TRIM23 mediates cGAS-induced autophagy in anti-HSV defense

Abstract

The cGAS-STING pathway, well-known to elicit interferon (IFN) responses, is also a key inducer of autophagy upon virus infection or other stimuli. Whereas the mediators for cGAS-induced IFN responses are well characterized, much less is known about how cGAS elicits autophagy. Here, we report that TRIM23, a unique TRIM protein harboring both ubiquitin E3 ligase and GTPase activity, is crucial for cGAS-STING-dependent antiviral autophagy. Genetic ablation of TRIM23 impairs autophagic control of HSV-1 infection. HSV-1 infection or cGAS-STING stimulation induces TBK1-mediated TRIM23 phosphorylation at S39, which triggers TRIM23 autoubiquitination and GTPase activity and ultimately elicits autophagy. Fibroblasts from a patient with herpes simplex encephalitis heterozygous for a dominant-negative, kinase-inactivating TBK1 mutation fail to activate autophagy by TRIM23 and cGAS-STING. Our results thus identify the cGAS-STING-TBK1-TRIM23 axis as a key autophagy defense pathway and may stimulate new therapeutic interventions for viral or inflammatory diseases.

Fig. 1. TRIM23 regulates HSV-1 pathogenesis and autophagy in vivo.

a–e Trim23^─/─ mice and wild type (WT) littermate controls of both sexes (8-week-old, sex-matched across treatments) were infected via intracerebral (I.C.) inoculation with 5 × 10⁵ PFU of mutHSV-1. a Kaplan–Meier survival curves of mutHSV-1-infected Trim23^─/─ and WT mice. b Viral titers in whole brain homogenates of Trim23^─/─ and WT mice, determined by plaque assay at day 3 post-infection. c HSV-1 ICP-0, ICP-8, UL36 and gB transcripts in whole brain homogenates, determined by RT-qPCR at day 3 post-infection. d HSV-1 antigen expression in the indicated areas of mid-sagittal brain sections of mutHSV-1-infected Trim23^─/─ and WT mice, determined at day 3 post-infection by immunohistochemistry. Scale bar, 3 mm (whole brain) or 50 µm (magnified areas). CP, caudate putamen; CB, cerebellum; HYP, hypothalamus; OB, olfactory bulb. e Endogenous p62 protein abundance in the whole brain lysates of mock-treated or mutHSV-1-infected Trim23^─/─ and WT mice, determined at day 3 post-infection by immunoblot (IB). #1–#3 represent individual mice. f Viral ICP-0 and ICP-8 transcript expression in Neuro2a (mouse neuroblast) cells that were transfected for 30 h with the indicated siRNAs and then infected for 36 h with mutHSV-1 at the indicated MOIs, determined by RT-qPCR. g Endogenous p62 protein abundance and LC3B-I-to-II conversion in the whole cell lysates (WCLs) of Neuro2a cells that were transfected for 30 h with the indicated siRNAs and then either mock-treated or infected with mutHSV-1 (MOI 1, 5, and 10) for 24 h, determined by IB. h Viral titers in SH-SY5Y (human neuroblastoma) cells that were transfected for 30 h with the indicated siRNAs and then infected for 24 h with mutHSV-1 at the indicated MOIs, determined by plaque assay. i Endogenous p62 protein abundance and LC3B-I-to-II conversion in the WCLs of SH-SY5Y cells that were transfected for 30 h with the indicated siRNAs and then either mock-treated or infected with mutHSV-1 (MOI 0.2, 1 and 2) for 20 h, determined by IB. j Viral ICP-0 and ICP-8 transcript expression in primary human dermal fibroblasts (HDFs) that were transfected for 30 h with the indicated siRNAs and then either mock-treated or infected with mutHSV-1 (MOI 0.02) for 24 h, determined by RT-qPCR. k Endogenous p62 protein abundance and LC3B-I-to-II conversion in the WCLs of HDFs that were transfected for 30 h with the indicated siRNAs and then either mock-treated (–) or infected with mutHSV-1 (MOI 10) for 16 h, determined by IB. All data (a to k) are representative of at least two independent experiments (n = 7 mice for WT and n = 11 mice for Trim23^─/─ (a); mean ± SD of n = 7 mice per condition (b); mean ± SD n = 8 mice for WT and n = 10 mice for Trim23^─/─ (c); mean ± SD of n = 3 biological replicates (f, h and j)). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Mantel–Cox log rank test (a), two-tailed Student’s t test with Welch’s correction (b, c), or two-tailed Student’s t test (f, h and j). Source data are provided as a Source Data file.

Fig. 2. TRIM23 is phosphorylated at Ser-39.

a Schematic representation of the domain organization of TRIM23 with the phospho-S39 (pS39) residue illustrated. RING, Really Interesting New Gene domain; BB1/2, B-Box 1 and 2; CC, coiled-coil domain; ARF, ADP-ribosylation factor domain. b Amino acid sequence of the region containing the S39 residue in TRIM23 from the indicated species. Numbers indicate amino acid positions. c Tandem mass spectra of the tryptic peptide K.VLECGVCEDVFpS₃₉LQGDKVPR.L of TRIM23-FLAG affinity-purified from transiently transfected HEK293T cells that identified phosphorylation at S39. d Phosphorylation of TRIM23-FLAG WT and S39A mutant in transiently transfected HEK293T cells that were either mock-treated (–) or treated with calyculin A (Cal A, a phosphatase inhibitor), determined at 48 h post-transfection by immunoprecipitation (IP) with anti-FLAG and IB with anti-p-S39-TRIM23 antibody. e S39 phosphorylation and poly-ubiquitination of FLAG-TRIM23 stably expressed in HDFs that were infected with mutHSV-1 as indicated, determined at 8 h post-infection by IP with anti-FLAG and IB with anti-p-S39-TRIM23 or anti-ubiquitin (Ub). f S39 phosphorylation of endogenous TRIM23 in HDFs that were either mock-treated or infected with WT HSV-1 or mutHSV-1 (MOI 5 for both) for 16 h, determined by immunostaining with the indicated antibodies and confocal microscopy analysis. DAPI, nuclei (blue). Scale bar, 20 µm. g Quantification of p-S39-TRIM23-positive cells for the data shown in (f). h Time course analysis of endogenous TRIM23 S39 phosphorylation in HDFs that were either mock-treated or infected with mutHSV-1 (MOI 5) for the indicated times, determined as in (f). DAPI, nuclei (blue). Scale bar, 20 µm. i Quantification of p-S39-TRIM23-positive cells for the data shown in (h). j Upper: Ribbon representation of the crystal structure of the TRIM23-RING dimer (green/blue) in complex with Ub-loaded E2 (orange/red). The loops containing S39 are highlighted in pink. Lower: Close-up view of the RING S39 loop, stabilized in part by a bifurcated hydrogen bond (yellow), and its proximity to the Ub or E2 binding interface. k K27-linked auto-polyubiquitination of FLAG-tagged TRIM23 WT or mutants in transiently transfected HEK293T cells that co-expressed an HA-tagged ubiquitin mutant (HA-Ub-K27only), determined at 48 h post-transfection by IP with anti-FLAG and IB with anti-HA. Data in (c) are from one unbiased MS analysis, data in (d–i and k) are representative of at least two independent experiments (mean ± SD of n  = 4 biological replicates (g, i). ****p < 0.0001 (two-tailed Student’s t test (g, i). Source data are provided as a Source Data file.

Fig. 3. Phosphorylation at S39 regulates TRIM23-mediated autophagy and antiviral activity.

a Top: GTPase enzymatic activity of FLAG-tagged TRIM23 WT and indicated mutants, or FLAG-tagged Rab5α (positive control), that were affinity purified by IP using anti-FLAG from transiently transfected HEK293T cells, determined by GTPase Glo assay. Values for WT TRIM23 were set to 100%. Bottom: Expression of the indicated proteins determined by IB. b GFP-LC3B puncta formation in HeLa cells stably expressing GFP-LC3B (green) that were transfected with either empty vector or the indicated FLAG-tagged TRIM23 constructs (red), determined at 48 h post-transfection by immunostaining with anti-FLAG followed by confocal microscopy analysis. DAPI, nuclei (blue). Scale bar, 20 µm. c Quantification of the number of GFP-LC3B puncta for the data shown in (b). d Top: Endogenous LC3B-I-to-II conversion in HEK293T cells that were transiently transfected with either empty vector or the indicated TRIM23-FLAG constructs, determined at 48 h post-transfection by IB with the indicated antibodies. Stimulation with rapamycin (3 μM for 4 h) served as a positive control. Bottom: Densitometric analysis of the LC3B-II signal intensities, normalized to the respective actin protein abundances, for the data shown on top. Values for the empty vector control (without rapamycin) were set to 1. e Co-localization of transiently expressed myc-tagged TRIM23 (green) with endogenous p62 (red) in HeLa cells, determined at 48 h post-transfection by immunostaining with anti-myc and anti-p62 followed by confocal microscopy analysis. DAPI, nuclei (blue). Scale bar, 20 μm. f Quantification of p62-TRIM23 colocalization for the data shown in (e), determined by Pearson’s correlation coefficient. g Viral titers in the supernatant of HEK293T cells that were transfected for 24 h with either empty vector or FLAG-tagged TRIM23 WT or mutants and then either mock-treated (–) or infected with mutHSV-1 (MOI 1) for 24 h, determined by plaque assay. h Representative expression of TRIM23-FLAG WT and mutant proteins for the experiment in (g), determined in the WCLs at 24 h post-transfection by IB with anti-FLAG and anti-actin (loading control). All data (a–h) are representative of at least two independent experiments (mean ± SD of n = 6 (a), n = 4 (g) biological replicates; mean ± SEM of n = 37, 30, 32, 34, 32 cells respectively for vector, WT, S39A, S39D, and S39E (c); n = 14 cells (f). ***p < 0.001; ****p < 0.0001 (two-tailed Student’s t test with Welch’s correction (a, c, f), or two-tailed Student’s t test (g). NS not significant. ND not detected. Source data are provided as a Source Data file.

Fig. 4. TBK1 phosphorylates TRIM23 at S39.

a S39 phosphorylation of TRIM23-FLAG in HEK293T cells that were co-transfected with the indicated siRNAs, determined at 48 h post-transfection by IP with anti-FLAG and IB with anti-p-S39-TRIM23. b S39 phosphorylation of TRIM23-FLAG in HEK293T cells that co-expressed either empty vector (–) or increasing amounts of HA-TBK1 or HA-IKKβ, determined at 48 h post-transfection by IP with anti-FLAG and IB with anti-p-S39-TRIM23. c S39 phosphorylation of TRIM23-FLAG in HEK293T cells that co-expressed the indicated HA-tagged TBK1 constructs, determined at 48 h post-transfection by IP with anti-FLAG and IB with anti-p-S39-TRIM23. d Top: In vitro kinase assay assessing S39 phosphorylation of purified GST-TRIM23 upon co-incubation with purified GST-TBK1, determined by IB with anti-p-S39-TRIM23, anti-TRIM23 and anti-TBK1. Bottom: Densitometric analysis of the p-S39-TRIM23 signal intensities, normalized to the respective TRIM23 protein abundances, for the data shown on top. e S39 phosphorylation and ubiquitination of TRIM23-FLAG in transiently transfected HEK293T cells that were  treated with either DMSO or increasing doses of BX795 (0.5, 1 and 2 µM) for 4 h, determined at 48 h post-transfection by IP with anti-FLAG and IB with the indicated antibodies. f Endogenous TRIM23 S39 phosphorylation in HDFs that were either mock-treated or infected with mutHSV-1 (MOI 5) for 12 h and then treated for 4 h with either DMSO or BX795 (5 µM), determined by immunostaining with the indicated antibodies and confocal microscopy analysis. DAPI, nuclei (blue). Scale bar, 20 µm. g Quantification of p-S39-TRIM23-positive cells for the data shown in (f). h Top: Representative confocal microscopy images showing the colocalization of endogenous p-S39-TRIM23 with p-S172-TBK1 in HDFs that were either mock-treated or infected with WT HSV-1 or mutHSV-1 (MOI 0.3 for both) for 8 h, determined by Proximity Ligation Assay (PLA). Bottom: Quantification of the PLA signals for the data shown on top. PLA signal (magenta), HSV-1 ICP27 (green), nuclei (DAPI, blue). Scale bar, 20 μm. i S39 phosphorylation of TRIM23-FLAG in transiently transfected TBK1 KO single cell clones (Cl-1 and Cl-2) and WT HEK293T cells, determined at 48 h post-transfection by IP with anti-FLAG and IB with anti-pS39-TRIM23. j S39 phosphorylation of FLAG-tagged TRIM23 WT or S39A in TBK1 KO HEK293T cells that were co-transfected with either empty vector (–) or HA-TBK1, determined at 48 h post-transfection by IP with anti-FLAG and IB with anti-pS39-TRIM23. k GFP-LC3B puncta formation in TBK1 KO and WT A549 cells that were transiently transfected with GFP-LC3B (green) and either empty vector or the indicated FLAG-tagged TRIM23 constructs (red), assessed at 48 h post-transfection by immunostaining with anti-FLAG followed by confocal microscopy analysis. DAPI, nuclei (blue), scale bar, 20 µm. l Quantification of GFP-LC3B puncta for the data shown in (k). All data (a–l) are representative of at least two independent experiments (mean ± SD of n = 4 blots (d), n = 4 biological replicates (g); mean ± SEM of n  = 49, 48, 52, 51 and 49 cells respectively for the data plots from left to right (h); n  = 26 cells (l). **p < 0.01, ***p < 0.001, ****p < 0.0001 (two-tailed Student’s t test (d, g), two-tailed Student’s t test with Welch’s correction (h, l). NS not significant. Source data are provided as a Source Data file.

Fig. 5. cGAS signaling induces TRIM23 S39 phosphorylation via TBK1 to elicit autophagy.

a Endogenous p62 protein abundance in the WCLs of HDFs that were transfected for 30 h with the indicated siRNAs and then either mock-treated (–) or infected with mutHSV-1 (MOI 10) for 20 h, determined by IB with anti-p62 and actin (loading control). WCLs were further immunoblotted with anti-HSV-1 to confirm efficient infection. b Endogenous TRIM23 S39 phosphorylation in HDFs that were either mock-treated or stimulated with poly(dG:dC)-LyoVec (3 µg/mL), ISD-LyoVec (5 µg/mL) or poly(I:C)-LyoVec (1.5 µg/mL) for 16 h, determined by immunostaining with the indicated antibodies and confocal microscopy analysis. DAPI, nuclei (blue). Scale bar, 20 µm. c Quantification of p-S39-TRIM23-positive cells for the data shown in (b). d S39 phosphorylation and ubiquitination of TRIM23-myc in transiently transfected HEK293T cells that also co-expressed empty vector, FLAG-cGAS and FLAG-STING, or FLAG-RIG-I, determined at 20 h post-transfection by IP with anti-myc and IB with anti-p-S39-TRIM23 or anti-Ub. e S39 phosphorylation and ubiquitination of FLAG-TRIM23 stably expressed in HDFs that were either mock-treated (–) or transfected with poly(dG:dC)-LyoVec (3 µg/mL) for 20 h, determined by IP with anti-FLAG and IB with anti-p-S39-TRIM23 or anti-Ub. f GFP-LC3B puncta formation in HDFs that were transfected for 24 h with the indicated siRNAs and then co-transfected for 12 h with GFP-LC3B (green) followed by mock treatment or stimulation with poly(dG:dC)-LyoVec (3 µg/mL) for 20 h, determined by confocal microscopy analysis. DAPI, nuclei (blue). Scale bar, 20 µm. g Quantification of GFP-LC3B puncta for the data shown in (f). h Endogenous LC3B-I-to-II conversion in HDFs that were transfected for 30 h with either si.C or si.TRIM23 and then stimulated with poly(dG:dC)-LyoVec (1 and 3 µg/mL) for 20 h, determined by IB with anti-LC3B and anti-actin (loading control). Viperin, an ISG, was included as a control to confirm efficient poly(dG:dC)-LyoVec stimulation. i Endogenous p62 phosphorylation (at S403) in HDFs that were transfected for 30 h with either si.C or si.TRIM23 and then stimulated with poly(dG:dC)-LyoVec (1 and 3 µg/mL) for 20 h, determined by IB with anti-p-S403-p62. In this assay, cells were treated with a combination of Pepstatin A and E64D (10 µg/mL each) for 4 h prior to harvesting to block autophagic flux. j Phosphorylation of endogenous p62 (at S403) in HEK293T cells that were transfected for 30 h with the indicated siRNAs and then co-transfected for 20 h with either empty vector or plasmids expressing FLAG-tagged cGAS and STING, determined in the WCLs by IB with the indicated antibodies. Autophagic flux was blocked as in (i). k Representative confocal microscopy images showing the colocalization of endogenous p-S39-TRIM23 with endogenous p-S172-TBK1 in HDFs that were either mock-treated or transfected with poly(dG:dC)-Lyovec (3 µg/mL) for 16 h, determined by PLA. PLA signal (magenta); nuclei (DAPI, blue). Scale bar, 20 μm. l Quantification of the PLA signals for the data shown in (k). All data (a–l) are representative of at least two independent experiments (mean ± SD of n = 4 biological replicates (c); mean ± SEM of n = 120 cells (g); n = 60, 42, 41, 92, 36, 44, 57, 92 cells respectively for the data plots from left to right (l). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (two-tailed Student’s t test (c), or two-tailed Student’s t test with Welch’s correction (g, l). NS, not significant. Source data are provided as a Source Data file.

Fig. 6. TRIM23, TBK1 and STING form a complex after HSV-1 infection or dsDNA stimulation.

a Co-localization of endogenous p-S39-TRIM23, p-S172-TBK1 and STING in primary HDFs that were either mock-treated, infected with mutHSV-1 (MOI 5) or treated with poly(dG:dC)-LyoVec (3 µg/mL) for 16 h, determined by immunostaining with the indicated antibodies and confocal microscopy analysis. DAPI, nuclei (white). Scale bar, 20 μm. b Quantification of co-localization of the indicated proteins for the data shown in (a), determined by Pearson’s correlation coefficient. c Schematic representation of the human STING domain organization as well as the mutant constructs used for the interaction mapping studies. d Binding of V5-tagged TRIM23 to FLAG-tagged STING WT and mutants in transiently transfected HEK293T cells, determined at 48 h post-transfection by IP with anti-FLAG and IB with anti-V5. Data (a, b, and d) are representative of at least two independent experiments (mean ± SEM of n = 14 cells (b)). ****p < 0.0001 (two-tailed Student’s t test with Welch’s correction). Source data are provided as a Source Data file.

Fig. 7. HSE patient fibroblasts carrying a TBK1 ‘null’ mutation exhibit impaired TRIM23 activation.

a Phosphorylation of endogenous p62 (at S403) and TBK1 (at S172) in the WCLs of either healthy control (HC) or patient fibroblasts carrying the heterozygous TBK1 mutation G159A (TBK1(p.G159A/WT)) that were either mock-treated (–) or infected with mutHSV1 (MOI 1) for 16 h, determined by IB using the indicated antibodies. b S39 phosphorylation and ubiquitination of TRIM23-FLAG in transiently transfected TBK1 KO HEK293T cells that were reconstituted with HA-tagged TBK1 WT or mutants, determined at 48 h post-transfection by IP with anti-FLAG and IB with the indicated antibodies. c Endogenous LC3B-I-to-II conversion and p62 phosphorylation (at S403) in TBK1 KO HEK293T cells that were transiently transfected with HA-tagged TBK1 WT or mutants, determined at 24 h post-transfection in the WCLs by IB with the indicated antibodies. d GFP-LC3B puncta formation in HC or patient fibroblasts that were transiently transfected with GFP-LC3B (green) together with either empty vector or the indicated FLAG-tagged TRIM23 constructs (red), assessed at 24 h post-transfection by immunostaining with anti-FLAG and confocal microscopy analysis. DAPI, nuclei (blue). Scale bar, 20 µm. e Quantification of GFP-LC3B puncta for the data shown in (d). f Colocalization of FLAG-TRIM23 (green) and endogenous p62 (red) in HC and patient fibroblasts that were transfected with empty vector (control) or FLAG-tagged TRIM23 for 20 h and then either mock-treated, transfected with poly(dG:dC)-LyoVec (3 µg/mL) or infected with mutHSV-1 (MOI 5) for 20 h, determined by confocal microscopy after immunostaining with the indicated antibodies. DAPI, nuclei (blue). Scale bar, 20 µm. g Quantification of FLAG-TRIM23 colocalization with p62 for the data shown in (f), determined by Pearson’s correlation coefficient. All data (a–g) are representative of at least two independent experiments (mean ± SEM of n = 26 cells (e), n = 20 cells (g). ****p < 0.0001 (two-tailed Student’s t test with Welch’s correction (e, g). NS, not significant. Source data are provided as a Source Data file.