A novel transcript isoform of STING that sequesters cGAMP and dominantly inhibits innate nucleic acid sensing

Abstract

STING is a core adaptor in innate nucleic acid sensing in mammalian cells, on which different sensing pathways converge to induce type I interferon (IFN) production. Particularly, STING is activated by 2'3'-cGAMP, a cyclic dinucleotide containing mixed phosphodiester linkages and produced by cytoplasmic DNA sensor cGAS. Here, we reported on a novel transcript isoform of STING designated STING-β that dominantly inhibits innate nucleic acid sensing. STING-β without transmembrane domains was widely expressed at low levels in various human tissues and viral induction of STING-β correlated inversely with IFN-β production. The expression of STING-β declined in patients with lupus, in which type I IFNs are commonly overproduced. STING-β suppressed the induction of IFNs, IFN-stimulated genes and other cytokines by various immunostimulatory agents including cyclic dinucleotides, DNA, RNA and viruses, whereas depletion of STING-β showed the opposite effect. STING-β interacted with STING-α and antagonized its antiviral function. STING-β also interacted with TBK1 and prevented it from binding with STING-α, TRIF or other transducers. In addition, STING-β bound to 2'3'-cGAMP and impeded its binding with and activation of STING-α, leading to suppression of IFN-β production. Taken together, STING-β sequesters 2'3'-cGAMP second messenger and other transducer molecules to inhibit innate nucleic acid sensing dominantly.

Figure 1.

Expression of STING-β mRNA and protein. (A) Genome and domain structure of STING-β. Boxes represent exons and the lines represent introns. Positions of isoform-specific primers used in RT-PCR are indicated. Also shown are transmembrane (TM) and cytoplasmic domains of STING-α and STING-β. Transcription of STING-β mRNA is driven by an alternative promoter located within intron 5 of STING-α. Compared to exon 6 of STING-α, exon 1 of STING-β contains an extra piece of sequence at the 5′ end, resulting in an additional 25 amino acids at the N-terminus. This unique N-terminal sequence is highlighted. (B) Expression of STING-α and STING-β transcripts in human tissues. Human MTC™ Panel I (Clontech, USA) containing the cDNA templates of various human tissues were used for RT-PCR (left panel) and RT-qPCR (right panel) analysis. The STING-β primers will amplify a fragment of the correct size from STING-β cDNA only, but neither STING-α cDNA nor genomic DNA. Likewise, the STING-α primers are specific to STING-α cDNA. mRNA level was obtained by the comparative Ct method. (C) Expression of STING-β transcript in virus-challenged THP-1 cells. A DNA virus HSV-1-ΔICP0 (5 M.O.I.), in which one major IFN antagonist named ICP0 is deleted, was used to stimulate type I IFN production in THP-1 cells. Samples were harvested at the indicated time points for RNA extraction. Temporal expression profile of STING-β was determined by RT-qPCR. (D) Expression of STING-β transcript in peripheral blood mononuclear cells of SLE patients. cDNA templates were prepared from peripheral blood mononuclear cells of SLE patients (n = 24) and healthy individuals (n = 24). Expression of STING-α and STING-β in theses samples was detected by RT-qPCR. Whereas there was no significant statistical difference (n.s.) in STING-α mRNA expression between the SLE and healthy groups, the levels of STING-β mRNA were significantly lower (^∗∗P < 0.01) in SLE samples versus cells from healthy people. Statistical analysis was performed using Student's t test. (E) Detection of recombinant STING-β protein expressed in HEK293T cells by STING-β-specific antibodies. STING-α-HA, STING-β-HA, MRP-HA and STING-β-GFP were overexpressed in HEK293T cells. After 48 h, cells were lysed for western blotting using the indicated antibodies. Rabbit anti-STING-β antiserum specifically recognizes STING-β but not STING-α or MRP. (F) Detection of endogenous STING-β protein by STING-β-specific antibodies. THP-1, MT2, Jurkat and U937 cells were lysed and incubated with cGAMP agarose for 2 h at 4°C. After washing with PBS containing 100 μM ATP for six times, sample loading buffer was added. The samples were boiled for 10 min and then further analyzed by SDS-PAGE and western blotting with anti-STING-β antibodies. Results in each panel are representative of three independent experiments.

Figure 2.

STING-β antagonizes the antiviral immunity of STING-α. (A) HEK293 cells were transfected with increasing doses (10, 50, 200, and 450 ng) of STING-β (lanes 2–5) or STING-α (lanes 6–9) plasmid together with IFNβ-luc (40 ng), IRF3-luc (40 ng), ISRE-luc (10 ng), κB-luc (10 ng) or SV40-luc (10 ng) reporter. Cells in lane 1 received pcDNA6 empty vector as control. The graphs show the means ± SD (n = 3). The differences between the indicated group and the control in lane 1 were statistically significant as judged by Student's t test (^∗P < 0.05, ^∗∗P < 0.01 and ^∗∗∗P < 0.001). (B) STING-α (50 ng, lanes 3–7) were co-transfected into HEK293 cells together with increasing doses of STING-β plasmid (20, 100, 200 and 400 ng for lanes 4–7). Cells in lane 1 received pcDNA6 empty vector. Cells in lane 2 received STING-β plasmid alone. Additionally, 10 ng of pRL-TK reporter was added as an internal control. pcDNA6 empty vector was used to balance the total amount of transfected DNA. Dual luciferase assays were performed 36 h after transfection. The differences between the indicated group and the control in lane 1 were statistically significant as judged by Student's t test (^∗P < 0.05, ^∗∗P < 0.01 and ^∗∗∗P < 0.001). (C, D) pcDNA6 empty vector (500 ng; lane 1), STING-β plasmid (500 ng; lane 2), STING-α plasmid (50 ng; lane 3) or STING-α (50 ng) + STING-β (450 ng) plasmids were transfected into HEK293T cells. After 36 h, cells were infected with VSV-GFP (0.1 M.O.I.) or HSV-1-GFP (1 M.O.I.) for another 12 h. The cells were then lysed for western blot analysis and the supernatant was collected for plaque assays. The differences between the indicated two groups were statistically significant as judged by Student's t test (^∗P < 0.05 and ^∗∗P < 0.01). (E, F) Fluorescent microscopic analysis of the proviral effect of STING-β in VSV-GFP- or HSV-1-GFP-infected HeLa cells. pcDNA6 empty vector (500 ng), STING-β plasmid (500 ng), STING-α plasmid (50 ng), STING-α (50 ng) + STING-β (450 ng) plasmids were transfected into HeLa cells. After 36 h, cells were infected with VSV-GFP (0.1 M.O.I.) (E) or HSV-1-GFP (1 M.O.I.) (F). Cells were analyzed by fluorescent microscopy 12 h after infection for VSV-GFP and 24 h after infection for HSV-1-GFP. Results in each panel are representative of three independent experiments.

Figure 3.

STING-β inhibits CDN- and DNA-induced activation of IFN-β promoter. (A, B) In HEK293T cells, STING-α plasmid (50 ng for lanes 4, 5 and 7–10) and increasing doses of STING-β plasmid (50, 50, 50, 200, 300 and 450 ng for lanes 2, 6 and 7–10) were co-transfected with pRL-TK (10 ng) and IFNβ-luc (40 ng) reporter plasmids as indicated. After 24 h, cells were stimulated by c-di-GMP (500 ng for lanes 3 and 5–10) (A) or cGAMP (500 ng for lanes 3 and 5–10) (B). The graphs show the means ± SD (n = 3). The differences between the indicated group and the control in lane 5 were statistically significant as judged by Student's t test (^∗P < 0.05, ^∗∗P < 0.01 and ^∗∗∗P < 0.001). (C) STING-α plasmid (50 ng for lanes 4, 5 and 7–10), cGAS plasmid (50 ng for lanes 3, 5–10) and STING-β plasmid (50, 50, 50, 200, 300 and 450 ng for lanes 2, 6 and 7–10) were co-transfected into HEK293T cells with pRL-TK (10 ng) and IFNβ-luc (40 ng) reporter plasmids. The differences between the indicated group and the control in lane 5 were statistically significant as judged by Student's t test (^∗∗P < 0.01). (D) STING-α plasmid (50 ng for lane 2, 6, 9, 11 and 13–16), cGAS plasmid (50 ng for lanes 4 and 8–16) plus STING-β plasmid (50 ng for lanes 3, 7, 10, 12 and 13 as well as 200, 300 and 350 ng for lanes 14–16) were co-transfected with pRL-TK (10 ng) and IFNβ-luc (40 ng) reporter plasmids. After 24 h, cells were stimulated with an immunostimulatory DNA known as HSV-1-60mer (500 ng for lanes 5–8 and 11–16) for another 12 h. Cells were then harvested for dual-luciferase assays. The differences between the indicated group and the control in lane 11 were statistically significant as judged by Student's t test (^∗∗∗P < 0.001). Results in each panel are representative of three independent experiments.

Figure 4.

Depletion of STING-β by siRNA potentiates innate immune activation. (A) THP-1 cells cultured in six-well plates were transfected with 1 μl of siRNA (100 mM) targeting no human gene (siNS as a non-specific control), STING-α or STING-β. After 48 h, THP-1 cells were transfected with HSV-1-60mer (1000 ng/ml) or infected with 5 M.O.I. of HSV-1-ΔICP0 for another 9 h. THP-1 cells were then collected for RT-qPCR and the conditioned media of these samples were stored at –80°C for ELISA and subsequent analysis of antiviral activity. The differences between the indicated group and the siNS control group in lane 2 or 7 were statistically significant as judged by Student's t test (^∗P < 0.05, ^∗∗P < 0.01 and ^∗∗∗P < 0.001). (B, C) Antiviral activity of conditioned media collected from siRNA-treated THP-1 cells. Conditioned media collected from THP-1 samples in (A) were used to stimulate HEK293T and HeLa cells by incubation for 3 h. After stimulation, HEK293T cells and HeLa cells were cultured for another 12 h and then infected with VSV-GFP (0.1 M.O.I.) or HSV-1-GFP (1 M.O.I.). The media from infected HEK293T cells were used for plaque assay (B) and GFP-positive HeLa cells can be observed under fluorescent microscope (C). The differences between the indicated group and the siNS control group in lane 2 in (A) were statistically significant as judged by Student's t test (^∗P < 0.05 and ^∗∗P < 0.01). Results in each panel are representative of three independent experiments.

Figure 5.

STING-β interacts with and inhibits STING-α and TBK1. (A, B) Interaction of STING-β with STING-α. Expression plasmids for STING-β-HA (9000 ng) and STING-α-FLAG (1000 ng) or STING-α-FLAG (1000 ng) and STING-β-HA (9000 ng) were co-transfected into HEK293T cells. After 48 h, cells were harvested and lysed. Immunoprecipitation (IP) was carried out with mouse anti-FLAG or anti-HA antibody. (C) Interaction of STING-β with MAVS, TBK1 and IKKϵ. Plasmids (1000 ng) expressing FLAG-tagged RIG-I, MDA5, MAVS, TBK1, IKKϵ and MyD88 were transfected individually into HEK293T cells together with STING-β-HA plasmid (9000 ng). Immunoprecipitation was performed with mouse anti-FLAG antibody. Input proteins were analyzed by western blotting with mouse anti-FLAG or anti-HA antibody. Immunoprecipitates were probed with rabbit anti-HA antibodies. (D) TBK1-FLAG plasmid (1000 ng) was co-transfected with STING-α-V5 plasmid (1000 ng) and increasing doses of STING-β-HA (4000 ng in lanes 2 and 4 as well as 8000 ng in lane 5) into HEK293T cells. Immunoprecipitation was carried out with mouse anti-FLAG antibody. (E) TBK1-FLAG plasmid (1000 ng) was co-transfected with TRIF-V5 plasmid (1000 ng) and increasing doses of STING-β-HA (4000 ng in lanes 2 and 4 as well as 8000 ng in lane 5) into HEK293T cells. Cells were harvested and lysed after 48 h. Immunoprecipitation was carried out with mouse anti-FLAG. Input proteins were probed with mouse anti-FLAG, anti-V5 or anti-HA antibody. Immunoprecipitates were probed with rabbit anti-V5, anti-HA or anti-FLAG antibodies. (F) Colocalisation of STING-β with STING-α and TBK1. STING-α-GFP plus STING-β-HA or STING-β-GFP plus TBK1-FLAG plasmids were co-transfected into HeLa cells. After 36 h, HeLa cells were fixed with 4% paraformaldehyde and then blocked with 5% bovine serum albumin. The cells were incubated with mouse anti-HA or anti-FLAG antibody and then goat anti-mouse IgG conjugated to TRITC (red) was used to stain for STING-β or TBK1. Nuclear morphology was revealed with DAPI (blue). Results in each panel are representative of three independent experiments.

Figure 6.

STING-β inhibits phosphorylation of TBK1 and IRF3. (A–C) HEK293T cells were transfected with 450 ng of STING-β plasmid. After 24 h cells were infected with SeV (80 HA/ml), VSV-GFP (0.1 M.O.I.) or HSV-1-ΔICP0 (5 M.O.I.) for another 9 h. Cells were lysed for western blotting. (D, E) HEK293T cells were transfected with 450 ng of STING-β plasmid together with 50 ng of MAVS or TRIF plasmid. After 36 h, cells were lysed for western blotting. (F) STING-β plasmid (450 ng in lanes 2, 4, 6 and 8) was co-transfected with either empty vector or STING-α plasmid (50 ng in lanes 5–8) into HEK293T cells. After 24 h, 500 ng of cGAMP was transfected to stimulate the cells for another 12 h. Cells were then harvested and lysed for western blot analysis with anti-p-TBK1, anti-p-IRF3, anti-FLAG, anti-HA and anti-β-actin antibodies. Gel images in each panel are representative of three independent experiments.

Figure 7.

STING-β impedes the binding of cGAMP to STING-α. (A) Endogenous STING-α and STING-β could be pulled down by cGAMP agarose. THP-1 cells were lysed and incubated with cGAMP agarose (lanes 2, 4 and 6) or control agarose (lanes 1, 3 and 5) for 2 h at 4°C. After washing with PBS containing 100 μM ATP for six times, sample loading buffer was added. The samples were boiled for 10 min and further analyzed by SDS-PAGE followed by silver staining (lanes 1–2) or western blotting with either anti-STING-α (lanes 3–4) or anti-STING-β (lanes 5–6). Arrows point to STING-α and STING-β bands of expected sizes. (B) Recombinant STING-α and STING-β could be pulled down by cGAMP agarose. STING-α-HA (1000 ng) and STING-β-HA (9000 ng) plasmids were transfected into HEK293T cells. After 48 h, cells were harvested and lysed. cGAMP pull-down (cGAMP ↓) assay was carried out with cGAMP agarose or control agarose. Input proteins and cGAMP pull-down products were analyzed by western blotting with mouse anti-HA or anti-FLAG antibody. (C) Overexpression of STING-β prevented STING-α from binding to cGAMP. STING-α-HA plasmid (1000 ng) and increasing doses of STING-β-HA plasmid (300 ng for lane 2 and 9000 ng for lane 3) were co-transfected into HEK293T cells. After 48 h, cells were lysed and cGAMP pull-down (cGAMP ↓) assay was carried out. (D) STING-β inhibits cGAMP-induced aggregation of STING-α. HEK293T cells were transfected with STING-α plasmid (50 ng for lanes 1–4), cGAMP (200 ng for lanes 2–4), GFP plasmid (200 ng for lane 3) and STING-β plasmid (200 ng for lane 4). The protein samples were analyzed with SDD-AGE, native PAGE and SDS-PAGE. (E-G) Knockdown of STING-β augments cGAMP-induced activation of IFN-β and ISG production. THP-1 cells cultured in six-well plates were transfected with either 1 μl of negative control siRNA (siNS; 100 mM) or 0.5 μl of siSTING-β1 (100 mM) plus 0.5 μl of siSTING-β2 (100 mM), denoted as siSTING-β1+2. After 48 h, THP-1 cells were transfected with cGAMP using Lipofectamine 2000 (Invitrogen). After another 12 h, THP-1 cells were collected for RT-qPCR analysis. The statistical differences between the indicated groups were judged by Student's t test (^∗P < 0.05, ^∗∗P < 0.01 and ^∗∗∗P < 0.001). Results in each panel are representative of three independent experiments.