The Mechanism Of Duck E3 Ubiquitin Ligase TRIM35 Regulating Duck RLRs Signal Pathway

Tripartite motif protein 35(TRIM35)is a member of the E3 ubiquitin ligase tripartite motif family protein,also known as HLS5 or MAIR.It has the function of E3 ubiquitin ligase and plays an important role in regulating the RIG-I signaling pathway.Many members of the TRIM family of proteins have been reported to be important regulators in innae immunity and antiviral responses,and play an important role in the induction of interferons and pro-inflammatory cytokines.Mammalian TRIM35 has been demonstrated to play crucial roles in cancer,cell death and innate antiviral immune response.It not only induced apoptosis in transfected cells,but also inhibited tumor cell growth,clonogenicity,and tumorigenicity.However,the role of avian TRIM35 in the antiviral innate immune response remains unclear.This study aims to clone duck TRIM35(du TRIM35)gene,and reveal the role of du TRIM35 in regulating duck antiviral innate immune response,and enrich the molecular mechanism of duckderived virus escaping innate immune response.The main contents are as follows:1.Cloning of du TRIM35 and its effect on the proliferation of VSV and DTMUVIn this study,the full-length c DNA of du TRIM35 was cloned for the first time.Using amino acid sequence alignment and genetic evolution analysis,it was found that du TRIM35 had the closest genetic relationship with geese.Fluorescence quantitative RT-PCR analysis showed that du TRIM35 is highly expressed in liver,heart,cerebellum and pancreas.Fluorescence quantitative RT-PCR,Western Blot and TCID50 analysis proved that the expression level of du TRIM35 was significantly upregulated after Se V,VSV and DTMUV were infected with duck embryo fibroblasts(DEFs).Overexpression of du TRIM35 significantly increased the proliferation levels of VSV and DTMUV in DEFs,while interference with endogenous du TRIM35 significantly reduced the proliferation titer of VSV and DTMUV in DEFs.2.Du TRIM35 inhibits Se V/du RIG-I-induced IFN-β productionDual luciferase experiment and fluorescence quantitative RT-PCR analysis showed that du TRIM35 significantly inhibited Se V-induced IFN-β production and the expression of ISG such as Viperin,PKR and CCL5.In order to further screen the key targets of du TRIM35 inhibiting RLRs signaling pathway,the eukaryotic expression plasmids of du TRIM35 and the eukaryotic expression plasmid of the key signaling molecules in the RLRs signaling pathway(du RIG-I,du MDA5,du MAVS,du TBK1,du IKKε,du IRF7)were co-transfected into DEFs,and it was found that du TRIM35 significantly inhibited du RIG-I-induced IFN-β,IRF7 and NF-κB promoter activity in a dose-dependent manner.3.Du TRIM35 inhibits IFN-β production by targeting du RIG-ICo-immunoprecipitation experiments found that du TRIM35 interacted with du RIG-I.By constructing truncated mutants of each domain of du TRIM35 and du RIGI,it was found that the RING domain of the N-terminal of du TRIM35 protein and the CARD domain of du RIG-I were the key domains for their interaction.4.Du TRIM35 interferes with the binding of du RIG-I and ds RNA,thereby antagonizing the production of IFN-βIn this study,the eukaryotic expression plasmid of du TRIM35 was co-transfected with the eukaryotic expression plasmids of the key signal molecules in the RLRs signaling pathway(du RIG-I,du MDA5,du MAVS,du TBK1,du IKKε,du IRF7)into293T cells.Western Blot analysis revealed that du TRIM35 did not degrade du RIG-I through the ubiquitin-proteasome pathway.However,RNA binding experiments found that du TRIM35 protein can interfere with the binding of du RIG-I and ds RNA,and inhibit du RIG-I-induced IFN-β production.In summary,this study revealed for the first time the role of du TRIM35 in regulating duck innate immune response,and found that du TRIM35 can promote the proliferation of viruses such as VSV and DTMUV.We found that du TRIM35 inhibited Se V/du RIG-I-induced IFN-β production by targeting du RIG-I and du TRIM35 interfered with the binding of du RIG-I and ds RNA,thereby antagonizing IFN-βproduction.This study revealed the role of du TRIM35 in regulating duck antiviral innate immune response and enriched the molecular mechanism of duck-derived virus escaping the innate immune response.