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

Mammalian viperin is a typical interferon (IFN)-induced antiviral protein. Fish have viperin homologs; however, little is known about the expression regulation of fish viperins. In this study, we report the expression regulation and antiviral function of a fish viperin from crucian carp Carassius auratus during IFN response. Crucian carp viperin is induced at mRNA and protein levels by fish IFNs and IFN stimuli such as poly(I:C). Consistently, this gene promoter contains multiple transcription factor binding sites including IFN-stimulated response elements (ISRE) and IFN gamma activation sequences (GAS), and is activated by two types of fish IFNs and also by the intracellular and extracellular poly(I:C). Activation of crucian carp viperin promoter by the intracellular poly(I:C) is mediated by retinoic acid-inducing gene I (RIG-I)-like receptors (RLR)-triggered IFN signaling pathway, which is further verified by the findings that each signaling molecule of RLR pathway is able to induce the expression of crucian carp viperin at mRNA and protein levels. Finally, overexpression of crucian carp viperin in cultured fish cells confers significant protection against infection of grass carp reovirus (GCRV). These data suggest that similar to mammalian homologs, crucian carp viperin exerts a conserved function through RLR-triggered IFN signaling pathway.

Wang B ，Zhang YB ，Liu TK ，Shi J ，Sun F ，Gui JF ... -  《-》

Gig1, a novel antiviral effector involved in fish interferon response.

Vertebrate interferon (IFN) response defenses against viral infection through the induction of hundreds of IFN-stimulated genes (ISGs). Most ISGs are conserved across vertebrates; however, little is known about the species-specific ISGs. In this study, we reported that grass carp reovirus (GCRV)-induced gene 1 (Gig1), previously screened as a virus-induced gene from UV-inactivated GCRV-infected crucian carp (Carassius auratus) blastulae embryonic (CAB) cells, was a typical fish ISG, which was significantly induced by intracellular poly(I:C) through retinoic acid-inducible gene I (RIG-I)-like receptors-triggered IFN signaling pathway. Transient or stable overexpression of Gig1 prevented GCRV replication efficiently in cultured fish cells. Strikingly, Gig1 homologs were found exclusively in fish species forming a novel gene family. These results illustrate that there exists a Gig1 gene family unique to fish species and the founding gene mediates a novel fish IFN antiviral pathway.

Sun F ，Zhang YB ，Jiang J ，Wang B ，Chen C ，Zhang J ，Gui JF ... -  《-》

Fish MAVS is involved in RLR pathway-mediated IFN response.

Mammalian mitochondrial antiviral signaling protein (MAVS) is an essential adapter involved in retinoic acid-inducible gene-I (RIG-I)-like receptor (RLR)-triggered interferon (IFN) antiviral immunity. Fish MAVS homologues have been identified in several fish species; however, the signaling pathway involving fish MAVS-mediated IFN response remains to be investigated. In the present study, we report identification of a fish MAVS orthologue from crucian carp Carassius auratus blastulae embryonic (CAB) cells and its function role in fish RLR signaling. Crucian carp MAVS is constitutively expressed in CAB cells and is not transcriptionally induced by cytosolic poly (I:C) and IFN. Overexpression of crucian carp MAVS results in activation of fish IFN promoter and ISRE-containing promoter as well as transcriptional expression of IFN and ISGs including PKR and Mx1, which is impaired by functional blockade of signaling molecules TBK1 and IRF3/7. Either cytosolic poly (I:C)-induced or RIG-I-induced IFN response is attenuated by functional blockade of crucian carp MAVS. These results together indicate that fish MAVS contributes to IFN antiviral immunity downstream of cytosolic poly (I:C) and RIG-I and upstream of TBK1 and IRF3/7. Moreover, we provide evidence that apart from crucian carp MAVS, crucian carp MITA is also involved in cytosolic poly (I:C)- and RIG-I-induced IFN response.

Zhang J ，Zhang YB ，Wu M ，Wang B ，Chen C ，Gui JF ... -  《-》

Fish virus-induced interferon exerts antiviral function through Stat1 pathway.

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.

Yu FF ，Zhang YB ，Liu TK ，Liu Y ，Sun F ，Jiang J ，Gui JF ... -  《-》

Identification, characterization and immunological response analysis of stimulator of interferon gene (STING) from grass carp Ctenopharyngodon idella.

Stimulator of interferon gene (STING), an important adapter responsible for RLR pathway, plays a pivotal role in both viral RNA- and DNA-triggered induction of IFNs in mammals. To understand the roles of STING in piscine immune system, STING gene (CiSTING) was identified from grass carp (Ctenopharyngodon idella). The genomic sequence of CiSTING was of 8548 base pairs (bp), including 899 bp 5' flank region, 7 exons and 6 introns. Promoter region was predicted and promoter activity was verified. The CiSTING cDNA was of 1358 bp with an open reading frame of 1185 bp, encoding a polypeptide of 394 amino acids with a signal peptide and three transmembrane motifs in the N-terminal region. mRNA expression of CiSTING was widespread in fifteen tissues investigated, and was up-regulated by GCRV in vivo and in vitro. Meanwhile, the transcription of CiSTING was inhibited at early stage, and then up-regulated at late phase upon poly(I:C) or PGN stimulation in vitro. Interestingly, CiSTING had little impact on LPS in vitro. In CiSTING over-expression cells, CiTBK1, CiIRF3 and CiIRF7 were significantly up-regulated post GCRV or viral/bacterial PAMPs stimulation. In addition, post GCRV or PGN stimulation, the transcription of CiIFN-I was remarkably inhibited while CiMx1 was up-regulated; as for poly(I:C) stimulation, mRNA expressions of CiIFN-I and CiMx1 were inhibited at early stage while enhanced at late phrase; after LPS stimulation, both CiIFN-I and CiMx1 were inhibited. Furthermore, antiviral activity of CiSTING was manifested by the inhibition of GCRV yield. Taken together, these results demonstrated that CiSTING may be involved in board innate immune responses via the TBK1-IRF3/IRF7 cascade, responding to not only dsRNA analogue in an IFN-dependent pathway, but also virus and bacterial PAMPs in an IFN-independent pathway. This study provided novel insights into the essential role of STING in innate immunity.

Feng X ，Yang C ，Zhang Y ，Peng L ，Chen X ，Rao Y ，Gu T ，Su J ... -  《-》