The E3 ligase ASB3 downregulates antiviral innate immunity by targeting MAVS for ubiquitin-proteasomal degradation

Abstract

E3 ubiquitin ligases are very important for regulating antiviral immunity during viral infection. Here, we discovered that Ankyrin repeat and SOCS box-containing protein 3 (ASB3), an E3 ligase, are upregulated in the presence of RNA viruses, particularly influenza A virus (IAV). Notably, overexpression of ASB3 inhibits type I IFN (IFN-I) responses induced by Sendai virus (SeV) and IAV, and ablation of ASB3 restores SeV and H9N2 infection-mediated transcription of IFN-β and its downstream interferon-stimulated genes (ISGs). Interestingly, animals lacking ASB3 presented decreased susceptibility to H9N2 and H1N1 infections. Mechanistically, ASB3 interacts with MAVS and directly mediates K48-linked polyubiquitination and degradation of MAVS at K297, thereby inhibiting the phosphorylation of TBK1 and IRF3 and downregulating downstream antiviral signaling. These findings establish ASB3 as a critical negative regulator that controls the activation of antiviral signaling and describe a novel function of ASB3 that has not been previously reported.

Fig. 1. ASB3 expression induced by IAV infection.

A, B A549 cells were infected with SeV and HSV-1 (MOI = 0.1) or transfected with poly(I:C) (2 μg/ml) and 2’3’cGAMP (2 μg/ml) for 0, 6, 12, or 18 h. Then, the protein levels of ASB3 in A549 cells were detected via immunoblot analysis. C, D A549 cells were infected with H9N2 or H1N1 (0.1 MOI or 1 MOI) for 0, 6, 12, or 18 h, and the protein levels of ASB3 were analyzed via immunoblot analysis. E, F A549 cells were infected with H9N2 (0.1 MOI) or H1N1 (0.1 MOI) for the indicated times. The subcellular localization of endogenous ASB3 and NP was analyzed via fluorescence microscopy.

Fig. 2. ASB3 negatively regulates type I IFN production.

A HEK293T cells were transfected with IFN-β-Luc, ISRE-Luc (100 ng), or Flag-tagged cGAS and STING and pRL-TK plasmids (50 ng) along with increasing HA-ASB3-expressing plasmids (50, 100, or 200 ng). After 24 h, the indicated cells were infected with SeV (MOI = 0.1) or transfected with poly(I:C) (2 μg/ml). B HEK293T cells were transfected with IFN-β-Luc, ISRE-Luc (100 ng), or Flag-tagged TRIF and pRL-TK plasmids (50 ng) along with HA-ASB3-expressing plasmids (200 ng). After 24 h, the indicated cells were infected with SeV (MOI = 0.1). Promoter activation was determined via a dual-luciferase reporter gene assay. C HEK293T cells were transfected with 1 μg of HA empty vector (EV) or HA-ASB3 expression plasmid. After 24 h, the cells were infected with SeV (MOI = 0.1) for an additional 12 h. The mRNA expression of IFNB1, IFIT2, ISG15, and OASL was measured by qPCR. D, E HEK293T cells were transfected with 1 μg of HA empty vector (EV) or HA-ASB3 expression plasmid. After 24 h, the cells were infected with H9N2 or H1N1 (MOI = 0.1) for an additional 12 h. The mRNA expression levels of IFNB1, CXCL10, IL-6, and TNF-α and protein lysates were measured via qPCR and immunoblot analysis. F, G A549 cells were transfected with 1 μg of HA empty vector (EV) or HA-ASB3 expression plasmid. After 24 h, the cells were infected with H9N2 or H1N1 (MOI = 0.1) for an additional 12 h. The mRNA expression levels of IFNB1, CXCL10, IL-6, and TNF-α and protein lysates were measured via qPCR and immunoblot analysis. H Fluorescence microscopy analysis and flow cytometric analysis of the replication of VSV-GFP in HEK293T cells transfected with EV or increasing HA-ASB3 expression plasmid at the indicated dose for 16 h, followed by treatment with or without VSV-GFP (MOI = 0.1) infection at the indicated times. I Viral titers in A549 cells were transfected with the ASB3 plasmid and infected with the H9N2 virus or H1N1 virus (n = 3). J Viral titers in HEK293T cells transfected with the ASB3 plasmid and infected with H9N2 virus or H1N1 virus (MOI = 0.1) for 24 h (n = 3).

Fig. 3. ASB3 deficiency enhances the type I IFN response.

A, B Volcano plots and heatmap of RNA-seq data showing upregulated or downregulated expression of genes in lung tissues from ASB3^+/+ (n = 3) and ASB3^-/- mice (n = 3) infected with H9N2 (1 × 10⁶ EID₅₀/mouse) or H1N1 (1 × 10⁵ EID₅₀/mouse) for 5 d. C, D Bone marrow-derived macrophages (BMDMs) and primary kidney epithelial cells isolated from ASB3^+/+ and ASB3^-/- mice were infected with H9N2 (MOI = 0.1) for 12 h. The mRNA expression of Ifnb1, Isg15, Isg56, Cxcl10, Ccl5, and Mx1 was measured via qPCR. The protein expression of pIRF3, IRF3, pIκBα, IκBα, pP65, P65, HA, and β-actin was measured via immunoblot analysis. E, F Primary kidney epithelial cells were infected with H9N2 (MOI = 0.1) for 12 h. The mRNA expression levels of Ifnb1, Isg15, Isg56, Cxcl10, Ccl5, and Mx1 were measured via qPCR. The protein expression of pIRF3, IRF3, pIκBα, IκBα, pP65, P65, HA, and β-actin was measured via immunoblot analysis.

Fig. 4. ASB3 specifically interacts with MAVS.

A, B HEK293T cells were transfected with the indicated plasmids (RIG-I-N, MDA5-N, MAVS, TBK1, and IRF3-5D) along with the control vector or increased amounts of ASB3 expression plasmids. IFN-β and ISRE promoter activity was assessed 24 h after transfection. C, D Co-IP analysis of the interaction of ASB3 with MAVS in HEK293T cells transfected with the indicated plasmids (RIG-I, MDA5, MAVS, TBK1, IRF3, TRAF3, and ASB3). E HEK293T cells were transfected with the indicated plasmids (MAVS and TRAF3) along with the ASB3 expression plasmid. Then, the sections were stained with a Flag antibody or HA antibody and a secondary antibody. DAPI (blue), Flag-MAVS or TRAF3 (red), and HA-ASB3 (green). Scale bars, 5 μm. F Co-IP analysis of the interaction of ASB3 with MAVS in HEK293T cells transfected with the indicated plasmids (MAVS, cGAS, STING, and ASB3). G In vitro transcription pull-down analysis of the interaction of ASB3 with MAVS. H, I Semiendogenous and endogenous Co-IP analysis of the interaction of ASB3 with MAVS in HEK293T or A549 cells infected with H9N2 or H1N1 (MOI = 0.1). J Co-IP analysis of the interaction of MAVS with ASB3 and its truncation mutants in HEK293T cells.

Fig. 5. ASB3 potentiates K48-linked polyubiquitination and degradation of MAVS.

A Immunoblot analysis of HEK293T cells transfected with the indicated plasmids (MAVS, TBK1, and IRF3) and different concentrations of ASB3 plasmids. B HEK293T or A549 cells were transfected with the HA-ASB3 vector together with increasing amounts (wedge) of the HA-ASB3 expression vector, and the protein was harvested for immunoblot analyses. C HEK293T cells were transfected with the indicated plasmids for 20 h and then treated with 3-methyladenine (3-MA) (10 mM), NH₄Cl (20 mM), MG132 (10 μM), or ZVAD (20 μM) for 6 h. The cell lysates were then analyzed by immunoblotting with the indicated antibodies. D Co-IP analysis of the polyubiquitination of MAVS in HEK293T cells transfected with Flag-MAVS, Myc-Ub, or its mutants K48 and K63 [K at the indicated residues and K at other residues were simultaneously mutated to arginines] and ASB3 plasmids and treated with MG132. E, F Co-IP analysis of the polyubiquitination of MAVS in HEK293T cells transfected with Myc-MAVS, Flag-Ub, or its mutants K48 and ASB3 plasmids and treated with or without MG132. G Semiendogenous Co-IP analysis of the polyubiquitination of MAVS in HEK293T cells transfected with the Myc-MAVS and ASB3 plasmids and treated with or without MG132. H, I Co-IP analysis of the polyubiquitination of MAVS in HEK293T cells transfected with Myc-MAVS, ASB3, or its truncation mutant plasmids and treated with or without MG132. J Luciferase reporter assays analyzing the IFN-β promoter activity of HEK293T cells transfected with HA-ASB3 and its deletions along with Flag-MAVS or infected with SeV (MOI = 0.1). K qPCR assays analyzing IFNB1 gene transcription levels in HEK293T cells transfected with HA-ASB3 and its truncation plasmids and infected with H9N2 (MOI = 0.1).

Fig. 6. ASB3-mediated MAVS degradation at residue K297.

A Immunoblot analysis of HEK293T cells transfected with MAVS or its mutants and ASB3 plasmids. B IFN-β and ISRE promoter activation analysis of MAVS^-/- HEK293T cells transfected with MAVS or its mutants and ASB3 plasmids. C MAVS^-/- HEK293T cells were transfected with MAVS or its mutants and ASB3 plasmids. The protein expression of pIRF3, IRF3, pIκBα, IκBα, Myc, HA, and β-actin was measured through immunoblot analysis. D, E Viral titers in MAVS^-/- HEK293T cells transfected with MAVS or its mutants and ASB3 plasmids and infected with H9N2 (MOI = 0.1) (D) or H1N1 (MOI = 0.1) (E) for 24 h (n = 3). F Molecular model of the MAVS domain generated by PyMOL (PDB: 3J6C). G Co-IP analysis of the polyubiquitination of MAVS in HEK293T cells transfected with Myc-MAVS or its truncation mutants, Flag-K48, and HA-ASB3 plasmids and treated with MG132. H Co-IP analysis of the polyubiquitination of MAVS in HEK293T cells transfected with the Myc-MAVS and ASB3 plasmids and treated with or without MG132.

Fig. 7. Loss of ASB3 enhances in vivo antiviral immunity.

A, B Weight loss rates and survival rates of ASB3^+/+ and ASB3^-/- mice (n = 5 mice/group) infected with H9N2 (1 × 10⁶ EID₅₀/mouse) via intranasal infection. D, E Weight loss rates and survival rates of ASB3^+/+ and ASB3^-/- mice (n = 5 mice/group) infected with H1N1 (1 × 10⁵ EID₅₀/mouse) via intranasal infection. C, F Gloss lesions of lungs from infected ASB3^+/+ or ASB3^-/- mice at 0, 3, and 5 d. The images are from one of seven representative mice. G–I Representative H&E-stained images of lung sections from ASB3^+/+ and ASB3^-/- mice infected with H9N2 or H1N1 for 3 d or 5 d. Scale bars, 50 or 100 µm. J A TCID₅₀ assay was used to test the H9N2 titer in mouse lungs. K A TCID₅₀ assay was used to test the H1N1 titer in mouse lungs.

Fig. 8. ASB3 deficiency eliminates MAVS degradation in the lungs.

A, B Immunoblot analysis of pTBK1, TBK1, pIRF3, IRF3, pIκBα, IκBα, MAVS, NP, and β-actin expression in the lungs of ASB3^+/+ and ASB3^-/- mice infected with H9N2 or H1N1 for 3 d and 5 d. C Lungs of ASB3^+/+ and ASB3^-/- mice were analyzed on d 5 of infection with H1N1 and imaged by confocal microscopy after immunofluorescence staining for MAVS (green), NP (red) and DAPI (blue). Scale bar, 20 μm. D, E qPCR analysis of Ifnb1 and Isg15 mRNA expression in the lungs of ASB3^+/+ and ASB3^-/- mice infected with H9N2 or H1N1 for 3 d and 5 d. F Co-IP analysis of the interaction of MAVS with ASB3 in the lungs of ASB3^+/+ mice infected with H9N2 or H1N1 for 5 d. G Co-IP analysis of the polyubiquitination of MAVS in the lungs of ASB3^+/+ and ASB3^-/- mice infected with H9N2 or H1N1 for 3 d and 5 d.

Fig. 9. A working model of ASB3 inhibition of antiviral immunity.

Upon RNA virus infection, ASB3 expression is upregulated. ASB3 associates with MAVS and mediates its K48-linked polyubiquitination at K297. This modification blocked the interaction of MAVS and TBK1, which inhibited the phosphorylation of TBK1 and IRF3, thereby impairing the transcriptional activation of IFN-I.