Fish MITA Serves as a Mediator for Distinct Fish IFN Gene Activation Dependent on IRF3 or IRF7

Abstract

In mammals, cytosolic sensors retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) activate multiple signaling cascades initiating IFN-α/β expression. IFN regulatory factor 3 (IRF3) is required for the activation of IFN-β, which, in turn, primes the expression of most IFN-α genes by IFN-induced IRF7 through the STAT1 pathway. In fish, RIG-I overexpression inhibits virus infection by induction of IFN response; however, the subtle signaling cascade mechanism remains to be identified. In this study, we clone an ortholog of MITA, a recently identified adaptor responsible for RLR pathway, from crucian carp (Carassius auratus L.), and demonstrate its ability to suppress viral replication through IRF3/7-dependent IFN response. The pivotal signaling molecules of RLR pathway, including RIG-I, melanoma differentiation-associated gene 5, laboratory of genetics and physiology 2, and TANK-binding kinase 1, are also cloned and characterized, confirming that the RLR-mediated IFN activation is conserved from fish to mammals. Further characterization of distinct IFN gene activation reveals that zebrafish IFN1 and IFN3 are induced by the MITA pathway but are dependent on distinct transcription factors. Whereas fish IFN genes cannot be classified into IFN-α or IFN-β, zebrafish IFN1 is primarily regulated by IRF3, thereby resembling that of IFN-β, and zebrafish IFN3 is regulated by IRF7, thereby resembling of those of IFN-αs. In contrast with mammalian IFN-α/β, zebrafish IFN1 and IFN3 are induced by the basally expressed IRF3 or IRF7, both of which are upregulated by IFN and virus infection. Collectively, these data suggest that IFN genes in fish and mammals have evolved independently to acquire a similar mechanism triggering their expression.

Expression regulation of zebrafish interferon regulatory factor 9 by promoter analysis

“…Real-time PCR was performed in a DNA Engine Chromo 4 realtime system (BioRad) with SYBR green real-time PCR master mix (TOYOBO) according to previous studies (Shi et al, 2012;Sun et al, 2011). All samples were analyzed in triplicates and the expression values were calculated as relative to b-actin with the 2 (ÀDDC(T)) method.…”

“…The purified fusion protein was applied to immunize rabbits to raise a polyclonal anti-IRF9 antiserum according to previous reports (Jiang et al, 2009;Yu et al, 2010). Western blots were performed according to previous reports (Jiang et al, 2009;Liu et al, 2011;Sun et al, 2010Sun et al, , 2011. Briefly, protein samples were separated on 12% SDS-PAGE gels and then electrophoretically transferred to a PVDF membrane (Millipore).…”

Fish MAVS is involved in RLR pathway-mediated IFN response

“…Characterization of these genes individually suggests that they all are involved in fish IFN response. For example, overexpression of salmon RIG-I or rainbow trout MDA5 results in upregulation of IFN and ISGs and also establish host antiviral states [21,22]; ectopic expression of crucian carp IRF3 or MITA triggers the expression of both IFN and ISGs [23,24]. Fish MAVS genes are first identified from Atlantic salmon (Salmo salar) and zebrafish (Danio rerio), both of which exhibit an ability to induce transcriptional expression of IFN and confer full protection against both RNA and DNA virus on cultured fish cells [21,25].…”

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

“…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.…”

“…Similar to mammals, virus infection elicits a conserved fish IFN antiviral response that is initiated by TLR pathway (Matsuo et al, 2008) or RLR pathway (Biacchesi et al, 2009;Chang et al, 2011;Sun et al, 2011). Two types of fish IFN genes have been identified in fish genomes, one most similar to mammalian type I IFNs and the other orthologous to mammalian type II IFN (Altmann et al, 2003;Hamming et al, 2011;Stolte et al, 2008;Zou et al, 2005).…”