Molecular Mechanism Of Duck TRIM29 Protein Regulating The Production Of Type Ⅰ Interferon In RIG-Ⅰ Signaling Pathway

The Retinoic acid-inducible gene I(RIG-I)-like receptors are major pattern recognition receptor in the host.The RIG-I-like receptor-mediated type I interferon signaling pathway plays a key role in antiviral innate immunity.TRIM family proteins are regulators in innate immune signaling pathways.TRIM29 is a member of the TRIM family.Mammalian TRIM29 plays an important role in regulating antiviral innate immunity,but the function of duck TRIM29 in antiviral innate immunity is still unknown.In view of this,this study explored the molecular mechanism of duck TRIM29 regulating the production of type I interferon in the RIG-I-mediated signaling pathway.In order to clone and identify duck TRIM29 gene,duck TRIM29 was amplified from duck embryo fibroblasts cell by RT-PCR.The results showed that the open reading frame of duck TRIM29 gene was 1716 bp and encoded 571 amino acids.The nucleotide sequence homology of duck TRIM29 and goose TRIM29 was 98.7%,the homology with chicken TRIM29 was 94.4%,the homology with human,mice and other mammals ranged from 69.3%to 70.9%.Domain prediction revealed that the duck TRIM29 mainly included the B-box domain(241-277 amino acid)and the Coiled-coil domain(278-368 amino acid).To investigate the role of duck TRIM29 in the duck RIG-I-mediated signaling pathway,the function of TRIM29 in the RIG-I-mediated signaling pathway was analyzed by quantitative RT-PCR and luciferase reporter gene assay.Quantitative RT-PCR results showed that the duck TRIM29 significantly down-regulated the expression of IFN-β,IRF7,Mx,and IL-6.The results of the luciferase reporter gene assay showed that duck TRIM29 significantly inhibited the avian IFN-β and IRF7 promoters in a dose-dependent manner.This suggests that duck TRIM29 inhibited RIG-I-mediated type I interferon production.To further explore how duck TRIM29 regulated the RIG-I signaling pathway,its mechanism was analyzed systematically by co-immunoprecipitation,quantitative RT-PCR and luciferase reporter gene assay.The co-immunoprecipitation results showed that duck TRIM29 interacted with MAVS,an adaptor protein of the RIG-I signaling pathway.Quantitative RT-PCR and luciferase reporter gene assay results showed that duck TRIM29 significantly down-regulated the expression of IFN-β,IRF7,Mx,PKR,OAS and IL-6.And duck TRIM29 significantly inhibited the avian IFN-β and IRF7 promoters induced by MAVS.This suggests that duck TRIM29 negatively regulated RIG-I-mediated type I interferon production by targeting MAVS.To clarify the mechanism of the interaction between duck TRIM29 and duck MAVS,their interaction was analyzed by co-immunoprecipitation and laser confocal.Coimmunoprecipitation and laser confocal results showed that duck TRIM29 interacted with MAVS and co-localized in the cytoplasm.The C-terminal region(369-571 amino acid)of duck TRIM29 was the key region for interaction between duck TRIM29 and duck MAVS.The 426-614 amino acids region of duck MAVS protein was the key region for their interaction.Duck TRIM29 could ubiquitinate duck MAVS protein,thereby inhibited avian IFN-β promoter induced by MAVS.In summary,duck TRIM29 inhibited RIG-I-mediated type I interferon production by interacted with duck MAVS.The C-terminal region(369-571 amino acid)of duck TRIM29 protein was the key region for interaction between duck TRIM29 and duck MAVS.The 426-614 amino acid of duck MAVS protein was the key region for their interaction.In addition,duck TRIM29 protein could ubiquitinate duck MAVS protein,thereby inhibited avian IFN-βpromoter induced by MAVS.Therefore,duck TRIM29 ubiquitinated duck MAVS protein to negatively regulated the production of type I interferon in the RIG-I signaling pathway.