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Previously, we reported that azamollugin, an aza-derivative of mollugin, exhibited potent inhibitory activity on NO production in LPS-stimulated RAW 264.7 cells. Further investigations in this study revealed that azamollugin not only suppressed iNOS gene expression regulated by NF-κB, but also inhibited LPS-induced IFN-β expression, which is known to be regulated by IRF3. Azamollugin exhibited an inhibitory activity on LPS-induced IRAK1 activation, suggesting inhibitory effect on the MyD88-dependent pathway. Furthermore, azamollugin inhibited LPS-induced phosphorylation of IRF3 and its upstream factor, TBK1/IKKε, suggesting an inhibitory effect on the TRIF-dependent pathway via TLR4. In addition, azamollugin also suppressed poly(I:C)-induced phosphorylation of TBK1 and IRF3, suggesting an inhibitory effect on the TRIF-dependent pathway via TLR3. These results suggest that azamollugin has inhibitory activity against both the MyD88-dependent and TRIF-dependent pathways, respectively. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (M. tb) is a significant intracellular pathogen responsible for numerous infectious disease‐related deaths worldwide. It uses ESX‐1 T7SS to damage phagosomes and to enter the cytosol of host cells after phagocytosis. During infection, M. tb and host mitochondria release dsDNA, which activates the CGAS‐STING1 pathway. This pathway leads to the production of type I interferons and proinflammatory cytokines and activates autophagy, which targets and degrades bacteria within autophagosomes. However, the role of type I IFNs in immunity against M. tb is controversial. While previous research has suggested a protective role, recent findings from cgas‐sting1 knockout mouse studies have contradicted this. Additionally, a study using knockout mice and non‐human primate models uncovered a new mechanism by which neutrophils recruited to lung infections form neutrophil extracellular traps. Activating plasmacytoid dendritic cells causes them to produce type I IFNs, which interfere with the function of interstitial macrophages and increase the likelihood of tuberculosis. Notably, M. tb uses its virulence proteins to disrupt the CGAS‐STING1 signaling pathway leading to enhanced pathogenesis. Investigating the CGAS‐STING1 pathway can help develop new ways to fight tuberculosis. [ABSTRACT FROM AUTHOR]

Simple Summary: A238L, a non-structural protein of the African swine fever virus (ASFV), inhibits the activation of NF-κB by suppressing the HAT activity of p300. Whether A238L also affects the transcriptional activity of IRF3 remains unexplored. Here we first confirmed the ability of A238L to suppress NF-κB-activity in L929 cells. In contrast, A238L did not inhibit but rather increased TBK1 and IRF3 phosphorylation and enhanced innate antiviral immunity in the absence or presence of poly d (A:T) or poly (I:C) stimulation, or herpes simplex virus type 1 (HSV-1) or Sendai virus (SeV) infection. This study reveals an unrecognized role for A238L in promoting antiviral immune responses by activating the TBK1-IRF3 pathway. African swine fever virus (ASFV) is a double-stranded DNA virus with an envelope. ASFV has almost the largest genome among all DNA viruses, and its mechanisms of immune evasion are complex. Better understanding of the molecular mechanisms of ASFV genes will improve vaccine design. A238L, a nonstructural protein of ASFV, inhibits NF-κB activation by suppressing the HAT activity of p300. Whether A238L also affects the transcriptional activity of IRF3 remains unexplored. Here we first confirmed the ability of A238L to suppress NF-κB-activity in L929 cells. A238L inhibits the expression of proinflammatory cytokine genes. In contrast, A238L increased the phosphorylation levels of TBK1 and IRF3 in three different cell lines. A238L increases the IRF3-driven promoter activity and induces IRF3 nuclear translocation. Furthermore, A238L enhanced innate antiviral immunity in the absence or presence of poly d (A:T) or poly (I:C) stimulation, or herpes simplex virus type 1 (HSV-1) or Sendai virus (SeV) infection. This study reveals a previously unrecognized role of A238L in promoting antiviral immune responses by TBK1-IRF3 pathway activation. [ABSTRACT FROM AUTHOR]

Background & aimsThe stimulator of interferon genes (STING)/TANK-binding kinase 1 (TBK1) pathway is vital in mediating innate immune and inflammatory responses during oxidative/endoplasmic reticulum (ER) stress. However, it remains unknown whether macrophage thioredoxin-interacting protein (TXNIP) may regulate TBK1 function and cell death pathways during oxidative/ER stress.MethodsA mouse model of hepatic ischaemia/reperfusion injury (IRI), the primary hepatocytes, and bone marrow-derived macrophages were used in the myeloid-specific TXNIP knockout (TXNIPM-KO) and TXNIP-proficient (TXNIPFL/FL) mice.ResultsThe TXNIPM-KO mice were resistant to ischaemia/reperfusion (IR) stress-induced liver damage with reduced serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST) levels, macrophage/neutrophil infiltration, and pro-inflammatory mediators compared with the TXNIPFL/FL controls. IR stress increased TXNIP, p-STING, and p-TBK1 expression in ischaemic livers. However, TXNIPM-KO inhibited STING, TBK1, interferon regulatory factor 3 (IRF3), and NF-κB activation with interferon-β (IFN-β) expression. Interestingly, TXNIPM-KO augmented nuclear factor (erythroid-derived 2)-like 2 (NRF2) activity, increased antioxidant gene expression, and reduced macrophage reactive oxygen species (ROS) production and hepatic apoptosis/necroptosis in IR-stressed livers. Mechanistically, macrophage TXNIP deficiency promoted cylindromatosis (CYLD), which colocalised and interacted with NADPH oxidase 4 (NOX4) to enhance NRF2 activity by deubiquitinating NOX4. Disruption of macrophage NRF2 or its target gene 2',5' oligoadenylate synthetase-like 1 (OASL1) enhanced Ras GTPase-activating protein-binding protein 1 (G3BP1) and TBK1-mediated inflammatory response. Notably, macrophage OASL1 deficiency induced hepatocyte apoptotic peptidase activating factor 1 (APAF1), cytochrome c, and caspase-9 activation, leading to increased caspase-3-initiated apoptosis and receptor-interacting serine/threonine-protein kinase 3 (RIPK3)-mediated necroptosis.ConclusionsMacrophage TXNIP deficiency enhances CYLD activity and activates the NRF2-OASL1 signalling, controlling IR stress-induced liver injury. The target gene OASL1 regulated by NRF2 is crucial for modulating STING-mediated TBK1 activation and Apaf1/cytochrome c/caspase-9-triggered apoptotic/necroptotic cell death pathway. Our findings underscore a novel role of macrophage TXNIP-mediated CYLD-NRF2-OASL1 axis in stress-induced liver inflammation and cell death, implying the potential therapeutic targets in liver inflammatory diseases.Lay summaryLiver inflammation and injury induced by ischaemia and reperfusion (the absence of blood flow to the liver tissue followed by the resupply of blood) is a significant cause of hepatic dysfunction and failure following liver transplantation, resection, and haemorrhagic shock. Herein, we uncover an underlying mechanism that contributes to liver inflammation and cell death in this setting and could be a therapeutic target in stress-induced liver inflammatory injury.

Background: Rheumatoid arthritis (RA) is an autoimmune inflammation disease characterized by imbalance of immune homeostasis. p53 mutants are commonly described as the guardian of cancer cells by conferring them drug-resistance and immune evasion. Importantly, p53 mutations have also been identified in RA patients, and this prompts the investigation of its role in RA pathogenesis. Methods: The cytotoxicity of disease-modifying anti-rheumatic drugs (DMARDs) against p53 wild-type (WT)/mutant-transfected RA fibroblast-like synoviocytes (RAFLSs) was evaluated by MTT assay. Adeno-associated virus (AAV) was employed to establish p53 WT/R211* adjuvant-induced arthritis (AIA) rat model. The arthritic condition of rats was assessed by various parameters such as micro-CT analysis. Knee joint samples were isolated for total RNA sequencing analysis. The expressions of cytokines and immune-related genes were examined by qPCR, ELISA assay and immunofluorescence. The mechanistic pathway was determined by immunoprecipitation and Western blotting in vitro and in vivo. Results: Among p53 mutants, p53R213* exhibited remarkable DMARD-resistance in RAFLSs. However, AAV-induced p53R211* overexpression ameliorated inflammatory arthritis in AIA rats without Methotrexate (MTX)-resistance, and our results discovered the immunomodulatory effect of p53R211* via suppression of T-cell activation and T helper 17 cell (Th17) infiltration in rat joint, and finally downregulated expressions of pro-inflammatory cytokines. Total RNA sequencing analysis identified the correlation of p53R211* with immune-related pathways. Further mechanistic studies revealed that p53R213*/R211* instead of wild-type p53 interacted with TANK-binding kinase 1 (TBK1) and suppressed the innate immune TBK1–Interferon regulatory factor 3 (IRF3)–Stimulator of interferon genes (STING) cascade. Conclusions: This study unravels the role of p53R213* mutant in RA pathogenesis, and identifies TBK1 as a potential anti-inflammatory target. [ABSTRACT FROM AUTHOR]

African swine fever virus (ASFV) is a double-stranded DNA virus with an envelope. ASFV has almost the largest genome among all DNA viruses, and its mechanisms of immune evasion are complex. Better understanding of the molecular mechanisms of ASFV genes will improve vaccine design. A238L, a nonstructural protein of ASFV, inhibits NF-κB activation by suppressing the HAT activity of p300. Whether A238L also affects the transcriptional activity of IRF3 remains unexplored. Here we first confirmed the ability of A238L to suppress NF-κB-activity in L929 cells. A238L inhibits the expression of proinflammatory cytokine genes. In contrast, A238L increased the phosphorylation levels of TBK1 and IRF3 in three different cell lines. A238L increases the IRF3-driven promoter activity and induces IRF3 nuclear translocation. Furthermore, A238L enhanced innate antiviral immunity in the absence or presence of poly d (A:T) or poly (I:C) stimulation, or herpes simplex virus type 1 (HSV-1) or Sendai virus (SeV) infection. This study reveals a previously unrecognized role of A238L in promoting antiviral immune responses by TBK1-IRF3 pathway activation.

Swine acute diarrhea syndrome (SADS) is first reported in January 2017 in Southern China. It subsequently causes widespread outbreaks in multiple pig farms, leading to economic losses. Therefore, it is an urgent to understand the molecular mechanisms underlying the pathogenesis and immune evasion of Swine acute diarrhea syndrome coronavirus (SADS‐CoV). Our research discovered that SADS‐CoV inhibited the production of interferon‐β (IFN‐β) during viral infection. The nonstructural protein 1 (nsp1) prevented the phosphorylation of TBK1 by obstructing the interaction between TBK1 and Ub protein. Moreover, nsp1 induced the degradation of CREB‐binding protein (CBP) through the proteasome‐dependent pathway, thereby disrupting the IFN‐β enhancer and inhibiting IFN transcription. Finally, we identified nsp1‐Phe39 as the critical amino acid that downregulated IFN production. In conclusion, our findings described two mechanisms in nsp1 that inhibited IFN production and provided new insights into the evasion strategy adopted by SADS‐CoV to evade host antiviral immunity. [ABSTRACT FROM AUTHOR]

Objective To explore the expression and role of stimulator of interferon gene (STING)-TANK-binding kinase 1 (TBK1)-interferon regulatory factor 3 (IRF3) signaling pathway in the brain of chronic stress mice. Methods Mice were divided into control (CON) group and chronic restraint stress (RST) group. Mice in the RST group were given chronic restraint stress stimulation (6 hours per day, 14 days). After 14 days, the mRNA expressions of pro-inflammatory cytokines CCL2, CXCL10, IL-1β, IL-6, IL-10, and TNFα in the brain were detected and analyzed by qRT-PCR; protein expression of STING, TBK1, p-TBK1, IRF3, and p-IRF3 were detected and analyzed by immunofluorescence staining and Western blotting. Results Compared to the CON group, the mRNA expressions of pro-inflammatory cytokines in the RST group were significantly increased (P<0.05). STING and microglia marker Iba-1 were highly co-located and the expression of STING was decreased as detected by immunofluorescence staining. Moreover, the protein expressions of STING, p-TBK1, and p-IRF3 were significantly decreased (all P<0.01). Conclusion Chronic restraint stress triggers a neuroinflammatory response and the STING-TBK1-IRF3 pathway in the brain of the RST mice is significantly inhibited. [ABSTRACT FROM AUTHOR]

Growing evidence highlights that tripartite motif (TRIM) family members exert crucial functions in host defense upon infection with microbial pathogens. Of note, the roles of a subfamily of fish-specific TRIM (FTR, also named as finTRIM) which have no true orthologues in mammals were largely unexplored. In this study, a novel FTR gene from grouper (EcFTR14) was identified, and its effects on fish virus infection and host interferon response were investigated. EcFTR14 contained RING, B-Box, and PRY-SPRY domains, and shared 54.81% identity to zebrafish FTR14. The transcription levels of EcFTR14 were markedly induced upon fish virus infection. EcFTR14 was mainly localized in the cytoplasm as small spots or bright aggregates. The overexpression of EcFTR14 could markedly enhance the replication of RGNNV or SGIV. Moreover, overexpression of EcFTR14 reduced the mRNA level of IFN-associated immune signaling molecules, suggesting that the antiviral action of EcFTR14 was due to the negative regulation on IFN response. EcFTR14 was found to interact with EcTBK1, EcIRF3 and EcIRF7, but not with EcSTING. Overexpression of EcFTR14 markedly suppressed the transcription levels of IFN-signaling related molecules evoked by EcSTING and EcTBK1. In addition, overexpression of EcFTR14 could degrade EcTBK1, EcIRF3 and EcIRF7 in a dose-dependent manner, that resulted in the suppression of antiviral effects induced by EcIRF3 and EcTBK1. Collectively, our findings illuminated that EcFTR14, a novel member of FTR family, acted as a pro-viral factor through negatively regulating targeting TBK1-IRF3 activated antviral action. The present study will for the first time shed light on the regulatory roles of FTR14 in innate immune response upon fish virus infection. • EcFTR14 was a novel FTR protein which shared 54.81% identity to zebrafish FTR14. • EcFTR14 were significantly up-regulated upon SGIV and RGNNV infection. • EcFTR14 acted as a pro-viral factor during SGIV and RGNNV infection. • EcFTR14 interacted with EcTBK1, EcIRF3, EcIRF7, and negatively regulated IFN response. • EcFTR14 weakened the antiviral effects induced by EcIRF3 and EcTBK1. [ABSTRACT FROM AUTHOR]

Cytosolic RNA/DNA sensing elicits primary defense against viral pathogens. Interferon regulatory factor 3 (IRF3), a key signal mediator/transcriptional factor of the antiviral-sensing pathway, is indispensible for interferon production and antiviral defense. However, how the status of IRF3 activation is controlled remains elusive. Through a functional screen of the human kinome, we found that mammalian sterile 20-like kinase 1 (Mst1), but not Mst2, profoundly inhibited cytosolic nucleic acid sensing. Mst1 associated with IRF3 and directly phosphorylated IRF3 at Thr75 and Thr253. This Mst1-mediated phosphorylation abolished activated IRF3 homodimerization, its occupancy on chromatin, and subsequent IRF3-mediated transcriptional responses. In addition, Mst1 also impeded virus-induced activation of TANK-binding kinase 1 (TBK1), further attenuating IRF3 activation. As a result, Mst1 depletion or ablation enabled an enhanced antiviral response and defense in cells and mice. Therefore, the identification of Mst1 as a novel physiological negative regulator of IRF3 activation provides mechanistic insights into innate antiviral defense and potential antiviral prevention strategies.

Abstract TANK-binding kinase 1 (TBK1), a core kinase of antiviral pathways, activates the production of interferons (IFNs). It has been reported that deacetylation activates TBK1; however, the precise mechanism still remains to be uncovered. We show here that during the early stage of viral infection, the acetylation of TBK1 was increased, and the acetylation of TBK1 at Lys241 enhanced the recruitment of IRF3 to TBK1. HDAC3 directly deacetylated TBK1 at Lys241 and Lys692, which resulted in the activation of TBK1. Deacetylation at Lys241 and Lys692 was critical for the kinase activity and dimerization of TBK1 respectively. Using knockout cell lines and transgenic mice, we confirmed that a HDAC3 null mutant exhibited enhanced susceptibility to viral challenge via impaired production of type I IFNs. Furthermore, activated TBK1 phosphorylated HDAC3, which promoted the deacetylation activity of HDAC3 and formed a feedback loop. In this study, we illustrated the roles the acetylated and deacetylated forms of TBK1 play in antiviral innate responses and clarified the post-translational modulations involved in the interaction between TBK1 and HDAC3.

Abstract Background Amlexanox (ALX), a TBK1 inhibitor, can modulate immune responses and has anti-inflammatory properties. To investigate its role in regulating the progression of experimental autoimmune encephalomyelitis (EAE), we studied the effect of ALX on the maturation of dendritic cells (DCs) and the responses of effector and regulatory T cells (Tregs). Methods In vitro, bone marrow-derived DCs (BMDCs) were cultured and treated with ALX. Their proliferation, maturation, and their stimulatory function to induce T cells responses were detected. In vivo, the development of EAE from different groups was recorded. At the peak stage of disease, HE, LFB, and electronic microscope (EM) were used to evaluate inflammation and demyelination. Maturation of splenic DC and Th1/Th17/Treg response in the CNS and peripheral were also detected. To further explore the mechanism underlying the action of ALX in DC maturation, the activation of TBK1, IRF3, and AKT was analyzed. Results Our data indicated that ALX significantly inhibited the proliferation and maturation of BMDCs, characterized by the reduced MHCII, a co-stimulatory molecule, IL12, and IL-23 expression, along with morphological alterations. Co-culture of ALX-treated BMDCs inhibited allogeneic T cell proliferation and MOG-specific T cell response. In EAE mice, ALX significantly attenuated the EAE development by decreasing inflammatory infiltration and demyelination in the spinal cords, accompanied by reduced frequency of splenic pathogenic Th1 and Th17 cells and increased Tregs. Moreover, ALX treatment decreased Th1 and Th17 cytokines, but increased Treg cytokines in the CNS and spleen. Notably, ALX treatment reduced the frequency and expression of CD80 and CD86 on splenic DCs and lowered IL-12 and IL-23 secretion, further supporting an impaired maturation of splenic DCs. In addition, ALX potently reduced the phosphorylation of IRF3 and AKT in BMDC and splenic DCs, both of which are substrates of TBK1 and associated with DC maturation. Conclusions ALX, a TBK1 inhibitor, mitigated EAE development by inhibiting DC maturation and subsequent pathogenic Th1 and Th17 responses while increasing Treg responses through attenuating the TBK1/AKT and TBK1/IRF3 signaling.

TANK-binding kinase 1 (TBK1), a core kinase of antiviral pathways, activates the production of interferons (IFNs). It has been reported that deacetylation activates TBK1; however, the precise mechanism still remains to be uncovered. We show here that during the early stage of viral infection, the acetylation of TBK1 was increased, and the acetylation of TBK1 at Lys241 enhanced the recruitment of IRF3 to TBK1. HDAC3 directly deacetylated TBK1 at Lys241 and Lys692, which resulted in the activation of TBK1. Deacetylation at Lys241 and Lys692 was critical for the kinase activity and dimerization of TBK1 respectively. Using knockout cell lines and transgenic mice, we confirmed that a HDAC3 null mutant exhibited enhanced susceptibility to viral challenge via impaired production of type I IFNs. Furthermore, activated TBK1 phosphorylated HDAC3, which promoted the deacetylation activity of HDAC3 and formed a feedback loop. In this study, we illustrated the roles the acetylated and deacetylated forms of TBK1 play in antiviral innate responses and clarified the post-translational modulations involved in the interaction between TBK1 and HDAC3. [ABSTRACT FROM AUTHOR]

TANK-binding kinase 1 (TBK1), an IKK-related serine/threonine kinase, is pivotal for the induction of antiviral type I interferon (IFN) by TLR and RLR signaling pathways. In a previous study, we demonstrated that TBK1 spliced isoforms (TBK1_tv1 and TBK1_tv2) from zebrafish were dominant negative regulators in the RLR antiviral pathway by targeting the functional TBK1–IRF3 complex formation. In this study, we show that the third TBK1 isoform (namely TBK1_tv3) inhibits zebrafish type I IFN production by promoting TBK1 and IRF3 degradation. First, ectopic expression of TBK1_tv3 suppresses poly(I:C)- and Spring viremia of carp virus-induced type I IFN response, and also inhibits the up-regulation of IFN promoter activities stimulated by RIG-I, MDA5, MAVS, TBK1, and IRF3. Second, TBK1_tv3 targets TBK1 and IRF3 to impair the formation of TBK1 dimer, TBK1–IRF3 complex, and IRF3 dimer. Notably, TBK1_tv3 promotes the degradation of TBK1 through the ubiquitin–proteasome pathway and the degradation of IRF3 through the lysosomal pathway. Further analysis demonstrates that TBK1_tv3 promotes the degradation of TBK1 for K48-linked ubiquitination by targeting the K251, K256, and K271 sites of TBK1. Collectively, our results suggest a novel TBK1 isoform-mediated negative regulation mechanism, which serves to balance the production of type I IFN and ISGs.

Background: STK4 deficiency due to homozygous mutations in the STK4 gene encoding the STK4/MST1 kinase was first described in 2012. STK4/MST1 kinase regulates cell proliferation, survival, differentiation, and immune responses through canonical and non-canonical Hippo signaling pathways. Objective: We describe an 11-year-old girl with a clinical presentation consisting of severe recurrent herpes zoster, chronic warts, and recurrent pneumonias, as well as a somatic phenotype with hypothyroidism and low stature. Whole exome sequencing revealed STK4 deficiency due to homozygosity for a novel frameshift variant in STK4, c.523dupA, p.(L174fsTer45), resulting in a premature stop codon within the kinase domain. Methods: We performed a thorough investigation of the genetics and innate and adaptive immunological abnormalities in STK4 deficiency. Results: We show significantly impaired type I, II, and III interferon (IFN) responses and partly reduced proinflammatory cytokine responses to ligands of Toll-like receptor (TLR)3, TLR9, and the cytosolic RNA and DNA sensors as well as to microorganisms. Impaired IFN responses could be attributed to reduced phosphorylation of TBK1 and IRF3. Moreover, virus infection induced enhanced cell death by apoptosis. Importantly, autophagy pathways were slightly disturbed, with enhanced LC3B-Ito LCB3-II conversion at the single cell level but normal overall formation of LCB3 punctae. Finally, the patient displayed some indicators of impaired adaptive immunity in the form of insufficient vaccination responses, T cell lymphopenia, and reduced Treg fractions, although with largely normal T cell proliferation and normal IFNg production. Conclusion: Here, we demonstrate disturbances in various immune cell populations and pathways involved in innate immune responses, cell death, autophagy, and adaptive immunity in a patient homozygous for a novel STK4 frameshift mutation. [ABSTRACT FROM AUTHOR]

TANK-binding kinase 1 (TBK1), an IKK-related serine/threonine kinase, is pivotal for the induction of antiviral type I interferon (IFN) by TLR and RLR signaling pathways. In a previous study, we demonstrated that TBK1 spliced isoforms (TBK1_tv1 and TBK1_tv2) from zebrafish were dominant negative regulators in the RLR antiviral pathway by targeting the functional TBK1–IRF3 complex formation. In this study, we show that the third TBK1 isoform (namely TBK1_tv3) inhibits zebrafish type I IFN production by promoting TBK1 and IRF3 degradation. First, ectopic expression of TBK1_tv3 suppresses poly(I:C)- and Spring viremia of carp virus-induced type I IFN response, and also inhibits the up-regulation of IFN promoter activities stimulated by RIG-I, MDA5, MAVS, TBK1, and IRF3. Second, TBK1_tv3 targets TBK1 and IRF3 to impair the formation of TBK1 dimer, TBK1–IRF3 complex, and IRF3 dimer. Notably, TBK1_tv3 promotes the degradation of TBK1 through the ubiquitin–proteasome pathway and the degradation of IRF3 through the lysosomal pathway. Further analysis demonstrates that TBK1_tv3 promotes the degradation of TBK1 for K48-linked ubiquitination by targeting the K251, K256, and K271 sites of TBK1. Collectively, our results suggest a novel TBK1 isoform-mediated negative regulation mechanism, which serves to balance the production of type I IFN and ISGs. [ABSTRACT FROM AUTHOR]

Singapore grouper iridovirus (SGIV), belonging to genus Ranavirus , family Iridoviridae , is a highly pathogenic agent and causes heavy economic losses in the global grouper aquaculture. Recent studies demonstrated that SGIV infection attenuated antiviral immune and inflammatory response induced by poly (I:C) in vitro. However, little was known about the potential functions of the immune regulatory proteins encoded by SGIV. Here, we identified the detailed roles of VP20 and clarified the potential mechanism underlying its immune regulatory function during SGIV infection. Our results showed that VP20 was an IE gene, and partially co-localized with Golgi apparatus and lysosomes in grouper cells. Overexpression of VP20 enhanced SGIV replication, demonstrated by the increase in the transcription levels of viral core genes and the protein synthesis of MCP. Reporter gene assays showed that SGIV VP20 overexpression significantly reduced the IFN promoter activity induced by poly (I:C), grouper stimulator of interferon genes (EcSTING) and TANK-binding kinase 1 (EcTBK1). Consistently, the transcription levels of IFN related genes were significantly decreased in VP20 overexpressing cells compared to those in control cells. Co-IP assay and confocal microscopy observations indicated that VP20 co-localized and interacted with EcTBK1 and EcIRF3, but not EcSTING. In addition, VP20 was able to degrade EcIRF3 and attenuate the antiviral action of EcIRF3, while had no effect on EcTBK1. Together, SGIV VP20 was speculated to promote viral replication through attenuating the IFN response mediated by TBK1-IRF3 in vitro. Our findings provided new insights into the immune regulatory function of SGIV encoded unknown proteins. • SGIV VP20 was partially localized with Golgi apparatus and lysosomes. • VP20 overexpression promoted the SGIV replication in vitro. • SGIV VP20 interacted and co-localized with EcTBK1 and EcIRF3. • SGIV VP20 degraded EcIRF3 and attenuated the antiviral action of EcIRF3. [ABSTRACT FROM AUTHOR]

ABSTRACT TANK-binding kinase 1 (TBK1) is a key component of the antiviral immunity signaling pathway. It activates downstream interferon regulatory factor 3 (IRF3) and subsequent type I interferon (IFN-I) production. Herpes simplex virus type 1 (HSV-1) can antagonize host antiviral immune responses and lead to latent infection. Here, HSV-1 tegument protein UL46 was demonstrated to downregulate TBK1-dependent antiviral innate immunity. UL46 interacted with TBK1 and reduced TBK1 activation and its downstream signaling. Our results showed that UL46 impaired the interaction of TBK1 and IRF3 and downregulated the activation of IRF3 by inhibiting the dimerization of TBK1 to reduce the IFN-I production induced by TBK1 and immunostimulatory DNA. The IFN-I and its downstream antiviral genes induced by UL46-deficient HSV-1 (ΔUL46 HSV-1) were higher than those of wild-type HSV-1 (WT HSV-1). In addition, the stable knockdown of TBK1 facilitated the replication of ΔUL46 HSV-1, but not WT HSV-1. Together, these findings reveal a novel mechanism of immune evasion by HSV-1. IMPORTANCE HSV-1 has evolved multiple strategies to evade host antiviral responses and establish a lifelong latent infection, but the molecular mechanisms by which HSV-1 interrupts antiviral innate immunity are not completely understood. As TBK1 is very critical for antiviral innate immunity, it is of great interest to reveal the immune evasion mechanism of HSV-1 by targeting TBK1. In the present study, HSV-1 UL46 was found to inhibit the activation of IFN-I by targeting TBK1, suggesting that the evasion of TBK1 mediated antiviral innate immunity by HSV-1 UL46. Findings in this study will provide new insights into the host-virus interaction and help develop new approaches against HSV-1 infection.

Early detection of viral invasion by pattern recognition receptors (PRR) is crucial for the induction of a rapid and efficient immune response. Cytosolic DNA sensors are the most recently described class of PRR, and induce transcription of type I interferons (IFN) and proinflammatory cytokines via the key adaptor protein stimulator of interferon genes (STING). Herpesviruses are a family of large DNA viruses widely known for their immense arsenal of proteins dedicated to manipulating and evading host immune responses. Tantamount to the significant role played by DNA sensors and STING in innate immune responses, herpesviruses have in turn evolved a range of mechanisms targeting virtually every step of this key signaling pathway. Strikingly, some herpesviruses also take advantage of this pathway to promote their own replication. In this review, we will summarize the current understanding of DNA sensing and subsequent induction of signaling and transcription, and showcase the close adaptation of herpesviruses to their host reflected by the myriad of viral proteins dedicated to modulating this critical innate immune pathway. [ABSTRACT FROM AUTHOR]

Class II HDACs, such as HDAC4, are critical regulators of the immune response in various immune cells; however, its role in innate immunity remains largely unknown. Here, we report that the overexpression of HDAC4 suppresses the production of type I interferons triggered by pattern-recognition receptors (PRRs). HDAC4 repressed the translocation of transcription factor IRF3 to the nucleus, thereby decreasing IRF3-mediated IFN-β expression. In particular, we also determined that HDAC4 can be phosphorylated and simultaneously block the phosphorylation of IRF3 at Ser386 and Ser396 by TBK1 and IKKε, respectively, by interacting with the kinase domain of TBK1 and IKKε. Furthermore, IFN-β may stimulate the expression of HDAC4. Our findings suggest that HDAC4 acts as a regulator of PRR signaling and is a novel mechanism of negative feedback regulation for preventing an over-reactive innate immune response. [ABSTRACT FROM AUTHOR]

Type I interferon (IFN) induction is a critical component of innate immune response to viral and bacterial infection, including S. aureus, but whether it activates the signaling in macrophages and the regulation mechanisms is less well understood. Here we show that S. aureus infection promoted the IFN-β mRNA expression and stimulator of IFN genes (STING)/TANK-binding kinase 1 (TBK1)/interferon regulatory factor 3 (IRF3)-dependent production of IFN-β. Infection with S. aureus induced caspase recruitment domain and membrane-associated guanylate kinase-like domain protein 3 (CARMA3) expression at both the mRNA and protein levels. The heat-killed bacteria failed to trigger IRF3 phosphorylation and upregulation of CARMA3 expression. However, overexpression of CARMA3 did not affect phosphorylation of TBK1 or IRF3 in RAW264.7 cells, J774A.1 macrophages, and mouse embryonic fibroblast (MEF) cells. In conclusion, S. aureus infection induces STING/TBK1/IRF3-mediated IFN-β production in a CARMA3-independent manner.

The golden pompano (Trachinotus blochii), a pivotal commercial marine species in China, has gained significant popularity worldwide. However, accompanied with rapid growth and high density aquaculture, golden pompano has been seriously threatened by Nervous necrosis virus (NNV), while its molecular biology research regarding the innate immune system remains unexplored, which is crucial for understanding the activation of interferon (IFN) production and antiviral responses. In this study, we aimed to identify the characterization and function of golden pompano TANK-binding kinase 1 (gpTBK1), thereby providing evidence of the conservation of this classical factor in the RLR pathway among marine fish. Initially, we found the expression of gpTBK1 upregulation in diseased golden pompano with NNV infection and we successfully cloned the full-length open reading frame (ORF) of gpTBK1, consisting of 2172 nucleotides encoding 723 amino acids, from the head kidney. Subsequent analysis of the amino acid sequence revealed homology between gpTBK1 and other fish TBK1 proteins, with conserved N-terminal Serine/Threonine protein kinases catalytic domain (S_TKc) and C-terminal coiled coil domain (CCD). Moreover, the expression pattern showed that gpTBK1 exhibited ubiquitous expression across all evaluated tissues. Furthermore, functional identification experiments indicated that gpTBK1 activated interferon promoters' activity in golden pompano and induced the expression of downstream IFN-stimulated genes (ISGs). Notably, gpTBK1 was found to co-localize and interact with gpIRF3 in the cytoplasm. Collectively, these data provide a comprehensive analysis of the characterization and functional role of gpTBK1 in promoting interferon production. This research may facilitate the further study of the innate antiviral response, particularly the anti-NNV mechanisms, in golden pompano. • gpTBK1 is highly homologous and the N-terminal S_TKc and C-terminal CCD domains are conserved. • The expression of gpTBK1 is upregulated in diseased golden pompano with NNV infection. • gpTBK1 is ubiquitously expressed in the evaluated tissues of the golden pompano. • gpTBK1 activates gpIFN promoters' activity and induces IFN-stimulated genes expression. • gpTBK1 is localized in the cytoplasm and has co-localization and interaction with gpIRF3. [ABSTRACT FROM AUTHOR]

Mutations of Ubiquilin 2 (UBQLN2) or TANK-binding kinase 1 (TBK1) are associated with amyotrophic lateral sclerosis and frontotemporal degeneration (ALS/FTD). However, the mechanisms whereby UBQLN2 or TBK1 mutations lead to ALS and FTD remain unclear. Here, we explored the effect of UBQLN2 on TBK1 in HEK-293T cells or in CRISPR–Cas9-mediated IRF3 and IRF7 knockout (KO) cells. We found an interaction between TBK1 and UBQLN2, which was affected by ALS/FTD-linked mutations in TBK1 or UBQLN2. Co-expression of UBQLN2 with TBK1 elevated the protein level of TBK1 as well as the phosphorylation of TBK1 and IRF3 in a UBQLN2 dose-dependent manner, and this phosphorylation was reduced by mutant UBQLN2. In addition, the cellular production of IFN1 and related pro-inflammatory cytokines was substantially elevated when UBQLN2 and TBK1 were co-expressed, which was also decreased by mutant UBQLN2. Functional assay revealed that mutant UBQLN2 significantly reduced the binding affinity of TBK1 for its partners, including IRF3, (SQSTM1)/p62 and optineurin (OPTN). Moreover, complete loss of IRF3 abolished the induction of IFN1 and related pro-inflammatory cytokines enhanced by UBQLN2 in HEK-293T cells, whereas no significant change in IRF7 knockout cells was observed. Thus, our findings suggest that UBQLN2 promotes IRF3 phosphorylation via TBK1, leading to enhanced IFN1 induction, and also imply that the dysregulated TBK1-IRF3 pathway may play a role in UBQLN2-related neurodegeneration.

Porcine epidemic diarrhea virus (PEDV) causes a porcine disease associated with swine epidemic diarrhea. The type I interferon (IFN-I or IFN α/β) is a key mediator of innate antiviral response during virus infection. Different antagonistic strategies have been identified and determined as to how PEDV infection inhibits the host’s IFN responses to escape the host innate immune pathway, but the pathogenic mechanisms of PEDV infection are not fully elucidated. Our preliminary results revealed that endogenous TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3), the key components in the IFN signaling pathway were downregulated in PEDV infected IPEC-J2 cells by iTRAQ analysis. In this study, we screened nsp15 as the most important viral encoded protein involved in TBK1 and IRF3 reduction. Endoribonuclease (EndoU) activity has been well determined for coronavirus nsp15. Three residues (H226, H241, and K282) of PEDV nsp15 were identified as critical amino acids for PEDV EndoU but not D265, which was not well correlated with published results of other coronaviruses, such as severe acute respiratory syndrome virus (SARS-CoV). Moreover, PEDV nsp15 can directly degrade the RNA levels of TBK1 and IRF3 dependent on its EndoU activity to suppress IFN production and constrain the induction of IFN stimulated genes (ISGs), by which PEDV antagonizes the host innate response to facilitate its replication. Collectively, these results have confirmed that PEDV nsp15 was capable of subverting the IFN response by the RNA degradation of TBK1 and IRF3.

Abstract Zika virus (ZIKV) is a mosquito-borne positive-sense single-stranded RNA virus in the family of Flaviviridae. Unlike other flaviviruses, ZIKV infection of pregnant women may result in birth defects in their newborns, such as microcephaly or vision problem. ZIKV is known to antagonize the interferon (IFN) production in infected cells. However, the exact mechanism of this interference is not fully understood. Here, we demonstrate that NS5 protein of ZIKV MR766 strain antagonizes IFN production through inhibiting the activation of TANK-binding kinase 1 (TBK1), which phosphorylates the transcription activator IFN regulatory factor 3 (IRF3). Mechanistically, NS5 interacts with the ubiquitin-like domain of TBK1 and results in less complex of TBK1 and TNF (tumor necrosis factor) receptor-associated factor 6 (TRAF6), leading to dampened TBK1 activation and IRF3 phosphorylation. Our study provides insights into the mechanism of ZIKV evasion of IFN-mediated innate immunity. Highlights • ZIKV NS5 antagonizes IFN production through inhibiting the activation of TBK1. • NS5 interacts with the ubiquitin-like domain of TBK1. • Both MT and RdRp domains of NS5 are needed for NS5 to interact with TBK1. • NS5 interaction with TBK1 leads to less TBK1 in complex with TRAF6. [ABSTRACT FROM AUTHOR]

Nontypeable Haemophilus influenzae (NTHI) is one of the leading causes of acute exacerbations of COPD (AECOPD). Although the immunoregulation function of NTHI outer member protein and endotoxin were confirmed, the role of NTHI DNA in activating immune responses remains to be elucidated. In this study, we found expression of IFN-β and IFN stimulated gene CXCL10 in host cells was forcefully elevated after treating with NTHI and NTHI DNA. Interestingly, we tested increased level of STING in NTHI infected mice lung. Meanwhile, STING expression in lung of mimic COPD murine model was higher than healthy mice after NTHI infection. Importantly, knockout of STING or overexpression of STING, TBK1 and IRF3 respectively impaired or enhanced IFN-β and CXCL10 expression during treating with NTHI and NTHI DNA. NTHI and NTHI DNA-induced I-IFN response appeared to be mediated by cGAS. Collectively, we suggested that NTHI DNA as a PAMP triggered I-IFN response, which was STING/TBK1/IRF3 dependent. Summary NTHI is the leading cause of acute exacerbations of COPD (AECOPD). Since AECOPD is an immune event, it is meaningful to elucidate the mechanism under NTHI induced immune response. It has been revealed that lipooligosaccharides and protein of NTHI could induce host immune response, but the function of NTHI nuclide acid during infection is unclear. In this research, we demonstrate NTHI DNA is a trigger for I-IFN expression, and the STING/TBK1/IRF3 pathway plays an integral role in sensing NTHI DNA to induce I-IFN expression. Moreover, by long-term intrabronchial infection of LPS, we constructed a mimic COPD murine model, in which the STING expression in lung tissues were higher than healthy mice after NTHI infection, which led us to surmise that NTHI cause AECOPD by inducing I-IFN production via STING signal pathway. [ABSTRACT FROM AUTHOR]

During viral infection, pattern recognition receptors (PRRs) and their associated adaptors recruit TANK-binding kinase 1 (TBK1) to activate interferon regulatory factor 3 (IRF3), resulting in production of type I interferons (IFNs). ICP0 and ICP34.5 are among the proteins encoded by herpes simplex virus 1 (HSV-1) that modulate type I IFN signaling. We constructed a recombinant virus (ΔXX) that lacks amino acids 87 to 106, a portion of the previously described TBK1-binding domain of the γ34.5 gene (D. Verpooten, Y. Ma, S. Hou, Z. Yan, and B. He, J Biol Chem 284: 1097-1105, 2009, https://doi.org/10.1074/JBC.M805905200). These 20 residues are outside the γ34.5 beclin1-binding domain (BBD) that interacts with beclin1 and regulates autophagy. Unexpectedly, ΔXX showed no deficit in replication in vivo in a variety of tissues and showed virulence comparable to that of wild-type and marker-rescued viruses following intracerebral infection. ΔXX was fully capable of mediating the dephosphorylation of eIF2α, and the virus was capable of controlling the phosphorylation of IRF3. In contrast, a null mutant in γ34.5 failed to control IRF3 phosphorylation due to an inability of the mutant to sustain expression of ICP0. Our data show that while γ34.5 regulates IRF3 phosphorylation, the TBK1-binding domain itself has no impact on IRF3 phosphorylation or on replication and pathogenesis in mice. [ABSTRACT FROM AUTHOR]

Ubiquitination, as one of the most prevalent posttranslational modifications of proteins, enables a tight control of host immune responses. Many viruses hijack the host ubiquitin system to regulate host antiviral responses for their survival. Here, we found that the fish pathogen nervous necrosis virus (NNV) recruited Lateolabrax japonicus E3 ubiquitin ligase ring finger protein 34 (LjRNF34) to inhibit the RIG-I-like receptor (RLR)-mediated interferon (IFN) response via ubiquitinating Lateolabrax japonicus TANK-binding kinase 1 (LjTBK1) and interferon regulatory factor 3 (LjIRF3). Ectopic expression of LjRNF34 greatly enhanced NNV replication and prevented IFN production, while deficiency of LjRNF34 led to the opposite effect. Furthermore, LjRNF34 targeted LjTBK1 and LjIRF3 via its RING domain. Of note, the interactions between LjRNF34 and LjTBK1 or LjIRF3 were conserved in different cellular models derived from fish. Mechanically, LjRNF34 promoted K27- and K48-linked ubiquitination and degradation of LjTBK1 and LjIRF3, which in turn diminished LjTBK1-induced translocation of LjIRF3 from the cytoplasm to the nucleus. Ultimately, NNV capsid protein (CP) was found to bind with LjRNF34, CP induced LjTBK1 and LjIRF3 degradation, and IFN suppression depended on LjRNF34. Our finding demonstrates a novel mechanism by which NNV CP evaded host innate immunity via LjRNF34 and provides a potential drug target for the control of NNV infection. IMPORTANCE Ubiquitination plays an essential role in the regulation of innate immune responses to pathogens. NNV, a type of RNA virus, is the causal agent of a highly destructive disease in a variety of marine and freshwater fish. A previous study reported NNV could hijack the ubiquitin system to manipulate the host's immune responses; however, how NNV utilizes ubiquitination to facilitate its own replication is not well understood. Here, we identified a novel distinct role of E3 ubiquitin ligase LjRNF34 as an IFN antagonist to promote NNV infection. NNV capsid protein utilized LjRNF34 to target LjTBK1 and LjIRF3 for K27- and K48-linked ubiquitination and degradation. Importantly, the interactions between LjRNF34 and CP, LjTBK1, or LjIRF3 are conserved in different cellular models derived from fish, suggesting it is a general immune evasion strategy exploited by NNV to target the IFN response via RNF34. [ABSTRACT FROM AUTHOR]

• Screening of 12 ASFV ORFs that modulate the cGAS-STING pathway. • M1249L inhibits the IFN-β promoter activity and transcriptional activity. • M1249L suppresses the phosphorylation of TBK1 mediated by cGAS and STING. • M1249L degrades endogenous and exogenous IRF3 by lysosomal pathways. Cyclic GMP-AMP synthase (cGAS) is a major DNA sensor. The recognition of cytosolic DNA by cGAS triggers a robust innate immune response that restricts the replication of diverse viral pathogens through the type I interferon (IFN) and nuclear factor-κB (NF-κB) pathways. African swine fever virus (ASFV) is a large and complex DNA virus reported to strongly inhibit the cGAS-STING signaling pathway. Herein, 12 ASFV structural proteins were screened to determine their effects on the cGAS-STING pathway. Ectopic expression of the ASFV caspid protein M1249L significantly inhibited the IFN-β promoter activity induced by the cGAS-STING pathway in a dose-dependent manner. And it could also downregulate the levels of IFN-β and several interferon-stimulating genes (ISGs) induced by cGAS-STING and 2'3'-cGAMP. Moreover, ASFV M1249L also suppressed phosphorylation of TBK1 by cGAS and STING overexpression. Further study showed that M1249L co-localized and interacted with interferon regulatory factor 3 (IRF3), which led to induce IRF3 degradation by lysosomal pathway. Taken together, our study revealed a novel strategy utilized by ASFV for cGAS-STING-related immune evasion. [ABSTRACT FROM AUTHOR]

[Display omitted] The interferon regulatory factor (IRF) family of transcription factors play a vital role in the human innate antiviral immune responses with production of interferons (IFNs) as a hallmark outcome of activation. In recent years, IRF3 has been considered a principal early regulator of type I IFNs (TI-IFNs) directly downstream of intracellular virus sensing. Despite decades of research on IRF-activating pathways, many questions remain on the regulation of IRF3 activation. The kinases IκB kinase epsilon (IKKε) and TANK-binding kinase-1 (TBK1) and the scaffold proteins TRAF family member-associated NF-kappa-B activator (TANK), NF-kappa-B-activating kinase-associated protein 1 (NAP1) and TANK-binding kinase 1-binding protein 1 (TBKBP1)/similar to NAP1 TBK1 adaptor (SINTBAD) are believed to be core components of an IRF3-activation complex yet their contextual involvement and complex composition are still unclear. This review will give an overview of antiviral signaling pathways leading to the activation of IRF3 and discuss recent developments in our understanding of its proximal regulation. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) play an important role in induction of innate immune responses. The stimulation of TLRs by microbial components triggers two branches of downstream signaling pathways: myeloid differential factor 88 (MyD88)- and toll-interleukin-1 receptor domain-containing adapter inducing interferon-β (TRIF)-dependent signaling pathways. Auranofin, a sulfur-containing gold compound (Au[I]), has been widely used for the treatment of rheumatoid arthritis. Since dysregulation of TLRs can lead to severe systemic inflammatory and joint destructive process in rheumatoid arthritis, auranofin-mediated modulation of TLR activation may have therapeutic potential against such diseases. Previously, we demonstrated that auranofin suppressed TLR4 signaling pathway by inhibiting TLR4 dimerization induced by LPS. Here, we examined the effect of auranofin on signal transduction via the TRIF-dependent pathway induced by a TLR3 agonist. Auranofin inhibited nuclear factor-κB and interferon (IFN) regulatory factor 3 (IRF3) activation induced by polyinosinic-polycytidylic acid (poly[I:C]). Auranofin inhibited poly[I:C]-induced phosphorylation of IRF3 as well as IFN-inducible genes such as IFN inducible protein-10. Furthermore, auranofin inhibited TBK1 kinase activity in vitro. All the results suggest that auranofin suppress TLR signaling at multiple steps. [ABSTRACT FROM AUTHOR]

Missense mutations of the tumor suppressor Neurofibromin 2 (NF2/Merlin/schwannomin) result in sporadic to frequent occurrences of tumorigenesis in multiple organs. However, the underlying pathogenicity of NF2-related tumorigenesis remains mostly unknown. Here we found that NF2 facilitated innate immunity by regulating YAP/TAZ-mediated TBK1 inhibition. Unexpectedly, patient-derived individual mutations in the FERM domain of NF2 (NF2m) converted NF2 into a potent suppressor of cGAS-STING signaling. Mechanistically, NF2m gained extreme associations with IRF3 and TBK1 and, upon innate nucleic acid sensing, was directly induced by the activated IRF3 to form cellular condensates, which contained the PP2A complex, to eliminate TBK1 activation. Accordingly, NF2m robustly suppressed STING-initiated antitumor immunity in cancer cell-autonomous and -nonautonomous murine models, and NF2m-IRF3 condensates were evident in human vestibular schwannomas. Our study reports phase separation-mediated quiescence of cGAS-STING signaling by a mutant tumor suppressor and reveals gain-of-function pathogenesis for NF2-related tumors by regulating antitumor immunity. [Display omitted] • Activated IRF3 directly binds and induces mutant NF2 to form cellular condensates • The PP2A complex in the NF2m condensates eliminates TBK1 activation • Disease-causing NF2 mutants potently inhibit cGAS-STING and RLR-MAVS signaling • NF2m phase separation abolishes STING-initiated antitumor immunity in mice Much is still unknown about tumor suppressors in immune regulation. Meng et al. identify an intriguing function of innate immunity that triggers liquid-liquid phase separation of disease-causing tumor suppressor mutants. This elegant gain-of-function effect via signal-induced condensation sequesters these major innate immune pathways and compromises antitumor immunity. [ABSTRACT FROM AUTHOR]