Zebrafish TRIF, a Golgi-Localized Protein, Participates in IFN Induction and NF-κB Activation

Abstract

The antiviral immune responses were triggered by the innate immune recognition of viral infection. The type I IFNs (IFN-β and IFN-α) are the key cytokines produced upon viral infection and consequently link innate immunity with adaptive immunity. A main antiviral system in mammals is TRIF-dependent TLRs pathway, but the TRIF-independent RIG-I pathway, has also been discovered recently. In this manuscript, our study focuses on the functional characterization of zebrafish TRIF based on the comparison of its sequence and functional evolution from zebrafish to mammals. Our experimental results show that the full length cDNA of zebrafish TRIF cloned by RACE-PCR approach encodes a protein of 556 amino acids. Luciferase reporter assay confirms that zebrafish TRIF is able to induce the IFN promoter as well as activate NF-κB response promoter. The IFN induction function of zebrafish TRIF is abolished when Ala359 is mutated to Pro or His. Laser confocal microscopy shows that zebrafish TRIF is colocalized with a Golgi apparatus marker, implying its unique subcellular localization in Golgi apparatus. In zebrafish, the mRNA expression of molecules participating in RIG-I pathway are much more sensitive and specific to polyinosine-polycytidylic acid induction compared with those in TRIF-dependent antiviral pathway. The TRIF-dependent TLR4 IFN induction signaling appears not to be functional in zebrafish, since IFN expression cannot be up-regulated by LPS. These two striking findings from de novo ligand induction experiments suggest a novel antiviral mechanism in zebrafish.

Identification and functional characterizations of a novel TRIF gene from grass carp (Ctenopharyngodon idella)

“…The response of amphioxus TRIF gene to poly(I:C) is significantly but not dramatically up-regulated . Zebrafish TRIF also has significantly but not strongly up-regulation in response to poly(I:C) (Fan et al, 2008).…”

“…Channel catfish TRIF expression is in widespread tissues, highly in ovary and spleen (Baoprasertkul et al, 2006). Zebrafish TRIF also expresses in extensive tissues, abundantly in liver and blood (Fan et al, 2008). In amphioxus, transcripts of TRIF are strongly detected in the epithelial cells of gut, skin, and gill, almost undetectable in the ovary and spermatozoa .…”

“…The N-terminal region harbors tumor necrosis factor (TNF) receptor-associated factor (TRAF) family proteins and forms a complex containing IRF3-activating kinase TBK1 (TANK-binding kinase-1), which is crucial for activating IFN-b (Sharma et al, 2003;Oganesyan et al, 2006). TIR domain contains three highly conserved regions (Box 1-3), which is an intracellular signaling domain that mediates proteinprotein interactions between the TLRs and signal-transduction components with TIR domain (Fan et al, 2008;Lu et al, 2008). The C-terminal region recruits receptor-interacting protein-1 (RIP-1), and this event is followed by the activation of other effectors (Meylan et al, 2004).…”

“…However, in teleosts, complete coding sequence of TRIF is just reported in channel catfish (Ictalurus punctatus) (Baoprasertkul et al, 2006), zebrafish (Danio rerio) (Fan et al, 2008) and T. rubripes (accession No., AB197917) in GenBank. Piscine TRIF plays an important role in immune response to bacteria (Baoprasertkul et al, 2006).…”

Characterization of SIGIRR/IL-1R8 Homolog from Zebrafish Provides New Insights into Its Inhibitory Role in Hepatic Inflammation

“…At 24 h after microinjection, the embryos with strong and GFP expression were used for further analysis. Real-time PCR was performed to measure the mRNA levels of IFN regulatory factor (IRF)3 and IFN1 as described earlier (33,34).…”