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

Abstract

Material

Fish IRF6 is a positive regulator of IFN expression and involved in both of the MyD88 and TBK1 pathways

“…In mammals, IRF5 participates in the MyD88 pathway to activate IFN expression, and fish IRF1 is downstream of MyD88 as a pivotal regulator of IFN [21]. On the other hand, IRF3 is a crucial transcription factor that is phosphorylated by TBK1 in both mammals and fish [15]. In most studies of mammals, only IRF3 and IRF7 is activated by both MyD88 and TBK1 to promote IFN transcription.…”

Fish viperin exerts a conserved antiviral function through RLR-triggered IFN signaling pathway

“…Similarly, the putative GAS sites might be important for type II IFN stimulation of crucian carp viperin, because type II IFN stimulation results in formation of STAT1 homodimers that binds to the GAS sites triggering some ISG transcription (Stark, 2007). Actually, our previous results have shown that the ISRE site is required for the induction of crucian carp Gig1, Gig2 and IRF3 by crucian carp IFN (Jiang et al, 2009;Li et al, 2012;Sun et al, 2014Sun et al, , 2010 , and the GAS is indispensable for the induction of zebrafish fish IRF9 by type II fish IFN (Shi et al, 2013). It is also noted that unlike other type I and type II zebrafish IFNs, DrIFN4 and DrIFNc1 cannot induce viperin expression (Fig.…”

“…For overexpression, wild type gene plasmids crucian carp RIG-I, MDA5, LGP2, MITA, TBK1, IRF3, IRF7, IFN, IRF9, STAT2, dominant negative mutant plasmids IRF3-DN and IRF7-DN, wild type gene plasmids zebrafish IFN1, IFN2, IFN3, IFNc1 (IFNrel) and IFNc2 were previously described (Shi et al, 2013;Sun et al, 2010Sun et al, , 2011. Wild type viperin plasmid was generated by cloning the open reading frame (ORF) of crucian carp viperin (Genbank accession No.…”

“…Transfection and luciferase assays were performed as described previously Sun et al, 2010). In brief, CAB or CO cells seeded in 6-well or 24-well plates overnight were transfected with a mixture containing of 2 lg or 0.5 lg plasmids and 4 ll or 1 ll lipofectamine 2000 (Invitrogen) in 500 ll or 100 ll FCS-free opti-MEM (Invitrogen) per well.…”

“…Western blots were performed as previously described Sun et al, 2010). Briefly, equal amounts of protein samples were separated by 12% SDS-PAGE gels and then electrophoretically transferred to a PVDF membrane (Millipore, Bedford, MA).…”