STING-Mediated IFI16 Degradation Negatively Controls Type I Interferon Production

Article

STING-Mediated IFI16 Degradation Negatively Controls Type I Interferon Production Graphical Abstract

Authors Dapei Li, Rongsheng Wu, Wen Guo, ..., Yanghua Qin, Feng Xu, Feng Ma

Correspondence [email protected]

In Brief Li et al. show that STING mediates negative feedback regulation of IFI16 and restricts type I IFN overproduction during immune responses to viruses such as HSV-1.

Highlights d

Overexpression of STING facilitates IFI16 degradation

d

E3 ligase TRIM21 plays a role in STING-mediated IFI16 degradation

d

IFI16-K3/4/6R mutation stabilizes IFI16 protein

d

IFI16-K3/4/6R facilitates type I IFN production

Li et al., 2019, Cell Reports 29, 1249–1260 October 29, 2019 ª 2019 Suzhou Institute of Systems Medicine. https://doi.org/10.1016/j.celrep.2019.09.069

Cell Reports

Article STING-Mediated IFI16 Degradation Negatively Controls Type I Interferon Production Dapei Li,1,2 Rongsheng Wu,1,2 Wen Guo,1,2 Lifen Xie,1,2 Zigang Qiao,1,2 Shengchuan Chen,1,2,3 Jingfei Zhu,1,2 Chaohao Huang,1,2,3 Jian Huang,4 Bicheng Chen,3 Yanghua Qin,5 Feng Xu,6 and Feng Ma1,2,7,* 1Center

for Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China 2Suzhou Institute of Systems Medicine, Suzhou 215123, China 3Department of Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China 4Department of Emergency, The First Affiliated Hospital of Soochow University, Suzhou 215006, China 5Department of Laboratory Diagnosis, Changhai Hospital of the Second Military Medical University, Shanghai 200433, China 6Department of Infectious Diseases, The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310009, China 7Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.celrep.2019.09.069

SUMMARY

g-interferon-inducible protein-16 (IFI16), a key DNA sensor, triggers downstream STING-dependent type I interferon (IFN-I) production and antiviral immunity. However, it is still unclear how to negatively regulate IFI16 to avoid excessive IFN-I production and autoimmunity. Here, we find that STING directly interacts with IFI16 and facilitates IFI16 degradation via the ubiquitin-proteasome pathway by recruiting the E3 ligase TRIM21. The 1-pyrin region of IFI16 is responsible for the IFI16-STING interaction, and the first three lysines in the N-terminal region of IFI16 are the key sites that lead to STING-mediated IFI16 ubiquitination and degradation. Compared to wildtype IFI16, a higher level of viral DNA triggered IFNb and antiviral IFN-stimulated gene expression, and thus less HSV-1 infection, was observed in the cells transfected with IFI16-K3/4/6R, an IFI16 mutant that is resistant to degradation. STING-mediated negative feedback regulation of IFI16 restricts IFN-I overproduction during antiviral immunity to avoid autoimmune diseases. INTRODUCTION g-interferon-inducible protein-16 (IFI16) has been identified as one of the most important innate immune sensors in eukaryotic cells (Almine et al., 2017; Goubau et al., 2013; Jønsson et al., 2017; Kerur et al., 2011; Monroe et al., 2014; Orzalli et al., 2015; Unterholzner et al., 2010). IFI16 recognizes both cytosolic and nuclear double-stranded DNA (dsDNA) from invaded DNA viruses such as vaccinia virus (VACV), herpes simplex virus 1 (HSV-1), and Kaposi sarcoma-associated herpesvirus (KSHV) (Ansari et al., 2015; Kerur et al., 2011; Unterholzner et al., 2010). The single-stranded DNA (ssDNA) from HIV-infected CD4+ T cells and the nuclear damaged DNA from etoposide-treated keratino-

cytes are also sensed by IFI16 (Dunphy et al., 2018; Monroe et al., 2014). DNA recognition by IFI16 triggers downstream stimulator of interferon genes-TANK-binding kinase 1-interferon regulatory factor 3 (STING-TBK1-IRF3) signaling to induce type I interferon (IFN-I) or apoptosis-associated speck-like protein containing a CARD (ASC)-caspase 1-dependent inflammasome to produce interleukin-1b (IL-1b) (Dunphy et al., 2018; Kerur et al., 2011; Unterholzner et al., 2010). IFN-I, including IFN-b and numerous IFN-a, play critical roles in the host immunity against viral infection by inducing >500 IFN-stimulated genes (ISGs) (Sadler and Williams, 2008). However, excessive production of IFN-I and overactivation of IFN-a/b receptor (IFNAR) downstream signaling lead to autoimmune diseases such as systemic lupus erythematosus (SLE) and Sjo¨gren syndrome (Biggioggero et al., 2010; Iwamoto et al., 2012; Nezos et al., 2015). Given that IFI16 itself is an ISG (Dawson and Trapani, 1995), IFI16-triggered IFN-I production induces more IFI16 protein by a positive feedback loop, which worsens the IFN-I-related autoimmune diseases. The level of IFI16 mRNA in the peripheral blood mononuclear cells (PBMCs) of patients with SLE is significantly higher than that in healthy people (Kimkong et al., 2009). Autoantibodies against IFI16 are detected in the sera from 29% of the patients with SLE (Seelig et al., 1994). In addition, high levels of circulating IFI16 and anti-IFI16 antibodies are prevalent in the sera of patients with Sjo¨gren syndrome (Alunno et al., 2015; Baer et al., 2016), as a result of nuclear export and secretion to the extracellular milieu as an autoantigen. Leaked IFI16 interacts with neighboring cells and functions as damage-associated molecular patterns (DAMPs) that lead to severe inflammatory damage (Bawadekar et al., 2015). Fine control of IFI16-triggered IFN-I production upon viral DNA recognition is necessary to properly clear the invading viruses without causing autoimmunity. To avoid the excessive accumulation of IFI16 protein and break the IFI16-IFN-I-IFI16 positive feedback loop, negative regulators directly targeting IFI16 should be identified. The ubiquitin-proteasome system (UPS) plays a critical role in modulating IFN-I production and downstream signaling during host innate immunity against viral infection (Bhoj and Chen, 2009; Heaton et al., 2016; Liu et al., 2018). Tripartite motif

Cell Reports 29, 1249–1260, October 29, 2019 ª 2019 Suzhou Institute of Systems Medicine. 1249 This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

(TRIM) proteins, a family of RING-finger ubiquitin E3 ligases, are widely recognized as key regulators in antiviral host defenses by targeting proteins involved in the induction of IFN-I and ISGs (Rajsbaum et al., 2014; van Gent et al., 2018). It has been reported that DDX41, an intracellular dsDNA and cyclic-di-GMP sensor in myeloid dendritic cells, is ubiquitinated at the Lys9/ 115 and degraded by TRIM21 (Parvatiyar et al., 2012; Zhang et al., 2011, 2013). Cyclic GMP-AMP synthase (cGAS), another essential cytosolic DNA virus sensor that triggers IFN-I signaling, undergoes robust K48-linked ubiquitination at Lys414, which is a recognition signal for p62-dependent selective autophagic degradation in resting cells (Chen et al., 2016; Sun et al., 2013). However, TRIM14, induced by IFN-I, stabilizes cGAS by recruiting USP14 to cleave the ubiquitin chains of cGAS at Lys414 (Chen et al., 2016). In addition, TRIM56 induces the Lys335 monoubiquitination of cGAS, resulting in a robust increase in its dimerization, DNA-binding activity, and cyclic guanosine monophosphate-AMP (cGAMP) production (Seo et al., 2018). STING, as both a direct cytosolic DNA sensor and an adaptor protein in IFN-I signaling, is also targeted and degraded by TRIM proteins, including TRIM30a and TRIM29 (Li et al., 2018; Wang et al., 2015). However, it is unclear whether TRIM proteinmediated UPS also negatively regulates the IFI16 protein level to maintain proper immune responses. In this study, we find that STING is not only a downstream adaptor protein of IFI16 to activate IFN-I transcription but it also negatively regulates upstream IFI16 stability by recruiting the E3 ligase TRIM21. STING-mediated degradation of IFI16 benefits the host cells to avoid excessive IFN-I production during antiviral innate immunity. RESULTS STING Negatively Controls IFI16 Protein Levels IFI16 recruits STING to activate downstream TBK1-IRF3 signaling and IFN-I production during viral DNA stimulation (Unterholzner et al., 2010). However, it is still unclear how IFI16 and STING interact and what the consequences are of the interaction. To investigate the precise molecular mechanism modulating the interaction between IFI16 and STING, we coexpressed STING with two isotypic variants of IFI16 in human embryonic kidney HEK293T cells. Unexpectedly, we found that the overexpression of STING significantly reduced the protein level of both IFI16 isoform1 and isoform2 in a STING dosedependent manner (Figure 1A). However, the expression of the other two well-known cytosolic DNA sensors upstream of STING, cGAS and DDX41, did not decline in STING-overexpressed cells (Figure S1A). IFI16 is inducible in the cells treated with IFNs and inflammatory stimuli such as IFN-g and IFN-b (Dawson and Trapani, 1995; Vanhove et al., 2015). Endogenous IFI16 protein levels were significantly induced by these stimuli in the human keratinocyte cell line HaCaT and human non-small-cell lung cancer A549 cells, while the overexpression of STING suppressed the accumulation of IFI16 induced by these stimuli (Figures 1B, 1C, and S1B). In HaCaT cells, IFN-g-induced IFI16 protein, but not IFI16 mRNA, declined 12 h post-IFN-g exposure (Figures 1B, S1B, and S1C), indicating that IFI16 protein is degraded after

1250 Cell Reports 29, 1249–1260, October 29, 2019

gene induction. Similarly, VACV- and HSV-1-induced IFI16 proteins were also degraded and more dramatically after STING overexpression, at the late stage of infection in HaCaT cells (Figures 1D and 1E). Nutlin-3, an antagonist of murine double minute 2 (Mdm2) E3 ligase of p53 that also acts as an IFI16 protein inducer (Shi et al., 2015), synergistically with IFN-g-induced IFI16 expression in a human osteosarcoma cell line, U2OS; STING also suppressed the IFI16 induction in these cells in a dose-dependent manner (Figure S1D). To determine whether STING also affects IFI16 transcription, we measured the IFI16 mRNA level in HEK293T cells transfected with different amounts of STING and found that the overexpression of STING did not decrease IFI16 transcripts (Figure 1F). The induction of IFI16 mRNA by IFN-g was also not downregulated by the overexpression of STING in HaCaT cells (Figure 1G). IFI16 degradation could be effectively rescued by proteasome inhibitor MG132 treatment, by STING small hairpin RNA/ small interfering RNA (shRNA/siRNA) transfection, or in STING knockout cells (Figures 1H, 1I, S1E, and S1F). STING negatively controls basal, inducible, and overexpressed IFI16 proteins in multiple cell lines without inhibiting IFI16 transcription. STING Facilitates IFI16 Degradation via the UPS We used an in vitro degradation system to confirm the hypothesis that STING facilitates IFI16 protein degradation. Lysates of STING-overexpressed HEK293T cells were incubated with purified FLAG-IFI16 proteins, and a similar reduction pattern of IFI16 was observed as in the STING-expressed HEK293T or HaCaT cells (Figures 1J and S1G). Furthermore, we measured the half-life of IFI16 through the cycloheximide (CHX) chase experiment and found that the half-life of IFI16 was obviously shortened in the STING-overexpressed cells (Figures 1K and 1L); this suggests that STING facilitates IFI16 protein degradation. Eukaryotic intracellular proteins are mainly degraded through lysosomal and proteasomal pathways. To investigate how STING facilitates IFI16 protein turnover, inhibitors targeting lysosomes or proteasomes were used to pretreat the cells. Consistent with the results in Figure S1E, the proteasome inhibitor MG132 effectively restored exogenous IFI16 levels in STINGoverexpressed HEK293T cells (Figure 2A), indicating that STING-mediated IFI16 degradation occurred through the proteasome pathway. However, the lysosome inhibitors also restored the exogenous IFI16 in HEK293T cells and the basal endogenous IFI16 in THP1 cells (Figures 2A and S2A), while the pretreatment of proteasome inhibitors MG132 and PS341 did not affect the basal protein level of IFI16 (Figure S2A). These results suggest that the accumulated or high-level IFI16 is degraded through both lysosomal and proteasomal pathways and the basal or low-level IFI16 is degraded mainly via the lysosomal system. Ubiquitination analysis showed that STING promoted the ubiquitination of IFI16 (Figures 2B and 2C), and this effect was significantly enhanced in the cells stimulated with IFN-g or Nutlin-3 (Figure 2D), which indicates that STING facilitates the ubiquitination of IFI16 and thus leads to IFI16 entry into proteasome-dependent degradation. The ubiquitination of the DNA sensor DDX41 was not increased by STING (Figure S2B). STING facilitated the interaction between IFI16 and 20S proteasome a4

Figure 1. STING Reduces IFI16 Stability (A) IFI16 isoform1-FLAG/isoform2-HA plasmids (2 mg) were co-transfected with gradually increasing amounts of Myc-STING vectors (0–1.5 mg) into HEK293T cells seeded in 6-well plates for 36 h, and the whole cell lysates were subjected to immunoblotting (IB). (B and C) Total cell lysates from HaCaT (B) or A549 (C) cells in 12-well plates were transfected with empty vectors (EVs) or STING vectors (0.5 mg) for 24 h, treated with IFN-g (20 ng/mL) for the indicated times (B) or with IFN-b (1000 U/mL) for 24 h (C), and then were subjected to IB analysis. (D and E) HaCaT cells in 12-well plates were transfected with EV or STING vectors (0.5 mg) for 20 h and then challenged with VACV (D) or HSV-1 (E) for the indicated times, virus MOI 1. Total cell lysates were subjected to IB analysis. (F) IFI16-FLAG vectors (2 mg) were co-transfected with gradually increasing amounts of Myc-STING vectors (0 mg, 0.25 mg, 0.5 mg) into HEK293T cells in 6-well plates for 30 h. The indicated mRNA levels were measured by RT-qPCR. The experiment was repeated 3 times, and the data are expressed as mean ± SD replicates of a representative experiment (n = 2; n.s., not significant; unpaired Student’s t test). (G) HaCaT cells in 12-well plates were transfected with EV or STING vectors (0.5 mg) for 20 h and then stimulated with IFN-g (20 ng/mL) for 12 h. The IFI16 mRNA levels were measured by RT-qPCR. The experiment was repeated 3 times, and the data are expressed as mean ± SD replicates of a representative experiment (n = 2; *p < 0.05, ***p < 0.001; n.s., not significant; unpaired Student’s t test). (H) HaCaT cells in 12-well plates were transfected with shSTING or shControl plasmids (1 mg); 48 h later, these cells were stimulated with IFN-g (20 ng/mL) for another 12 h. Total cell lysates were subjected to IB with the indicated antibodies. (I) WT or STING
/
HaCaT cells in 12-well plates were treated with IFN-g (20 ng/mL) for 12 h. Total cell lysates were subjected to IB analysis. (J) Anti-FLAG-conjugated agarose beads were purified, followed by elution with FLAG peptides. FLAG-IFI16 protein was incubated with EV or cell lysates from HEK293T cells transfected with hemagglutinin (HA)-tagged STING vectors, for the indicated times. The whole reaction mixture was subjected to IB analysis. (K and L) IFI16-FLAG plasmids (2 mg) were co-transfected with EV or Myc-STING vectors (1 mg) in 6-well seeded HEK293T cells. After CHX (100 mg/mL) treatment, cells were harvested at the indicated time points, and total cell lysates were detected by IB (K). The optical density of the IFI16 protein bands was acquired with ImageJ software (L). Protein levels obtained from three western blotting (WB) images were normalized to a-tubulin. The data are expressed as mean ± SD replicates of a representative experiment (n = 3; *p < 0.05, **p < 0.01, ***p < 0.001; n.s., not significant; unpaired Student’s t test).

subunit PSMA7, further confirming the role of STING in assisting in IFI16 degradation through the proteasome pathway, as IFI16 ubiquitination levels were also enhanced by the proteasome inhibitor MG132 (Figures S2C and S2D). There are seven types of poly-ubiquitination linkages involving protein degradation, activation, or localization. Further experiments showed that STING mainly enhanced K27, K48, and K63-linked poly-ubiquitination of IFI16, which suggests that STING-mediated various linkages of IFI16 ubiquitination may control different aspects of IFI16, including IFI16 stability (Figure 2E). Therefore, STING is not only an adaptor protein downstream of IFI16 to activate the TBK1-IRF3-IFN-a/b signaling axis but it also negatively feedback regulates the excessive upstream IFI16 protein via the UPS.

E3 Ligase TRIM21 Is Required for STING-Mediated IFI16 Degradation The ubiquitin E3 ligase targeting IFI16 is still unknown, although we have observed that STING facilitates IFI16 ubiquitination and subsequent degradation. To identify the E3 ligase that catalyzes IFI16 ubiquitination, FLAG-IFI16-interacting proteins were immunoprecipitated and analyzed by nano-liquid chromatographyelectrospray ionization-tandem mass spectrometry (nano-LCESI-MS/MS) assay. The overexpression of STING contributed to more interactions between IFI16 and its binding proteins (Figure 3A, first and second lanes) and also more ubiquitin modification of IFI16 (Figure S3A). Eight differential protein bands were analyzed by MS (Table S1). TRIM21 was identified as an

Cell Reports 29, 1249–1260, October 29, 2019 1251

Figure 2. STING Promotes the Ubiquitination and Proteasomal Degradation of IFI16 (A) FLAG-IFI16 plasmids (2 mg) were transfected with EV or Myc-STING (0.5 mg) in 6-well plates seeded with HEK293T cells for 24 h, followed by MG132 (5 or 10 mM) treatment for 12 h. Total cell lysate was subjected to IB with the indicated antibodies. (B–D) EV or IFI16 vectors (4 mg) were co-transfected into HEK293T cells in a 60-mm dish with STING (0.5 mg) or ubiquitin (Ub)/Ub (G76A, with a mutation on glycine 76, which is responsible for binding to the lysine of target protein, to alanine) (2 mg) plasmids. After 24 h, MG132 (10 mM) without (B and C) or with (D) Nutlin-3 (30 mM) and IFN-g (20 ng/mL) were added into the cell culture medium for 12 h (B) or 8 h (C and D). The whole cell lysates (WCLs) were subjected to analysis by immunoprecipitation (IP), followed by IB detection. (E) EV or IFI16 plasmids (4 mg) were co-transfected into HEK293T cells in a 60-mm dish with STING (0.5 mg) or Ub (2 mg) plasmids. After 30 h, IFN-g (20 ng/mL) and MG132 (10 mM) were exposed to cells for 8 h, and the WCLs were subjected to analysis by IP, followed by IB detection.

IFI16-interacted E3 ligase in STING-overexpressed HEK293T cells, with a confidence level as high as 99% (Figure 3B; Table S1). To verify whether TRIM21 is the E3 ubiquitin ligase of IFI16, we co-expressed IFI16 with different doses of TRIM21 in HEK293T cells and found a gradually decrease in the IFI16 protein level in a TRIM21 dose-dependent manner (Figure 3C). Endogenous IFI16 in HaCaT cells also decreased during TRIM21 expression (Figure 3D). When TRIM21 was knocked down by an siRNA (Figures S3B and S3C), exogenously expressed IFI16 in HEK293T cells as well as endogenous IFI16 in HaCaT cells were both well restored (Figures 3E, 3F, and S3D), suggesting that TRIM21 directly regulates the IFI16 pro-

1252 Cell Reports 29, 1249–1260, October 29, 2019

tein level. Further ubiquitination analysis in the IFI16-overexpressed HEK293T cells showed that the overexpression of TRIM21 significantly enhanced IFI16 ubiquitination and reduced the IFI16 protein level (Figure 3G). STING promoted TRIM21-induced IFI16 ubiquitination and the IFI16-TRIM21 interaction (Figure 3G). Similarly, IFN-g treatment also promoted TRIM21-induced IFI16 ubiquitination and the IFI16-TRIM21 interaction (Figure S3E), which was consistent with the results indicated in Figure 2D that IFN-g treatment enhanced IFI16 ubiquitination. The ubiquitination of endogenous IFI16 in HaCaT cells induced by STING was obviously attenuated in the TRIM21 knockdown cells (Figure 3H), and STINGmediated IFI16 degradation was also rescued after TRIM21 siRNA transfection (Figures 3F and 3H). These results indicate that STING-mediated IFI16 degradation via the ubiquitinproteasome pathway is TRIM21 dependent. TRIM21 is identified as a ubiquitin E3 ligase of IFI16. The First Three Lysines in the N-Terminal Region of IFI16 Are Critical for Degradation To map the critical region that is required for STING-mediated IFI16 ubiquitination and degradation, we made a series of IFI16 truncated mutants to test their stability (Figure 4A). Overexpression of STING led to the degradation of all of these mutants, including the one containing the 127 amino acids (aa) of the IFI16 N-terminal fragment (Figure 4B). We fused GFP with the

Figure 3. TRIM21 Is Required for the STINGMediated IFI16 Degradation (A and B) EV, FLAG-IFI16 WT, or FLAG-IFI16 K3/4/ 6R plasmids (8 mg) were co-transfected into HEK293T cells in a 100-mm dish with HA-STING (1.0 mg). After 24 h, IFN-g (20 ng/mL) and MG132 (10 mM) were administered for another 8 h, and immunoprecipitates separated by anti-FLAG antibody from total lysates were analyzed by silver staining after SDS-PAGE (A). The peptides, including 187NFLVEEEQR195 of TRIM21, were identified through nano-LC-ESI-MS/MS analysis (B). See also Table S1. (C) FLAG-IFI16 vectors (2 mg) were co-transfected into HEK293T cells in 6-well plates with increasing doses of HA-TRIM21 plasmids (0–2 mg) for 30 h. The total cell lysates were subjected to IB detection. (D) Endogenous IFI16 protein levels were analyzed by IB from HaCaT cells transfected with HATRIM21 plasmid (1–2 mg) in 6-well plates for 26 h, with or without IFN-g (20 ng/mL) treatment for 8 h. (E and F) TRIM21 siRNA (10 nM for E, 20 nM for F) or negative control (NC) siRNA was transfected into HEK293T (E) or HaCaT (F) cells pre-seeded in a 12-well plate overnight. After 24 h, FLAG-IFI16 isoform1/isoform2 (1 mg) (E) or HA-STING (0.5 mg) (F) vectors were transfected into the corresponding groups for 40 h with (F) or without (E) IFN-g (20 ng/mL) treatment for 8 h. The total cell lysates were subjected to IB detection. (G) The ubiquitination of transiently expressed IFI16 underwent IP and IB analysis in HEK293T (60-mm dish) transfected with FLAG-IFI16 (4 mg), HA-TRIM21 (2 mg), and HA-STING (0.5 mg) plasmids for 30 h. MG132 (10 mM) was added into the cell culture medium 8 h before harvest. (H) The ubiquitination of endogenous IFI16 underwent IP and IB analysis in HaCaT cells (100-mm dish) transfected with HA-STING (1 mg) and EV (1 mg) plasmids for 48 h or TRIM21 siRNA (10 nM) for 72 h. MG132 (10 mM) and IFN-g (20 ng/mL) were added into the cell culture medium 8 h before harvest.

first 127 aa of IFI16, termed GFP-127 aa. Much less GFP fluorescence was detected in the GFP-127 aa-transfected cells than in the wild-type (WT) GFP-transfected cells (Figure S4A). The overexpression of STING further decreased GFP fluorescence in the GFP-127 aa-transfected cells, while it did not affect it in the WT GFP-transfected cells (Figure S4A). Given that the degradation of excessive IFI16 occurs mainly through the UPS, we next screened all of the possible ubiquitination sites located in the first 127 aa region of IFI16 by point mutations. All of the candidate lysines in the 1-127 aa were mutated to arginine in a series of IFI16 point mutant constructs. The overexpression of STING led to the degradation of most of these mutants in HEK293T cells, except the IFI16-K3/4/6R, where the first three lysines in the N-terminal region of IFI16 were mutated (Figures 4C, S4B, and S4C), suggesting that the K3/4/6 are the core sites for IFI16 stability. TRIM21 was identified as a ubiquitin E3 ligase of IFI16 in Figure 3, mediating the ubiquitination and degradation of IFI16 efficiently. Consistent with our expecta-

tions, we found that the IFI16-K3/4/6R mutant was more resistant to degradation by TRIM21 (Figure 4D). In addition, less ubiquitination of IFI16-K3/4/6R was detected compared to IFI16-WT, and STING overexpression did not affect the ubiquitination of IFI16-K3/4/6R (Figure 4E). Further validation was supported by the CHX chase experiment. The protein translated from IFI16K3/4/6R was much more stable and displayed a longer half-life than that from IFI16-WT (Figures 4F and 4G). Like IFI16-WT, the IFI16-K3/4/6R mutant associated with STING (Figure S4D). These results therefore indicate that the first three lysines—K3/ 4/6—in the N-terminal region of IFI16 are core sites for IFI16 ubiquitination, which is critical for STING-mediated degradation. The 1-Pyrin Region of IFI16 Is Responsible for the IFI16STING Interaction IFI16 associates with STING in both the cells stimulated with VACV 70-mer DNA and a cell-free system (Jakobsen and Paludan, 2014; Unterholzner et al., 2010). However, the region of

Cell Reports 29, 1249–1260, October 29, 2019 1253

Figure 4. K3/4/6 Are Critical Sites for STING-Mediated IFI16 Degradation (A–C) IFI16 truncated mutants containing N-terminal fragments (1 mg) (B) or lysine-to-arginine (KR) mutants (1 mg) in which lysine has been mutated to arginine (C) were co-transfected with EV (0.5 mg) or STING (0.5 mg) in HEK293T cells seeded in 12-well plates for 36 h, and total cell lysates were analyzed by IB. Schematic diagram of IFI16 domains (Ni et al., 2016) and the mutation sites is shown in (A). (D) Two IFI16 isoforms and the K3/4/6R mutant plasmids (1 mg) were co-transfected with gradually increasing amounts of HA-TRIM21 vectors (0–1 mg) in HEK293T cells seeded in 12-well plates for 30 h. Total cell lysates were subjected to IB analysis. (E) HEK293T cells in a 60-mm dish were transfected with IFI16 (4 mg), EV (0.5 mg), or STING (0.5 mg) vectors for 30 h. MG132 (10 mM) and Nutlin-3 (30 mM) were added into the cell culture medium 8 h before harvest. Anti-FLAG immunoprecipitates from total cell lysates were subjected to IB analysis. (F and G) FLAG-IFI16 WT or K3/4/6R mutant plasmids (2 mg) were transfected into HEK293T cells in 6-well plates. After CHX (100 mg/mL) treatment, cells were harvested at the indicated time points, and total cell lysates were detected by IB (F). The optical density of IFI16 or K3/4/6R protein bands in WB was acquired with ImageJ software. Protein levels obtained from 2 WB images were normalized to a-tubulin. The data are expressed as mean ± SD replicates of a representative experiment (n = 2; **p < 0.01; unpaired Student’s t test) (G).

IFI16 that is responsible for the IFI16-STING interaction is unknown. To identify this region in IFI16, we first overexpressed two isoforms of IFI16 with STING in HEK293T cells and found that both isoforms interacted with STING and were degraded by STING expression very well (Figures 5A and S5A). The endogenous IFI16 also associated with STING well in human macrophages THP1 cells (Figure 5B). In addition, an in vitro binding assay also indicated the significant binding between IFI16 and STING (Figure S5B). An in situ proximity ligation assay (PLA) further showed direct interaction of endogenous IFI16STING in HaCaT cells. IFI16-STING complexes significantly increased during the first 4 h and gradually decreased later (Figures 5C–5E), which is consistent with the degradation of IFI16 mediated by STING to avoid continuous downstream signaling activation. The pyrin domain (PYD) of IFI16 is thought to mediate proteinprotein interaction, such as ASC and BRCA1 (Jakobsen and Paludan, 2014). IFI16 mutants lacking the first 84 aa and 127 aa were constructed, called IFI16(D1-PYD) and IFI16(D1-127), respectively (Figure 4A). No interaction was observed between STING and two IFI16 mutants (Figure 5F). IFI16(D1-PYD) and IFI16(D1-127) mutants also failed to be degraded by STING

1254 Cell Reports 29, 1249–1260, October 29, 2019

overexpression (Figure 5G). The ubiquitin modification of these two IFI16 mutants was not enhanced by STING (Figure S5C). These results show that the 1-PYD region of IFI16 is responsible for interacting with STING, and this region also contains the K3/4/6 mediating IFI16 ubiquitination and degradation by STING. STING-Mediated IFI16 Degradation Prevents Excessive IFN-I Production The IFI16-K3/4/6R mutant is more resistant to STING-mediated degradation. Therefore, we thought that this mutant would trigger stronger STING downstream signaling and more IFN-I production. As we expected, IFI16-K3/4/6R was able to activate higher transcription activity of the IFN-b promoter in the presence of STING than IFI16-WT did (Figure 6A). IFI16-WT or IFI16-K3/4/6R were transiently transfected into the IFI16
/
HaCaT cells which were defective in IFN-b signals (Figures 6B and S6A). More phosphorylated STAT1(Tyr701) and the ISG MX1 were detected in the IFI16-K3/4/6R-transfected cells than in the IFI16-WT-transfected cells (Figures 6B and S6B). Similar results were obtained from the assays performed in the pancreatic tumor PANC1 cells (Figure S6C). Next, IFI16
/
HaCaT cells were stably restored with IFI16-WT and IFI16-K3/4/6R mutant (Figure 6C), and these cells were activated by transfection with

Figure 5. 1-PYD Domain of IFI16 Is Responsible for Interaction with STING (A and B) Immunoprecipitation analysis of the association of exogenous IFI16-STING in HEK293T cells in a 60-mm dish transfected with IFI16 (4 mg), EV (0.5 mg), or STING (0.5 mg) vectors for 30 h (A) and endogenous IFI16-STING in THP1 cells stimulated with (B) or without (A) lipopolysaccharide (LPS) (100 ng/mL) for 12 h. (C–E) Endogenous IFI16-STING interaction under natural conditions or IFN-g (10 ng/mL) stimulated HaCaT cells by in situ PLA technology. The schematic diagram indicates the principle of in situ PLA measuring endogenous IFI16-STING protein interactions in cells (C). The experimental groups (0–8 h) were detected with anti-IFI16 and antiSTING antibodies, and the NC group was only added with anti-IFI16 antibody (D), followed by subsequent amplification reactions. The red spots represent the positive interaction complexes. The data are expressed as mean ± SD replicates of a representative experiment. Quantification of IFI16-STING interaction complexes from 3 independent fields contains at least 100 cells. Error bars represent SDs; **p < 0.01; unpaired Student’s t test (E). (F and G) Mutants of FLAG-tagged D1-127 and D1PYD that contain N-terminal fragment deletion (4 mg) were constructed and co-transfected with Myc-STING (0.5 mg) in HEK293T cells in a 60-mm dish for 30 h. Anti-FLAG immunoprecipitated (F) and the WCL (G) were detected by IB.

viral dsDNA mimic herring testis DNA (HT-DNA). More IFN-b and ISG56 mRNA were induced in the IFI16-K3/4/6R-restored cells than in the IFI16-WT-restored cells (Figures 6D and 6E). Consistent results were obtained when these cells were activated by the HSV-1 dsDNA mimic HSV 60-mer (Figures 6F and 6G). We also compared the IFN-I activation ability of IFI16-WT and IFI16-K3/4/6R mutant in the human macrophage THP1 cells differentiated by phorbol 12-myristate 13-acetate (PMA) stimulation for 48 h. Consistent with the results from HaCaT cells, the IFI16-K3/4/6R-transfected IFI16
/
THP1 cells transcribed more IFN-b and ISG56 mRNA than IFI16-WT-transfected cells, when these cells were activated by transfected HT-DNA and HSV 60-mer (Figures S6D–S6G).

To compare the antiviral activity of IFI16-WT and IFI16-K3/4/6R during DNA virus HSV-1 infection, we challenged the IFI16
/
HaCaT cells which were restored with IFI16-WT and IFI16-K3/4/ 6R with HSV-1, and found that much stronger activation of IFN-I downstream signaling included higher phosphorylated (p-)STAT1(Tyr701) (Figure 6H) and antiviral ISG OAS1 induction (Figure S6H), suggesting the better antiviral activity of IFI16-K3/4/6R than of IFI16-WT. A similar result was obtained in the IFI16-K3/4R cell lines, one of the clones closer to K3/4/6R mutation, which showed higher IFN-b and antiviral ISG MX1 mRNA induction ability (Figures 6I and S6I), indicating a critical role for K3/4 in IFI16 function. As we expected, less HSV-1-luciferase reporter expression was detected in the IFI16-K3/4/6R-restored cells than in the IFI16-WT-restored cells (Figure 6J). IFI16-K3/4/6R, a more stable mutant of IFI16, is able to trigger more IFN-I production and IFN-I-dependent antiviral activity. However, excessive IFN-I production is detrimental to the host cells. Therefore, STING-mediated IFI16 degradation could prevent excessive IFN-I production and benefit the host cells. In summary, we have described that STING-mediated IFI16 degradation negatively regulates viral DNA-induced IFN-I

Cell Reports 29, 1249–1260, October 29, 2019 1255

Figure 6. STING-Mediated Degradation of IFI16 Attenuates IFN-b Signaling (A) HEK293T cells in 24-well plates were cotransfected with a firefly IFN-b-luciferase reporter (150 ng), renilla luciferase transfection control (20 ng), and the indicated dose of FLAG-IFI16/HASTING expressing plasmids for 24 h. Relative firefly luciferase activity was quantified 24 h post-transfection. The experiment was repeated 3 times, and the data are expressed as mean ± SD replicates of a representative experiment (n = 2; **p < 0.01; unpaired Student’s t test). (B) Total cellular supernatant from IFI16
/
HaCaT cells transfected with EV, FLAG-IFI16-WT, or FLAG-IFI16-K3/4/6R plasmids (1 mg) in 12-well plates for 28 h, and stimulated by IFN-g (20 ng/mL) for 20 h, were subjected to IB detection with the indicated antibodies. (C–G) EV, FLAG-IFI16-WT, and FLAG-IFI16-K3/4/ 6R plasmids (6 mg) were transfected into a 100-mm dish of cultivated IFI16
/
HaCaT cells, which were screened with G418 (1,000 mg/mL) antibiotics 40 h later. After 1 week, protein expressions were identified by IB (C). HT-DNA (D and E) or HSV 60-mer (F and G) (2 mg/mL) were transfected for IFN-b signal stimulation, and the mRNA levels of IFN-b and ISG56 were detected by RT-qPCR 8 h later. The experiment was repeated 3 times, and the data are expressed as mean ± SD replicates of a representative experiment (n = 2; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t test). (H) EV, FLAG-IFI16-WT, and FLAG-IFI16-K3/4/6R (1 mg) transiently expressing IFI16
/
HaCaT cells in 12-well plates for 18 h were infected by HSV-1 (MOI = 0.5) virus for the indicated times. The STAT1 phosphorylation was identified by IB. (I) IFI16-WT, IFI16-KO, and IFI16-K3/4R HaCaT cell clones were infected with HSV-1 (MOI = 1) for 12 h. The mRNA levels of IFN-b and MX1 were detected by RT-qPCR. The experiment was repeated 3 times, and the data are expressed as mean ± SD replicates of a representative experiment (n = 2; *p < 0.05, ***p < 0.001; unpaired Student’s t test). (J) HEK293T cells in 24-well plates were cotransfected with EV, FLAG-IFI16-WT, and FLAGIFI16-K3/4/6R plasmids (600 ng), and after 8 h, they were infected with HSV-1 (MOI = 1) carrying a firefly luciferase reporter. Relative firefly luciferase activity was quantified 24 h post-infection. The experiment was repeated 3 times, and the data are expressed as mean ± SD replicates of a representative experiment (n = 4; ***p < 0.001; n.s., not significant; unpaired Student’s t test).

production to avoid excessive antiviral immunity. We have outlined a negative feedback regulation pathway that controls the excessive induction of IFI16 during DNA virus infection (Figure 7). DISCUSSION It has been well established that IFI16 plays an important role in inhibiting tumorigenesis by cooperating with p53 (Choubey and Panchanathan, 2016; Fujiuchi et al., 2004; Lin et al., 2017). Several studies have also described IFI16 as one of the critical

1256 Cell Reports 29, 1249–1260, October 29, 2019

DNA sensors in the recognition of pathogenic DNA and nuclear damaged DNA (Almine et al., 2017; Dunphy et al., 2018; Orzalli et al., 2015; Unterholzner et al., 2010). IFI16 shuttles between the cytoplasm and the nucleus and recognizes both dsDNA and ssDNA (Ansari et al., 2015; Monroe et al., 2014). Although it was known that IFI16 is inducible in cells stimulated with IFN-I or IFN-II or infected with HSV-1 (Dawson and Trapani,
ska et al., 2018), it is still unclear whether there is 1995; Jab1on an intrinsic pathway that negatively controls IFI16 levels to maintain normal immunity against infection. The IFI16 protein level is

Figure 7. Working Model As a DNA sensor, IFI16 recognizes the genomic DNA from invaded viruses, which triggers downstream STING-dependent IFN-I and ISGs production, including IFI16 protein. To eliminate the excessive IFI16, STING directly interacts with IFI16 and facilitates IFI16 ubiquitination on the lysine3/4/6 and degradation via the ubiquitin-proteasome pathway by recruiting the ubiquitin E3 ligase TRIM21. This negative feedback regulation of IFI16 restricts IFN-I overproduction during antiviral immunity to avoid autoimmune diseases.

not increasing constantly under IFN-g stimulation, although IFI16 transcription is continuously induced by IFN-g in HaCaT cells. DNA viruses such as HSV-1 and VACV do not induce IFI16 expression constantly at the late stage of viral infection, and the overexpression of STING promotes IFI16 protein degradation at the late stage of infection. Proteasome inhibitor MG132 inhibits both inducible and overexpressed IFI16 degradation. These results suggest that an intrinsic mechanism controls IFI16 accumulation to avoid excessive or continuous activation. The present study has demonstrated that STING, a downstream adaptor protein of IFI16, negatively regulates the IFI16 protein level by facilitating its K48-linked ubiquitination and degradation. In addition, our unbiased screening via the IP and nano-LC-ESIMS/MS assays has identified that TRIM21 is a ubiquitin E3 ligase promoting IFI16 turnover. The viral ubiquitin ligase ICP0 from HSV-1 has been identified as inducing IFI16 degradation (Orzalli et al., 2012). However, Cuchet-Lourenc¸o et al. (2013) believe that ICP0 is neither sufficient nor necessary for the degradation of IFI16 during HSV-1 infection. We have shown that TRIM21 is identified as an endogenous E3 ligase, which is recruited by STING to catalyze IFI16 ubiquitination. It has been reported that TRIM21 is also an IFN-inducible E3 ligase that mediates the K48-linked ubiquitination and degradation of DDX41 and negatively regulates the innate immune response to intracellular dsDNA (Zhang et al., 2013), which suggests a universal role for TRIM21 in modulating the cellular levels of cytosolic DNA sensors.

IFI16 functions as DAMPs or induces anti-IFI16 antibody in sera, which leads to severe inflammatory damage and thus autoimmunity (Alunno et al., 2015; Baer et al., 2016; Bawadekar et al., 2015). IFI16-triggered overproduction of IFN-I via the IFI16STING-TBK1-IRF3 signaling axis also highly correlates with autoimmune diseases such as SLE and Sjo¨gren syndrome (Nezos et al., 2015). To eliminate the high risk of persistent IFI16 induction or excessive IFI16 accumulation, negative regulators are required to directly target IFI16 degradation. Our results have indicated that TRIM21 is recruited by STING contributing to a negative feedback regulation of IFI16. Therefore, based on our conclusions from the present study, CRISPR activation (CRISPRa) system targeting and driving TRIM21 transcription is a promising strategy to inhibit the excessive IFI16 (Gilbert et al., 2014; Konermann et al., 2015). In addition, the TrimAway system, which uses TRIM21 cooperating with IFI16 antibody, is also able to remove the native IFI16 proteins in multiple cells (Clift et al., 2017, 2018). We have identified that the first three lysines (K3/4/6) located at the N-terminal of IFI16 are critical for its stability. Mutation of K3/4/6 significantly abolishes STING-mediated IFI16 ubiquitination and degradation. Consistently, IFI16-K3/4/6R mutation extends the half-life of IFI16 protein, induces more STINGdependent IFN-I and downstream ISG expression, and thus makes the cells more resistant to DNA virus such as HSV-1 infection. Mutation of K3/4 by CRISPR-Cas9 technology in HaCaT cells leads to further induction of IFN-b and MX1 during HSV-1 infection. In a cohort of patients with genital herpes and healthy controls, the minor G allele of the IFI16 SNP rs2276404, which locates in the 50 UTR of IFI16, is associated with resistance to HSV-2 infection. Furthermore, the combination of this allele with the C allele of rs1417806 is significantly overrepresented in uninfected individuals (Eriksson et al., 2017). It will be interesting to investigate whether any SNPs locate or affect K3/4/6 of IFI16 and control IFI16 protein stability. Moreover, in future studies, we will investigate whether the IFI16-K3/4/6R transgenic mice protect themselves better than do WT mice during DNA virus infection, or even appear autoimmune because of the higher levels of IFI16 and IFN-I. There are nuclear localization sequences and acetylation sites in the region linking the HINa and HINb domains of IFI16 (Li et al., 2012); and our results show that ubiquitination sites also exist ahead of the PYD domain, which suggest that the non-structural regions of IFI16 play important roles in IFI16 post-translational modification and functions. IFI16 interacts with many proteins involved in host antiviral immunity, inflammasomes, and cell apoptosis pathways, such as STING, AIM2, ASC, p53, and BRCA1 (Jakobsen and Paludan, 2014; Veeranki and Choubey, 2012). HIN-200 or PYD domains are responsible for the interaction between IFI16 and these proteins (Veeranki and Choubey, 2012). However, it is unclear which region of IFI16 is required for the IFI16-STING interaction, although we and other groups believe that there is robust interaction between these two proteins (Jakobsen and Paludan, 2014; Unterholzner et al., 2010). Here, we have described that PYD is necessary for IFI16 binding with STING, and the first six amino acids located at the N-terminal of IFI16 potentially regulate the IFI16-STING interaction. Thus, we suggest that the

Cell Reports 29, 1249–1260, October 29, 2019 1257

1-PYD region of IFI16 could activate downstream signaling by interacting with STING, similar to the two CARD domains of retinoic acid-inducible gene I (RIG-I) interacting with mitochondrial antiviral-signaling protein (MAVS) to activate downstream TBK1IRF3 signaling (Kawai et al., 2005; Meylan et al., 2005; Seth et al., 2005). Fine regulation of the IFI16 expression level during DNA virus infection is important. A high level of IFI16 triggers strong IFN-I-dependent antiviral immunity, following with the overactivation of host innate immunity, which leads to autoimmunity. Negative feedback regulation of IFI16 mediated by STINGTRIM21 attenuates antiviral immunity and protects cells from self-injury. The present study provides targets and strategies to maintain proper protein levels of IFI16, and thus avoids autoimmune diseases.

AUTHOR CONTRIBUTIONS

STAR+METHODS

Almine, J.F., O’Hare, C.A., Dunphy, G., Haga, I.R., Naik, R.J., Atrih, A., Connolly, D.J., Taylor, J., Kelsall, I.R., Bowie, A.G., et al. (2017). IFI16 and cGAS cooperate in the activation of STING during DNA sensing in human keratinocytes. Nat. Commun. 8, 14392.

Detailed methods are provided in the online version of this paper and include the following: d d d

d

d d

KEY RESOURCES TABLE LEAD CONTACT AND MATERIALS AVAILABILITY EXPERIMENTAL MODEL AND SUBJECT DETAILS B Cell lines B Plasmids and Generation of IFI16 Mutants METHOD DETAILS B Cells transfection and stimulation B Generation of STING stably expressing U2OS cells
/
B Generation of STING HaCaT cells B Generation of IFI16-K3/4R HaCaT cells B Immunoprecipitation and Immunoblot Analysis B In vitro degradation/binding assay B Quantitative real-time PCR (RT-qPCR) B CHX-chase Assays B RNAi B Luciferase assay B HSV-luciferase assay B In Situ Proximity Ligation Assay (In Situ PLA) QUANTIFICATION AND STATISTICAL ANALYSIS DATA AND CODE AVAILABILITY

SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/10.1016/j. celrep.2019.09.069. ACKNOWLEDGMENTS We appreciate the excellent technical support from the RNA technology platform of the Suzhou Institute of Systems Medicine. We also thank Dr. Leonie Unterholzner (University of Dundee) for providing the control and IFI16
/
HaCaT cells, and Dr. M.R. Jakobsen (University Medical Center Utrecht) for providing the control and IFI16
/
THP1 cells. This work was supported by the National Key Research and Development Program of China (2018YFA0900803); the NFSC (31800760, 81471606, 31670883, and 31771560); National Thousand Youths Talents Program (to F.M.); CAMS Initiative for Innovative Medicine (2016-I2M-1-005); the non-profit Central Research Institute Fund of CAMS (2016ZX310189, 2016ZX310194, and 2017NL31004); NSF of Jiangsu Province (BK20170408); and the Shanghai Pujiang Program (16PJD001).

1258 Cell Reports 29, 1249–1260, October 29, 2019

F.M. and D.L. conceived the idea for the study and designed the experiments. D.L., R.W., W.G., S.C., L.X., Z.Q., J.Z., and C.H. performed all of the experiments. J.H., B.C., F.X., and Y.Q. provided the reagents and suggestions. F.M. and D.L. analyzed the data and wrote the manuscript. DECLARATION OF INTERESTS The authors declare no competing interests. Received: January 20, 2019 Revised: July 22, 2019 Accepted: September 20, 2019 Published: October 29, 2019 REFERENCES

Alunno, A., Caneparo, V., Carubbi, F., Bistoni, O., Caterbi, S., Bartoloni, E., Giacomelli, R., Gariglio, M., Landolfo, S., and Gerli, R. (2015). Interferon gamma-inducible protein 16 in primary Sjo¨gren’s syndrome: a novel player in disease pathogenesis? Arthritis Res. Ther. 17, 208. Ansari, M.A., Dutta, S., Veettil, M.V., Dutta, D., Iqbal, J., Kumar, B., Roy, A., Chikoti, L., Singh, V.V., and Chandran, B. (2015). Herpesvirus Genome Recognition Induced Acetylation of Nuclear IFI16 Is Essential for Its Cytoplasmic Translocation, Inflammasome and IFN-b Responses. PLoS Pathog. 11, e1005019. Baer, A.N., Petri, M., Sohn, J., Rosen, A., and Casciola-Rosen, L. (2016). Association of Antibodies to Interferon-Inducible Protein-16 With Markers of More Severe Disease in Primary Sjo¨gren’s Syndrome. Arthritis Care Res. (Hoboken) 68, 254–260. Bawadekar, M., De Andrea, M., Gariglio, M., and Landolfo, S. (2015). Mislocalization of the interferon inducible protein IFI16 by environmental insults: implications in autoimmunity. Cytokine Growth Factor Rev. 26, 213–219. Bhoj, V.G., and Chen, Z.J. (2009). Ubiquitylation in innate and adaptive immunity. Nature 458, 430–437. Biggioggero, M., Gabbriellini, L., and Meroni, P.L. (2010). Type I interferon therapy and its role in autoimmunity. Autoimmunity 43, 248–254. Chen, M., Meng, Q., Qin, Y., Liang, P., Tan, P., He, L., Zhou, Y., Chen, Y., Huang, J., Wang, R.F., and Cui, J. (2016). TRIM14 Inhibits cGAS Degradation Mediated by Selective Autophagy Receptor p62 to Promote Innate Immune Responses. Mol. Cell 64, 105–119. Choubey, D., and Panchanathan, R. (2016). IFI16, an amplifier of DNA-damage response: role in cellular senescence and aging-associated inflammatory diseases. Ageing Res. Rev. 28, 27–36. Chu, V.T., Weber, T., Wefers, B., Wurst, W., Sander, S., Rajewsky, K., and €hn, R. (2015). Increasing the efficiency of homology-directed repair for Ku CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat. Biotechnol. 33, 543–548. Clift, D., McEwan, W.A., Labzin, L.I., Konieczny, V., Mogessie, B., James, L.C., and Schuh, M. (2017). A Method for the Acute and Rapid Degradation of Endogenous Proteins. Cell 171, 1692–1706.e18. Clift, D., So, C., McEwan, W.A., James, L.C., and Schuh, M. (2018). Acute and rapid degradation of endogenous proteins by Trim-Away. Nat. Protoc. 13, 2149–2175. Cuchet-Lourenc¸o, D., Anderson, G., Sloan, E., Orr, A., and Everett, R.D. (2013). The viral ubiquitin ligase ICP0 is neither sufficient nor necessary for degradation of the cellular DNA sensor IFI16 during herpes simplex virus 1 infection. J. Virol. 87, 13422–13432.

Dawson, M.J., and Trapani, J.A. (1995). IFI 16 gene encodes a nuclear protein whose expression is induced by interferons in human myeloid leukaemia cell lines. J. Cell. Biochem. 57, 39–51. Dunphy, G., Flannery, S.M., Almine, J.F., Connolly, D.J., Paulus, C., Jonsson, K.L., Jakobsen, M.R., Nevels, M.M., Bowie, A.G., and Unterholzner, L. (2018). Non-canonical Activation of the DNA Sensing Adaptor STING by ATM and IFI16 Mediates NF-kappaB Signaling after Nuclear DNA Damage. Mol. Cell 71, 745–760.e5. €ter, K., Tunba¨ck, P., Nordstro¨m, I., Eriksson, K., Svensson, A., Hait, A.S., Schlu Padyukov, L., Liljeqvist, J.A., Mogensen, T.H., and Paludan, S.R. (2017). Cutting Edge: Genetic Association between IFI16 Single Nucleotide Polymorphisms and Resistance to Genital Herpes Correlates with IFI16 Expression Levels and HSV-2-Induced IFN-b Expression. J. Immunol. 199, 2613–2617. Fujiuchi, N., Aglipay, J.A., Ohtsuka, T., Maehara, N., Sahin, F., Su, G.H., Lee, S.W., and Ouchi, T. (2004). Requirement of IFI16 for the maximal activation of p53 induced by ionizing radiation. J. Biol. Chem. 279, 20339–20344. Gilbert, L.A., Horlbeck, M.A., Adamson, B., Villalta, J.E., Chen, Y., Whitehead, E.H., Guimaraes, C., Panning, B., Ploegh, H.L., Bassik, M.C., et al. (2014). Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell 159, 647–661. Goubau, D., Deddouche, S., and Reis e Sousa, C. (2013). Cytosolic sensing of viruses. Immunity 38, 855–869. Heaton, S.M., Borg, N.A., and Dixit, V.M. (2016). Ubiquitin in the activation and attenuation of innate antiviral immunity. J. Exp. Med. 213, 1–13. Hsu, P.D., Lander, E.S., and Zhang, F. (2014). Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278. Iwamoto, S., Kido, M., Aoki, N., Nishiura, H., Maruoka, R., Ikeda, A., Okazaki, T., Chiba, T., and Watanabe, N. (2012). IFN-g is reciprocally involved in the concurrent development of organ-specific autoimmunity in the liver and stomach. Autoimmunity 45, 186–198.
ska, A., Studzin
ska, M., Suski, P., Kalinka, J., and Paradowska, E. Jab1on (2018). Enhanced expression of IFI16 and RIG-I in human third-trimester placentas following HSV-1 infection. Clin. Exp. Immunol. 193, 255–263. Jakobsen, M.R., and Paludan, S.R. (2014). IFI16: at the interphase between innate DNA sensing and genome regulation. Cytokine Growth Factor Rev. 25, 649–655. Jønsson, K.L., Laustsen, A., Krapp, C., Skipper, K.A., Thavachelvam, K., Hotter, D., Egedal, J.H., Kjolby, M., Mohammadi, P., Prabakaran, T., et al. (2017). IFI16 is required for DNA sensing in human macrophages by promoting production and function of cGAMP. Nat. Commun. 8, 14391. Kawai, T., Takahashi, K., Sato, S., Coban, C., Kumar, H., Kato, H., Ishii, K.J., Takeuchi, O., and Akira, S. (2005). IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction. Nat. Immunol. 6, 981–988. Kerur, N., Veettil, M.V., Sharma-Walia, N., Bottero, V., Sadagopan, S., Otageri, P., and Chandran, B. (2011). IFI16 acts as a nuclear pathogen sensor to induce the inflammasome in response to Kaposi Sarcoma-associated herpesvirus infection. Cell Host Microbe 9, 363–375. Kimkong, I., Avihingsanon, Y., and Hirankarn, N. (2009). Expression profile of HIN200 in leukocytes and renal biopsy of SLE patients by real-time RT-PCR. Lupus 18, 1066–1072. Konermann, S., Brigham, M.D., Trevino, A.E., Joung, J., Abudayyeh, O.O., Barcena, C., Hsu, P.D., Habib, N., Gootenberg, J.S., Nishimasu, H., et al. (2015). Genome-scale transcriptional activation by an engineered CRISPRCas9 complex. Nature 517, 583–588. Li, T., Diner, B.A., Chen, J., and Cristea, I.M. (2012). Acetylation modulates cellular distribution and DNA sensing ability of interferon-inducible protein IFI16. Proc. Natl. Acad. Sci. USA 109, 10558–10563. Li, Q., Lin, L., Tong, Y., Liu, Y., Mou, J., Wang, X., Wang, X., Gong, Y., Zhao, Y., Liu, Y., et al. (2018). TRIM29 negatively controls antiviral immune response through targeting STING for degradation. Cell Discov. 4, 13. Lin, W., Zhao, Z., Ni, Z., Zhao, Y., Du, W., and Chen, S. (2017). IFI16 restoration in hepatocellular carcinoma induces tumour inhibition via activation of p53

signals and inflammasome. Cell Prolif. Published online October 8, 2017. https://doi.org/10.1111/cpr.12392. Liu, Q., Wu, Y., Qin, Y., Hu, J., Xie, W., Qin, F.X., and Cui, J. (2018). Broad and diverse mechanisms used by deubiquitinase family members in regulating the type I interferon signaling pathway during antiviral responses. Sci. Adv. 4, eaar2824. Ma, F., Liu, S.Y., Razani, B., Arora, N., Li, B., Kagechika, H., Tontonoz, P., Nu´n˜ez, V., Ricote, M., and Cheng, G. (2014). Retinoid X receptor a attenuates host antiviral response by suppressing type I interferon. Nat. Commun. 5, 5494. Meylan, E., Curran, J., Hofmann, K., Moradpour, D., Binder, M., Bartenschlager, R., and Tschopp, J. (2005). Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus. Nature 437, 1167–1172. Monroe, K.M., Yang, Z., Johnson, J.R., Geng, X., Doitsh, G., Krogan, N.J., and Greene, W.C. (2014). IFI16 DNA sensor is required for death of lymphoid CD4 T cells abortively infected with HIV. Science 343, 428–432. Nezos, A., Gravani, F., Tassidou, A., Kapsogeorgou, E.K., Voulgarelis, M., Koutsilieris, M., Crow, M.K., and Mavragani, C.P. (2015). Type I and II interferon signatures in Sjogren’s syndrome pathogenesis: contributions in distinct clinical phenotypes and Sjogren’s related lymphomagenesis. J. Autoimmun. 63, 47–58. Ni, X., Ru, H., Ma, F., Zhao, L., Shaw, N., Feng, Y., Ding, W., Gong, W., Wang, Q., Ouyang, S., et al. (2016). New insights into the structural basis of DNA recognition by HINa and HINb domains of IFI16. J. Mol. Cell Biol. 8, 51–61. Orzalli, M.H., DeLuca, N.A., and Knipe, D.M. (2012). Nuclear IFI16 induction of IRF-3 signaling during herpesviral infection and degradation of IFI16 by the viral ICP0 protein. Proc. Natl. Acad. Sci. USA 109, E3008–E3017. Orzalli, M.H., Broekema, N.M., Diner, B.A., Hancks, D.C., Elde, N.C., Cristea, I.M., and Knipe, D.M. (2015). cGAS-mediated stabilization of IFI16 promotes innate signaling during herpes simplex virus infection. Proc. Natl. Acad. Sci. USA 112, E1773–E1781. Parvatiyar, K., Zhang, Z., Teles, R.M., Ouyang, S., Jiang, Y., Iyer, S.S., Zaver, S.A., Schenk, M., Zeng, S., Zhong, W., et al. (2012). The helicase DDX41 recognizes the bacterial secondary messengers cyclic di-GMP and cyclic di-AMP to activate a type I interferon immune response. Nat. Immunol. 13, 1155–1161. Rajsbaum, R., Garcı´a-Sastre, A., and Versteeg, G.A. (2014). TRIMmunity: the roles of the TRIM E3-ubiquitin ligase family in innate antiviral immunity. J. Mol. Biol. 426, 1265–1284. Sadler, A.J., and Williams, B.R. (2008). Interferon-inducible antiviral effectors. Nat. Rev. Immunol. 8, 559–568. Sanjana, N.E., Shalem, O., and Zhang, F. (2014). Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods 11, 783–784. Seelig, H.P., Ehrfeld, H., and Renz, M. (1994). Interferon-gamma-inducible protein p16. A new target of antinuclear antibodies in patients with systemic lupus erythematosus. Arthritis Rheum. 37, 1672–1683. Seo, G.J., Kim, C., Shin, W.J., Sklan, E.H., Eoh, H., and Jung, J.U. (2018). TRIM56-mediated monoubiquitination of cGAS for cytosolic DNA sensing. Nat. Commun. 9, 613. Seth, R.B., Sun, L., Ea, C.K., and Chen, Z.J. (2005). Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3. Cell 122, 669–682. Shalem, O., Sanjana, N.E., Hartenian, E., Shi, X., Scott, D.A., Mikkelson, T., Heckl, D., Ebert, B.L., Root, D.E., Doench, J.G., and Zhang, F. (2014). Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343, 84–87. Shi, X.L., Yang, J., Mao, N., Wu, J.H., Ren, L.F., Yang, Y., Yin, X.L., Wei, L., Li, M.Y., and Wang, B.N. (2015). Nutlin-3-induced redistribution of chromatinbound IFI16 in human hepatocellular carcinoma cells in vitro is associated with p53 activation. Acta Pharmacol. Sin. 36, 252–258. Sun, L., Wu, J., Du, F., Chen, X., and Chen, Z.J. (2013). Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science 339, 786–791.

Cell Reports 29, 1249–1260, October 29, 2019 1259

Unterholzner, L., Keating, S.E., Baran, M., Horan, K.A., Jensen, S.B., Sharma, S., Sirois, C.M., Jin, T., Latz, E., Xiao, T.S., et al. (2010). IFI16 is an innate immune sensor for intracellular DNA. Nat. Immunol. 11, 997–1004. van Gent, M., Sparrer, K.M.J., and Gack, M.U. (2018). TRIM Proteins and Their Roles in Antiviral Host Defenses. Annu. Rev. Virol. 5, 385–405. Vanhove, W., Peeters, P.M., Staelens, D., Schraenen, A., Van der Goten, J., Cleynen, I., De Schepper, S., Van Lommel, L., Reynaert, N.L., Schuit, F., et al. (2015). Strong Upregulation of AIM2 and IFI16 Inflammasomes in the Mucosa of Patients with Active Inflammatory Bowel Disease. Inflamm. Bowel Dis. 21, 2673–2682. Veeranki, S., and Choubey, D. (2012). Interferon-inducible p200-family protein IFI16, an innate immune sensor for cytosolic and nuclear double-stranded DNA: regulation of subcellular localization. Mol. Immunol. 49, 567–571.

1260 Cell Reports 29, 1249–1260, October 29, 2019

Wang, Y., Lian, Q., Yang, B., Yan, S., Zhou, H., He, L., Lin, G., Lian, Z., Jiang, Z., and Sun, B. (2015). TRIM30a Is a Negative-Feedback Regulator of the Intracellular DNA and DNA Virus-Triggered Response by Targeting STING. PLoS Pathog. 11, e1005012. Yang, K., Shi, H.X., Liu, X.Y., Shan, Y.F., Wei, B., Chen, S., and Wang, C. (2009). TRIM21 is essential to sustain IFN regulatory factor 3 activation during antiviral response. J. Immunol. 182, 3782–3792. Zhang, Z., Yuan, B., Bao, M., Lu, N., Kim, T., and Liu, Y.J. (2011). The helicase DDX41 senses intracellular DNA mediated by the adaptor STING in dendritic cells. Nat. Immunol. 12, 959–965. Zhang, Z., Bao, M., Lu, N., Weng, L., Yuan, B., and Liu, Y.J. (2013). The E3 ubiquitin ligase TRIM21 negatively regulates the innate immune response to intracellular double-stranded DNA. Nat. Immunol. 14, 172–178.

STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Antibodies ANTI-FLAG M2 Affinity Gel

Sigma

Cat# A2220, RRID:AB_10063035

Mouse monoclonal Anti-HA
Agarose antibody

Sigma

Cat# A2095, RRID:AB_257974

Rabbit monoclonal IFI16 (D8B5T) antibody

Cell Signaling Technology

Cat# 14970S, RRID:AB_2798669

Mouse monoclonal IFI16 antibody

Santa Cruz Biotechnology

Cat# sc-8023, RRID:AB_627775

Rabbit monoclonal STING antibody

Cell Signaling Technology

Cat# 13647, RRID:AB_2732796

Rabbit IgG antibody

Cell Signaling Technology

Cat# 2729, RRID:AB_1031062

Rabbit monoclonal p-STAT1 (Tyr701) antibody

Cell Signaling Technology

Cat# 9167, RRID:AB_561284

Rabbit monoclonal STAT1 antibody

Cell Signaling Technology

Cat# 14994, RRID:AB_2737027

Rabbit monoclonal MX1 antibody

Cell Signaling Technology

Cat# 37849, RRID:AB_2799122

Rabbit monoclonal OAS1 antibody

Cell Signaling Technology

Cat# 14498, RRID:AB_2798498

Mouse monoclonal a-Tubulin antibody

Sigma

Cat# T5168, RRID:AB_477579

HRP conjugated Rabbit monoclonal GAPDH (14C10) antibody

Cell Signaling Technology

Cat# 3683, RRID:AB_1642205

Mouse monoclonal ANTI-FLAG M2-HRP antibody

Sigma

Cat# A8592, RRID:AB_439702

Mouse monoclonal Ubiquitin (P4D1) HRP antibody

Cell Signaling Technology

Cat# 14049S, RRID:AB_2798376

Mouse monoclonal HA-Tag (6E2)-HRP antibody

Cell Signaling Technology

Cat# 2999S, RRID:AB_1264166

Rabbit polyclonal anti-c-Myc
HRP antibody

Sigma

Cat# A5598, RRID:AB_439682

Anti-rabbit IgG, HRP-linked Antibody

Cell Signaling Technology

Cat# 7074S, RRID:AB_2099233

Anti-mouse IgG, HRP-linked Antibody

Cell Signaling Technology

Cat# 7076S, RRID:AB_330924

HSV-1 strain 17

Ma et al., 2014

N/A

VACV

ATCC

Cat# VR-1354

MG132

Sigma

Cat# M8699

Pepstatin A

Sigma

Cat# P5318

Concanamycin A

Enzo Life Sciences

Cat# ALX-380-034-C025

Bortezomib (PS341)

Selleck

Cat# S1013

Cycloheximide (CHX)

Sigma

Cat# C7698

Lipopolysaccharides (LPS)

Sigma

Cat# L3024

Recombinant human IFN-b

R&D system

Cat# 8499-IF

Recombinant human IFN-g

InvivoGen

Cat# rcyec-hifng

Nutlin-3

Selleck

Cat# S1061

Lipofectamine 2000

Invitrogen

Cat# 11668019

Polyethylenimine

Polysciences

Cat# 23966

INTERFERin

Polyplus-transfection

Cat# n409-10

Polybrene

EMD Millipore

Cat# TR-1003

Puromycin

InvivoGen

Cat# ant-pr-1

Bacterial and Virus Strains

Chemicals, Peptides, and Recombinant Proteins

Geneticin Selective Antibiotic (G418 Sulfate)

Thermo Fisher Scientific

Cat# 10131035

Herring testis DNA (HT-DNA)

Sigma

Cat# D6898

DMEM

GIBCO

Cat# 11965-092

Fetal bovine serum

GIBCO

Cat# 10100147

Penicillin+Streptomycin

GIBCO

Cat# 15140122

cOmplete Protease Inhibitor Cocktail Tablets

Roche

Cat# 11697498001

PhosSTOP

Roche

Cat# 4906837001 (Continued on next page)

Cell Reports 29, 1249–1260.e1–e4, October 29, 2019 e1

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

Cell Line Nucleofector Kit V

Lonza

Cat# VCA-1003

Dual-Luciferase Reporter Assay System

Promega

Cat# E1910

Duolink In Situ PLA Probe Anti-Mouse MINUS

Sigma

Cat# DUO92004

Duolink In Situ PLA Probe Anti-Rabbit PLUS

Sigma

Cat# DUO92002

Duolink In Situ Detection Reagents Red

Sigma

Cat# DUO92008

Duolink In Situ Wash Buffers, Fluorescence

Sigma

Cat# DUO82049

Critical Commercial Assays

ProteoSilver Silver Stain Kit

Sigma

Cat# PROT-SIL1

DNAiso Reagent

Takara

Cat# 9770A

RNeasy Mini Kit

QIAGEN

Cat# 74104

PrimeScript RT Master Mix (Perfect Real Time)

Takara

Cat# RR036B

TB Green Premix Ex Taq (Tli RNaseH Plus)

Takara

Cat# RR420B

Western Chemiluminescent HRP Substrate (ECL)

EMD Millipore

Cat# WBKLS0500

Muta-direct TM kit

Sbsgene

Cat# SDM-15

shRNA targeting STING: 50 -GATCCGGTCATATTACAT CGGATATCC TTCCTGTCAGAGATATCCGATGTAATATGACCTTTTT-30 (sense)

This paper

N/A

shRNA targeting STING: 50 -AATTCAAAAAGGTCATA TTACATCGGA TATCTCTGACAGGAAGGATATCCGATGTAATATGACC-30 (anti-sense)

This paper

N/A

siRNA targeting STING: siSTING-1: 50 -GGTCATA TTACATCGGATA-30

This paper

N/A

siRNA targeting STING: siSTING-2: 50 -GCATTACA ACAACCTGCTA-30

This paper

N/A

siRNA targeting TRIM21: 50 -GCAGGAGUUGGCUGAGAAG-30

Yang et al., 2009

N/A

siRNA control: 50 -UUCUCCGAACGUGUCACGU-30

Genepharma

Cat# A06001

sgRNA targeting IFI16: #1 50 -TATACCAACGCTTGAAGACC-30

This paper

N/A

sgRNA targeting IFI16: #2 50 -TTTGACAGTGCTGCTTGTGG-30

This paper

N/A

sgRNA targeting STING: #1 50 - AGAGCACACTCTCCGGTACC-30

This paper

N/A

sgRNA targeting STING: #2 50 -AAGGGCGGGCCGACCGCATT-30

This paper

N/A

GraphPad Prism 8

GraphPad software

https://www.graphpad.com

ImageJ

ImageJ software

https://imagej.nih.gov/ij/

Oligonucleotides

Software and Algorithms

Other LEICA TCA SP8 confocal microscope

Leica Biosystems

N/A

LCS software package

Leica Biosystems

N/A

Roche LightCycler 480 II

Roche

Product No.: D 100 03

LEAD CONTACT AND MATERIALS AVAILABILITY Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Dr. Feng Ma ([email protected]). This study did not generate new unique reagents. EXPERIMENTAL MODEL AND SUBJECT DETAILS Cell lines Human embryonic kidney HEK293T cells, human non-small cell lung cancer A549 cells, and human macrophages THP1 cells were purchased from American Type Culture Collection (ATCC). WT and IFI16
/
keratinocytes HaCaT cells were generously provided by Professor Leonie Unterholzner (University of Edinburgh, UK). WT and IFI16
/
THP1 cells were kindly provided by Professor M.R. Jakobsen (University Medical Center Utrecht, Netherlands). Human osteosarcoma U2OS cells were from National Infrastructure Cell Line Resource (Shanghai, China). THP1 cells were cultured in RPMI1640 and the rest cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum, 100 Units/mL penicillin, and 100 mg/mL streptomycin, at 37 C and 10% CO2.

e2 Cell Reports 29, 1249–1260.e1–e4, October 29, 2019

Plasmids and Generation of IFI16 Mutants All the IFI16 and STING plasmids were gifts from Professor Genhong Cheng (University of California, Los Angeles). Flag-PSMA7 vector was gifted from Professor Cao Cheng (Beijing Institute of Biotechnology, Beijing, China). HA-TRIM21, HA-Ub and HA-Ub mutant vectors were kindly provided by Professor Cui Jun (Sun Yat-Sen University, China). IFI16-truncated and IFI16-pointed mutants were generated by PCR amplification using WT IFI16 isoform2 plasmid as templates by subcloning or according to the protocol of Muta-direct TM kit (Sbsgene). The sequence of all constructs was verified by Sanger DNA sequencing. METHOD DETAILS Cells transfection and stimulation Plasmids transient transfection was performed with Lipofectamine 2000 or Polyethylenimine, and siRNAs was delivered into the HEK293T, HaCaT or THP1 cells by INTERFERin according to the manufacturer’s instructions. HSV60-mer (Table S2) and HT-DNA was transfected into HaCaT cells at the ratio of 2.5 ml:1 mg (reagents/DNA) by using Lipofectamine 2000. Cells were treated with MG132, Pep A, PS341, Con A, Nutlin-3, LPS, IFN-g or IFN-b as indicated in figure legends. Generation of STING stably expressing U2OS cells The STING stably expressing cells were generated on the U2OS cell lines. In brief, the HA-STING expressing plasmid with a pcDNA3.1 backbone was transfected into U2OS cells. Transfected cells were selected with 800 mg/mL G418 at 48 hr post transfection. After 4 weeks selection single cell clones were established and subjected to western blotting to confirm the expression of STING of each cell clones. Generation of STING
/
HaCaT cells HaCaT cells population lacking STING genes were generated using CRISPR/Cas9 technology (Sanjana et al., 2014; Shalem et al., 2014). Two separate sgRNAs targeting STING were inserted into the lentiCRISPRv2 vectors expressing a Cas9 gene (Addgene#52961). The sgRNA sequences were shown in Key Resources Table. Lenti-CRISPR virions were produced by transfecting HEK293T cells with the following plasmids: CRISPR/Cas9 vector, pMD.2G, pRSV-REV, and pMDlg/p-RRE. Viral supernatants were harvested after 72 hr and used to infect HaCaT cells in the presence of 6 mg/mL polybrene. Transduced cells were selected with 3 mg/ mL puromycin at 48 hr post transduction. After 10 days selection, the population cells were subjected to western blotting to confirm the deletion of STING expression. Generation of IFI16-K3/4R HaCaT cells HaCaT IFI16-K3/4R cells were generated using CRISPR/Cas9 technology (Chu et al., 2015; Hsu et al., 2014). Two separate sgRNAs targeting IFI16 were inserted into the lentiCRISPRv2 vectors expressing a Cas9 gene. The sgRNA sequences were shown in Key Resources Table. Lenti-CRISPR virions were produced by transfecting HEK293T cells with the following plasmids: CRISPR/Cas9 vector, pMD.2G, pRSV-REV, and pMDlg/p-RRE. Viral supernatants were harvested after 72 hr and used to infect HaCaT cells, which has been transfected with 0.5 mg/mL donor DNA fragment for 8 hr, in the presence of 6 mg/mL polybrene. Transduced cells were maintained in complete medium supplied with 1 mM SCR7 inhibitor and selected with 3 mg/mL puromycin at 48 hr post transduction. After 4 weeks selection single cell clones were established and subjected to genotyping. IFI16-WT, IFI16-KO and IFI16-K3/4R HaCaT cell clones were obtained for antiviral signal induction analysis. Immunoprecipitation and Immunoblot Analysis Cell lysates were prepared in lysis buffer (50 mM Tris-HCl, pH7.5, 150 mM NaCl, 1% (v/v) Nonidet P-40, 0.5 mM EDTA) supplemented with complete protease inhibitor cocktail (for phosphorylation analysis, the phosphatase inhibitor PhosSTOP was added into lysis buffer according to the manufacturer’s instructions). Soluble protein was subjected to immunoprecipitation with anti-Flag antibody, anti-HA antibody, anti-IFI16 antibody, anti-STING antibody or anti-rabbit IgG antibody. An aliquot of the total lysate was included as a control. Immunoblot analysis was performed with anti-IFI16 antibody, anti-STING antibody, anti-p-STAT1 (Tyr701) antibody, anti-STAT1 antibody, anti-MX1 antibody, anti-OAS1 antibody, anti-a-Tubulin antibody, anti-b-Actin, or HRP conjugated anti-Flag antibody, HRP conjugated anti-Ub antibody, HRP conjugated anti-HA antibody, HRP conjugated anti-Myc antibody and HRP conjugated anti-GAPDH antibody. The antigen-antibody complexes were visualized by chemiluminescence (ECL, Millipore). In vitro degradation/binding assay Flag-IFI16 plasmids (8 mg), EV (2 mg) or HA-STING (2 mg) vectors were transfected into 100 mm dish and the cells were harvested after 30 hr. Cells were lysed in 500 mL lysis buffer (50 mM Tris-HCl, pH7.5, 150 mM NaCl, 1% (v/v) Nonidet P-40, 0.5 mM EDTA). IFI16 expressing lysates were subjected to immunoprecipitation with anti-Flag antibody conjugated beads (50 mL). Anti-Flag immune precipitates were eluted by Flag peptides (Sigma, 200 mL). Eluted Flag-IFI16 protein (2 mg) or Flag-IFI16 protein conjugated beads (10 mL) was incubated with EV or HA-STING vectors transfected HEK293T cell lysates (100 mL) for indicated time. The whole reaction mixtures were subjected to IB analysis.

Cell Reports 29, 1249–1260.e1–e4, October 29, 2019 e3

Quantitative real-time PCR (RT-qPCR) RNA was extracted using the RNeasy Mini Kit from QIAGEN, and cDNA was synthesized using the PrimeScript RT Master Mix (Takara). Real-time PCR amplification was performed using TB Green Premix Ex Taq (Tli RNaseH Plus) on a Roche LightCycler 480 II system. Primer sequences for target genes were shown in Key Resources Table and Table S2. CHX-chase Assays HEK293T cells were transfected with plasmids for 20 hr (Figure 1) or 24 hr (Figure 5). After that, CHX (100 mg/mL) was added into fresh cell culture medium. Cells were harvested at indicated time points, and total cell lysates were subjected to SDS-PAGE and western blot detection. Optical density of IFI16 protein normalized to a-Tubulin bands was acquired by ImageJ software. RNAi The negative control siRNA and the double strand siRNAs targeting STING or TRIM21 (sequences seen in Key Resources Table) were obtained from Genepharma. Human monocytes THP1, human keratinocytes HaCaT or HEK293T cells were transfected with siRNA (the dose were indicated in the figure legends) using INTERFERin according to the manufacturer’s instructions. Cells were stimulated with indicated drugs 48 hr after siRNA transfection. The RNAi efficiency was tested by WB or RT-qPCR. Luciferase assay HEK293T were seeded in 24-well plates at a density of 1.5*105 cells per well and cultured for 24 hr. The cells were transfected with a mixture of IFN-b promoter firefly luciferase reporter plasmid, Renilla luciferase transfect control and other indicated plasmids using Polyethylenimine transfection reagents. 24 hr post transfection, cells were lysed and luciferase activity was measured using the DualLuciferase Reporter Assay System according the manufacturer’s instruction. HSV-luciferase assay HEK293T were seeded in 24-well plates at a density of 1.5*105 cells per well and cultured for 24 hr. The cells were transfected with indicated plasmids using Polyethylenimine transfection reagents. 8 hr post transfection, cells were infected by HSV-luc virus (MOI = 1) for 24 hr. The cells were lysed and the firefly luciferase activity was measured using the Dual-Luciferase Reporter Assay System according the manufacturer’s instruction. In Situ Proximity Ligation Assay (In Situ PLA) Duolink in situ PLA (Duolink Detection kit) was used to detect interactions between IFI16 and STING. Briefly, HaCaT cells plated on glass coverslips in the presence or absence of IFN-g (10 ng/mL) for 1-8 hr were fixed using 4% formaldehyde. The fixed cells were incubated with mouse anti-IFI16 and rabbit anti-STING primary antibodies or with only anti-IFI16 antibody as a control. The Duolink system provides oligonucleotide-labeled secondary antibodies (PLA probes) to each of the primary antibodies that, in combination with a DNA amplification-based reporter system, generate a signal only when the two primary antibodies are in close enough proximity. The signal from each detected pair of primary antibodies was visualized as a spot according to the manufacturer’s instructions. Slides were evaluated using a LEICA TCA SP8 confocal microscope. Cell images obtained were exported using the LCS software package in TIF format for further analysis. Interaction analysis per cell was determined by ImageJ software. QUANTIFICATION AND STATISTICAL ANALYSIS Number of experimental repeats are shown in the figure legend. All bar graphs are means with SD. Statistical analysis was performed with Student’s t test in GraphPad Prism 8 software. P value less than 0.05 was considered significant. *p < 0.05, **p < 0.01, and ***p < 0.001. DATA AND CODE AVAILABILITY This study did not generate any unique datasets or code.

e4 Cell Reports 29, 1249–1260.e1–e4, October 29, 2019