Regulation of IFN Response in Vertebrates IFN-Inducible Protein Reveals Evolving Characterization of Fish IRF3 as an

In mammals, IFN regulatory factor (IRF) 3 is a critical player in modulating transcription of type I IFN and IFN-stimulated genes (ISGs). In this study, we describe the roles of crucian carp (Carassius auratus L.) IRF3 in activating fish IFN and ISGs. Fish IRF3 exhibits a large sequence divergence from mammalian orthologs. Whereas mammalian IRF3 is constitutively expressed, fish IRF3 protein is significantly upregulated by IFN, poly-IC, and other stimuli known as IFN inducers in mammals. The IFN-inducible property of fish IRF3 is consistent with the comparative analysis of 59 flanking regulatory region of vertebrate IRF3 genes, which reveals the presence of typical IFN-stimulated response elements in fish and amphibians, but an absence in tetrapods. Furthermore , either IFN or poly-IC induces phosphorylation and cytoplasmic-to-nuclear translocation of IRF3, which seems essential for its function in that phosphomimic active IRF3 exhibits stronger transactivation than wild type IRF3. Finally, overexpression of fish IRF3 activates production of IFN that in turn triggers ISG transcription through Stat1 pathway, whereas transfection of dominant negative mutant IRF3-DN abrogates poly-IC induction of ISGs, probably owing to blockade of IFN production. Therefore, regulation of IFN response by vertebrate IRF3 is another ancient trait. These data provide evidence of the evolving function of vertebrate IRF3 on regulating IFN response. I n mammals, IFN response is the first line of defense against virus infection and is generally initiated through the recognition of viral products rapidly by germline-encoded pattern recognition receptors (1). The best-characterized pattern recognition receptors include the TLRs, such as TLR3, that recognize viral motifs presented at the cell surface or within the endosomal compartment in immune cell lineages, and the RIG-I–like receptors (such as RIG-I and MDA5) that mediate cytosolic viral component recognition in most cell types (1). Such recognition events trigger distinct signaling pathways that converge on the activation of the viral activated kinase, TBK1, which phosphor-ylates IFN regulatory factor 3 (IRF3), leading to the induction of type I IFN and subsequent downstream antiviral genes (2, 3). IRF3 belongs to the IRF family that includes 9 members in mammals, 10 members in birds, and 11 members in fish (4, 5). Structurally , all IRF members share extensive homology in the N-terminal DNA binding domain (DBD), characterized by five tryptophan repeat elements located within the first 115 aa of the proteins. The DBD forms a helix-turn-helix structure that mediates specific binding to a DNA sequence corresponding to the …