has been shown in recent years to mediate constitutive as well as inducible host defenses against a variety of viruses. Fine-tuning of these ancient IRF1-mediated host defenses, and countering strategies by pathogens to disarm IRF1, play crucial roles in pathogenesis and determining the outcome of infection.

Fig 1. The discovery and recognition mechanism of IRF1.

(A) The four positive regulatory domains (PRDs) within IFNβ promoter (nucleotides -105 to +19 relative to the transcriptional start site). (B) Key features of the IRF family members. DBD, DNA binding domain; IAD, IRF-association domain; AR, auto-inhibitory region; α1–3, α helices 1–3; β1–4, β sheets 1–4; L1-3, loops 1–3. (C) The detailed structural study of DNA-binding by IRF proteins involved the DBD of IRF1. Trp11, Trp38 and Trp58 of IRF1 strategically straddle the major groove of the ‘GAAA’ segment of the DNA, forming hydrogen bonds and van der Waals contacts with the sugar-phosphate backbone (blue dashed lines). Arg82, Cys83, Asn86 and Ser87, all within the α3 recognition helix, mediate direct contacts between the DBD and GAAA (red dashed lines). With high structural similarities of the DBD, IRF family members recognize consensus ‘AANNGAAA’ sequence, yet they display slightly different binding specificities [120].

Fig 2. Evolutionary conservation of IRF1.

(A) Phylogenetic occurrence of IRF- and IFN-dependent innate immune response. The selection of individual species is based on literature indicating certain effects and is not intended to be comprehensive. In cnidarians and bilaterians, which diverged from the common eumetazoan ancestor ~600–630 million years ago, IRFs are proposed to be separated into IRF1 and IRF4 subfamilies. (B) Evolutionary comparison of vertebrate IRF1 and invertebrate IRF1-like genes within the DBD. *, C. gigas ‘IRF1’ gene that has been reported. Three other predicted transcript variants of Pacific oyster IRF1 exist (LOC105343806; NCBI Accession numbers: XM_011451290.2 for X1, XM_011451291.2 for X2 and XM_011451292.2 for X3). The predicted amino acid sequence shares 53% identity with H. sapiens IRF1 at the DBD, and contains all five tryptophans as well as the conserved GAAA-contacting residues. LOC105343805 thus might not represent bona fide C. gigas IRF1. **, B. belcheri ‘IRF1’ does not present a rigid one-to-one ortholog relationship with vertebrate IRF1. Rather, it is linked to the vertebrate IRF1 subfamily based on phylogenetic analysis.

Fig 3. IRF1 in IFN-dependent host defenses.

(A) RIG-I-like receptors (RLRs) including retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) associate with mitochondrial antiviral signaling protein (MAVS) to activate the nuclear factor-kB (NF-κB), which induces immediate transcription of IRF1 mRNA. Newly synthesized IRF1 protein acts downstream to induce interferons and pro-inflammatory cytokines. (B) Toll-like receptors (TLRs), except TLR3, interact with the adaptor MYD88 protein, which also interacts with IRF1 so as to license rapid IRF1 migration into the nucleus. (C) Interferon receptors (IFNRs) activate Janus kinases (JAKs)- and STAT-dependent signaling cascades, leading to the formation of IFN-stimulated gene factor 3 (ISGF3, a heterotrimeric complex composed of phosphorylated STAT1 and STAT2 with IRF9) and GAF (the STAT1 homodimer complex), which induce IRF1 expression in a positive feedback loop, ultimately augmenting induction of IFN-stimulated genes (ISGs). (D) TNF receptors initiate signaling cascades that activate NF-κB, leading to IRF1 transcription and in turn the expression of type I interferons, but at much lower levels than RLR or TLR signaling pathways. (E) Type I IFN-dependent IRF1 expression leads to AIM2- and NLRP3-dependent inflammasome responses to Francisella novicida and influenza A virus, respectively. Epigenetic modification including histone methylation and acetylation in the promoter-proximal regions also contributes to dynamic regulation of chromatin states, affecting binding of IRF1 and other transcription factors.

Fig 4. Basal antiviral activity of IRF1 and the downstream effectors.

(A) Nuclear IRF1 mediates constitutive expression of diverse classes of antiviral effectors acting on lipid metabolism, protein degradation pathways, in addition to driving basal expression of conventional antiviral ISGs such as OAS2. IRF1 target genes are underlined. (B) Antiviral spectrum of IRF1 effector genes based on siRNA knockdown assays in immortalized hepatocytes (PH5CH8) infected with hepatitis A virus (HAV), hepatitis C virus (HCV), or flaviviruses (dengue or Zika viruses), or BEAS-2B cells infected with vesicular stomatitis virus.

Fig 5. IRF1 contribution to dynamic transcriptional activation networks.

IRF1 binds to GAAA sequences within IRF-E and ISRE enhancers, driving transcriptional activation of target genes. In the case of ISRE binding, IRF1 can form homo- or hetero dimers with other members of the IRF family. In genes with multiple cis-regulatory elements, coordinate binding of IRF1 and other transcription factors such as NF-κB, ISGF3 or GAF to the enhancer region often has a synergistic impact on gene expression. This can be driven by protein-protein interaction-directed formation of enhanceosomes, or co-localization of transcription factors binding to individual cis-elements. IRF1 both modulates and is regulated by activating and repressive epigenetic marks on chromatin, including histone methylation (such as H3K4me3 and H3K27me3) or acetylation at promoter proximal sites.

Fig 6. Regulation of IRF1 responses by virus and host.

Various pathways cross-regulate IRF1 responses, altering the expression level and activation state of IRF1. (A) Transcription of IRF1 is mediated largely by NF-κB and GAF, which are antagonized by HCV core protein. IRF1 transcription is also is regulated epigenetically by histone H3 methylation and acetylation. While both HAV and HCV encode proteases that disable MAVS signaling, HCV and EV71 disrupt the nuclear translocation of GAF. HCMV usurps the host Roquin protein to reduce IRF1 mRNA levels by binding to the 5′ UTR of IRF1 transcripts, while other viruses activate EGFR signaling to downregulate IRF1 mRNA. miRNAs also mediate downregulation of IRF1 mRNA levels. (B) IFNγ receptor signaling activates Mnk1/eIF4E to ensure translation of IRF1 mRNA, whereas NLRX1 promotes IRF1 protein synthesis by counteracting PKR-mediated global protein translation. The host gene encoding miR-205 associates with the promoters of its target genes at Alu sequences in proximity of IRF-E motif, thereby suppressing IRF1 binding. Activity of IRF1 protein is also regulated post-translationally via ubiquitination, SUMOylation, phosphorylation and acetylation.