β-Catenin Is Required for the cGAS/STING Signaling Pathway but Antagonized by the Herpes Simplex Virus 1 US3 Protein

Abstract

The cGAS/STING-mediated DNA-sensing signaling pathway is crucial for interferon (IFN) production and host antiviral responses. Herpes simplex virus I (HSV-1) is a DNA virus that has evolved multiple strategies to evade host immune responses. Here, we demonstrate that the highly conserved β-catenin protein in the Wnt signaling pathway is an important factor to enhance the transcription of type I interferon (IFN-I) in the cGAS/STING signaling pathway, and the production of IFN-I mediated by β-catenin was antagonized by HSV-1 US3 protein via its kinase activity. Infection by US3-deficienct HSV-1 and its kinase-dead variants failed to downregulate IFN-I and IFN-stimulated gene (ISG) production induced by β-catenin. Consistent with this, absence of β-catenin enhanced the replication of US3-deficienct HSV-1, but not wild-type HSV-1. The underlying mechanism was the interaction of US3 with β-catenin and its hyperphosphorylation of β-catenin at Thr556 to block its nuclear translocation. For the first time, HSV-1 US3 has been shown to inhibit IFN-I production through hyperphosphorylation of β-catenin and to subvert host antiviral innate immunity.IMPORTANCE Although increasing evidence has demonstrated that HSV-1 subverts host immune responses and establishes lifelong latent infection, the molecular mechanisms by which HSV-1 interrupts antiviral innate immunity, especially the cGAS/STING-mediated cellular DNA-sensing signaling pathway, have not been fully explored. Here, we show that β-catenin promotes cGAS/STING-mediated activation of the IFN pathway, which is important for cellular innate immune responses and intrinsic resistance to DNA virus infection. The protein kinase US3 antagonizes the production of IFN by targeting β-catenin via its kinase activity. The findings in this study reveal a novel mechanism for HSV-1 to evade host antiviral immunity and add new knowledge to help in understanding the interaction between the host and HSV-1 infection.

Keywords: HSV-1; US3; type I IFN; β-catenin.

FIG 1

β-Catenin is required for optimal IFN-β production in the DNA-sensing signaling pathway. (A) Western blot analysis of endogenous β-catenin using cell lysates of HEK293T and β-catenin-KO-293T cells. (B and C) HEK293T and β-catenin-KO-293T cells were cotransfected with IFN-β promoter plasmid (IFN-β–Luc) (B) or (pRDIII-I)4-Luc reporter (C), along with Renilla luciferase (pRL-TK) reporter plasmid, cGAS-Flag, and STING-HA plasmids. Luciferase activity was measured 24 h posttransfection in the cell lysates. (D to F) HEK293T and β-catenin-KO-293T cells were cotransfected with cGAS-Flag and STING-HA plasmids. At 24 h posttransfection, cells were harvested and subjected to qRT-PCR analysis. The data represent the results of one of the triplicate experiments. The error bars represent SD of three independent experiments. Statistical analysis was performed using Student's t test with GraphPad Prism 5.0 software. *, 0.01 < P < 0.05; **, 0.001 < P < 0.01.

FIG 2

US3 inhibits the activation of IFN-β by targeting β-catenin. (A) β-catenin-KO-293T cells were transfected with IFN-β promoter plasmid, along with pRL-TK plasmid, IRF3/5D-Flag, β-catenin–Myc, and US3-Flag plasmids or empty vector. Luciferase activity was measured 24 h posttransfection in the cell lysates. (B) β-catenin-KO-293T cells were transfected with IFN-β promoter plasmid, along with pRL-TK plasmid, or IRF3/5D-Flag or IRF3/5D-Flag S175A, β-catenin–Myc, and US3-Flag plasmids or empty vector for 24 h. Cells were harvested and subjected to DLR assay. (C) β-catenin-KO-293T cells were transfected with IRF3/5D-Flag or IRF3/5D-Flag S175A, β-catenin–Myc, and US3-Flag plasmids, or empty vector for 24 h. Cells were harvested and subjected to qRT-PCR analysis. (D and E) HEK293T and β-catenin-KO-293T cells were infected with WT HSV-1 (D) or ΔUS3 HSV-1 (E) (MOI = 1). The cells were harvested at the indicated time points for viral plaque assay. The data represent the results of one of the triplicate experiments. The error bars represent SD of three independent experiments. Statistical analysis was performed using Student's t test with GraphPad Prism 5.0 software. *, 0.01 < P < 0.05; **, 0.001 < P < 0.01; ***, P < 0.001.

FIG 3

HSV-1 US3 inhibits β-catenin-mediated IFN-β activation, depending on its kinase activity. (A) β-catenin-KO-293T cells were transfected with β-catenin–Myc or IRF3/5D-Flag S175A and US3-Flag plasmids or empty vector for 24 h. Cells were harvested and subjected to DLR assay. (B to D) β-catenin-KO-293T cells were transfected with IRF3/5D-Flag S175A, β-catenin–Myc, US3-Flag, US3-Flag K220M, or US3-Flag D305A expression plasmid or empty vector for 24 h. Cells were harvested and subjected to qRT-PCR analysis. (E to G) β-catenin-KO-293T cells were infected with WT HSV-1, ΔUS3 HSV-1, K220M HSV-1, or D305A HSV-1 (MOI = 2) following transfection with β-catenin–Myc and IRF3/5D-Flag S175A for 24 h. Cells were harvested and subjected to qRT-PCR analysis. The data represent the results of one of the triplicate experiments. Statistical analysis was performed using Student's t test with GraphPad Prism 5.0 software. *, 0.01 < P < 0.05; **, 0.001 < P < 0.01.

FIG 4

US3 blocks the nuclear translocation of β-catenin. (A to F) L929 cells were transfected with empty vector, US3-Flag, US3-Flag K220M, or US3-Flag D305A expression plasmid for 12 h (A, B, and E), and L929 cells were infected with WT HSV-1, ΔUS3 HSV-1, K220M HSV-1, or D305A HSV-1 (MOI = 2) for 6 h (C, D, and F), and then the cells were treated with LiCl for 20 h. (E and F) Cells were harvested and subjected to WB analysis. (A to D) Cells were fractionated, and the cytosolic (B and D) and nuclear (A and C) fractions were analyzed by WB analysis for β-catenin levels. Immunoblotting of β-actin and lamin B1 served as internal loading controls for the cytosolic and nuclear fractions, respectively. (G) L929 cells were infected with WT HSV-1, ΔUS3 HSV-1, K220M HSV-1, or D305A HSV-1 (MOI = 2), and then the cells were treated with LiCl for 20 h. Cells were stained with mouse anti-β-catenin antibody. FITC-conjugated goat anti-mouse was used as the secondary antibody. Cell nuclei (blue) were stained with Hoechst 33258. The images were obtained by fluorescence microscopy using a 40× objective lens. (H) A549 cells were cotransfected with US3-Myc and IRF3/5D-Flag S175A. After 12 h, the cells were treated with LiCl for 20 h. Cells were harvested and subjected to qRT-PCR analysis. The data represent the results of one of the triplicate experiments. Statistical analysis was performed using Student's t test with GraphPad Prism 5.0 software. *, 0.01 < P < 0.05.

FIG 5

US3 phosphorylates β-catenin at the Thr556 site to inhibit IFN-β production induced by β-catenin. (A) HEK293T cells were cotransfected with β-catenin–Myc, along with US3-Flag, for 36 h. IP was performed using anti-Myc antibody and immunoblotting with anti-Myc or anti-Flag antibody. (B) L929 cells were transfected with US3-Flag, and then the cells were treated with LiCl for 20 h. IP was performed using anti-Flag antibody and immunoblotting with anti-Flag or anti-β-catenin antibody. (C) HEK293T cells were cotransfected with β-catenin–Myc ΔN, along with US3-Flag, K220M-Flag, D305A-Flag, or vector plasmid for 36 h and subjected to WB. (D) HEK293T cells were transfected with β-catenin–Myc ΔN or β-catenin–Myc ΔN T556A plasmid, along with US3-Flag or empty plasmid, for 36 h and subjected to WB. (E to H) β-catenin-KO-293T cells were transfected with β-catenin–Myc or β-catenin–Myc T556A, US3-Flag, K220M-Flag, D305A-Flag, or vector plasmid for 36 h. The cells were fractionated, and the cytosolic and nuclear fractions were analyzed by WB for β-catenin levels. Immunoblotting of β-actin and lamin B1 served as internal loading controls for the cytosolic and nuclear fractions, respectively. (I) β-catenin-KO-293T cells were transfected with IFN-β promoter plasmid, along with pRL-TK, IRF3/5D-Flag S175A, β-catenin–Myc, β-catenin–Myc T556A, or US3-Flag plasmid or empty vector for 24 h. Cells were harvested and subjected to DLR assay. (J) β-catenin-KO-293T cells were transfected with IRF3/5D-Flag S175A, along with β-catenin–Myc or β-catenin–Myc T556A and US3-Flag plasmids or empty vector for 24 h. Cells were harvested and subjected to qRT-PCR analysis. The data represent the results of one of the triplicate experiments. Statistical analysis was performed using Student's t test with GraphPad Prism 5.0 software. *, 0.01 < P < 0.05; **, 0.001 < P < 0.01.

FIG 6

Working model of the roles of β-catenin and US3 in the DNA-sensing signaling pathway. β-Catenin is required for optimal IFN-β production in the DNA-sensing signaling pathway. The HSV-1 protein kinase US3 interacts with β-catenin, hyperphosphorylates it to block the nuclear translocation of β-catenin, and then inhibits the production of IFN-β induced by β-catenin via its kinase activity.