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The type IV IFN (IFN-υ) is reported in vertebrates from fish to primary mammals with IFN-υR1 and IL-10R2 as receptor subunits. In this study, the proximal promoter of IFN-υ was identified in the amphibian model, Xenopus laevis, with functional IFN-sensitive responsive element and NF-κB sites, which can be transcriptionally activated by transcription factors, such as IFN regulatory factor (IRF)1, IRF3, IRF7, and p65. It was further found that IFN-υ signals through the classical IFN-stimulated gene (ISG) factor 3 (ISGF3) to induce the expression of ISGs. It seems likely that the promoter elements of the IFN-υ gene in amphibians is similar to type III IFN genes, and that the mechanism involved in IFN-υ induction is very much similar to type I and III IFNs. Using recombinant IFN-υ protein and the X. laevis A6 cell line, >400 ISGs were identified in the transcriptome, including ISGs homologous to humans. However, as many as 268 genes were unrelated to human or zebrafish ISGs, and some of these ISGs were expanded families such as the amphibian novel TRIM protein (AMNTR) family. AMNTR50, a member in the family, was found to be induced by type I, III, and IV IFNs through IFN-sensitive responsive element sites of the proximal promoter, and this molecule has a negative role in regulating the expression of type I, III, and IV IFNs. It is considered that the current study contributes to the understanding of transcription, signaling, and functional aspects of type IV IFN at least in amphibians., (Copyright © 2023 by The American Association of Immunologists, Inc.)

Interferons (IFNs) are critical soluble factors in the immune system and are composed of three types, (I, II and III) that utilize different receptor complexes IFN-αR1/IFN-αR2, IFN-γR1/IFN-γR2, and IFN-λR1/IL-10R2, respectively. Here we identify IFN-υ from the genomic sequences of vertebrates. The members of class II cytokine receptors, IFN-υR1 and IL-10R2, are identified as the receptor complex of IFN-υ, and are associated with IFN-υ stimulated gene expression and antiviral activity in zebrafish (Danio rerio) and African clawed frog (Xenopus laevis). IFN-υ and IFN-υR1 are separately located at unique and highly conserved loci, being distinct from all other three-type IFNs. IFN-υ and IFN-υR1 are phylogenetically clustered with class II cytokines and class II cytokine receptors, respectively. Therefore, the finding of this IFN ligand-receptor system may be considered as a type IV IFN, in addition to the currently recognized three types of IFNs in vertebrates., (© 2022. The Author(s).)

Interferon regulatory factors (IRFs) are a family of transcriptional factors capable of regulating the expression of distinct subsets of interferon (IFN)-stimulated genes by binding to their promoters. IRF1 was the first member identified for its ability to regulate the IFNβ gene and has now been revealed to exhibit remarkable functional diversity in the regulation of different cellular responses. In the present study, the IRF1 gene was identified and characterized in Japanese eel, Anguilla japonica (AjIRF1). The open reading frame of AjIRF1 was 804 bp in length, encoding a protein of 267 amino acids (aa) that encompasses a conserved N-terminal DNA binding domain (DBD). Sequence alignment shows the presence of six highly conserved tryptophan (W) residues in the DBD of IRF1, IRF2 and IRF11, while other IRF members have only five tryptophans. Expression analysis showed that AjIRF1 was significantly upregulated in all tested organs/tissues in response to Poly I:C stimulation or Edwardsiella tarda infection. Furthermore, the functional activity of AjIRF1 was confirmed in driving the transcription of AjIFN promoters, which depends on the highly conserved residues within DBD. Subcellular distribution analysis revealed that AjIRF1 was localized exclusively in the nucleus, which is cooperatively regulated by a bipartite NLS embedded within the DBD and a monopartite NLS located immediately downstream of the DBD. Taken together, this study presents the expression profile of AjIRF1 and defines the functional motifs required for its nuclear import and its role in activating IFN promoters, thus providing helpful information for further research on the regulatory mechanisms of teleost IRF1., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Two members of type II IFNs have been identified in fish, i.e. an IFN-γ gene as in other vertebrates and a unique IFN-γ related (IFN-γ rel) gene being solely present in fish. However, the signalling pathways involved in the down-stream signalling of type II IFNs in fish remains poorly described. In this study, the type II IFNs mediated IRF1 was investigated in zebrafish, and the true homologous gene of mammalian IRF1 in fish was revealed despite the report of so-called IRF1a and IRF1b in zebrafish. As revealed in overexpression analysis, zebrafish IFN-γ had a higher induction ability than IFN-γ rel in relation with the expression of IRF1. IFN-γ stimulated the expression level of STAT1a and also STAT1b, but they had opposite trends with the increase of time; enhancement of STAT1a waned after 12 h post injection of plasmids; whereas STAT1b expression increased continuously. Zebrafish IRF1 gene promoter contained several putative transcription factor binding sites, including GAS and NF-κB motifs. Luciferase assay revealed that the GAS site was essential in the IFN-γ triggered IRF1 expression. In contrast, IRF11 contained neither GAS nor NF-κB elements, and did not respond to IFN-γ induction. It is considered that STAT1a and STAT1b are structurally and functionally similar to STAT1α and STAT1β in mammal respectively, and that IRF11, although used to be nominated as IRF1a, is not the orthologue of mammalian IRF1, but IRF1b in zebrafish should be the orthologue., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Interferon regulatory factor (IRF) 3 and IRF7 have been identified as regulators of type I interferon (IFN) gene expression in mammals. In the present study, the two genes were cloned and characterized in the European eel, Anguilla anguilla. The full-length cDNA sequence of IRF3 and IRF7 in the European eel, named as AaIRF3 and AaIRF7 consists of 2879 and 2419 bp respectively. Multiple alignments showed that the two IRFs have a highly conserved DNA binding domain (DBD) in the N terminus, with the characteristic motif containing five tryptophan residues, which is a feature present in their mammalian homologues. But, IRF7 has only four of the five residues in other species of fish. The expression of AaIRF3 and AaIRF7 both displayed an obvious dose-dependent manner following polyinosinic:polycytidylic acid (PolyI:C) challenge. In vivo expression analysis showed that the mRNA level of AaIRF3 and AaIRF7 was significantly up-regulated in response to PolyI:C stimulation in all examined tissues/organs except in muscle, with a lower level of increase observed in response to lipopolysaccharide (LPS) challenge and Edwardsiella tarda infection, indicating that AaIRF3 and AaIRF7 may be more likely involved in antiviral immune response. In addition, some pattern recognition receptors genes related with the production of type I IFNs and those genes in response to type I IFNs were identified in the European eel genome database, indicating a relatively conserved system in the production of type I IFN and its signalling in the European eel., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

We demonstrate for the first time in vertebrates, that alternative splicing of interferon (IFN) genes can lead to a functional intracellular IFN (iIFN). Fish IFN genes possess introns and in rainbow trout three alternatively spliced transcripts of the IFN1 gene exist. Two of the encoded IFNs are predicted to lack a signal peptide. When overexpressed these iIFNs induce antiviral responses. Variants of the two IFNR receptor chains (IFNAR1 and IFNAR2) lacking a signal peptide are also present in trout. Transfection of HEK 293T cells with the iIFN and iIFNR molecules results in STAT phosphorylation and induction of antiviral genes. These results show that fish possess a functioning iIFN system that may act as a novel defence to combat viral infection.

Highlights • PCV2 induces cGAS up-regulation and cGAMP production. • PCV2 induces STING expression, dimerization and translocation into the nucleus. • Knockdown of cGAS or STING decreases IFN-β production. • Knockdown of cGAS or STING decreases the viral load and replication of PCV2. Abstract PCV2 is a single-stranded DNA virus that we previously found to induce IFN-β production via RIG-I and MDA-5. cGAS is known to be the most important DNA sensor for the recognition of cytosolic DNA; however, it remains unclear whether the interferon production induced by PCV2 is associated with cGAS. In the present study, PCV2 infection was found to increase the level of cGAS and STING expression, promote the release of cyclic dinucleotide cGAMP, and induce STING dimerization and translocation into the nucleus of PK-15 cells. These findings indicate that PCV2 infection activates both cGAS and STING. Furthermore, the knockdown of cGAS and STING decreased both the mRNA expression and promoter activity of IFN-β, demonstrating that the cGAS/STING signaling pathway contributes to the production of IFN-β. In addition, a knockdown of cGAS and STING also decreased the PCV2 viral load and infectivity. Therefore, PCV2 infection activates the cGAS/STING signaling pathway to induce IFN-β production and the knockdown of cGAS and STING decreases viral replication in PK-15 cells. These results provide further insight into the relationship between PCV2 and host innate immunity. [ABSTRACT FROM AUTHOR]

Vertebrate gamma-interferon inducible lysosomal thiol reductase (GILT) is an IFN-γ-inducible protein and is involved in MHCII-restricted antigen processing and cross-presentation of MHCI-restricted antigens in adaptive immunity. Outside of the endocytic MHC pathway, GILT regulates the cellular redox state, inhibits T cell activation, neutralizes extracellular pathogens and is also a host factor of some bacterial pathogens. In this report, we isolated and characterized three divergent GILT -like genes, GILT1 , GILT2 and GILT3 , which share only 30.9–40.4% identities in a crustacean mud crab Scylla paramamosain . Whilst the crab GILT1 and GILT3 possess four and five exons, respectively, the GILT2 is intronless, suggesting that GILT2 may arise from a recent retroposition event. The invertebrate GILT -like genes have diverse gene organizations and may be evolved in a species/lineage-specific manner as suggested by phylogenetic tree analysis. The amino acid sequences equivalent to human mature GILT are well conserved, including the GILT signature and nine of the ten cysteine residues that potentially form 5 disulfide bonds in human GILT, across the animal kingdom. However, most invertebrate GILT -like molecules lack the human-type N-terminal propeptide, as well as the human-type C-terminal with a conserved cysteine residue, suggesting differences in post translational processing and mode of action. All the three GILT -like genes are highly expressed in the hepatopancreas and up-regulated by pathogenic bacterial infection suggesting a role in immune defense against bacterial diseases. This study may provide the basis for further investigation of the expanding functions of GILT -like molecules in immunity and other physiological processes in mud crabs and other animals. [ABSTRACT FROM AUTHOR]

Teleost fish are unique in having type I and type II interferons (IFNs) only, and the type I IFNs are classified into Group one and Group two based on the presence of two or four cysteines respectively, and are further classified into seven subgroups. In the present study, three distinct type I IFNs, IFNc, IFNd and IFNh, have been identified in the genome sequences of a perciform fish, the mandarin fish Siniperca chuatsi . These IFNs are induced following the stimulation of Polyinosinic polycytidylic acid (poly(I:C)) and Resiquimod (R848) either in vivo or in vitro . But, the infectious spleen and kidney necrosis virus (ISKNV) infection caused a delayed response of IFNs, which may be resulted from the viral inhibition of type I IFN production and related signalling. The three receptor subunits, cytokine receptor family B 1 (CRFB1), CRFB2 and CRFB5 are also expressed in a similar manner as observed for the IFNs, and IFNc, IFNd and IFNh use preferentially the receptor complex, CRFB2 and CRFB5, CRFB1 and CRFB5, CRFB1 and CRFB5 respectively for their effective signalling in the induction of IFN-stimulated genes (ISGs). Moreover, the IFNs are able to induce their own expression, and also the IRF3 and IRF7 expression, leading to the amplification of IFN cascade. It is further revealed that these three IFNs are transcribed differently by IRF7 and IRF3. The composition, function, signalling and transcription of type I IFNs have been investigated in detail in a teleost fish. [ABSTRACT FROM AUTHOR]

IFN-λ (IFNL), i.e. type III IFN genes were found in a conserved gene locus in tetrapod vertebrates. But, a unique locus containing IFNL was found in avian. In turtle and crocodile, IFNL genes were distributed in these two separate loci. As revealed in phylogenetic trees, IFN-λs in these two different loci and other amniotes were grouped into two different clades. The conservation in gene presence and gene locus was also observed for the receptors of IFN-λ, IFN-λR1 and IL-10RB in tetrapods. It is further revealed that in North American green anole lizard Anolis carolinensis , a single IFNL gene was situated collinearly in the conserved locus as in other tetrapods, together with its receptors IFN-λR1 and IL-10RB also identified in this study. The IFN-λ and its receptors were expressed in all examined organs/tissues, and their expression was stimulated following the injection of polyI:polyC. The ISREs in promoter of IFN-λ in lizard were responsible to IRF3 as demonstrated using luciferase report system, and IFN-λ in lizard functioned through the receptors, IFN-λR1 and IL-10RB, as the up-regulation of ISGs was observed in ligand-receptor transfected, and also in recombinant IFN-λ stimulated, cell lines. Taken together, it is concluded that the mechanisms involved in type III IFN ligand-receptor system, and in its signalling pathway and its down-stream genes may be conserved in green anole lizard, and may even be so in tetrapods from xenopus to human. [ABSTRACT FROM AUTHOR]

Highlights: [•] The IFN-γ gene has conserved genome and motifs in Chinese soft-shelled turtle. [•] IFN-γ signaling pathway molecule STAT1 was up-regulated in IFN-γ overexpressed cells. [•] IRF1 and IRF7, and Mx and PKR were also up-regulated in overexpressed cells. [•] Cells overexpressing IFN-γ resulted in the reduced iridovirus replication. [•] Significant increase in morality was observed in IFN-γ transfected cells upon the virus infection. [Copyright &y& Elsevier]

Abstract: The interferon regulatory factor 5 (IRF-5) is known to be involved in the innate immune response and in the regulation of DNA damage-induced apoptosis. In the present study, the cDNA and genomic sequences of IRF-5 were identified in grass carp (Ctenopharyngodon idella). The cDNA of grass carp IRF-5 (gcIRF-5) contains an open reading frame (ORF) of 1560 nucleotides, encoding a putative 519 amino acid protein, which showed 34.5–83.9% identity to IRF-5 homologues from mammals, amphibian, avian and fish, and 96.2% and 95.0% identity to zebrafish IRF-5 in the DNA-binding domain (DBD) and IRF association domain (IAD), respectively. The genomic DNA sequence of gcIRF-5 contains 6075bp consisting of 9 exons and 8 introns. The expression of gcIRF-5 was observed in all organs examined. The analysis of real-time quantitative RT-PCR revealed that grass carp reovirus (GCRV) induced the expression of gcIRF-5 in spleen and head kidney. [Copyright &y& Elsevier]