Avian TRIM13 attenuates antiviral innate immunity by targeting MAVS for autophagic degradation

Abstract

MAVS (mitochondrial antiviral signaling protein) is a crucial adaptor in antiviral innate immunity that must be tightly regulated to maintain immune homeostasis. In this study, we identified the duck Anas platyrhynchos domesticus TRIM13 (ApdTRIM13) as a novel negative regulator of duck MAVS (ApdMAVS) that mediates the antiviral innate immune response. Upon infection with RNA viruses, ApdTRIM13 expression increased, and it specifically binds to ApdMAVS through its TM domain, facilitating the degradation of ApdMAVS in a manner independent of E3 ligase activity. Furthermore, ApdTRIM13 recruits the autophagic cargo receptor duck SQSTM1 (ApdSQSTM1), which facilitates its interaction with ApdMAVS independent of ubiquitin signaling, and subsequently delivers ApdMAVS to phagophores for degradation. Depletion of ApdSQSTM1 reduces ApdTRIM13-mediated autophagic degradation of ApdMAVS, thereby enhancing the antiviral immune response. Collectively, our findings reveal a novel mechanism by which ApdTRIM13 regulates type I interferon production by targeting ApdMAVS for selective autophagic degradation mediated by ApdSQSTM1, providing insights into the crosstalk between selective autophagy and innate immune responses in avian species.Abbreviation: 3-MA: 3-methyladenine; ATG5: autophagy related 5; baf A1: bafilomycin A1; BECN1: beclin 1; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CARD: caspase recruitment domain; co-IP: co-immunoprecipitation; DEFs: duck embryonic fibroblasts; DTMUV: duck Tembusu virus; eGFP: enhanced green fluorescent protein; hpi: hours post infection; IFIH1/MDA5: interferon induced with helicase C domain 1; IFN: interferon; IKBKE/IKKε: inhibitor of nuclear factor kappa B kinase subunit epsilon; IP: immunoprecipitation; IRF7: interferon regulatory factor 7; ISRE: interferon-stimulated response element; mAb: monoclonal antibody; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAVS: mitochondrial antiviral signaling protein; MOI: multiplicity of infection; NBR1: NBR1 autophagy cargo receptor; NFKB: nuclear factor kappa B; pAb: polyclonal antibody; poly(I:C): Polyriboinosinic polyribocytidylic acid; RIGI: RNA sensor RIG-I; RLR: RIGI-like-receptor; SeV: sendai virus; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TAX1BP1: Tax1 binding protein 1; TBK1: TANK binding kinase 1; TCID50: 50% tissue culture infectious dose; TM: tansmembrane; TOLLIP: toll interacting protein; TRIM: tripartite motif containing; UBA: ubiquitin-associated domain; Ub: ubiquitin; VSV: vesicular stomatitis virus; WT: wild type.

Keywords: Antiviral immunity; ApdMAVS; ApdTRIM13; SQSTM1; autophagic degradation.

Figure 1.

ApdTRIM13 negatively regulates rlr-mediated signaling pathways. (A-D) DEFs were infected with SeV (A), DTMUV (B) or stimulated with poly(i:c) (C) or IFNB (D). At the indicated time points, cell lysates were analyzed by western blotting with the indicated antibodies. (E and F) DEFs were transfected with IFNB, IRF7, or NFKB reporter plasmids, together with increasing amounts of flag-ApdTRIM13 expression plasmid, and then infected with SeV (E) or transfected with poly(i:c) (F) for 16 h, respectively. Promoter activity in response to IFNB, IRF7, or NFKB was analyzed in cell lysates. (G-I) DEFs were transfected with flag-ApdTRIM13 or an empty vector for 24 h, followed by infection with SeV (G), DTMUV (H), or transfection with poly(i:c) (I), respectively. The levels of IFNB, CCL5, and RSAD2 mRNAs were measured by rt-qPCR at the indicated time points. (J and K) DEFs were transfected with flag-ApdTRIM13 or an empty vector for 24 h, followed by DTMUV infection (MOI = 0.1). At the indicated time points, viral RNA and viral titer were determined by rt-qPCR (J) and TCID₅₀ (K). (L and M) DEFs were transfected with flag-ApdTRIM13 or an empty vector for 24 h, followed by vsv-eGFP infection (MOI = 0.01). At 12 h post-infection, cells were subjected to fluorescence microscopy analyses (BF, bright field) (L) and flow cytometry analyses (M). Scale bars, 200 μm. In panels E-K, data are represented as mean ± SEM of three independent experiments. *p < 0.05 and **p < 0.01 (unpaired Student’s t-test).

Figure 2.

ApdTRIM13 deficiency enhances antiviral responses. (A) DEFs were transfected with IFNB or ISRE reporter plasmids, together with negative siRNA or specific siRNA targeting ApdTRIM13 followed by infection with SeV or transfection with poly(i:c), respectively. After 16 h, IFNB- or isre-promoter activity was analyzed in the cell lysates. (B-D) DEFs were transfected with either negative siRNA or specific siRNA targeting ApdTRIM13, followed by infection with SeV (B), DTMUV (C), or transfection with poly(i:c) (D), respectively. The levels of IFNB, CCL5, and RSAD2 mRNAs were measured by rt-qPCR at the indicated time points. (E and F) DEFs transfected with either negative siRNA or specific siRNA targeting ApdTRIM13 were infected with DTMUV (MOI = 0.1). Viral RNA and viral titer were determined by rt-qPCR (E) and TCID₅₀ (F) at the indicated time points. (G and H) DEFs transfected with either negative siRNA or specific siRNA targeting ApdTRIM13 were infected with vsv-eGFP (MOI = 0.01). At 12 h post-infection, cells were subjected to fluorescence microscopy analyses (BF, bright field) (G) and flow cytometry analyses (H). Scale bars, 200 μm. In panels A-F, data are represented as mean ± SEM of three independent experiments. *p < 0.05 and **p < 0.01 (unpaired Student’s t-test).

Figure 3.

ApdTRIM13 inhibits RLR signaling by targeting ApdMAVS. (A) schematic representation of the ApdRIGI- or ApdIFIH1-mediated IFNB signaling pathway. (B-G) DEFs were co-transfected with flag-tagged ApdRIGI, ApdIFIH1, ApdMAVS, ApdTBK1, ApdIKBKE, ApdIRF7 or an empty vector and HA-ApdTRIM13, along with pRL-TK and IFNB-Luc or ISRE-Luc. Luciferase assays were performed 30 h post-transfection. (H) DEFs were co-transfected with flag-ApdMAVS and increasing amounts of HA-ApdTRIM13, along with pRL-TK and IFNB-Luc or ISRE-Luc. Luciferase assays were performed 30 h post-transfection. (I) DEFs were co-transfected with an empty vector or the ha-tagged mutants of ApdTRIM13 and flag-ApdMAVS, along with pRL-TK and IFNB-Luc or ISRE-Luc. Luciferase assays were performed 30 h post-transfection. Three independent experiments were performed, and data are expressed as mean ± SEM (**p < 0.01; ns, no significant difference).

Figure 4.

ApdTRIM13 interacts with ApdMAVS. (A) HEK-293T cells were co-transfected with plasmids encoding flag-ApdMAVS together with HA-ApdTRIM2, HA-ApdTRIM8, or HA-ApdTRIM13. At 28 h post-transfection, cell lysates were immunoprecipitated with an anti-flag antibody, followed by western blotting with the indicated antibodies. (B) HEK-293T cells were co-transfected with plasmids encoding flag-ApdMAVS and HA-ApdTRIM13. At 28 h post-transfection, cell lysates were immunoprecipitated with an anti-ha antibody, followed by western blotting with the indicated antibodies. (C) purified GST-ApdTRIM13 was used to pull down flag-ApdMAVS from HEK-293T cell lysates. GST was used as a negative control. (D) DEFs were transfected with poly(i:c) for 12 h, and cell lysates were immunoprecipitated with an anti-ApdTRIM13 antibody, followed by western blotting with the indicated antibodies. (E) HeLa cells were co-transfected with plasmids expressing HA-ApdMAVS and flag-ApdTRIM13. At 28 h post-transfection, the cells were fixed for immunofluorescence assays to detect ApdMAVS (green) and ApdTRIM13 (blue) with anti-ha and anti-flag antibodies, respectively. Mitochondria were stained with MitoTracker (red). (F) Co-immunoprecipitation analysis of the interaction of flag-ApdMAVS with ha-tagged ApdTRIM13 or its truncation mutants in HEK-293T cells. (G) Co-immunoprecipitation analysis of the interaction of HA-ApdTRIM13 with flag-tagged ApdMAVS or its truncation mutants in HEK-293T cells.

Figure 5.

ApdTRIM13 promotes autophagic degradation of ApdMAVS. (A and B) DEFs were transfected with flag-ApdMAVS, together with empty vector or increasing amounts of plasmid expressing HA-ApdTRIM13, and cell lysates were analyzed by western blotting with the indicated antibodies (A). Total RNA was extracted for rt-qPCR analysis of ApdMAVS mRNA (B). (C) DEFs were transfected with either negative siRNA or specific siRNA targeting ApdTRIM13 for 12 h, followed by transfection with poly(i:c). At the indicated time points, cell lysates were analyzed by western blotting with the indicated antibodies. (D) DEFs were transfected with flag-ApdMAVS, together with an empty vector or HA-ApdTRIM13. At 24 h post-transfection, the cells were treated with CHX (100 μg/mL). At the indicated time points, cell lysates were analyzed by western blotting with the indicated antibodies. (E) ApdMAVS expression levels were quantitated by measuring the band intensities using ImageJ software. ApdMAVS protein levels were normalized to ACTB, and the relative ApdMAVS protein level at 0 h was set as 1. (F) DEFs were transfected with flag-ApdMAVS, together with an empty vector or HA-ApdTRIM13. At 24 h post-transfection, the cells were treated with MG132 (10 μM), 3-MA (10 mm), baf A₁ (10 μM), or NH₄Cl (20 mm) for 6 h. Cell lysates were analyzed by western blotting with the indicated antibodies. (G) DEFs were transfected with flag-ApdMAVS, together with an empty vector or HA-ApdTRIM13. At 24 h post-transfection, the cells were cultured in EBSS for the indicated time points. Cell lysates were analyzed by western blotting with the indicated antibodies. (H and I) DEFs were co-transfected with siApdATG5 (H) or siApdBECN1 (I) and the indicated plasmids. At 24 h post-transfection, cell lysates were analyzed by western blotting with the indicated antibodies. (J) DEFs were transfected with flag-ApdMAVS, together with an empty vector or increasing amounts of plasmid expressing HA-ApdTRIM13. After 28 h, cell lysates were analyzed by western blotting with the indicated antibodies. (K) HeLa cells were co-transfected with flag-ApdTRIM13 and GFP-LC3B, along with HA-ApdMAVS for 24 h. Cells were then fixed and subjected to immunofluorescence assays using anti-flag and anti-ha antibodies. The fluorescent signals were visualized using confocal immunofluorescence microscopy.

Figure 6.

ApdTRIM13 enhances the recognition of ApdMAVS by the cargo receptor ApdSQSTM1. (A and B) HEK-293T cells were co-transfected with plasmids encoding HA-ApdTRIM13 (A) or HA-ApdMAVS (B), along with the indicated Flag-tagged cargo receptors. At 28 h post-transfection, cell lysates were immunoprecipitated with an anti-Flag antibody, followed by western blotting with the indicated antibodies. (C and D) DEFs were co-transfected with plasmids encoding HA-ApdMAVS and Flag-ApdSQSTM1 (C), or Flag-ApdTOLLIP (D), together with or without MYC-ApdTRIM13, followed by treatment with baf A₁ (10 μM). Cell lysates were then immunoprecipitated with an anti-Flag antibody and subjected to immunoblot analysis with the indicated antibodies. (E and F) DEFs were co-transfected with specific siRNA targeting ApdTRIM13 and plasmids encoding Flag-ApdMAVS and HA-ApdSQSTM1 (E), or HA-ApdTOLLIP (F). At 28 h post-transfection, cell lysates were immunoprecipitated with an anti-Flag antibody, followed by western blotting with the indicated antibodies. (G-J) DEFs were transfected with siApdTOLLIP (G, H) or siApdSQSTM1 (I, J), together with HA-ApdTRIM13 alone or Flag-ApdMAVS and HA-ApdTRIM13. At 24 h post-transfection, cell lysates were subjected to immunoblot analysis with the indicated antibodies. (K and L) DEFs were co-transfected with siNegative or siApdSQSTM1, HA-ApdTRIM13 and Flag-ApdMAVS, along with pRL-TK and IFNB-Luc (K) or ISRE-Luc (J). Luciferase assays were performed at 24 h post-transfection. In panels K and L, data are represented as mean ± SEM of three independent experiments. *p < 0.05 and **p < 0.01 (unpaired Student’s t-test).

Figure 7.

ApdTRIM13 links ApdSQSTM1 and ApdMAVS through its TM domain. (A) DEFs were co-transfected with ha-ubiquitin (ub) and flag-ApdMAVS, with or without MYC-ApdTRIM13, and then treated with baf A₁ (10 μM). Cell lysates were then immunoprecipitated with an anti-flag antibody and subjected to immunoblot analysis with the indicated antibodies. (B) DEFs were co-transfected with HA-Ub and flag-ApdTRIM13 or its mutants, with or without MYC-ApdTRIM13, and then treated with baf A₁ (10 μM). Cell lysates were then immunoprecipitated with an anti-flag antibody and subjected to immunoblot analysis with the indicated antibodies. (C) Co-immunoprecipitation analysis of the interaction of HA-ApdSQSTM1 with flag-tagged ApdTRIM13 or its mutants in HEK-293T cells. (D) Co-immunoprecipitation analysis of the interaction of flag-ApdTRIM13 with HA-ApdSQSTM1 or its truncation mutant in HEK-293T cells. (E) DEFs were transfected with the indicated plasmids for 28 h, and cell lysates were used for immunoblot analysis with the indicated antibodies. (F) Co-immunoprecipitation analysis of the interaction of flag-ApdSQSTM1 with HA-ApdTRIM13 or its mutants in HEK-293T cells. (G) Schematic representation of ApdSQSTM1 and its truncation mutants. (H) Co-immunoprecipitation analysis of the interaction of flag-ApdTRIM13 with HA-ApdSQSTM1 or its truncation mutants in HEK-293T cells.

Figure 8.

A proposed working model illustrating how ApdTRIM13 negatively regulates the type I IFN signaling pathway during RNA virus infection.