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. 2019 Oct;16(10):800-809.
doi: 10.1038/s41423-018-0057-4. Epub 2018 Jul 4.

The methyltransferase PRMT6 attenuates antiviral innate immunity by blocking TBK1-IRF3 signaling

Hua Zhang  1 , Chaofeng Han  1 , Tianliang Li  1 , Nan Li  1 , Xuetao Cao  2   3
Affiliations

The methyltransferase PRMT6 attenuates antiviral innate immunity by blocking TBK1-IRF3 signaling

Hua Zhang et al. Cell Mol Immunol. 2019 Oct.

Erratum in

Abstract

Protein arginine methyltransferases (PRMTs) play diverse biological roles and are specifically involved in immune cell development and inflammation. However, their role in antiviral innate immunity has not been elucidated. Viral infection triggers the TBK1-IRF3 signaling pathway to stimulate the production of type-I interferon, which mediates antiviral immunity. We performed a functional screen of the nine mammalian PRMTs for regulators of IFN-β expression and found that PRMT6 inhibits the antiviral innate immune response. Viral infection also upregulated PRMT6 protein levels. We generated PRMT6-deficient mice and found that they exhibited enhanced antiviral innate immunity. PRMT6 deficiency promoted the TBK1-IRF3 interaction and subsequently enhanced IRF3 activation and type-I interferon production. Mechanistically, viral infection enhanced the binding of PRMT6 to IRF3 and inhibited the interaction between IRF3 and TBK1; this mechanism was independent of PRMT6 methyltransferase activity. Thus, PRMT6 inhibits antiviral innate immunity by sequestering IRF3, thereby blocking TBK1-IRF3 signaling. Our work demonstrates a methyltransferase-independent role for PRMTs. It also identifies a negative regulator of the antiviral immune response, which may protect the host from the damaging effects of an overactive immune system and/or be exploited by viruses to escape immune detection.

Keywords: IFN; IRF3; PRMT6; TBK1; antiviral innate immunity.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
Identification of PRMT6 as a negative regulator of type-I interferon expression. a Luciferase activity in HEK293T cells transfected with IFN-β luciferase reporter, the indicated signal proteins, and PRMT1–9. The results are expressed relative to Renilla luciferase activity. b Real-time PCR analysis of Ifna and Ifnb mRNA in control Raw264.7 cells and Raw264.7 cells stably overexpressing PRMT6 infected with VSV for the indicated lengths of time. The results are normalized to Gapdh expression. c Immunoblot analysis of total and phosphorylated IRF3 in control Raw264.7 cells and PRMT6-overexpressing Raw264.7 cells infected with VSV for the indicated lengths of time. d–h Immunoblot analysis of PRMT6 expression in mouse peritoneal macrophages (d, e), THP-1 cells (f, g), and A549 cells (h) infected with the virus for the indicated lengths of time. i Immunoblot analysis of PRMT6 expression in HBV-transfected HepG2.2.15 cells. Data in a and b are presented as the mean ± SEM of three independent experiments. Data in c–i are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 2
Fig. 2
PRMT6 deficiency enhances antiviral innate immunity in vivo. a Survival of Prmt6+/+ and Prmt6–/– mice after intraperitoneal injection of VSV (1.5 × 108 plaque-forming units per gram of body weight) (n = 7). b Determination of VSV load in the liver, spleen, and lungs of Prmt6+/+ and Prmt6–/– mice 18 h after infection with VSV, as measured by TCID50 assay. c Real-time PCR analysis of VSV RNA levels in liver, spleen, and lungs of Prmt6+/+ and Prmt6–/– mice 18 h after intraperitoneal injection with a medium or VSV. d Hematoxylin-and-eosin staining of lung sections from mice in C. Scale bar, 50 μm. e ELISA demonstrating serum cytokine levels in Prmt6+/+ and Prmt6–/– mice after intraperitoneal injection of VSV for the indicated lengths of time. Data in a and d are representative of three independent experiments. Data in b, c, and e are presented as the mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01
Fig. 3
Fig. 3
PRMT6 deficiency promotes IRF3 activation and increases IFN-I production. a, b Real-time PCR analysis of Ifna, Ifnb, and Il6 mRNA in Prmt6+/+ and Prmt6–/– BMDMs infected with VSV (a) and HSV-1(b) for the indicated lengths of time. c ELISA demonstrating cytokine levels in supernatants from Prmt6+/+ and Prmt6–/– BMDMs infected with VSV for the indicated lengths of time. d ELISA demonstrating cytokine levels in supernatants from Prmt6+/+ and Prmt6–/– BMDMs infected with HSV-1. e Immunoblot analysis of phosphorylation of the indicated molecules in Prmt6+/+ and Prmt6–/– BMDMs infected with VSV for the indicated lengths of time. Data in a–d are presented as the mean ± SEM of three independent experiments. Data in e are representative of three independent experiments. **p < 0.01
Fig. 4
Fig. 4
PRMT6 inhibits IFN-I expression in a methyltransferase-independent manner. a Luciferase activity in HEK293T cells transfected with the IFN-β luciferase reporter, the indicated adaptor proteins, and increasing amounts of PRMT6. The results are expressed relative to Renilla luciferase activity. b Luciferase activity in HEK293T cells transfected with the IFN-β luciferase reporter, the indicated adaptor proteins, and the empty vector (EV), PRMT6, or the catalytically inactive PRMT6 (PRMT6(dead)). The results are expressed relative to Renilla luciferase activity. Data in a and b are presented as the mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 5
Fig. 5
PRMT6 targets the TBK1–IRF3 signaling complex. a Luciferase activity (top) and immunoblot analysis (below) of HEK293T cells transfected with IFN-β luciferase reporter and the indicated vectors. b In vitro kinase assay demonstrating the levels of phosphor-IRF3 (Ser396) and total IRF3 in HEK293T cells transfected with GST–IRF3(380–427) as the substrate and the indicated combinations of TBK1–Myc and PRMT6–V5. Immunoblot analysis was used to assess proteins immunoprecipitated with an anti-Myc antibody or GST-fused IRF3 peptide (top) as well as whole-cell lysates (below). c Co-immunoprecipitation and immunoblot analysis of Prmt6+/+ and Prmt6–/– BMDMs infected with VSV for the indicated lengths of time. Data in a–c are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 6
Fig. 6
PRMT6 binds and sequesters IRF3 to disrupt assembly of the TBK1–IRF3 signaling complex. a Co-immunoprecipitation and immunoblot analysis of HEK293T cells transfected with PRMT6–V5 and Myc-tagged TBK1, IKKε, IRF3, or IRF7. b Co-immunoprecipitation and immunoblot analysis of HEK293T cells transfected with PRMT6–V5 and IRF3-Flag. c Co-immunoprecipitation and immunoblot analysis of BMDMs infected with VSV for the indicated lengths of time. d Co-immunoprecipitation and immunoblot analysis of HEK293T cells transfected with IRF3-Flag, TBK1–Myc, and PRMT6 or PRMT6(dead). e Co-immunoprecipitation and immunoblot analysis of BMDMs infected with VSV for the indicated lengths of time. Data in a–e are representative of three independent experiments
Fig. 7
Fig. 7
PRMT6 blocks TBK1–IRF3 signaling in a manner dependent on its 189–318 amino acid domain. a Domain structure and domain deletion mutants of PRMT6. b Co-immunoprecipitation and immunoblot analysis of HEK293T cells transfected with IRF3–Myc and PRMT6 mutants. c Luciferase activity in HEK293T cells transfected with IFN-β luciferase reporter, the indicated adaptor proteins, and empty vector (EV), PRMT6, or PRMT6 mutants. The results are expressed relative to Renilla luciferase activity. d Co-immunoprecipitation and immunoblot analysis of HEK293T cells transfected with IRF3-Flag, TBK1–Myc, and PRMT6 or PRMT6 mutants. e Luciferase activity in HEK293T cells transfected with IFN-β luciferase reporter, IRF3-Flag, TBK1–Myc, and PRMT6(WT), PRMT6(Δ189–318), or PRMT6(189–318). The results are expressed relative to Renilla luciferase activity. f Co-immunoprecipitation and immunoblot analysis of HEK293T cells transfected with IRF3-Flag, TBK1–Myc, and PRMT6 or PRMT6 mutants. Data in c and e are presented as the mean ± SEM of three independent experiments. Data in b, d, and f are representative of three independent experiments. ***p < 0.001
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