Zebrafish TRIF, a Golgi-localized protein, participates in IFN induction and NF-kappaB activation.
ABSTRACT The antiviral immune responses were triggered by the innate immune recognition of viral infection. The type I IFNs (IFN-beta and IFN-alpha) 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-kappaB 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.
Article: Effect of viral RNA mimics and ISA virus infection on expression of key genes of the Atlantic salmon interferon system[show abstract] [hide abstract]
ABSTRACT: We have studied the innate immune responses of Atlantic salmon and how this responds to stimulation and infection by the aquatic orthomyxovirus Infectious salmon anemia virus (ISAV). This work has focused on the Type I interferon (IFN) system which is critically important in the vertebrate immune response against viral infections. As a part of this work we have studied the Atlantic salmon IFN regulatory factor 7 (IRF7). In mammals IRF7 is the major regulator of IFN transcription in response to viral infections, and our results show that the salmon homolog performs similar tasks. Furthermore, we have investigated the stimulatory effect of two synthetic ligands that induce the IFN system by mimicking viral infections in the cell and compared this to the immune response against ISAV. Our results indicate that both specialized immune cells and non-immune cells induce the IFN system in response to these treatments, but we also show that ISAV is able to interfere with the host` IFN response in order to successfully replicate. Finally, our studies show that the ISAV genome encodes two genes, S7ORF1 and S8ORF2, which is involved in antagonizing the IFN response during infections. The papers of the thesis are not available in Munin: 1. Kileng, Ø., Brundtland, M. I. and Robertsen, B.: «Infectious salmon anemia virus is a powerful inducer of key genes of the type I interferon system of Atlantic salmon, but is not inhibited by interferon», Fish & Shellfish immunology 23(2007); 378-389 (Elsevier - publisher's restriction). Available at http://dx.doi.org/10.1016/j.fsi.2006.11.011 2. Kileng, Ø., Albuquerque, A. and Robertsen, B.: «Induction of interferon system genes in Atlantic salmon by the imidazoquinoline S-27609, a ligand for Toll-like receptor 7», Fish & Shellfish immunology 24(2007); 514-522 (Elsevier - publisher's restriction). Available at http://dx.doi.org/10.1016/j.fsi.2007.10.005 3. GarcÃa-Rosado, E., Markussen, T., Kileng, Ø., Baekkevold, E. S., Robertsen, B., Mjaaland, S. and Rimstad, E.: «Molecular and functional characterization of two infectious salmon anaemia virus (ISAV) proteins with type I interferon antagonizing activity», Virus Research 133(2008); 228-238 (Elsevier - publisher's restriction). Available at http://dx.doi.org/10.1016/j.virusres.2008.01.008 4. Kileng, Ø., Workenhe, S. T., Bergan, V. and Robertsen, B.: «An Atlantic salmon IRF-7 gene identified by analysis of sequence and function and its expression compared with a STAT1 gene». Manuscript, later published in Developmental and Comparative Immunology 33(2009); 18-27 (Elsevier), published version available at http://dx.doi.org/10.1016/j.dci.2008.07.020
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ABSTRACT: African swine fever virus (ASFV) encodes proteins that manipulate important host antiviral mechanisms. Bioinformatic analysis of the ASFV genome revealed ORF I329L, a gene without any previous functional characterization as a possible inhibitor of TLR signaling. We demonstrate that ORF I329L encodes a highly glycosylated protein expressed in the cell membrane and on its surface. I329L also inhibited dsRNA-stimulated activation of NFκB and IRF3, two key players in innate immunity. Consistent with this, expression of I329L protein also inhibited the activation of interferon-β and CCL5. Finally, overexpression of TRIF reversed I329L-mediated inhibition of both NFκB and IRF3 activation. Our results suggest that TRIF, a key MyD88-independent adaptor molecule, is a possible target of this viral host modulation gene. The demonstration of an ASFV host evasion molecule inhibiting TLR responses is consistent with the ability of this virus to infect vertebrate and invertebrate hosts, both of which deploy innate immunity controlled by conserved TLR systems.Archives of Virology 01/2011; 156(4):597-609. · 2.11 Impact Factor
Article: Identification of a retinoic acid-inducible gene I from grass carp (Ctenopharyngodon idella) and expression analysis in vivo and in vitro.[show abstract] [hide abstract]
ABSTRACT: RIG-I (retinoic acid inducible gene-I) is a key mediator of antiviral immunity, able to couple detection of infection by RNA and DNA viruses to the induction of interferons. In the present study, a RIG-I gene from grass carp Ctenopharyngodon idella (CiRIG-I) was isolated and characterized. The full-length cDNA of CiRIG-I was of 3198 bp and encoded a polypeptide of 947 amino acids with an estimated molecular mass of 108,730 Da and a predicted isoelectric point of 5.85, including six main overlapping structural domains: two CARDs (caspase activation and recruitment domain), one ResIII (conserved restriction domain of bacterial type III restriction enzyme), one DEXDc (DEAD/DEAH box helicase domain), one HELICc (helicase superfamily c-terminal domain) and one RD (regulatory domain). The CiRIG-I mRNA was widespread expression in the tested 15 tissues by semi-quantitative RT-PCR (sqRT-PCR) assay. The CiRIG-I expressions in spleen and liver were significantly induced following grass carp reovirus (GCRV) infection. CiRIG-I mRNA expression was rapidly and significantly up-regulated in vitro after GCRV infection, and the CiRIG-I transcripts were also significantly enhanced in vitro post the synthetic double stranded RNA polyinosinic-polycytidylic potassium salt (poly(I:C)) stimulation. These results collectively suggested that CiRIG-I was an inducible protein, involved in the antiviral innate immune defense to GCRV in grass carp, and laid the foundation for the further mechanism research of RIG-I in fishes.Fish & Shellfish Immunology 02/2011; 30(3):936-43. · 3.32 Impact Factor