IRF3 Dimerization Research Articles

Sec13 is a positive regulator of VISA-mediated antiviral signaling.

Viral infection triggers the innate antiviral immune response that rapidly produces type I interferons in most cell types to combat viruses invading. Upon viral infection, the cytoplasmic RNA sensors RIG-I/MDA5 recognize viral RNA, and then RIG-I/MDA5 is transported to mitochondria interacting with VISA through the CARD domain. From there, VISA recruits downstream antiviral signaling pathways molecules, such as TRAFs and TBK1. Eventually, IRF3 is phosphorylated and type I IFNs are induced to fight as the first line of defense against viruses. However, it remains unclear how VISA acts as a scaffold to assemble the signalosome in RIG-I-mediated antiviral signaling. Here, we demonstrated Sec13 as a novel component that was involved in VISA-mediated antiviral signaling pathway. The co-immunoprecipitation assays showed that Sec13 specifically interacts with VISA. Overexpression of Sec13 increases VISA's aggregation and ubiquitination and significantly enhances the phosphorylation and dimerization of IRF3, facilitating the IFN-β production. Conversely, the knockdown of Sec13 attenuates Sendai virus-induced and VISA-mediated IRF3 activation and the production of IFNβ, thus weakens antiviral immune activity.

ZWINT positively regulates TBK1-mediated RLR antiviral signaling

TANK-Binding Kinase 1 (TBK1) is a serine/threonine kinase ubiquitously expressed in many tissues and plays an important role in the RLR antiviral signaling pathway. Upon viral infection, various virus components, including viral DNA, RNA are sensed by different pattern recognition receptors (PRRs). Among these PRRs, TLR3, RIG-I/MDA5, and cGAS recruit different adaptors including TRIF, VISA/ MAVS, and MITA/STING to activate TBK1. Activated TBK1 then promote the phosphorylation and homodimerization of IFN-regulatory factors 3 and 7 (IRF3/7), in turn promoting type-I Interferon (IFNI) production. In this study, we reported that ZW10 Interacting Kinetochore Protein (ZWINT) positively regulates RLR antiviral signaling by targeting RIG-I/VISA/TBK1. Ectopic expression of ZWINT potentiated Sendai virus-induced, as well as TBK1- or VISA/MAVS-mediated IFN-β promoter activation by enhancing dimerization of IRF3. In contrast, knocking down of ZWINT expression with siRNA resulted in reduced IFN-β promoter activation and IRF3 dimerization induced by Sendai virus, indicating that ZWINT positively regulates TBK1-mediated RLR antiviral signaling.

Accessory proteins 8b and 8ab of severe acute respiratory syndrome coronavirus suppress the interferon signaling pathway by mediating ubiquitin-dependent rapid degradation of interferon regulatory factor 3

Severe acute respiratory syndrome coronavirus (SARS-CoV) is an inefficient inducer of interferon (IFN) response. It expresses various proteins that effectively circumvent IFN production at different levels via distinct mechanisms. Through the construction of recombinant IBV expressing proteins 8a, 8b and 8ab encoded by SARS-CoV ORF8, we demonstrate that expression of 8b and 8ab enables the corresponding recombinant viruses to partially overcome the inhibitory actions of IFN activation to achieve higher replication efficiencies in cells. We also found that proteins 8b and 8ab could physically interact with IRF3. Overexpression of 8b and 8ab resulted in the reduction of poly (I:C)-induced IRF3 dimerization and inhibition of the IFN-β signaling pathway. This counteracting effect was partially mediated by protein 8b/8ab-induced degradation of IRF3 in a ubiquitin-proteasome-dependent manner. Taken together, we propose that SARS-CoV may exploit the unique functions of proteins 8b and 8ab as novel mechanisms to overcome the effect of IFN response during virus infection.

Endoplasmic Reticulum Transmembrane Proteins ZDHHC1 and STING Both Act as Direct Adaptors for IRF3 Activation in Teleost.

IFN regulatory factor (IRF)3 is a central regulator for IFN-β expression in different types of pathogenic infections. Mammals have various pathogenic sensors that are involved in monitoring pathogen intrusions. These sensors can trigger IRF3-mediated antiviral responses through different pathways. Endoplasmic reticulum-associated proteins stimulator of IFN gene (STING) and zinc finger DHHC-type containing 1 (ZDHHC1) are critical mediators of IRF3 activation in response to viral DNA infections. In this study, grass carp STING and ZDHHC1 were found to have some similar molecular features and subcellular localization, and both were upregulated upon stimulation with polyinosinic:polycytidylic acid, B-DNA, or Z-DNA. Based on these results, we suggest that grass carp STING and ZDHHC1 might possess some properties similar to their mammalian counterparts. Overexpression of ZDHHC1 and STING in Ctenopharyngodon idella kidney cells upregulated IFN expression, whereas knockdown of IRF3 inhibited IFN activation. In addition, coimmunoprecipitation and GST pull-down assays demonstrated that STING and ZDHHC1 can interact separately with IRF3 and promote the dimerization and nuclear translocation of IRF3. Furthermore, we also found that small interfering RNA-mediated knockdown of STING could inhibit the expression of IFN and ZDHHC1 in fish cells. Similarly, knockdown of STING resulted in inhibition of the IFN promoter. In contrast, ZDHHC1 knockdown also inhibited IFN expression but had no apparent effect on STING, which indicates that STING is necessary for IFN activation through ZDHHC1. In conclusion, STING and ZDHHC1 in fish can respond to viral DNA or RNA molecules in cytoplasm, as well as activate IRF3 and, eventually, trigger IFN expression.

Rubicon Modulates Antiviral Type I Interferon (IFN) Signaling by Targeting IFN Regulatory Factor 3 Dimerization.

Rubicon is part of a Beclin-1-Vps34-containing autophagy complex. Rubicon induces antimicrobial responses upon Toll-like receptor (TLR) stimulation and functions as a feedback inhibitor to prevent unbalanced proinflammatory responses depending on dectin-1 signaling. However, the role played by Rubicon during antiviral immune responses, particularly the type I interferon (IFN) responses, remains largely unknown. Here, we report that Rubicon acts as a negative regulator for virus-triggered IFN signaling. Knockdown of Rubicon promoted type I interferon signaling and inhibited virus replication, while overexpression of Rubicon had the opposite effect. Rubicon specifically interacts with the interferon regulatory factor (IRF) association domain (IAD) of IRF3, and this interaction leads to inhibition of the dimerization of IRF3, which negatively regulates IFN-mediated antiviral response. Thus, our findings suggest the novel additional role of Rubicon as a negative regulator that inhibits the IFN signaling and cellular antiviral responses, providing a novel cellular mechanism of IRF3 inhibition.IMPORTANCE The type I IFN system is a critical innate immune response that protects organisms against virus infection. However, type I IFN signaling must be tightly regulated to avoid excessive production of IFNs. Hence, negative regulatory mechanisms for type I IFN signaling are important, and to date, several related molecules have been identified. Here, we show that Rubicon is a major negative regulator of type I IFN signaling, and unlike previous reports of cellular molecules that inhibit IRF3 activation via proteasomal degradation or dephosphorylation of IRF3, we show that Rubicon interacts with IRF3 and that ultimately this interaction leads to inhibition of the dimerization of IRF3. Thus, we identified a novel negative regulator of type I IFN signaling pathways and a novel cellular mechanism of IRF3 inhibition. The results of this study will increase our understanding of the role of negative-feedback mechanisms that regulate type I IFN signaling and maintain immune homeostasis.

ERRα negatively regulates type I interferon induction by inhibiting TBK1-IRF3 interaction.

Estrogen-related receptor α (ERRα) is a member of the nuclear receptor superfamily controlling energy homeostasis; however, its precise role in regulating antiviral innate immunity remains to be clarified. Here, we showed that ERRα deficiency conferred resistance to viral infection both in vivo and in vitro. Mechanistically, ERRα inhibited the production of type-I interferon (IFN-I) and the expression of multiple interferon-stimulated genes (ISGs). Furthermore, we found that viral infection induced TBK1-dependent ERRα stabilization, which in turn associated with TBK1 and IRF3 to impede the formation of TBK1-IRF3, IRF3 phosphorylation, IRF3 dimerization, and the DNA binding affinity of IRF3. The effect of ERRα on IFN-I production was independent of its transcriptional activity and PCG-1α. Notably, ERRα chemical inhibitor XCT790 has broad antiviral potency. This work not only identifies ERRα as a critical negative regulator of antiviral signaling, but also provides a potential target for future antiviral therapy.

Involvement of IKKα in STAT1 phosphorylation in antiviral signaling

Abstract RIG-I-like receptors (RLRs) sense non-self RNA, inducing type I IFNs. Type I IFN promotes the expression of IFN-stimulated genes (ISGs), which requires the activation of STAT1. We previously reported that dsRNA induced STAT1 phosphorylation via a type I IFN-independent pathway in addition to the well-known type I IFN-dependent pathway. IKKα is involved in antiviral signaling induced by dsRNA in the RLR-mediated antiviral signaling; however, its role remains uncertain. In this study, we investigated the role of IKKα in RLR signaling, which is essential for antiviral innate immune responses in non-professional antigen-presenting cells, such as epithelial cells. Silencing of IKKα markedly decreased the level of IFN-β and STAT1 phosphorylation in response to dsRNA. However, the inhibition of IKKα did not alter the RLR signaling-mediated dimerization of IRF3 or activation of NFκB. These results suggest a non-canonical role of IKKα in RLR signaling. In addition, STAT1 phosphorylation was inhibited by IKKα knockdown in IFNAR-deficient cells. These results suggest that the dual regulation of STAT1 by IKKα in the RLR-mediated antiviral signaling.

MDA5 Induces a Stronger Interferon Response than RIG-I to GCRV Infection through a Mechanism Involving the Phosphorylation and Dimerization of IRF3 and IRF7 in CIK Cells.

Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are critical cytosolic sensors that trigger the production of interferons (IFNs). Though their recognition functions are well identified, their unique roles in the downstream signal transduction remain to be elucidated. Herein, we report the differential effect between grass carp (Ctenopharyngodon idella) MDA5 (CiMDA5) and CiRIG-I on the production of various IFNs upon grass carp reovirus (GCRV) infection in C. idella kidney (CIK) cell line. In CIK cells, grass carp IFN1 (CiIFN1) and CiIFN3 are relatively highly expressed while CiIFN2 and CiIFN4 are relatively slightly expressed. Following GCRV infection, CiMDA5 induces a more extensive type I IFN response than CiRIG-I. Further investigation reveals that both CiMDA5 and CiRIG-I facilitate the expression and total phosphorylation levels of grass carp IFN regulatory factor (IRF) 3 (CiIRF3) and CiIRF7 upon GCRV infection or poly(I:C) stimulation. However, the difference is that CiRIG-I decreases the threonine phosphorylation level of CiIRF7. As a consequence, CiMDA5 enhances the heterodimerization of CiIRF3 and CiIRF7 and homodimerization of CiIRF7, whereas CiRIG-I facilitates the heterodimerization but attenuates homodimerization of CiIRF7. Moreover, the present study suggests that CiIRF3 and CiIRF7 heterodimers and CiIRF7 homodimers are able to induce more extensive IFN-I responses than CiIRF3 homodimers under GCRV infection. Additionally, CiMDA5 induces a stronger type II IFN (IFN-II) response against GCRV infection than CiRIG-I. Collectively, these results demonstrate that CiMDA5 plays a more potent role than CiRIG-I in IFN response to GCRV infection through differentially regulating the phosphorylation and dimerization of CiIRF3 and CiIRF7.

Clarithromycin prevents human respiratory syncytial virus-induced airway epithelial responses by modulating activation of interferon regulatory factor-3

Macrolide antibiotics exert immunomodulatory activity by reducing pro-inflammatory cytokine production by airway epithelial cells, fibroblasts, vascular endothelial cells, and immune cells. However, the underlying mechanism of action remains unclear. Here, we examined the effect of clarithromycin (CAM) on pro-inflammatory cytokine production, including interferons (IFNs), by primary human nasal epithelial cells and lung epithelial cell lines (A549 and BEAS-2B cells) after stimulation by Toll-like receptor (TLR) and RIG-I-like receptor (RLR) agonists and after infection by human respiratory syncytial virus (RSV). CAM treatment led to a significant reduction in poly I:C- and RSV-mediated IL-8, CCL5, IFN-β and -λ production. Furthermore, IFN-β promoter activity (activated by poly I:C and RSV infection) was significantly reduced after treatment with CAM. CAM also inhibited IRF-3 dimerization and subsequent translocation to the nucleus. We conclude that CAM acts a crucial modulator of the innate immune response, particularly IFN production, by modulating IRF-3 dimerization and subsequent translocation to the nucleus of airway epithelial cells. This newly identified immunomodulatory action of CAM will facilitate the discovery of new macrolides with an anti-inflammatory role.

Uncovering the mechanism of E1A antagonism of the cGAS-STING pathway

Abstract The cGAS-STING pathway is critical for antiviral defense. Cytosolic DNA is detected by cGAS, which synthesizes the second messenger cGAMP that activates the signaling adaptor STING. Like the RNA sensing pathway, the cGAS-STING pathway converges on kinase TBK-1, which phosphorylates IRF-3, allowing it to translocate to the nucleus to induce production of type I interferons. Many viruses encode inhibitors of this pathway to avoid the ensuing antiviral state. Previously, we discovered that oncoproteins E7 and E1A encoded by human papillomavirus and adenovirus, respectively, specifically block the cGAS-STING pathway and that these proteins bind to STING. However, the mechanism underlying the antagonism of the cGAS-STING pathway is unknown. To dissect the mechanism for E1A, we used HEK 293 cells, which express E1A, to assess known events in the pathway: TBK-1 and IRF-3 phosphorylation, IRF-3 dimerization and IRF-3 nuclear translocation. Consistent with a specific block in the DNA-activated interferon response in HEK 293 cells, we found that IRF-3 dimerization and nuclear translocation were prevented in response to DNA, but not to RNA stimulation. Surprisingly, we found that early events were intact in response to DNA, including robust phosphorylation of IRF-3 on serine 386, which is required for dimerization. However, phosphorylation of serine 396, an event with unclear importance for IRF-3 activation, was impaired after DNA stimulation. This reveals that E1A blocks a potentially unappreciated late step in the pathway, preventing complete IRF-3 phosphorylation and subsequent dimerization. Ongoing work will address the temporal and spatial regulation of this novel step in STING-dependent IRF-3 activation and its antagonism by E1A.

The structural basis of IRF-3 recruitment, activation, and targeting in innate immunity

Abstract Type I interferons (IFNs) are key cytokines mediating innate antiviral immunity. cGAS, RLRs, and TLRs recognize microbial DNA, RNA, and LPS to induce the expression of type I IFNs. These signaling pathways converge at the recruitment and activation of IRF-3. STING, MAVS, and TRIF mediate the recruitment of IRF-3 through a conserved pLxIS motif. Here we show that the pLxIS motif of phosphorylated STING, MAVS, and TRIF binds to IRF-3 in a similar manner, while residues upstream of the motif confer specificity. The structure of IRF-3 phosphomimetic mutant S386/396E bound to CBP reveals that the pLxIS motif also mediates IRF-3 dimerization and activation. Moreover, rotavirus NSP1 employs a pLxIS motif to target IRF-3 for degradation. However, phosphorylation of NSP1 is not required for its activity. These results suggest a concerted mechanism for recruitment and activation of IRF-3 that can be subverted by viral proteins to evade innate immune response.

Nonstructural protein p39 of feline calicivirus suppresses host innate immune response by preventing IRF-3 activation

Feline calicivirus (FCV) is an important veterinary pathogen that causes acute upper respiratory tract diseases and, occasionally, highly contagious febrile hemorrhagic syndrome in cats. Many viruses have adopted mechanisms for evading IFN-α/β signaling, particularly by directly or indirectly suppressing activation of IRF-3. In this study, we investigated whether nonstructural proteins of FCV possess these mechanisms. When p39, a nonstructural protein of FCV, was transiently expressed in 293T cells, it suppressed IFN-β and ISG15 mRNA production induced by dsRNA. Expression of p39 also suppressed phosphorylation and dimerization of IRF-3 induced by dsRNA. These results suggest that p39 suppresses type 1 IFN production by preventing IRF-3 activation. This may become an important factor in understanding the pathogenesis and virulence of FCV.

ID: 53: cFLIPL-IRF3 interactions prevent IRF3 – Mediated gene expression

There are several cellular proteins whose over-expression is correlated to a poor prognosis for cancer survival. cFLIPL is one such protein, and one function that may be important in tumorigenesis is NF-kB activation. It previously was shown that cFLIPL inhibits the expression of the IFN-b gene, a gene controlled by NF-kB, IRF3 and AP1, suggesting that cFLIPL may affect other transcription factors. To solve this mystery, the effect of cFLIPL on IRF3 activation was interrogated. It was observed that ectopic expression of cFLIPL inhibited the transcription of a synthetic luciferase gene and cellular genes controlled by IRF3. cFLIPL expression did not prevent IRF3 phosphorylation, dimerization and nuclear localization. Instead, cFLIPL prevented IRF3:DNA interactions as detected by ChIP assays, and cFLIPL also co-immunoprecipitated with IRF3. Interestingly, cFLIPL acted in a multi-functional manner in the nucleus to inhibit IRF3 because cFLIPL also prevented IRF3:CBP interactions. Mutational analysis confirmed these interactions to be relevant. cFLIPS, an alternative splice form of cFLIP, did not disrupt IRF3:CBP interactions, prevent IRF3:DNA interactions or inhibit IRF3 activation. In contrast, the caspase-like domain (CLD) of cFLIPL was sufficient to inhibit all three of these events. These data show a novel function for cFLIPL in modulating innate immune responses To begin to understand whether cFLIP’s IRF3 inhibitory function may also contribute to tumorigenesis, we identified that the molecular mechanism of IRF3 inhibition occurs within the nucleus.

Phosphatidylinositol-3-kinase and Akt are required for RIG-I-mediated anti-viral signalling through cross-talk with IPS-1.

Retinoic acid-inducible gene I (RIG-I) is a cytosolic pattern-recognition receptor that recognizes viruses and triggers anti-viral immune responses. Activation of intracellular RIG-I signalling is mediated through interferon-β (IFN-β) promoter stimulator-1 (IPS-1), an adaptor of RIG-I, which induces IFN regulatory factor (IRF) 3 activation and type I IFN expression. The phosphatidylinositol-3-kinase (PI3K) and Akt pathway is activated in host immune cells upon viral infection. However, the mechanism as to how they work in RIG-I signalling has not been fully elucidated. Therefore, we investigated the role of PI3K and Akt in the regulation of RIG-I-mediated IRF3 activation and type I IFN expression in macrophages. Our results show that Sendai virus infection, which is recognized by RIG-I, led to IRF3 activation and IFN-β expression and these responses were attenuated by the PI3K inhibitor (LY294002) and an Akt dominant-negative mutant in the macrophage cell line(RAW264.7). IRF3 phosphorylation and dimerization as well as IFN-β expression induced by a synthetic RIG-I agonist, short poly(I:C), were suppressed by LY294002 or siRNA-Akt in bone marrow-derived macrophages. Suppression of PI3K and Akt using a dominant-negative mutant and siRNA knockdown resulted in attenuation of IRF3 activation and IFN-β expression induced by RIG-I itself or its adaptor, IPS-1. Association of Akt with IPS-1 increased with short poly(I:C) stimulation and required the pleckstrin homology domain of Akt and caspase-recruitment domain in IPS-1. Collectively, our results show that PI3K and Akt are required downstream of IPS-1 for RIG-I-mediated anti-viral immune responses. The results describe a novel, interactive relationship between RIG-I downstream signalling molecules resulting in efficient anti-viral immunity.

The SARS coronavirus papain like protease can inhibit IRF3 at a post activation step that requires deubiquitination activity.

BackgroundThe outcome of a viral infection is regulated by complex interactions of viral and host factors. SARS coronavirus (SARS-CoV) engages and regulates several innate immune response pathways during infection. We have previously shown that the SARS-CoV Papain-like Protease (PLpro) inhibits type I interferon (IFN) by inhibiting IRF3 phosphorylation thereby blocking downstream Interferon induction. This finding prompted us to identify other potential mechanisms of inhibition of PLpro on IFN induction.MethodsWe have used plasmids expressing PLpro and IRF3 including an IRF3 mutant that is constitutively active, called IRF3(5D). In these experiments we utilize transfections, chromatin immunoprecipitation, Electro-mobility Shift Assays (EMSA) and protein localization to identify where IRF3 and IRF3(5D) are inhibited by PLpro.ResultsHere we show that PLpro also inhibits IRF3 activation at a step after phosphorylation and that this inhibition is dependent on the de-ubiquitination (DUB) activity of PLpro. We found that PLpro is able to block the type I IFN induction of a constitutively active IRF3, but does not inhibit IRF3 dimerization, nuclear localization or DNA binding. However, inhibition of PLpro’s DUB activity by mutagenesis blocked the IRF3 inhibition activity of PLpro, suggesting a role for IRF3 ubiquitination in induction of a type I IFN innate immune response.ConclusionThese results demonstrate an additional mechanism that PLpro is able to inhibit IRF3 signaling. These data suggest novel innate immune antagonism activities of PLpro that may contribute to SARS-CoV pathogenesis.

MOV10 provides antiviral activity against RNA viruses by enhancing IFN induction through IKKε and IRF3. (INC8P.441)

Abstract MOV10 (Moloney leukemia virus 10, homolog) is an interferon-inducible putative DEAG-containing RNA helicase, which has been known to be associated with the RNA-induced silencing complex (RISC). Although, it is known to inhibit HIV-1 replication, analysis of its border antiviral activity and the mechanism has not been explored. Here, we report that MOV10 exhibits antiviral activity against a number of RNA viruses including EMCV, CVB, SeV and VSV by enhancing IFN induction. The antiviral activity of MOV10 is dependent on IFN signaling, and stable expression of MOV10 enhances IRF3 dimerization and IFN/ISG induction. Using various knockout cells combined with siRNA knockdowns, we show that MOV10-mediated IRF3 activation and IFN/ISG induction is not dependent on RIG-I/MAVS-mediated RNA sensing. Upon virus infection MOV10 specifically interacts and activates IKKε to induce IRF3 activation without involving TBK1. The important role of MOV10 in mediating antiviral signaling is further supported by our findings that CVB 3Cpro protease specifically targets MOV10 for degradation as a possible innate immune evasion mechanism. Taken together, these results establish MOV10, an evolutionary conserved protein, as an antiviral gene that uses a novel signaling pathway to enhance IFN response.

SARS coronavirus papain-like protease inhibits the type I interferon signaling pathway through interaction with the STING-TRAF3-TBK1 complex

SARS coronavirus (SARS-CoV) develops an antagonistic mechanism by which to evade the antiviral activities of interferon (IFN). Previous studies suggested that SARS-CoV papain-like protease (PLpro) inhibits activation of the IRF3 pathway, which would normally elicit a robust IFN response, but the mechanism(s) used by SARS PLpro to inhibit activation of the IRF3 pathway is not fully known. In this study, we uncovered a novel mechanism that may explain how SARS PLpro efficiently inhibits activation of the IRF3 pathway. We found that expression of the membrane-anchored PLpro domain (PLpro-TM) from SARS-CoV inhibits STING/TBK1/IKKε-mediated activation of type I IFNs and disrupts the phosphorylation and dimerization of IRF3, which are activated by STING and TBK1. Meanwhile, we showed that PLpro-TM physically interacts with TRAF3, TBK1, IKKε, STING, and IRF3, the key components that assemble the STING-TRAF3-TBK1 complex for activation of IFN expression. However, the interaction between the components in STING-TRAF3-TBK1 complex is disrupted by PLpro-TM. Furthermore, SARS PLpro-TM reduces the levels of ubiquitinated forms of RIG-I, STING, TRAF3, TBK1, and IRF3 in the STING-TRAF3-TBK1 complex. These results collectively point to a new mechanism used by SARS-CoV through which PLpro negatively regulates IRF3 activation by interaction with STING-TRAF3-TBK1 complex, yielding a SARS-CoV countermeasure against host innate immunity.

The SARS coronavirus papain like protease can inhibit IRF3 at a post activation step that requires deubiquitination activity

The outcome of a viral infection is regulated by complex interactions of viral and host factors. SARS coronavirus (SARS-CoV) engages and regulates several innate immune response pathways during infection. We have previously shown that the SARS-CoV Papain-like Protease (PLpro) inhibits type I interferon (IFN) by inhibiting IRF3 phosphorylation thereby blocking downstream Interferon induction. This finding prompted us to identify other potential mechanisms of inhibition of PLpro on IFN induction. We have used plasmids expressing PLpro and IRF3 including an IRF3 mutant that is constitutively active, called IRF3(5D). In these experiments we utilize transfections, chromatin immunoprecipitation, Electro-mobility Shift Assays (EMSA) and protein localization to identify where IRF3 and IRF3(5D) are inhibited by PLpro. Here we show that PLpro also inhibits IRF3 activation at a step after phosphorylation and that this inhibition is dependent on the de-ubiquitination (DUB) activity of PLpro. We found that PLpro is able to block the type I IFN induction of a constitutively active IRF3, but does not inhibit IRF3 dimerization, nuclear localization or DNA binding. However, inhibition of PLpro’s DUB activity by mutagenesis blocked the IRF3 inhibition activity of PLpro, suggesting a role for IRF3 ubiquitination in induction of a type I IFN innate immune response. These results demonstrate an additional mechanism that PLpro is able to inhibit IRF3 signaling. These data suggest novel innate immune antagonism activities of PLpro that may contribute to SARS-CoV pathogenesis.

The antiviral activity of poly-γ-glutamic acid, a polypeptide secreted by Bacillus sp., through induction of CD14-dependent type I interferon responses

Poly-γ-glutamic acid (γ-PGA) is an anionic polypeptide secreted by Bacillus sp. that has been shown to activate immune cells through interactions with toll-like receptor 4 (TLR4). However, its ability to induce the type I interferon (IFN) response has not yet been characterized. Here, we demonstrate that γ-PGA induces type I IFN signaling pathway via the TLR4 signaling pathway. The induction required both myeloid differentiation factor 2 (MD2) and the pattern-recognition receptor CD14, which are two TLR4-associated accessory proteins. The γ-PGA with high molecular weights (2000 and 5000 kDa) was able to activate the subsequent signals through TLR4/MD2 to result in dimerization of IRF-3, a transcription factor required for IFN gene expression, leading to increases in mRNA levels of the type I IFN-response genes, 2′–5′ OAS and ISG56. Moreover, γ-PGA (2000 kDa) displayed an antiviral activity against SARS coronavirus and hepatitis C virus. Our results identify high-molecular weight γ-PGA as a TLR4 ligand and demonstrate that γ-PGA requires both CD14 and MD2 for the activation of type I IFN responses. Our results suggest that the microbial biopolymer γ-PGA may have therapeutic potential against a broad range of viruses sensitive to type I IFNs.

TRIM11 Negatively Regulates IFNβ Production and Antiviral Activity by Targeting TBK1

The innate immune response is a host defense mechanism against infection by viruses and bacteria. Type I interferons (IFNα/β) play a crucial role in innate immunity. If not tightly regulated under normal conditions and during immune responses, IFN production can become aberrant, leading to inflammatory and autoimmune diseases. In this study, we identified TRIM11 (tripartite motif containing 11) as a novel negative regulator of IFNβ production. Ectopic expression of TRIM11 decreased IFNβ promoter activity induced by poly (I:C) stimulation or overexpression of RIG-I (retinoic acid-inducible gene-I) signaling cascade components RIG-IN (constitutively active form of RIG-I), MAVS (mitochondrial antiviral signaling protein), or TBK1 (TANK-binding kinase-1). Conversely, TRIM11 knockdown enhanced IFNβ promoter activity induced by these stimuli. Moreover, TRIM11 overexpression inhibited the phosphorylation and dimerization of IRF3 and expression of IFNβ mRNA. By contrast, TRIM11 knockdown increased the IRF3 phosphorylation and IFNβ mRNA expression. We also found that TRIM11 and TBK1, a key kinase that phosphorylates IRF3 in the RIG-I pathway, interacted with each other through CC and CC2 domain, respectively. This interaction was enhanced in the presence of the TBK1 adaptor proteins, NAP1 (NF-κB activating kinase-associated protein-1), SINTBAD (similar to NAP1 TBK1 adaptor) or TANK (TRAF family member-associated NF-κB activator). Consistent with its inhibitory role in RIG-I-mediated IFNβ signaling, TRIM11 overexpression enhanced viral infectivity, whereas TRIM11 knockdown produced the opposite effect. Collectively, our results suggest that TRIM11 inhibits RIG-I-mediated IFNβ production by targeting the TBK1 signaling complex.