Induction of interferons (IFNs) produces an innate immune response through activation of the JAK-STAT signaling pathway. Type I IFN signaling activates downstream gene expression through the IFN-stimulated gene factor 3 (ISGF3) complex, while type II IFN (IFN-γ) signaling is mediated through active STAT1 protein. The IFN target gene Mx is involved in the defense against viral infection. However, the mechanism by which Tetraodon (pufferfish) Mx is regulated by IFN signaling has not been identified. In this study, we describe the cloning and expression of Tetraodon STAT1, STAT2, and IFN regulatory factor 9 (IRF9). By combining constitutively-active STAT1 (STAT1-JH1) and STAT2 (STA2-JH1) fusion proteins with IRF9, we demonstrate that a constitutively-active ISGF3 complex increases the transcriptional activity of the Tetraodon Mx promoter via direct binding to two IFN-stimulated response element (ISRE) sites. In addition, a constitutively-active TnIRF9-S2C containing a fusion of the C-terminal region of STAT2 and IRF9 also activated the Mx promoter through binding to the ISRE sites. Furthermore, constitutively-active STAT1-JH1 elevates Mx promoter activity through two IFN gamma-activated sequence (GAS) elements. The Mx promoter is also activated by constitutively-active TnIRF9-S2C and STAT1-JH1 protein, as determined using an invivo luciferase assay. We conclude that the Tetraodon Mx gene is activated via Type I (IFN-1) and Type II (IFN-γ) signaling. These results provide mechanistic insights into the role of IFN signaling in teleosts, and the invivo luciferase assay may be suitable as a tool for studying induction and regulation by IFNs in teleost fish.
ASJC Scopus subject areas