TRIM30α Is a Negative-Feedback Regulator of the Intracellular DNA and DNA Virus-Triggered Response by Targeting STING

<div><p>Uncontrolled immune responses to intracellular DNA have been shown to induce autoimmune diseases. Homeostasis regulation of immune responses to cytosolic DNA is critical for limiting the risk of autoimmunity and survival of the host. Here, we report that the E3 ubiquitin ligase tripartite motif protein 30α (TRIM30α) was induced by herpes simplex virus type 1 (HSV-1) infection in dendritic cells (DCs). Knockdown or genetic ablation of TRIM30α augmented the type I IFNs and interleukin-6 response to intracellular DNA and DNA viruses. <i>Trim30α</i>-deficient mice were more resistant to infection by DNA viruses. Biochemical analyses showed that TRIM30α interacted with the stimulator of interferon genes (STING), which is a critical regulator of the DNA-sensing response. Overexpression of TRIM30α promoted the degradation of STING via K48-linked ubiquitination at Lys275 through a proteasome-dependent pathway. These findings indicate that E3 ligase TRIM30α is an important negative-feedback regulator of innate immune responses to DNA viruses by targeting STING.</p></div

TRIM30α knockdown promotes immune signaling by cytoplasmic DNA and DNA virus infection in D2SC cells.

<p>(A) Immunoblot analysis of the knockdown of exogenous TRIM30α in D2SC cells treated with control siRNA (SC) or TRIM30α siRNA (T3) for 24 h and then stimulated for 16 h with poly(dA:dT) (1 μg/ml). β-actin served as a loading control throughout the experiment. (B, C) ELISA of type I IFN and IL-6 in D2SC cells treated with siRNA SC or T3 and then stimulated for 16 h with poly(dA:dT) (1 μg/ml), ISD (1 μg/ml), genomic DNA (2 μg/ml), c-di-GMP (8 μg/ml) or poly(I:C) (5 μg/ml) (C) or with HSV-1 (MOI 10) or vaccinia virus (VACV) (MOI 10). (D) Real-time PCR of IP-10 mRNA in D2SC cells treated with siRNA SC or T3 and then stimulated for 8 h with ISD (1 μg/ml) or poly(I:C) (5 μg/ml). (E) Immunoblot analysis of p-IRF3, p-p65 and TRIM30α in lysates of D2SC cells treated with siRNA SC or T3 and then stimulated for 1–5 h with poly(dA:dT) (1 μg/ml). Densitometry analysis to quantify ratio of p-IRF3 or p-p65 to β-actin is shown on the right. The data are representative of three independent experiments and are presented as mean ± SEM. *<i>p</i> < 0.05, **<i>p</i> < 0.01 and ***<i>p</i> < 0.001.</p

TRIM30α deficiency protects the mice from DNA virus infection.

<p>(A, B) ELISA of IFN-β and IL-6 in wild-type and <i>Trim30α</i>^-/- CD11c⁺ splenocytes stimulated for 24 h with ISD (1 μg/ml), HSV-1 (MOI 10) or poly(I:C) (5 μg/ml). (C, D) Real-time PCR of IFN-β and TNF-α in the lung and liver from wild type and <i>Trim30α</i>^-/- mice infected with HSV-1 (2×10⁷ PFU) (i.v.) for the indicated times. (E) ELISA of IFN-β and IL-6 in serum of wild type and <i>Trim30α</i>^-/- mice 6 h after intravenous infection with HSV-1 (1.2×10⁷ PFU) (n = 5). (F) Survival of age- and sex-matched wild-type and <i>Trim30α</i>^<i>-/-</i> mice (n = 5 per group) infected with HSV-1 (2×10⁷ PFU) (i.v.) and monitored daily for 15 d. The data are representative of three independent experiments and are presented as mean ± SEM. *<i>p</i> < 0.05, **<i>p</i> < 0.01 and ***<i>p</i> < 0.001.</p

TRIM30α deficiency inhibits DNA virus infection.

<p>(A, B) Viral titers in L929 cells transfected with control siRNA SC and T3 (A), empty vector (Vec) or TRIM30α plasmid (B) for 24 h and then infected with HSV-1 (MOI 10) for 20 h. The titers of HSV-1 were determined by standard plaque assay. (C) Viral titers in wild type and <i>Trim30α</i>^-/- mice intraperitoneally injected with HSV-1 (1×10⁷ plaque-forming units (PFU)). HSV-1 titers were measured 20 h later by plaque assay of peritoneal wash fluid. (D) Real-time PCR of HSV-1 genomic DNA in the brain, lung and liver from wild type and <i>Trim30α</i>^-/- mice infected with HSV-1 (2×10⁷ PFU) (i.v.) for 2 days. The data are representative of three independent experiments and are presented as mean ± SEM. *<i>p</i> < 0.05, ***<i>p</i> < 0.001.</p

TRIM30α enhances the degradation of STING.

<p>(A) Immunoblot analysis of STING, TBK1, IRF3 and TRIM30α in lysates from wild type and <i>Trim30α</i>^-/- BMDCs stimulated for 2–24 h with poly(dA:dT) (1 μg/ml). (B) Immunoblot analysis of STING in lysates from wild type BMDCs stimulated for 2–24 h with poly(dA:dT) (1 μg/ml) in the presence or absence of 20 mM MG132. (C) Confocal microscopy of L929 cells transfected for 24 h with Flag-tagged STING and then mock treated or stimulated for 12 h with ISD (1 μg/ml). Immunofluorescence was performed using anti-Flag (red) and anti-20S proteasome β1 (green). Nuclei were stained with the DNA-intercalating dye DAPI. (D) Immunoblot analysis of STING in lysates of L929 cells transfected with increasing doses of Myc-tagged TRIM30α (0, 0.8 and 1.2 μg) and then treated for 6 h with DMSO (negative control) or 20 mM MG132. Densitometry analysis to quantify ratio of STING to β-actin is shown on the below. (E) Immunoblot analysis of STING in lysates of L929 cells transfected with full-length TRIM30α, C35A or ΔR (1μg) and then treated as in D.</p

TRIM30α interacts with STING.

<p>(A) Luciferase activity of MEF cells transfected with the IFN-β luciferase reporter, together with STING, TBK1, MDA5 or VISA, and the empty vector (Vec) or the TRIM30α plasmid. (B) Luciferase activity of MEF cells transfected with the NF-κB luciferase reporter, together with STING or VISA, and the empty vector (Vec) or the TRIM30α plasmid. (C and D) Luciferase activity of MEF cells cotransfected with the IFN-β promoter or the NF-κB promoter and empty vector or increasing amounts of TRIM30α (C) or C35A (D) (0.2, 0.4 and 0.6 μg) together with STING. (E) Immunoblot analysis of lysates from HEK293T cells transfected with Flag-TRIM30α together with HA-tagged vector, STING or TAK1 plasmids, followed by immunoprecipitation (IP) with anti-Flag Ab and immunoblot analysis with anti-HA Ab. (F, G) Immunoblot analysis in lysates of D2SC cells mock treated or stimulated with poly(dA:dT) (1 μg/ml) (F); or with HSV-1 (MOI 10) (G) for 8 h, followed by immunoprecipitation with anti-TRIM30α Ab and immunoblot analysis with anti-STING Ab. (H) A schematic presentation of full-length TRIM30α and its mutants. RING, ring-finger domain; BBOX, B-box domain; CC, coiled-coil domain; SPRY, SPRY domain. (I) Immunoblot analysis of lysates from HEK293T cells transfected with the indicated plasmids, followed by immunoprecipitation (IP) with anti-HA Ab and immunoblot analysis with anti-Flag Ab. (J) A schematic presentation of full-length STING and its mutants. (Transmembrane) TM1, 21-41aa; TM2, 47-67aa; TM3, 87-106aa; TM4, 115-135aa; CTD, carboxy-terminal domain; CTT, carboxy-terminal tail. (K) Immunoblot analysis of lysates from HEK293T cells transfected with the indicated plasmids and then performed as in I. (L) Confocal microscopy of L929 cells transfected for 24 h with Flag-tagged STING and HA-tagged full-length TRIM30α, ΔRING or ΔSPRY mutant of TRIM30α. Immunofluorescence was performed using anti-HA (red) and anti-Flag (green). (M) Confocal microscopy of L929 cells transfected for 24 h with cyan fluorescent protein-labeled TRIM30a (CFP-TRIM30α) and yellow fluorescent protein-labeled STING (YFP-STING) and then mock treated or stimulated for 4 h with poly(dA:dT) (1 μg/ml) or ISD (1 μg/ml). Nuclei were stained with the DNA-intercalating dye DAPI. Staining of calnexin served as a marker of the endoplasmic reticulum (ER). The data are representative of three independent experiments and are presented as mean ± SEM. *<i>p</i> < 0.05 and **<i>p</i> < 0.01.</p

TRIM30α deficiency promotes immune signaling by cytoplasmic DNA and DNA virus infection in BMDCs.

<p>(A) Immunoblot analysis of TRIM30α expression in the splenocytes of wild type (WT) and <i>Trim30α</i>^<i>-/-</i> (KO) mice. (B, C) ELISA of type I IFN and IL-6 in wild-type and <i>Trim30α</i>^-/- BMDCs mock treated or stimulated for 16 h by transfection with poly(dA:dT) (1 μg/ml), ISD (1 μg/ml), c-di-GMP (8 μg/ml), genomic DNA (2 μg/ml) or poly(I:C) (5 μg/ml) (B); or with HSV-1 (MOI 10) or VACV (MOI 10) (C). (D) Immunoblot analysis of phosphorylated IRF3 (p-IRF3), p-p65 and TRIM30α in lysates of wild type and <i>Trim30α</i>^-/- BMDCs stimulated for 1–5 h with poly(dA:dT) (1 μg/ml). Densitometry analysis to quantify ratio of p-IRF3 or p-p65 to β-actin is shown on the right. The data are representative of three independent experiments and are presented as mean ± SEM. *<i>p</i> < 0.05, **<i>p</i> < 0.01 and ***<i>p</i> < 0.001.</p

TRIM30α is an E3 ubiquitin ligase and targets STING for K48-mediated ubiquitination at Lys275.

<p>(A) Immunoblot analysis of lysates from HEK293T cells transfected with plasmids for Flag-STING, HA-ubiquitin and increasing concentrations of Myc-TRIM30α (0, 1, 1.5 and 2 μg), followed by immunoprecipitation with anti-Flag, and analyzed via immunoblot with anti-HA Ab. (B) Immunoblot analysis of lysates from HEK293T cells transfected with various combinations of plasmids for Myc-TRIM30α, Flag-STING, HA-K48-ubi and HA-K63-ubi and then performed as in A. (C) Immunoblot analysis of lysates from HEK293T cells transfected with the indicated plasmids and Myc-tagged TRIM30α (C35A), ΔR and then performed as in B. (D) Immunoblot analysis of lysates in wild-type and <i>Trim30α</i>^-/- BMDCs stimulated for 8 h with ISD (1 μg/ml), followed by immunoprecipitation with anti-STING Ab and analyzed with anti-Ub, anti-K48- and anti-K63-linked ubiquitin. (E) Immunoblot analysis of STING ubiquitination in vitro. TRIM30α and STING were quickly translated in vitro, and then, the biotin-ubiquitin E1 and indicated E2s were added for the ubiquitination assays. Ubiquitination was assessed by anti-ubi. (F) Immunoblot analysis of lysates from HEK293T cells transfected with Myc-tagged TRIM30α, Flag-tagged wild-type and mutant STING together with HA-tagged ubiquitin, then performed as in A. (G) Luciferase assays of MEF cells cotransfected with the IFN-β promoter and empty vector or TRIM30α together with wild-type and mutant STING plasmids. (H) Real-time PCR of IFN-β and IP-10 mRNA in L929 cells transfected with Flag-tagged vector, wild type STING and K275R mutant, 24 h after transfection, then infected the above cells with HSV-1 (MOI 10) for 10 h. The data are representative of three independent experiments and are presented as mean ± SEM. *<i>p</i> < 0.05, **<i>p</i> < 0.01 and ***<i>p</i> < 0.001.</p

ELISA to detect specific anti-HA IgG antibody at different time points in the sera of volunteers for the 2009 A/H1N1 influenza vaccine administration.

<p>The OD450 nm on different days were measured and results were displayed as ΔOD450 nm with each prevaccination (baseline) OD subtracted in every subject's serial samples. "pre": pre-vaccination. The OD value of the pre-vaccination sera was 0.76±0.06 (IgG baseline). The blank OD (no sera added) of the ELISA was 0.05. **: p<0.001, two-way Student's t-test.</p

Dynamic Changes of the Hemagglutination-Inhibition Titers against the Seasonal H1N1 Virus after Administration of the 2009 A/H1N1 Influenza Vaccine.

<p>Dynamic Changes of the Hemagglutination-Inhibition Titers against the Seasonal H1N1 Virus after Administration of the 2009 A/H1N1 Influenza Vaccine.</p