[show abstract][hide abstract] ABSTRACT: 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 5' 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.
The Journal of Immunology 11/2010; 185(12):7573-82. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Virus-induced interferons (IFNs) have been identified in various fish species and display antiviral activities similar to mammalian type I IFNs. However, apart from the mammalian IFN system, the IFN signaling pathway remains largely unknown. Using transient transfection and recombinant protein, we are reporting in this study that a crucian carp (Carassius auratus L.) IFN exhibits strong antiviral activity against grass carp hemorrhagic virus (GCHV) infection and also mediates Poly I:C-induced antiviral response, which correlates with its ability to induce a set of IFN-stimulated genes (ISGs). Strikingly, overexpression of wild-type Stat1 increases the effects of IFN on both the expression of ISGs and the inhibition of virus infection, whereas a dominant negative mutant of Stat1 (Stat1-Delta C), which lacks of the C-terminal transcriptional activation domain (TAD), inhibits the antiviral activity of IFN and reduces the expression of ISGs, demonstrating that fish IFN induces the expression of ISGs and host antiviral response through Stat1 pathway reminiscent that of mammalian IFNs. Significantly, unlike mammalian type I IFNs, recombinant fish IFN is able to upregulate IFN itself, which is enhanced by overexpression of Stat1 but impaired by knockdown of Stat1, indicating a positive feedback loop in regulation of IFN itself. These results provide strong evidence for the existence of an evolutionary conserved Stat1 pathway between fish and mammals, which is indispensable for fish virus-induced IFN antiviral response.
[show abstract][hide abstract] ABSTRACT: Grass carp hemorrhagic virus (GCHV)-induced gene 2 (Gig2) is a novel gene previously identified from UV-inactivated GCHV-treated Carassius auratus blastulae embryonic (CAB) cells, suggesting that it should play a pivotal role in the interferon (IFN) antiviral response. In this study, a polyclonal anti-Gig2 antiserum was generated and used to study the inductive expression pattern by Western blot analysis, showing no basal expression in normal CAB cells but a significant up-regulation upon UV-inactivated GCHV, polyinosinic:polycytidylic acid (Poly I:C) and recombinant IFN (rIFN). However, constitutive expression of Gig2 is observed in all tested tissues from grass carp (Ctenopharyngodon idellus), and Poly I:C injection increases the relative amount of Gig2 protein in skin, spleen, trunk kidney, gill, hindgut and thymus. Moreover, the genomic sequence covering the whole Gig2 ORF and the upstream promoter region were amplified by genomic walking. Significantly, the Gig2 promoter contains three IFN-stimulated response elements (ISREs), nine GAAA/TTTC motifs and five gamma-IFN activating sites (GAS), which are the characteristics of genes responsive to both type I IFN and type II IFN. Subsequently, the complete Gig2 promoter sequence was cloned into pGL3-Basic vector, and its activity was measured by luciferase assays in the transfected CAB cells. The Gig2 promoter-driven construct is highly induced in CAB cells after treatment with Poly I:C or rIFN, and the functional capability is dependent on IFN regulatory factor 7 (IRF7), because its activity can be stimulated by IRF7. Collectively, the data provide strong evidence that Gig2 is indeed a novel IFN inducible gene and its expression is likely dependent on IRF7 upon Poly I:C or IFN.