UFL1 promotes antiviral immune response by maintaining STING stability independent of UFMylation

Abstract

The precise regulation of STING homeostasis is essential for its antiviral function. Post-translational modification, especially ubiquitination, is important for the regulation of STING homeostasis. Previous studies have focused on how STING is degraded, but little is known about its maintenance. Here, we show that UFM1 specific ligase UFL1 promotes innate immune response by maintaining STING expression independent of UFMylation. Mechanistically, UFL1 inhibits TRIM29 to interact with STING, thereby reducing its ubiquitination at K338/K347/K370 and subsequent proteasomal degradation. DNA virus infection reduces the UFL1 expression, which may promote STING degradation and facilitate viral expansion. Our study identifies UFL1 as a crucial regulator for the maintenance of STING stability and antiviral function, and provides novel insights into the mechanistic explanation for the immunological escape of DNA virus.

Fig. 1. UFL1 promotes antiviral innate immunity.

A, B Ufl1 expression in PMs infected with HSV-1 or VACV detected by qRT-PCR (A) and Western blot (B). C Ifnb1, Il6 or Tnf mRNA expression in PMs infected with HSV-1 or VACV. D IFN-β and cytokines production in supernatants of PMs 24 hr post HSV-1 infection. E Ifnb1 and Il6 mRNA expression in PMs transfected with ISD or Poly dG:dC. F IFNB1 and IL6 mRNA expression in A549 cells transfected with Poly dA:dT. G Ifnb1 and Il6 mRNA expression in PMs infected with VSV or transfected with Poly I:C. H HSV-1 titers in supernatants of PMs by TCID50 assay. I Ifnb1 mRNA and HSV-1 TK RNA expression in Irf3^−/− PMs infected with HSV-1. Data are presented as means ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2. Loss of UFL1 in macrophages drives mice more susceptible to HSV-1 infection.

A Weight loss of Ufl1^fl/fl and Ufl1^fl/flLyz^cre+/− mice after intraperitoneal injection of HSV-1 (1 × 10⁸ pfu/g). B–E HSV-1 TK RNA expression (B), HSV-1 titers in lung homogenates (C), H&E of lung (D) and cytokines production in the serum (E) from Ufl1^fl/fl and Ufl1^fl/flLyz^cre+/− mice in response to i.v. infection with HSV-1. The scale bar is 100 µm (D). F, G Ifnb1, Il6, or Tnf mRNA expression in PMs from Ufl1^fl/fl and Ufl1^fl/flLyz^cre+/− mice infected with HSV-1 (F) or transfected with cGAMP and exogenous nucleic acid stimulations (G). H HSV-1 TK RNA expression in PMs from Ufl1^fl/fl and Ufl1^fl/flLyz^cre+/− mice infected with HSV-1. I IFN-β and cytokines production in supernatants of BMDMs from Ufl1^fl/fl and Ufl1^fl/flLyz^cre+/− mice infected with HSV-1 for 24 h. Data are presented as means ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3. UFL1 targets cGAS-STING signal pathway.

A IFN-β luciferase activity in HEK293 cells transfected with indicated molecules. B cGAS and STING triggered IFN-β luciferase activity in HEK293 cells, transfected with increasing amounts of UFL1. C, D Phosphorylation of the indicated molecules after HSV-1 infection in PMs (C) and BMDMs (D) from Ufl1^fl/fl and Ufl1^fl/flLyz^cre+/− mice. E, F Quantification of protein in PMs (E) and BMDMs (F) via Image J. Data are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4. UFL1 interacts with STING.

A V5-tagged UFL1 and Flag-tagged cGAS, STING, RIG-I, MAVS, TBK1, IRF3 were transfected into HEK293T cells as indicated, and their interaction was tested by V5 immunoprecipitation. B Flag-tagged STING or Flag-tagged cGAS were transfected with UFL1 into HEK293T cells, and their interaction was tested by Flag immunoprecipitation. C, D (Left) Interaction between STING and UFL1 induced by HSV-1 was examined by immunoprecipitation in PMs (C) and BMDMs (D). (Right) Quantification of UFL1 level normalized by STING in PMs (C) and BMDMs (D) via Image J. E Confocal analysis of endogenous UFL1 and ER co-localization in HELA cells. The bar in the picture stood for 10 µm. The “r” represents PCC value of UFL1 and ER co-localization. F (Above) Confocal analysis of UFL1 and STING expression and co-localization in HELA cells stimulated with HSV-1 or not. The bar in the picture stood for 10 µm. (Below) Quantification of the co-localization rate of UFL1 and STING via LAS X software. The “r” represents PCC value of UFL1 and STING co-localization. G PLA of STING and UFL1 interaction in 293T cells with HSV-1 stimulated or not. The bar in the picture stood for 5 µm. H (Above) Confocal analysis of UFL1 and ERGIC co-localization in HELA cells stimulated with HSV-1 or not. The bar in the picture stood for 10 µm. (Below) Quantification of the co-localization rate of UFL1 and ERGIC via LAS X software. The “r” represents PCC value of UFL1 and ERGIC co-localization. I Truncated UFL1 mutants were transfected into HEK293T cells as indicated, and STING-UFL1 interaction was tested by STING immunoprecipitation. J Truncated STING mutants were transfected into HEK293T cells as indicated, and UFL1-STING interaction was tested by UFL1 immunoprecipitation. Data shown are representative of three independent experiments with similar results (A–D, I, J).

Fig. 5. UFL1 inhibits K48-linked ubiquitination and proteasomal degradation of STING.

A IFN-β luciferase activity in HEK293 cells transfected with cGAS plus STING, and different truncated UFL1 mutants as indicated. Data are presented as means ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. B, C (Above) Expression of cGAS and STING post HSV-1 infection in PMs (B) and BMDMs (C) knockdown of UFL1 or not. (Below) Quantification of STING level normalized by GAPDH via Image J. D Expression of STING with increasing doses of V5-tagged UFL1 in MEF cells. E Expression of STING in the presence or absence of UFL1, treated with indicated inhibitors. F (Above) Expression of STING in PMs with UFL1 knockdown or not, treated with indicated inhibitors. (Below) Quantification of STING level normalized by ACTIN via Image J. G The ubiquitination of STING in HEK293T cells transfected with UFL1 and HA-tagged WT or mutant ubiquitin. H The endogenous K48-ubiquitination of STING in BMDMs from Ufl1^fl/fl and Ufl1^fl/flLyz^cre+/− mice. Data shown are representative of three independent experiments with similar results (B–H).

Fig. 6. UFL1 reduces STING ubiquitination at Lys338/347/370.

A, C Expression of different single site mutants (A) or multiple site mutants (C) of STING in HEK293 cells transfected with or without V5-tagged UFL1. B, D IFN-β luciferase activity in HEK293 cells transfected with different STING mutants as indicated. Data are presented as means ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. E The ubiquitination of different STING mutants in HEK293T cells transfected with V5-tagged UFL1, HA-tagged ubiquitin as indicated. Data shown are representative of three independent experiments with similar results (A, C, E).

Fig. 7. UFL1 reduces STING ubiquitination by competition with TRIM29.

A (Left) Co-immunoprecipitation and immunoblot analysis of TRIM29-STING and UFL1-STING interaction in 293T cells transfected with varying doses of UFL1. (Right) Quantification of UFL1-V5 and TRIM29-Flag level normalized by STING-Myc via Image J. B PLA of STING and TRIM29 interaction in 293T cells with ULF1 overexpressed or not. The bar in the picture stood for 5 µm. C, D WT (C) and K48-linked (D) ubiquitination of STING by TRIM29 in HEK293T cells transfected with UFL1 or not. E Interaction between UFL1, TRIM29 and STING induced by HSV-1 was examined by immunoprecipitation in BMDMs. F Ifnb and Il6 mRNA expression in L929 cells infected with HSV-1. G Phosphorylation of the indicated molecules and expression of STING in L929 cells transfected with Poly dA:dT. H The mechanism of UFL1 promoting antiviral immune response through reducing STING ubiquitination and degradation. Data shown are representative of three independent experiments with similar results (A, C–E, G).