STING1基因敲除HEK293细胞

Induction of autophagy is an ancient function of the cyclic GMP-AMP (cGAMP) synthase (cGAS)-stimulator of interferon genes (STING) pathway through which autophagic cargoes are delivered to lysosomes for degradation. However, whether lysosome function is also modulated by the cGAS-STING pathway remains unknown. Here, we discovered that the cGAS-STING pathway upregulated lysosomal activity by stimulating lysosome biogenesis independently of the downstream protein kinase TANK-binding kinase 1 (TBK1). STING activation enhanced lysosome biogenesis through inducing the nuclear translocation of transcription factor EB (TFEB) as well as its paralogs transcription factor E3 (TFE3) and microphthalmia-associated transcription factor (MITF). STING-induced lipidation of GABA type A receptor-associated protein (GABARAP), an autophagy-related protein, on STING vesicles was responsible for TFEB activation. Membrane-bound GABARAP sequestered the GTPase-activating protein folliculin (FLCN) and FLCN-interacting protein (FNIP) complex to block its function toward the Rag GTPases Ras-related GTP-binding C and D (RagC and RagD), abolishing mechanistic target of rapamycin (mTOR) complex 1 (mTORC1)-dependent phosphorylation and inactivation of TFEB. Functionally, STING-induced lysosome biogenesis within cells facilitated the clearance of cytoplasmic DNA and invading pathogens. Thus, our findings reveal that induction of lysosome biogenesis is another important function of the cGAS-STING pathway.

The cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS)-cyclic GMP-AMP (cGAMP)-stimulator of interferon genes (STING) pathway mediates antiviral innate immunity upon sensing cytosolic DNA. Here, we examined the impact of sphingomyelin phosphodiesterase acid-like 3B (SMPDL3B), a paralog of the LXR lipid metabolism-induced cGAMP-degrading enzyme SMPDL3A, on viral infection. We found that SMPDL3B was induced and stabilized by both viral infection and membrane-disturbing agents, suggesting a role in sensing membrane stress as an early signal of cellular danger. Deletion of SMPDL3B impaired DNA virus infection. Upon induction, SMPDL3B suppressed cGAS-STING signaling and downstream transcriptional pathways, including the interferon response. Mechanistically, SMPDL3B functioned as a cGAMP hydrolase; cGAMP-induced SMPDL3B dimerization enabled its hydrolase activity and a negative feedback loop that dampened STING signaling. SMPDL3B-deficient cells had elevated cGAMP concentrations, and Smpdl3b mice exhibited enhanced cGAMP accumulation, heightened immune activation, and reduced viral loads upon herpes simplex virus type 1 (HSV-1) infection. Thus, SMPDL3B links membrane stress to modulation of cGAS-STING signaling through cGAMP degradation, with potential implications in the contexts of inflammation or autoimmunity.

Pattern recognition receptor (PRR)-induced interferon (IFN) is critical for effective immunity. The PRRs Toll-like receptor (TLR) 3, TLR4 and cyclic GMP-AMP synthase (cGAS), together with the stimulator of IFN genes (STING), signal through TANK-binding kinase 1 (TBK1), which activates the type-I/III IFN-inducing transcription factor interferon-response factor 3 (IRF3). The mechanism by which these PRRs activate TBK1 remains unresolved. Here we show that lysine-11 (K11)-linked ubiquitination drives TBK1 activation by these PRRs. The E3 ligase ANKIB1 attaches K11-linked ubiquitin chains to components of the TLR3- and cGAS-STING-induced signalosomes. This facilitates Optineurin recruitment to these complexes, in turn enabling recruitment and activation of TBK1 and IRF3, defining an uncharacterized signalling axis. In mice, ANKIB1 deficiency dampens IFN induction via TLR3 and cGAS-STING, reducing interferonopathy and compromising protection against HSV-1, respectively. Together, our results demonstrate an unanticipated and critical role for ANKIB1-generated K11-linked ubiquitination in the immune response activated by cGAS-STING, TLR3 and TLR4.

Stimulator of interferon genes (STING) is activated in many pathophysiological conditions, leading to TBK1-dependent interferon production in higher organisms. However, primordial functions of STING independent of TBK1 are poorly understood. Here, through proteomics and bioinformatics approaches, we identify lysosomal biogenesis as an unexpected function of STING. Transcription factor EB (TFEB), an evolutionarily conserved regulator of lysosomal biogenesis and host defense, is activated by STING from multiple species, including humans, mice, and frogs. STING-mediated TFEB activation is independent of TBK1, but it requires STING trafficking and its conserved proton channel. GABARAP lipidation, stimulated by the channel of STING, is key for STING-dependent TFEB activation. STING stimulates global upregulation of TFEB-target genes, mediating lysosomal biogenesis and autophagy. TFEB supports cell survival during chronic sterile STING activation, a common condition in aging and age-related diseases. These results reveal a primordial function of STING in the biogenesis of lysosomes, essential organelles in immunity and cellular stress resistance.

Necroptosis is a pro-inflammatory, lytic cell death executed by a pseudokinase mixed lineage kinase-like protein MLKL. Upon necroptosis induction by various inflammatory signals, MLKL is phosphorylated by receptor-interacting serine/threonine-protein kinase 3 (RIPK3) and translocates from the cytosol to the plasma membrane, causing membrane disruption and the release of damage-associated molecular patterns (DAMPs). We report here that phosphor-MLKL also translocates to mitochondria and induces a microtubule-dependent release of mitochondrial DNA (mtDNA). The released mtDNA activates the cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) pathway, resulting in the upregulation of interferon-beta (Ifnb) expression. In a necroptosis-mediated inflammatory bowel disease (IBD) mouse model, interfering with the cGAS-STING pathway reduced inflammation and promoted intestinal recovery. Thus, MLKL induces inflammation not only in a cell non-autonomous fashion by releasing DAMP signals, but also in a cell-autonomous manner by causing mtDNA leakage into the cytosol, thereby activating the cGAS-STING pathway.

STING-dependent cytosolic DNA sensing in dendritic cells (DCs) initiates antitumor immune responses, but how STING signaling is metabolically regulated in the tumor microenvironment remains unknown. Here, we show that oxidative stress is required for STING-induced DC antitumor function through a process that directs SUMO-specific protease 3 (SENP3) activity. DC-specific deletion of Senp3 drives tumor progression by blunting STING-dependent type-I interferon (IFN) signaling in DCs and dampening antitumor immune responses. DC-derived reactive oxygen species (ROS) trigger SENP3 accumulation and the SENP3-IFI204 interaction, thereby catalyzing IFI204 deSUMOylation and boosting STING signaling activation in mice. Consistently, SENP3 senses ROS to facilitate STING-dependent DC activity in tissue samples from colorectal cancer patients. Our results reveal that oxidative stress as a metabolic regulator promotes STING-mediated DC antitumor immune responses and highlights SENP3 as an overflow valve for STING signaling induction in the metabolically abnormal tumor microenvironment.

The cGAS-STING pathway is a critical regulator of type I Interferon (IFN) and inflammation upon cytosolic DNA-sensing. cGAS-STING signaling termination is regulated by lysosomal-mediated degradation of STING; however, the mechanisms controlling the inhibitory targeting of STING are incompletely understood. Here, we identify the selective autophagy receptor TAX1BP1 as a negative regulator of the cGAS-STING pathway. TAX1BP1-deficient macrophages activated by cGAS or STING agonists accumulate higher-order STING aggregates, exhibit heightened STING signaling, and increased production of type I IFN and proinflammatory cytokines. Mechanistically, TAX1BP1 promotes STING degradation through microautophagy by facilitating the interaction of STING with the ESCRT-0 protein HGS. Furthermore, STING activation is associated with the swelling and fragmentation of the Golgi apparatus, and TAX1BP1 and p62/SQSTM1 are essential for the autophagic degradation of fragmented Golgi (Golgiphagy). Our findings suggest that STING activation at the Golgi is coupled to its downregulation by Golgiphagy to restrict innate immune responses.

Viruses rely on intracellular materials, including iron, to complete their life cycles and iron withholding may limit viral infections. However, the mechanisms through which viruses disrupt host iron homeostasis and the impact of intracellular iron on the host's antiviral defense aren't well studied. Here we show that viral infections facilitate the polyubiquitination and degradation of ferroportin (FPN1, the only cellular iron exporter) by upregulating the host E3 ubiquitin ligase DTX3L, leading to an elevation in cellular iron levels. Excessive ferrous iron suppresses type I IFN responses and autophagy by promoting TBK1 hydroxylation and STING carbonylation in macrophages. FPN1 deficiency suppresses host antiviral defense and facilitates viral replication in vitro and in vivo, while DTX3L deficiency has the opposite effect. These results reveal that viruses hijack host FPN1 to disrupt iron withholding and achieve immune escape, and suggest that iron homeostasis maintained by FPN1 is required for the optimal activation of TBK1- and STING-dependent antiviral responses.

As a core aptamer for anti-DNA viral immunity, STING1 (stimulator of interferon response cGAMP interactor 1) is tightly regulated to ensure the proper functioning of the natural antiviral immune response. However, many mechanisms underlying the regulation of STING1 remain largely unknown. In this study, we identify EXOC4/SEC8 (exocyst complex component 4) as a novel positive regulator of DNA virus-triggered type I interferon signaling responses through stabilizing STING1, thereby inhibiting DNA viral replication. Mechanistically, EXOC4 suppresses K27-linked ubiquitination of STING1 at K338, K347, and K370 catalyzed by the E3 ligase FBXL19 (F-box and leucine rich repeat protein 19), thereby preventing ubiquitinated-STING1 from recognition by SQSTM1 (sequestosome 1) for autophagic degradation. Importantly, mice conditionally knocked out for are more susceptible to herpes simplex virus type 1 (HSV-1) infection and exhibit more severe lung pathology compared to control mice. This further confirms the important role of EXOC4/SEC8 in antiviral natural immunity. Taken together, our study reveals the importance of EXOC4/SEC8 in promoting STING1-centered antiviral natural immunity and highlights its potential as an anti-DNA viral therapeutic target.: ACTB/β-actin: actin beta; BMDMs: bone marrow-derived macrophages; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; cGAMP: cyclic GMP-AMP; CQ: chloroquine; ER: endoplasmic reticulum; EXOC4/SEC8: exocyst complex component 4; CGAS: cyclic GMP-AMP synthase; HAdV-4: human adenovirus type 4; HSV-1: herpes simplex virus type 1; IFIT1: interferon induced protein with tetratricopeptide repeats 1; IFIT2: interferon induced protein with tetratricopeptide repeats 2; IFNB1: interferon beta 1; IRF3: interferon regulatory factor 3; IFN-I: type I interferon; ISGs: IFN-stimulated genes; ISRE: IFN-stimulated response element; MG132/Z-LLL-CHO: carbobenzoxy-Leu-Leu-leucinal; MOI: multiplicity of infection; MST: microscale thermophoresis; PMs: peritoneal macrophages; Poly(dA:dT): polydeoxyadenylic-thymidylic acid; qPCR: quantitative real-time PCR; shRNAs: short hairpin RNAs; siRNA: small interfering RNA; SQSTM1: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TBK1: TANK binding kinase 1; TCID: 50% tissue culture infectious dose; WT: wild-type.

STING1/MITA not only induces innate immune responses but also triggers macroautophagy/autophagy to selectively degrade signaling molecules. However, the molecular mechanisms regulating STING1-mediated selective autophagy remain unclear. Here, we first report that ATP2A2 directly interacts with STING1, regulating STING1-mediated innate immune response by modulating its polymerization and trafficking, thereby inhibiting DNA virus infection. Notably, while screening for reticulophagy receptors involved in STING1-mediated selective autophagy, we identified SEC62 as an important receptor protein in STING1-mediated reticulophagy. Mechanistically, SEC62 strengthens its interaction with STING1 upon activation and concurrently facilitates STING1-mediated reticulophagy upon starvation, which are dependent on ATP2A2. Furthermore, knocking down SEC62 in WT cells inhibits STING1-mediated MAP1LC3B/LC3B lipidation and autophagosome formation, an effect that is lost in knockout cells, suggesting that SEC62's role in STING1-mediated selective autophagy is ATP2A2 dependent. Thus, our findings identify the reticulophagy receptor SEC62 as a novel receptor protein regulating STING1-mediated selective autophagy, providing new insight into the mechanism regarding a reticulophagy receptor in the process of STING1-induced selective autophagy. aa: amino acids; AP-MS: affinity tag purification-mass spectrometry; ATP2A1: ATPase sarcoplasmic/endoplasmic reticulum Ca transporting 1; ATP2A2: ATPase sarcoplasmic/endoplasmic reticulum Ca transporting 2; ATP2A3: ATPase sarcoplasmic/endoplasmic reticulum Ca transporting 3; CANX: calnexin; CCPG1: cell cycle progression 1; CGAS: cyclic GMP-AMP synthase; ctDNA: calf thymus DNA; dsRNA: double-stranded RNA; diABZI: diamidobenzimidazole; ER: endoplasmic reticulum; ERGIC: ER-Golgi intermediate compartment; EBSS: Earle's Balanced Salt Solution; EV: empty vector; FL: full length; GOLGA2/GM130: golgin A2; HSV-1: herpes simplex virus type 1; IRF3: interferon regulatory factor 3; IFNs: type I interferons; ISD: interferon stimulatory DNA; KO: knockout; MAVS: mitochondrial antiviral signaling protein; MOI: multiplicity of infection; poly(I:C): polyinosinic-polycytidylic acid; NBR1: NBR1 autophagy cargo receptor; PRR: pattern recognition receptor; reticulophagy: selective autophagic degradation of the ER; RETREG1/FAM134B: reticulophagy regulator 1; RIGI: RNA sensor RIG-I; RTN3L: reticulon 3; SEC62: SEC62 homolog, preprotein translocation factor; SeV: Sendai virus; STIM1: stromal interaction molecule 1; STING1/MITA: stimulator of interferon response cGAMP interactor 1; TBK1: TANK binding kinase 1; TEX264: testis expressed 264, ER-phagy receptor; TMX1: thioredoxin related transmembrane protein 1; VSV: vesicular stomatitis virus; VACV: vaccinia virus; ZMPSTE24: zinc metallopeptidase STE24.

The stimulator of interferon genes (STING) is a critical mediator of innate immunity against cytosolic DNA pathogens, requiring precise regulation to balance antiviral defense and immune tolerance. While lipid metabolism influences immune signaling, the role of stearoyl-CoA desaturase 1 (SCD1), a key enzyme converting saturated fatty acids (SFAs) to monounsaturated fatty acids (MUFAs), in STING activation remains unexplored. Here, we identify SCD1 as a metabolic checkpoint that licenses STING activation through biophysical membrane remodeling. Mechanistically, SCD1-generated MUFAs incorporate into endoplasmic reticulum (ER) phospholipids, enhancing membrane curvature and fluidity. This biophysical remodeling facilitates cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) (cGAMP) binding to STING and promotes its dimerization, enabling downstream TBK1-IRF3 signaling and type I interferons (IFNs) production. Consequently, deficiency or pharmacological inhibition impairs STING activation, attenuates antiviral responses against herpes simplex virus-1 (HSV-1), and exacerbates viral replication in vitro and in vivo. Conversely, MUFAs supplementation rescues STING activation in -deficient models. Our findings establish SCD1-mediated lipid desaturation as a fundamental regulator of STING-driven immunity, highlighting its therapeutic potential for disorders of aberrant STING activation.

BACKGROUND:The innate immune system serves as the host's first line of defense against invading pathogens. Stimulator of interferon genes (STING) is a key component of this system, yet its relationship with glucose metabolism, particularly in antiviral immunity, remains underexplored. METHODS:Metabolomics analysis was used for detecting metabolic alterations in spleens from STING knockout (KO) and wild-type (WT) mice. Co-immunoprecipitation was employed for determining ubiquitination of TKT. Mass spectrometry was used for detecting interaction proteins of STING. Enzyme activity kits were used for detecting the activities of TKT and G6PD. RESULTS:In this study, we demonstrate that herpes simplex virus (HSV) infection activates the pentose phosphate pathway (PPP) in host cells, thereby initiating an antiviral immune response. Using STING-manipulated cells and systemic knockout mice, we show that STING positively regulates PPP, which, in turn, limits HSV infection. Inhibition of the PPP significantly reduced the production of antiviral immune factors and dampened STING-induced innate immune responses. Mechanistically, we discovered that STING interacts with transketolase (TKT), a key enzyme in the non-oxidative branch of the PPP, and reduces its ubiquitination via the E3 ubiquitin ligase UBE3A, stabilizing TKT. Silencing TKT or inhibiting its activity with oxythiamine diminished antiviral immune factor production. CONCLUSION:Our findings reveal that the PPP plays a synergistic role in generating antiviral immune factors during viral infection and suggest that PPP activation could serve as an adjunct strategy for antiviral therapy.

RIG-I and STING are critical for mediating the RIG-I and cGAS-STING signaling pathways that guard against viral infection. Here, we report that ubiquitin-specific peptidase 39 (USP39) positively regulates the RIG-I and cGAS-STING pathways to induce antiviral innate immunity in vitro and in vivo. The USP39 deficiency impaired the antiviral immune response of macrophages, leading to low type I IFNs expression, and high RNA and (e.g., VSV, H1N1 PR8) DNA virus (e.g., HSV-1) replication. Moreover, USP39-deficient mice were more sensitive to VSV or HSV-1 infection than control mice. Conversely, USP39 overexpression promoted the antiviral immune response. Mechanistically, we found that USP39 regulates RIG-I protein expression by promoting pre-RIG-I mRNA splicing and maturation. In addition, we also revealed that USP39 interacts with and stabilizes STING protein by deubiquitinating K48-linked polyubiquitin of STING at K288. These data show that USP39 positively regulates RNA and DNA-virus-induced RIG-I and cGAS-STING signaling, respectively, by promoting post-transcriptional control of RIG-I and stabilization of STING. These data provide new insights and potential therapeutic targets to control viral infections.

Histone deacetylase HDAC6 has been implicated in regulating antiviral innate immunity. However, its precise function in response to DNA virus infection remains elusive. Herein, we find that HDAC6 deficiency promotes the activation of cGAS-STING signaling and type I interferon (IFN) production, both in vitro and in vivo, resulting in a decrease in HSV-1 infection. Mechanistically, HDAC6 deacetylates tripartite motif protein 56 (TRIM56) at K110 in mice, thereby impairing the monoubiquitination cGAS and its DNA binding ability. Overexpression of TRIM56 K110Q protects mice against HSV-1 infection. Notably, different amino acids at position 110 of TRIM56 in human and mouse cause species-specific IFN responses. Further, we show that during early stages of HSV-1 infection, the viral protein US3 phosphorylates HDAC6 to inhibit the cGAS-mediated antiviral response. Our results suggest that HDAC6 inhibits cGAS activation through TRIM56 deacetylation in a species-specific manner.

Stimulator of interferon genes (STING) is essential for innate immune pathway activation in response to pathogenic DNA. Proper activation of STING signaling requires STING translocation and phosphorylation. Here, we show that dual serine/threonine and tyrosine protein kinase (DSTYK) directly phosphorylates STING Ser366 at late endosomes to promote the activation of STING signaling. We find that TBK1 promotes STING post-Golgi trafficking via its kinase activity, thereby enabling the interaction between DSTYK and STING. We also demonstrate that DSTYK and TBK1 can both promote STING phosphorylation at late endosomes. Using an in vivo Dstyk-knockout model, we showed that mice deficient in DSTYK demonstrate reduced STING signaling activation and are more susceptible to infection with a DNA virus. Together, we reveal the previously unknown cellular function of DSTYK in phosphorylating STING and our findings provide insights into the mechanism of STING signaling activation at late endosomes.

Stimulator of interferon genes (STING) is vital in the cytosolic DNA-sensing process and critical for initiating the innate immune response, which has important functions in host defense and contributes to the pathogenesis of inflammatory diseases. Zinc finger CCCH-type antiviral protein 1 (ZC3HAV1) specifically binds the CpG dinucleotides in the viral RNAs of multiple viruses and promotes their degradation. ZAPS (ZC3HAV1 short isoform) is a potent stimulator of retinoid acid-inducible gene I (RIG-I) signaling during the antiviral response. However, how ZC3HAV1 controls STING signaling is unclear. Here, we show that ZC3HAV1 specifically potentiates STING activation by associating with STING to promote its oligomerization and translocation from the endoplasmic reticulum (ER) to the Golgi, which facilitates activation of IRF3 and NF-κB pathway. Accordingly, Zc3hav1 deficiency protects mice against herpes simplex virus-1 (HSV-1) infection- or 5,6-dimethylxanthenone-4-acetic acid (DMXAA)-induced inflammation in a STING-dependent manner. These results indicate that ZC3HAV1 is a key regulator of STING signaling, which suggests its possible use as a therapeutic target for STING-dependent inflammation.

Endoplasmic reticulum (ER) autophagy (ER-phagy) is a vital homeostatic process triggered by multiple signals and plays a crucial role in regulating innate immunity and viral replication. However, the mechanisms by which host proteins utilize ER-phagy to regulate innate immune response during viral infection remains largely unclear. Here, we uncover the regulatory crosstalk between innate immune adapter, ER retention protein Stimulator of Interferon Genes (STING), and the G protein-coupled receptor ADGRE5/CD97 (Cluster of Differentiation 97). Our results demonstrate that CD97 suppresses the STING-mediated type-I interferon (IFN-I) response against DNA virus and cytosolic DNA, thereby promoting herpes simplex virus type 1 (HSV-1) replication in both cells and mice. CD97 facilitates the recruitment of the ER-phagy receptor, FAM134B (family with sequence similarity 134, member B), to initiate ER-phagy, resulting in the degradation of STING subsequent to DNA virus infection. Furthermore, Cd97-deficient mice exhibit higher IFN-I response and greater resistance to HSV-1 infection. Additionally, our findings reveal that inhibiting CD97 with sanguinarine effectively disrupts HSV-1 replication. These findings shed light on the role of CD97 in the innate immune response against DNA virus infections and offer valuable checkpoint for anti-viral STING activation.

Human cytomegalovirus (HCMV) has evolved diverse strategies for immune evasion. In this study, we identified HCMV-pUS2 as an indirect antagonist of the cGAS-STING pathway by promoting the degradation of lectin mannose-binding 2-like protein (LMAN2L), an unrecognized host factor involved in STING pathway. First, we discovered that HCMV, but not other DNA viruses such as HSV-1 and VACV, induces proteasomal degradation of LMAN2L during the immediate-early stage of infection. We then demonstrated that HCMV-pUS2 mediates LMAN2L degradation by recruiting the host E3 ubiquitin ligase RNF139 and E2 ubiquitin-conjugating enzyme UBE2G2, directing LMAN2L to the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway. LMAN2L knockout diminishes HCMV-induced expression of type I interferons and interferon-stimulated genes. Furthermore, LMAN2L co-localizes and interacts with STING. Though it does not affect STING dimerization or TBK1 recruitment, it is essential for STING translocation from the ER to the Golgi. Our findings uncover LMAN2L as a novel host regulator of the STING pathway and identify pUS2-mediated ERAD as a previously unrecognized viral immune evasion strategy.

The cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS)-dependent pathway is a key DNA-sensing pathway that recognizes cytosolic DNA and plays a crucial role in initiating innate immune responses against pathogenic microbes and cancer. Various molecules have been identified as regulators of the cGAS-dependent pathway that controls innate immune responses. However, despite the important roles of Stimulator-of-interferon genes (STING) in the cGAS-dependent pathway, the regulation of its activation has not been elucidated. Here, we show that the E3 ubiquitin ligase, RING finger protein 39 (RNF39), interacts with STING in macrophages and HERK293T cells. Moreover, RNF39 accelerates DNA-sensing pathways by promoting lysine (K)63-linked ubiquitination of STING, and then facilitating the formation of STING-TBK1 complex. Concordantly, Rnf39 deficiency inhibits innate immune responses triggered by DNA viral infection and accelerates viral replication. Furthermore, herpes simplex virus-1 (HSV-1) infection induces RNF39 expression in an IFN-I-dependent manner. Thus, we outline a novel mechanism for controlling STING activation and a feedback mechanism for controlling antiviral immune responses. RNF39 could be a priming intervention target for the prevention and treatment of viral diseases, especially DNA viral infections.

Stimulator of interferon genes (STING) is an endoplasmic reticulum (ER) protein that plays a crucial role in cytosolic DNA-mediated innate immunity. Both STING agonists and antagonists have demonstrated their ability to enhance mouse survival against coronavirus, however, the physiological role of endogenous STING in coronavirus infection remains unclear. Our research unveils that STING inhibits coronavirus replication by impeding the formation of the ER-derived double-membrane vesicles (DMVs), the organelles in which coronavirus replicates. We found that STING was still capable of inhibiting coronavirus OC43 infection in cells, regardless of the knockout of cGAS or MAVS, or blocking type I interferon receptor. Moreover, STING disrupted the interaction between two crucial proteins, NSP4 and NSP6, involved in DMV formation, leading to the disruption of DMV formation. Taken together, our study sheds light on a novel antiviral role of STING in coronavirus infection, elucidating how it disrupts the formation of viral replication organelles, thereby impeding the replication process.

Stimulator of interferon genes (STING) is a pivotal innate immune adaptor, and its functions during DNA virus infections have been extensively documented. However, its homeostatic regulation is not well understood. Our study demonstrates that Unc-93 homolog B1 (UNC93B1) is a crucial checker for STING to prevent hyperactivation. Ectopic expression of UNC93B1 attenuates IFN-β promoter activity and the transcriptions of IFN-β, ISG54, and ISG56 genes. Moreover, UNC93B1 also blocks the IRF3 nuclear translocation induced by ectopic expression of both cyclic GMP-AMP synthase (cGAS) and STING and reduces the stability of STING by facilitating its autophagy-lysosome degradation, which can be reversed by lysosome inhibitors. Mechanistically, UNC93B1 interacts with STING and suppresses STING-activated downstream signaling by delivering STING to the lysosomes for degradation, depending on its trafficking capability. UNC93B1 knockout in human embryonic kidney 293T cells facilitates IFN-β promoter activity, IFN-β, ISG54, and ISG56 transcriptions, and IRF3 nuclear translocation induced by ectopic expression of cGAS and STING. Infected with herpes simplex virus-1 (HSV-1), UNC93B1 knockdown BJ cells or primary peritoneal macrophages from Unc93b1-deficient (Unc93b1 ) mice show enhanced IFN-β, ISG54, and ISG56 transcriptions, TBK1 phosphorylation, and reduced STING degradation and viral replication. In addition, Unc93b1 mice exhibit higher IFN-β, ISG54, and ISG56 transcriptions and lower mortality upon HSV-1 infection in vivo. Collectively, these findings demonstrate that UNC93B1 attenuates the cGAS-STING signaling pathway by targeting STING for autophagy-lysosome degradation and provide novel insights into the function of UNC93B1 in antiviral innate immunity.