Phosphorylation Of IRF3 Research Articles

Structure and Ubiquitination-Dependent Activation of TANK-Binding Kinase 1

Upon stimulation by pathogen-associated inflammatory signals, TANK-binding kinase 1 (TBK1) induces type I interferon expression and modulates nuclear factor κB (NF-κB) signaling. Here, we describe the 2.4 Å-resolution crystal structure of nearly full-length TBK1 in complex with specific inhibitors. The structure reveals a dimeric assembly created by an extensive network of interactions among the kinase, ubiquitin-like, and scaffold/dimerization domains. An intact TBK1 dimer undergoes K63-linked polyubiquitination on lysines 30 and 401, and these modifications are required for TBK1 activity. The ubiquitination sites and dimer contacts are conserved in the close homolog inhibitor of κB kinase ε (IKKε) but not in IKKβ, a canonical IKK that assembles in an unrelated manner. The multidomain architecture of TBK1 provides a structural platform for integrating ubiquitination with kinase activation and IRF3 phosphorylation. The structure of TBK1 will facilitate studies of the atypical IKKs in normal and disease physiology and further the development of more specific inhibitors that may be useful as anticancer or anti-inflammatory agents.

Human kinome analysis reveals novel kinases contributing to virus infection and retinoic-acid inducible gene I-induced type I and type IIIIFNgene expression

Activation of host innate antiviral responses are mediated by retinoic-acid inducible gene I (RIG-I)-like receptors, RIG-I and melanoma differentiation-associated gene 5, and TLRs 3, 7, 8 and 9, recognising different types of viral nucleic acids. The major components of the RIG-I- and TLR pathways have putatively been identified, but previously unrecognised kinases may contribute to virus infection-induced activation of the IFN response. Here, we screened a human kinase cDNA library, termed the kinome, using an IFN-λ1 promoter-driven luciferase reporter assay in HEK293 cells during Sendai virus infection. Of the 568 kinases analysed, nearly 50 enhanced IFN-λ1 gene expression at least twofold in response to Sendai virus infection. The best activators were FYN (FYN oncogene related to SRC, FGR, YES), serine/threonine kinase 24, activin A receptor type 1 and SRPK1 (SFRS protein kinase 1). These kinases enhanced RIG-I-dependent IFN-λ1 promoter activation via IFN-stimulated response and NF-κB elements, as confirmed using mutant IFN-λ1 promoter constructs. FYN and SRPK1 enhanced IFN-λ1 and CXCL10 protein production via the RIG-I pathway, and stimulated RIG-I and MyD88-dependent phosphorylation of IRF3 and IRF7 transcription factors, respectively. We conclude that several previously unrecognised kinases, particularly FYN and SRPK1, positively regulate IFN-λ1 and similarly regulated cytokine and chemokine genes during viral infection.

Induction of Siglec-G by RNA Viruses Inhibits the Innate Immune Response by Promoting RIG-I Degradation

RIG-I is a critical RNA virus sensor that serves to initiate antiviral innate immunity. However, posttranslational regulation of RIG-I signaling remains to be fully understood. We report here that RNA viruses, but not DNA viruses or bacteria, specifically upregulate lectin family member Siglecg expression in macrophages by RIG-I- or NF-κB-dependent mechanisms. Siglec-G-induced recruitment of SHP2 and the E3 ubiquitin ligase c-Cbl to RIG-I leads to RIG-I degradation via K48-linked ubiquitination at Lys813 by c-Cbl. By increasing type I interferon production, targeted inactivation of Siglecg protects mice against lethal RNA virus infection. Taken together, our data reveal a negative feedback loop of RIG-I signaling and identify a Siglec-G-mediated immune evasion pathway exploited by RNA viruses with implication in antiviral applications. These findings also provide insights into the functions and crosstalk of Siglec-G, a known adaptive response regulator, in innate immunity.

Yohimbine Enhances Protection of Berberine against LPS-Induced Mouse Lethality through Multiple Mechanisms

Sepsis remains a major cause of mortality in intensive care units, better therapies are urgently needed. Gram-negative bacterial lipopolysaccharide (LPS) is an important trigger of sepsis. We have demonstrated that berberine (Ber) protects against lethality induced by LPS, which is enhanced by yohimbine (Y) pretreatment, and Ber combined with Y also improves survival in septic mice. However, the precise mechanisms by which Y enhances protection of Ber against LPS - induced lethality remain unclear. The present study confirmed that simultaneously administered Y also enhanced protection of Ber against LPS-induced lethality. Ber or/and Y attenuated liver injury, but not renal injury in LPS-challenged mice. Ber or/and Y all inhibited LPS-stimulated IκBα, JNK and ERK phosphorylation, NF-κB activation as well as TNF-α production. Ber also increased IL-10 production in LPS-challenged mice, which was enhanced by Y. Furthermore, Ber or/and Y all suppressed LPS-induced IRF3, TyK2 and STAT1 phosphorylation, as well as IFN-β and IP-10 mRNA expression in spleen of mice at 1 h after LPS challenge. Especially, Y enhanced the inhibitory effect of Ber on LPS-induced IP-10 mRNA expression. In vitro experiments further demonstrated that Y significantly enhanced the inhibitory effect of Ber on TNF-α production in LPS-treated peritoneal macrophages, Ber combined with Y promoted LPS-induced IL-10 production and LPS-stimulated IκBα, JNK, ERK and IRF3 phosphorylation and NF-κB activation were also suppressed by Ber or/and Y pretreatment in peritoneal macrophages. Taken together, these results demonstrate that Y enhances the protection of Ber against LPS-induced lethality in mice via attenuating liver injury, upregulating IL-10 production and suppressing IκBα, JNK, ERK and IRF3 phosphorylation. Ber combined with Y may be an effective immunomodulator agent for the prevention of sepsis.

Selective inhibition by simvastatin of IRF3 phosphorylation and TSLP production in dsRNA‐challenged bronchial epithelial cells from COPD donors

Statin treatment may ameliorate viral infection-induced exacerbations of chronic obstructive pulmonary disease (COPD), which exhibit Th2-type bronchial inflammation. Thymic stromal lymphopoietin (TSLP), a hub cytokine switching on Th2 inflammation, is overproduced in viral and dsRNA-stimulated bronchial epithelial cells from COPD donors. Hence, TSLP may be causally involved in exacerbations. This study tests the hypothesis that simvastatin inhibits dsRNA-induced TSLP. Epithelial cells, obtained by bronchoscopy from COPD (n = 7) and smoker control (n = 8) donors, were grown and stimulated with a viral infection and danger signal surrogate, dsRNA (10 μg·mL(-1) ). Cells were treated with simvastatin (0.2-5 μg·mL(-1) ), with or without mevalonate (13-26 μg·mL(-1) ), or dexamethasone (1 μg·mL(-1) ) before dsRNA. Cytokine expression and production, and transcription factor (IRF3 and NF-κB) activation were determined. dsRNA induced TSLP, TNF-α, CXCL8 and IFN-β. TSLP was overproduced in dsRNA-exposed COPD cells compared with control. Simvastatin, but not dexamethasone, concentration-dependently inhibited dsRNA-induced TSLP. Unexpectedly, simvastatin acted independently of mevalonate and did not affect dsRNA-induced NF-κB activation nor did it reduce production of TNF-α and CXCL8. Instead, simvastatin inhibited dsRNA-induced IRF3 phosphorylation and generation of IFN-β. Independent of mevalonate and NF-κB, previously acknowledged anti-inflammatory mechanisms of pleiotropic statins, simvastatin selectively inhibited dsRNA-induced IRF3 activation and production of TSLP and IFN-β in COPD epithelium. These data provide novel insight into epithelial generation of TSLP and suggest paths to be exploited in drug discovery aimed at inhibiting TSLP-induced pulmonary immunopathology.

Arsenic trioxide prevents nitric oxide production in lipopolysaccharide ‐stimulated RAW 264.7 by inhibiting a TRIF‐dependent pathway

Arsenic trioxide (ATO) is one of the most potent drugs in cancer chemotherapy, and is highly effective in treating both newly diagnosed and relapse patients with acute promyelocytic leukemia (APL). Despite a number of reports regarding the molecular mechanisms by which ATO promotes anti-tumor or pro-apoptotic activity in hematological and other solid malignancies, the effects of ATO on immune responses remain poorly understood. To further understand and clarify the effects of ATO on immune responses, we sought to examine whether ATO affects the production of nitric oxide (NO) in a lipopolysaccharide (LPS)-stimulated mouse macrophage cell line, RAW 264.7. Arsenic trioxide was found to prevent NO production in a dose-dependent manner. Arsenic trioxide significantly inhibited the increase in inducible nitric oxide synthase (iNOS) at both the mRNA and protein levels. Furthermore, our analyses revealed that the inhibitory effect of ATO on iNOS expression was ascribed to the prevention of IRF3 phosphorylation, interferon (IFN)-β expression, and STAT1 phosphorylation, but not the prevention of the MyD88-dependent pathway. Taken together, our results indicate that ATO prevents NO production by inhibiting the TIR-domain-containing adaptor protein inducing IFN-β (TRIF)-dependent pathway, thus highlighting an anti-inflammatory property of ATO in innate immunity.

Ndfip1 Negatively Regulates RIG-I–Dependent Immune Signaling by Enhancing E3 Ligase Smurf1-Mediated MAVS Degradation

Ndfip1 functions as both a recruiter and an activator of multiple HECT domain E3 ubiquitin ligases of the Nedd4 family. In this study, we demonstrate that Ndfip1 is involved in the ubiquitin-mediated degradation of mitochondrial antiviral signaling (MAVS), which is a key adaptor protein in RIG-I-like receptor-mediated immune signaling. We found that overexpression of Ndfip1 severely impaired MAVS and Sendai virus-mediated activation of IFN-stimulated response element, NF-κB, IFN-β promoter, and polyinosinic-polycytidylic acid or influenza virus RNA-stimulated IRF-3 phosphorylation, as well as the transcription of IFN-β. This functional interaction was confirmed by knockdown of Ndfip1, which facilitated MAVS-mediated downstream signaling and elevated MAVS protein levels. Further analysis indicated that Ndfip1 enhances both self-ubiquitination of HECT domain-containing E3 ubiquitin ligase Smurf1 and its interaction with MAVS, and eventually promotes MAVS degradation. In addition, the activation of IFN-β by MAVS, influenza virus RNA, polyinosinic-polycytidylic acid, and Sendai virus was enhanced in Ndfip1 knockdown cells. These results reveal that Ndfip1 is a potent inhibitor of MAVS-mediated antiviral response.

Endoplasmic Reticulum Stress Regulates the Innate Immunity Critical Transcription Factor IRF3

IFN regulatory factor 3 (IRF3) regulates early type I IFNs and other genes involved in innate immunity. We have previously shown that cells undergoing an endoplasmic reticulum (ER) stress response called the unfolded protein response produce synergistically augmented IFN-β when stimulated with pattern recognition receptor agonists such as LPS. Concomitant ER stress and LPS stimulation resulted in greater recruitment of the IRF3 transcription factor to ifnb1 gene regulatory elements. In this study, we used murine cells to demonstrate that both oxygen-glucose deprivation and pharmacologic unfolded protein response inducers trigger phosphorylation and nuclear translocation of IRF3, even in the absence of exogenous LPS. Different ER stressors used distinct mechanisms to activate IRF3: IRF3 phosphorylation due to calcium-mobilizing ER stress (thapsigargin treatment, oxygen-glucose deprivation) critically depended upon stimulator of IFN gene, an ER-resident nucleic acid-responsive molecule. However, calcium mobilization alone by ionomycin was insufficient for IRF3 phosphorylation. In contrast, other forms of ER stress (e.g., tunicamycin treatment) promote IRF3 phosphorylation independently of stimulator of IFN gene and TANK-binding kinase 1. Rather, IRF3 activation by tunicamycin and 2-deoxyglucose was inhibited by 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, a serine protease inhibitor that blocks activating transcription factor 6 processing. Interfering with ER stress-induced IRF3 activation abrogated IFN-β synergy. Together, these data suggest ER stress primes cells to respond to innate immune stimuli by activating the IRF3 transcription factor. Our results also suggest certain types of ER stress accomplish IRF3 phosphorylation by co-opting existing innate immune pathogen response pathways. These data have implications for diseases involving ER stress and type I IFN.

Phenylmethimazole Blocks dsRNA-Induced IRF3 Nuclear Translocation and Homodimerization

Previous studies revealed that phenylmethimazole (C10) inhibits IRF3 signaling, preventing dsRNA-induction of type 1 interferon gene expression, production, and downstream signaling. In the present study, we investigated the molecular basis for C10 inhibition of dsRNA-stimulated IRF3 signaling. IRF-3 Trans-AM assays were used to measure C10 effects on dsRNA induction of IRF3 DNA binding. Green fluorescent protein-labeled IRF3 was used to measure C10 effects on dsRNA-induced IRF3 nuclear translocation. Native PAGE, SDS PAGE, and western blotting were used to identify effects of C10 on IRF3 homodimer formation and phosphorylation, respectively. There was a significant impairment of dsRNA-induced IRF3 DNA binding activity in human embryonic kidney and pancreatic cancer cells with C10 treatment. C10 also blocked dsRNA-induced IRF3 nuclear translocation and homodimer formation without blocking serine 396 phosphorylation of IRF3. Together, these results indicate that C10 interferes with IRF3 signaling by blocking dsRNA-induced IRF3 homodimer formation, a prerequisite for nuclear translocation and DNA binding activities.

Tetraspanin 6 (TSPAN6) Negatively Regulates Retinoic Acid-inducible Gene I-like Receptor-mediated Immune Signaling in a Ubiquitination-dependent Manner

The recognition between retinoic acid-inducible gene I-like receptors (RLRs) and viral RNA triggers an intracellular cascade of signaling to induce the expression of type I IFNs. Both positive and negative regulation of the RLR signaling pathway are important for the host antiviral immune response. Here, we demonstrate that the tetraspanin protein TSPAN6 inhibits RLR signaling by affecting the formation of the adaptor MAVS (mitochondrial antiviral signaling)-centered signalosome. We found that overexpression of TSPAN6 impaired RLR-mediated activation of IFN-stimulated response element, NF-κB, and IFN-β promoters, whereas knockdown of TSPAN6 enhanced the RLR-mediated signaling pathway. Interestingly, as the RLR pathway was activated, TSPAN6 underwent Lys-63-linked ubiquitination, which promoted its association with MAVS. The interaction of TSPAN6 and MAVS interfered with the recruitment of RLR downstream molecules TRAF3, MITA, and IRF3 to MAVS. Further study revealed that the first transmembrane domain of TSPAN6 is critical for its ubiquitination and association with MAVS as well as its inhibitory effect on RLR signaling. We concluded that TSPAN6 functions as a negative regulator of the RLR pathway by interacting with MAVS in a ubiquitination-dependent manner.

P129 Endoplasmic reticulum stress activates interferon regulatory factor 3

Introduction Cells undergoing an endoplasmic reticulum (ER) stress response called the “Unfolded Protein Response” (UPR) produce synergistically augmented IFN-β when stimulated with pattern recognition receptor agonists such as lipopolysaccharide (LPS). Previous studies revealed increased IRF3 at ifnb1 genetic regulatory elements during ER stress; however the underlying mechanism was unclear [1] . In this study we sought to determine whether ER stress directly activated IRF3. Methods This study was conducted in murine macrophages (primary and RAW264.7) and STING−/− or +/+ murine embryonic fibroblasts. IRF3 activation was monitored by immunofluorescence microscopy and western blot detection of S386 and S396 respectively as well as IRF3 nuclear translocation. IRF3 regulated gene expression was detected by quantitative PCR. TBK1 family kinases were inhibited with MRT67307. Site-1 protease cleavage of ATF6 was blocked with AEBSF. Results Oxygen-glucose deprivation and multiple pharmacologic UPR inducers triggered the phosphorylation (S386) and nuclear translocation of IRF3, even in the absence of exogenous LPS. UPR induction also enhanced LPS-dependent phosphorylation of IRF3 at S396. Consistent with IRF3 activation, ER stress augmented LPS-dependent induction of several IRF3 regulated genes (IFN-β, IFN-α4, and CXCL10) but not others (ISG54, RANTES). Further mechanistic dissection revealed differences among UPR inducers: IRF3 phosphorylation due to calcium-mobilizing ER stress (thapsigargin or A23187) critically depended upon Stimulator of interferon gene (STING) and Tank-binding kinase (TBK1). However, calcium mobilization alone by ionomycin was insufficient for IRF3 phosphorylation. STING was also necessary for optimal synergistic induction of IFN-βby thapsigargin and LPS. In contrast, non-calcium-dependent forms of ER stress (e.g., tunicamycin treatment, oxygen glucose deprivation, 2-deoxyglucose) promoted IRF3 phosphorylation independently of STING and TBK1. Rather, IRF3 phosphorylation and synergistic IFN-βinduction by these UPR inducers was abrogated by AEBSF, an inhibitor of ATF6 processing. AEBSF had no effect on thapsigargin induced IRF3 phosphorylation or IFN-β synergy. Conclusion Discrete IRF3 activating pathways were identified for different types of ER stress that involved either STING/TBK1 or Site-1 specific proteases (e.g. ATF6). The activation of STING by ER stress has not been described. Although ER stress is not sufficient for expression of IRF3-regulated genes, activation of IRF3 by the UPR plays a critical role in augmenting LPS-dependent expression, as interference in this process severely impacted IFN-β induction. These data have implications for diseases involving ER stress and type I IFN.

P132 The human dead-box helicase 3 couples IKK-epsilon to IRF3 activation

Introduction The human DEAD-box RNA helicase DDX3 has been implicated in different cellular processes including transcription, splicing, mRNA nuclear export and translation. We and others previously showed that DDX3 is involved in innate immune signalling pathways which trigger the induction of type I interferon through the kinases IKKe or TBK1 [1] , [2] , [3] . We demonstrated that DDX3 interacts with IKKe, and that it contributes to IRF3 activation. Other studies have confirmed a role for DDX3 in IFN-s induction, but placed DDX3 upstream in the MAVS-mediated signalling pathway, or suggested that DDX3 binds to the IFN-s promoter directly. Therefore, our study here aimed at placing DDX3 more firmly in the signalling pathway(s) leading to IFN-s induction, by dissecting its molecular interaction with IKKe and the functional consequences of this interaction. Methods The DDX3, IKKe and IRF3 interaction studies were performed using co-immunoprecipitation (Co-IP) and His pulldown assays. In vitro kinase assays were employed for investigating DDX3 and IRF3 phosphorylation by IKKe. IKKe- phosphorylation sites in DDX3 were identified using Mass Spectrometry. Luciferase reporter assays were used to study effects of phospho-deficient and –mimetic DDX3 mutants on IFN-s promoter induction. Results We show here that DDX3 directly interacts with, and is phosphorylated by IKKe. Phosphorylation of DDX3 occurs mainly in its N-terminal region, which is required for IFN-s induction, and is targeted by the vaccinia virus protein K7 in a viral immune evasion strategy. In agreement with this, we found that K7 specifically inhibits DDX3-phosphorylation by IKKe. Furthermore, we have identified one serine residue in the N-terminus of DDX3 that is crucial for the effect of DDX3 on the IFN-s promoter. We also show that DDX3 directly enhances IKKe activity and phosphorylation of IRF3 in an in vitro reconstitution assay. In addition, we demonstrate that DDX3 directly binds to IRF3, and that this interaction is crucial for the DDX3 effect on IFN-s induction. Phosphorylation of DDX3 by IKKe facilitates the subsequent recruitment of IRF3. IRF3 and IKKe have adjacent and potentially overlapping, but not identical, binding sites in the N-terminus of DDX3. Conclusion Our study has shown that the N-terminus of DDX3 directly interacts with IKKe and IRF3 and that this mediates their activation and functional interaction. The interaction between DDX3 and IRF3 contributes to IRF3 activation and is required for the effect of DDX3 on IFN-s promoter induction. The effects of DDX3 on IKKe activation and IRF3 activation can be uncoupled by mutation of a single serine residue in the N-terminus of DDX3.Our work implicates DDX3 as a bridging adaptor for IKKe and IRF3 in RLH and TLR signalling pathways.

P146 TLR2-ligands induce NF-κB activation exclusively from endosomal compartments

Introduction Antiphospholipid antibody syndrome (APS) is an auto-immune disease associated with arterial or venous thrombosis and/or recurrent fetal loss and is caused by pathogenic antiphospholipid antibodies (aPLA). We recently demonstrated TLR2 as a crucial factor in recognizing aPLA by monocytes. While TLR4 is known to transmit signals from both the plasma membrane and endosomal compartments, less is known about the function of endosomal trafficking on TLR2 signaling. Methods Primary human monocytes and HEK-Blue2™ cells were treated with TLR2 ligands, i.e. LTA and Pam 3 CSK 4 , or with aPLA, in the presence of endocytosis inhibitors. Expression of TNF and tissue factor (TF) as well as NF- κ B activation were assessed. In order to evaluate the importance of receptor internalization, clathrin expression was reduced by siRNA and NF- κ B activation assessed after treatment with TLR2 ligands and aPLA. Endocytosis of biotinylated LTA and Pam 3 CSK 4 or aPLA was evaluated by flow cytometry. Results We demonstrate that blocking antibodies to TLR2, TLR1 or TLR6, as well as antibodies to the TLR co-receptor CD14, significantly decreased TNF and TF responses to LTA, Pam 3 CSK 4 and aPLA. TLR2 ligands internalization is observed in primary human monocytes and HEK-Blue2™ cells and we show that NF-kB-dependent cell activation is associated to ligand internalization. Pharmacological blockade and siRNA approaches revealed the importance of the clathrin/dynamin-dependent endocytic pathway in ligand-induced NF- κ B activation. In addition, LTA-, Pam 3 CSK 4 - and aPLA-beads, which could not be internalized, are unable to activate NF- κ B while soluble ligands and LPS-beads (a TLR4 ligand) are competent to trigger NF- κ B activation. Furthermore, in a similar manner as TLR4, we demonstrate that TLR2 ligands uptake is regulate by CD14. Unlike LPS, TLR2 ligands do not induce phosphorylation of IRF-3 or IFN β expression. Finally, CD14-dependent endocytosis is shown to regulate the activation of NF-kB induced by LTA, Pam 3 CSK 4 and aPLA in HEK-Blue2™ cells or primary human monocytes. Conclusion Our results reveal that TLR2-ligand complexes are internalized, via clathrin- and CD14-dependent endocytosis into primary human monocytes and HEK-Blue2™ cells; endocytosis of these complexes is required for NF- κ B activation. The evidence that TLR1 and TLR6 play a part in the pathogenicity of aPLA contributes to a better understanding of the antiphospholipid syndrome and provides insight for the development of novel therapies. In conclusion, our study reveals a new important aspect of the regulation of TLR2-dependent signaling. Although present at the cell surface, TLR2-ligands induce NF- κ B activation exclusively from endosomal compartment.

P037 Effects of vaccinia virus-expressed IRF-3 ON VACV replication in tissue culture and in atopic Nc/Nga mice

Introduction Vaccinia viruses (VACV) of several strains had been used in the eradication campaign against smallpox. Recently, vaccination of selected groups of professionals was reintroduced in certain countries, imposing a risk of an inadvertent spread of VACV to non-immune population. In individuals with a history of atopic dermatitis (AD), inoculation of VACV can result in eczema vaccinatum, an uncontrolled spread of VACV in the skin. Pathogenesis of eczema vaccinatum has not been completely understood, but Th2-skewed immune responses together with lower immigration of immune cell types, such as NK cells, plasmacytoic dendritic cells and neutrophils, and defective cytokine production and antimicrobial peptides have been suggested. Recently, increased production of IL-17 resulting in lower immigration of NK cells and increased VACV replication were demonstrated. VACV encodes many factors that interfere with host immune responses. Among others, VACV specifically inhibits phosphorylation of IRF-3 and expression of type I interferons at multiple levels. To overcome this block, we have generated a recombinant VACV expressing murine IRF-3. Methods We have cloned murine IRF-3 cDNA into pSC11, a vector expressing the recombinant gene under control of VACV early/late promoter p7.5 and allowing homologous recombination into the thymidine kinase gene of VACV. The recombinant VACV expressing IRF-3 (VACV-IRF3) was selected, and the insertion and expression of IRF-3 were confirmed by Southern blot analysis and RT-PCR, respectively. The growth of the virus in green monkey kidney derived BSC-40 cells and in mouse fibroblasts NIH-3T3 is characterized by serial dilutions and plaque assays, changes in host cell morphology by optical and fluorescent microscopy, expression of murine IFN-beta by RT-PCR. The growth of the virus and immune responses towards it are characterized in atopic Nc/Nga mice, using serial dilutions and plaque assays, ELISA and neutralization assays. Results Murine IRF-3 mRNA was found expressed by VACV-IRF3 in cells of different embryonic origins. Based on preliminary experiments, its expression does not seem to affect virus yields in tissue culture in comparison with VACV expressing luciferase (VACV-LUC). However, VACV-IRF3 seems to induce expression of murine IFN-beta and apoptosis in mouse NIH-3T3 fibroblasts while causing lytic infection of monkey BSC-40 cells. A study of the effects of IRF-3 expression on virulence, size of the lesions after a skin inoculation, and the titers of VACV-specific antibodies in the atopic Nc/Nga mice is currently under progress. Conclusion Expression of murine IRF-3 by a VACV recombinant seems to overcome VACV-mediated inhibition of IFN type I expression in murine cells. Increased production of IFN type I could decrease a risk of eczema vaccinatum and other post-vaccination complications that result from uncontrolled VACV growth. Apoptosis of VACV-IRF3-infected cells could affect mounting of VACV-specific immune responses.

I20 Linear postive and negative regulation of the interferon antiviral response

The cytosolic RIG-I pathway is activated by many RNA viruses - including influenza - via viral RNA replicative intermediates that contain short hairpin dsRNA and 5′ triphosphate structures. We characterized natural and synthetic RIG-I agonists based on sequences from the 5’UTR regions of distinct negative-strand viruses – vesicular stomatitis virus (VSV), Influenza, Rabies, Measles, and Sendai virus - and demonstrated potent stimulation of RIG-I antiviral responses at concentrations in the picomolar range. In human bronchial epithelial A549 cells, 5′pppRNA induced IRF3 phosphorylation and dimerization, STAT1 Tyr701 phosphorylation, as well as a >100-fold increase in the transcription of interferon stimulated genes (ISGs) and genes involved in inflammation. The magnitude and duration of ISG and inflammatory gene expression was evaluated by gene expression profiling, where 5’pppRNA triggered a sustained and diverse range of antiviral and inflammatory genes compared to treatment with IFN α , and bioinformatics analysis identified distinct nodes of IRF7, IRF1 and NF-kB gene activation. Pre-treatment of A549 cells with 5’pppRNA dramatically blocked H1N1 A/PR/8/34 Influenza virus replication; furthermore, intravenous delivery of 5′pppRNA to BALB/c mice generated an antiviral response in mouse lungs that protected animals from a lethal challenge with H1N1 A/PR/8/34. RNA agonist delivery inhibited virus replication in mouse lungs within the first 24h after H1N1 challenge and protected the lungs of infected animals from virus-induced pathology. Finally, 5’pppRNA pre-treatment also completely or partially blocked replication of Dengue, Vaccinia and HIV-1 replication. These results illustrate that naturally derived RIG-I agonists represent a potent stimulator of the innate antiviral response, with the capacity to block replication of multiple pathogenic human viruses. Termination of IFN signalling is likewise crucial to the proper maintenance of the innate and adaptive immune response to virus infection. We also identified an essential role for LUBAC-mediated linear ubiquitination of NEMO in the negative regulation of the RIG-I antiviral pathway through sequestration of TRAF3 from the MAVS adapter. LUBAC and NEMO-Ub constructs inhibited RIG-I signaling downstream of MAVS and upstream of TBK1; linearly ubiquitinated NEMO interacted physically with TRAF3, and disrupted the MAVS-TRAF3 complex, thus providing a mechanistic explanation for the downregulation of RIG-I signaling. Using SHARPIN deficient cpdm MEFs, we observed on the one hand, an increased and prolonged antiviral response, while on the other hand, an impaired NF- κ B activation, indicating that linear ubiquitination is required for NF- κ B activation downstream of RIG-I. Interestingly, an increase in apoptotic cell death was also detected in SHARPIN-deficient cpdm MEFs after VSV infection, potentially attributable to the absence of the anti-apoptotic activity of NF- κ B. These studies reveal a novel negative feedback mechanism used by host cells to regulate the IFN antiviral response.

Individual Interferon Regulatory Factor-3 Thiol Residues Are Not Critical for Its Activation Following Virus Infection

The interferon regulatory factor (IRF)-3 transcription factor plays a central role in the capacity of the host to mount an efficient innate antiviral immune defense, mainly through the regulation of type I Interferon genes. A tight regulation of IRF-3 is crucial for an adapted intensity and duration of the response. Redox-dependent processes are now well known to regulate signaling cascades. Recent reports have revealed that signaling molecules upstream of IRF-3, including the mitochondrial antiviral-signalling protein (MAVS) and the TNF receptor associated factors (TRAFs) adaptors, are sensitive to redox regulation. In the present study, we assessed whether redox regulation of thiol residues contained in IRF-3, which are priviledged redox sensors, play a role in its regulation following Sendai virus infection, using a combination of mutation of Cysteine (Cys) residues into Alanine and thiols alkylation using N-ethyl maleimide. Alkylation of IRF-3 on Cys289 appears to destabilize IRF-3 dimer in vitro. However, a detailed analysis of IRF-3 phosphorylation, dimerization, nuclear accumulation, and induction of target gene promoter in vivo led us to conclude that IRF-3 specific, individual Cys residues redox status does not play an essential role in its activation in vivo.

Mechanism of inhibition of lipopolysaccharide-induced interferon-β production by 2-aminopurine

2-Aminopurine (2-AP) is widely used as an inhibitor for double stranded RNA-dependent protein kinase (PKR). Previously, we reported that 2-AP inhibits Toll-like receptor (TLR) ligand-induced nitric oxide production through the prevention of interferon (IFN)-β production. In this study, we investigated the mechanisms for 2-AP inhibition of lipopolysaccharide (LPS)-induced IFN-β production. A reporter gene assay showed that LPS-induced IFN-β promoter, but not nuclear factor (NF)-κB, activation was significantly inhibited by 2-AP. IFN-β promoter activation induced by the overexpression of Toll/interleukin-1 receptor domain-containing adaptor inducing IFN-β (TRIF) was significantly inhibited by 2-AP in a dose-dependent manner, while TRIF- or myeloid differentiation primary response gene 88-dependent NF-κB activation was not inhibited. IFN-β promoter activation induced by expression of the downstream signaling molecules, tumor necrosis factor receptor-associated factor family member-associated NF-κB activator-binding kinase 1, inhibitor of NF-κB kinase i and a constitutively active mutant of interferon regulatory factor (IRF)-3, was also inhibited by 2-AP. Another PKR inhibitor harboring the imidazolo-oxindole structure, however, did not affect TRIF signaling molecules-induced IFN-β promoter activation, suggesting that the inhibition of IFN-β transcription by 2-AP is independent of PKR inhibition. Further, we examined the effect of 2-AP on LPS-induced IRF-3 activation by immunoblotting. While 2-AP did not affect LPS-induced phosphorylation of IRF-3, nuclear translocation of IRF-3 was inhibited. Moreover, we revealed that LPS-induced phosphorylation of Akt, another key molecule involved in IRF-3 activation, was inhibited by 2-AP. These results suggest that 2-AP inhibits nuclear translocation of phosphorylated-IRF-3 by inhibiting Akt activation.

PPM1B negatively regulates antiviral response via dephosphorylating TBK1

The production of type I interferon must be tightly regulated and aberrant production of type I interferon is harmful or even fatal to the host. TBK1 phosphorylation at Ser172 plays an essential role in TBK1-mediated antiviral response. However, how TBK1 activity is negatively regulated remains poorly understood. Using a functional genomics approach, we have identified PPM1B as a TBK1 phosphatase. PPM1B dephosphorylates TBK1 in vivo and in vitro. PPM1B wild-type but not its phosphatase-deficient R179G mutant inhibits TBK1-mediated antiviral response and facilitates VSV replication in the cells. Viral infection induces association of PPM1B with TBK1 in a transient fashion in the cells. Conversely, suppression of PPM1B expression enhances virus-induced IRF3 phosphorylation and IFNβ production. Our study identifies a previously unrecognized role for PPM1B in the negative regulation of antiviral response by acting as a TBK1 phosphatase.

Richard K. Olney, MD (1947–2012)

Sphingosine 1-phosphate (S1P) lyase (SPL) is an intracellular enzyme that mediates the irreversible degradation of the bioactive lipid S1P. We have previously reported that overexpressed SPL displays anti-influenza viral activity; however, the underlying mechanism is incompletely understood. In this study, we demonstrate that SPL functions as a positive regulator of IKKε to propel type I IFN–mediated innate immune responses against viral infection. Exogenous SPL expression inhibited influenza A virus replication, which correlated with an increase in type I IFN production and IFN-stimulated gene accumulation upon infection. In contrast, the lack of SPL expression led to an elevated cellular susceptibility to influenza A virus infection. In support of this, SPL-deficient cells were defective in mounting an effective IFN response when stimulated by influenza viral RNAs. SPL augmented the activation status of IKKε and enhanced the kinase-induced phosphorylation of IRF3 and the synthesis of type I IFNs. However, the S1P degradation-incompetent form of SPL also enhanced IFN responses, suggesting that SPL’s pro-IFN function is independent of S1P. Biochemical analyses revealed that SPL, as well as the mutant form of SPL, interacts with IKKε. Importantly, when endogenous IKKε was downregulated using a small interfering RNA approach, SPL’s anti-influenza viral activity was markedly suppressed. This indicates that IKKε is crucial for SPL-mediated inhibition of influenza virus replication. Thus, the results illustrate the functional significance of the SPL–IKKε–IFN axis during host innate immunity against viral infection.

MAVS ubiquitination by the E3 ligase TRIM25 and degradation by the proteasome is involved in type I interferon production after activation of the antiviral RIG-I-like receptors

BackgroundDuring a viral infection, the intracellular RIG-I-like receptors (RLRs) sense viral RNA and signal through the mitochondrial antiviral signaling adaptor MAVS (also known as IPS-1, Cardif and VISA) whose activation triggers a rapid production of type I interferons (IFN) and of pro-inflammatory cytokines through the transcription factors IRF3/IRF7 and NF-κB, respectively. While MAVS is essential for this signaling and known to operate through the scaffold protein NEMO and the protein kinase TBK1 that phosphorylates IRF3, its mechanism of action and regulation remain unclear.ResultsWe report here that RLR activation triggers MAVS ubiquitination on lysine 7 and 10 by the E3 ubiquitin ligase TRIM25 and marks it for proteasomal degradation concomitantly with downstream signaling. Inhibition of this MAVS degradation with a proteasome inhibitor does not affect NF-κB signaling but it hampers IRF3 activation, and NEMO and TBK1, two essential mediators in type I IFN production, are retained at the mitochondria.ConclusionsThese results suggest that MAVS functions as a recruitment platform that assembles a signaling complex involving NEMO and TBK1, and that the proteasome-mediated MAVS degradation is required to release the signaling complex into the cytosol, allowing IRF3 phosphorylation by TBK1.