Antiviral Innate Signaling Research Articles

Mutual Regulation of NOD2 and RIG-I in Zebrafish Provides Insights into the Coordination between Innate Antibacterial and Antiviral Signaling Pathways.

Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) and retinoic acid-inducible gene I (RIG-I) are two important cytosolic pattern recognition receptors (PRRs) in the recognition of pathogen-associated molecular patterns (PAMPs), initiating innate antibacterial and antiviral signaling pathways. However, the relationship between these PRRs, especially in teleost fish models, is rarely reported. In this article, we describe the mutual regulation of zebrafish NOD2 (DrNOD2) and RIG-I (DrRIG-I) in innate immune responses. Luciferase assays were conducted to determine the activation of NF-κB and interferon signaling. Morpholino-mediated knockdown and mRNA-mediated rescue were performed to further confirm the regulatory roles between DrNOD2 and DrRIG-I. Results showed that DrNOD2 and DrRIG-I shared conserved structural hallmarks with their mammalian counterparts, and activated DrRIG-I signaling can induce DrNOD2 production. Surprisingly, DrNOD2-initiated signaling can also induce DrRIG-I expression, indicating that a mutual regulatory mechanism may exist between them. Studies conducted using HEK293T cells and zebrafish embryos showed that DrRIG-I could negatively regulate DrNOD2-activated NF-κB signaling, and DrNOD2 could inhibit DrRIG-I-induced IFN signaling. Moreover, knocking down DrRIG-I expression by morpholino could enhance DrNOD2-initiated NF-κB activation, and vice versa, which could be rescued by their corresponding mRNAs. Results revealed a mutual feedback regulatory mechanism underlying NOD2 and RIG-I signaling pathways in teleosts. This mechanism reflects the coordination between cytosolic antibacterial and antiviral PRRs in the complex network of innate immunity.

USP18 recruits USP20 to promote innate antiviral response through deubiquitinating STING/MITA.

STING (also known as MITA) mediates the innate antiviral signaling and ubiquitination of STING is key to its function. However, the deubiquitination process of STING is unclear. Here we report that USP18 recruits USP20 to deconjugate K48-linked ubiquitination chains from STING and promotes the stability of STING and the expression of type I IFNs and proinflammatory cytokines after DNA virus infection. USP18 deficiency or knockdown of USP20 resulted in enhanced K48-linked ubiquitination and accelerated degradation of STING, and impaired activation of IRF3 and NF-κB as well as induction of downstream genes after infection with DNA virus HSV-1 or transfection of various DNA ligands. In addition, Usp18-/- mice were more susceptible to HSV-1 infection compared with the wild-type littermates. USP18 did not deubiquitinate STING in vitro but facilitated USP20 to catalyze deubiquitination of STING in a manner independent of the enzymatic activity of USP18. In addition, reconstitution of STING into Usp18-/- MEFs restored HSV-1-induced expression of downstream genes and cellular antiviral responses. Our findings thus uncover previously uncharacterized roles of USP18 and USP20 in mediating virus-triggered signaling and contribute to the understanding of the complicated regulatory system of the innate antiviral responses.

Ubiquitination Upregulates Influenza Virus Polymerase Function.

The influenza A virus polymerase plays an essential role in the virus life cycle, directing synthesis of viral mRNAs and genomes. It is a trimeric complex composed of subunits PA, PB1, and PB2 and associates with viral RNAs and nucleoprotein (NP) to form higher-order ribonucleoprotein (RNP) complexes. The polymerase is regulated temporally over the course of infection to ensure coordinated expression of viral genes as well as replication of the viral genome. Various host factors and processes have been implicated in regulation of the IAV polymerase function, including posttranslational modifications; however, the mechanisms are not fully understood. Here we demonstrate that ubiquitination plays an important role in stimulating polymerase activity. We show that all protein subunits in the RNP are ubiquitinated, but ubiquitination does not significantly alter protein levels. Instead, ubiquitination and an active proteasome enhance polymerase activity. Expression of ubiquitin upregulates polymerase function in a dose-dependent fashion, causing increased accumulation of viral RNA (vRNA), cRNA, and mRNA and enhanced viral gene expression during infection. Ubiquitin expression directly affects polymerase activity independent of nucleoprotein (NP) or ribonucleoprotein (RNP) assembly. Ubiquitination and the ubiquitin-proteasome pathway play key roles during multiple stages of influenza virus infection, and data presented here now demonstrate that these processes modulate viral polymerase activity independent of protein degradation. IMPORTANCE The cellular ubiquitin-proteasome pathway impacts steps during the entire influenza virus life cycle. Ubiquitination suppresses replication by targeting viral proteins for degradation and stimulating innate antiviral signaling pathways. Ubiquitination also enhances replication by facilitating viral entry and virion disassembly. We identify here an addition proviral role of the ubiquitin-proteasome system, showing that all of the proteins in the viral replication machinery are subject to ubiquitination and this is crucial for optimal viral polymerase activity. Manipulation of the ubiquitin machinery for therapeutic benefit is therefore likely to disrupt the function of multiple viral proteins at stages throughout the course of infection.

MAVS maintains mitochondrial homeostasis via autophagy.

Mitochondrial antiviral signalling protein (MAVS) acts as a critical adaptor protein to transduce antiviral signalling by physically interacting with activated RIG-I and MDA5 receptors. MAVS executes its functions at the outer membrane of mitochondria to regulate downstream antiviral signalling, indicating that the mitochondria provides a functional platform for innate antiviral signalling transduction. However, little is known about whether and how MAVS-mediated antiviral signalling contributes to mitochondrial homeostasis. Here we show that the activation of MAVS is sufficient to induce autophagic signalling, which may mediate the turnover of the damaged mitochondria. Importantly, we find MAVS directly interacts with LC3 through its LC3-binding motif ‘YxxI’, suggesting that MAVS might act as an autophagy receptor to mediate mitochondrial turnover upon excessive activation of RLR signalling. Furthermore, we provide evidence that both MAVS self-aggregation and its interaction with TRAF2/6 proteins are important for MAVS-mediated mitochondrial turnover. Collectively, our findings suggest that MAVS acts as a potential receptor for mitochondria-associated autophagic signalling to maintain mitochondrial homeostasis.

MSX1 Modulates RLR-Mediated Innate Antiviral Signaling by Facilitating Assembly of TBK1-Associated Complexes.

Recognition of viral dsRNA by the retinoic acid-inducible gene-1-like receptors (RLRs) triggers signaling cascades that lead to activation of the TBK1 kinase and transcription factor IFN regulatory factor 3, induction of downstream antiviral genes, and innate antiviral responses. In this study, we identified muscle segment homeobox1 (MSX1) as an important modulator of RLR-mediated signaling pathways. Knockdown or knockout of MSX1 significantly impaired Sendai virus-triggered activation of TBK1 and IFN regulatory factor 3, induction of downstream antiviral genes, and cellular antiviral responses. Interestingly, MSX1 was translocated from the nucleus to cytoplasm, particularly mitochondria upon infection of Sendai virus. Biochemcially, MSX1 was important for assembly of TBK1/IKK-related kinase-associated protein 1/TNFR-associated factor-associated NF-κB activator complexes. Our results suggest that MSX1 is an important component of RLR-mediated signaling and reveal mechanisms on innate immune responses against RNA viruses.

RNF123 has an E3 ligase-independent function in RIG-I-like receptor-mediated antiviral signaling.

Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are cytoplasmic sensors crucial for recognizing different species of viral RNAs, which triggers the production of type I interferons (IFNs) and inflammatory cytokines. Here, we identify RING finger protein 123 (RNF123) as a negative regulator of RIG-I and MDA5. Overexpression of RNF123 inhibits IFN-β production triggered by Sendai virus (SeV) and encephalomyocarditis picornavirus (EMCV). Knockdown or knockout of endogenous RNF123 potentiates IFN-β production triggered by SeV and EMCV, but not by the sensor of DNA viruses cGAS RNF123 associates with RIG-I and MDA5 in both endogenous and exogenous cases in a viral infection-inducible manner. The SPRY and coiled-coil, but not the RING, domains of RNF123 are required for the inhibitory function. RNF123 interacts with the N-terminal CARD domains of RIG-I/MDA5 and competes with the downstream adaptor VISA/MAVS/IPS-1/Cardif for RIG-I/MDA5 CARD binding. These findings suggest that RNF123 functions as a novel inhibitor of innate antiviral signaling mediated by RIG-I and MDA5, a function that does not depend on its E3 ligase activity.

Promoting proteasomal degradation of the viral RNA sensor RIG-I

The p97 complex attenuates antiviral signaling by recruiting the E3 ubiquitin ligase RNF125 to RIG-I.

Class A Scavenger Receptor-Mediated Double-Stranded RNA Internalization Is Independent of Innate Antiviral Signaling and Does Not Require Phosphatidylinositol 3-Kinase Activity.

dsRNA is a potent trigger of innate immune signaling, eliciting effects within virally infected cells and after release from dying cells. Given its inherent stability, extracellular dsRNA induces both local and systemic effects. Although the class A scavenger receptors (SR-As) mediate dsRNA entry, it is unknown whether they contribute to signaling beyond ligand internalization. In this study, we investigated whether SR-As contribute to innate immune signaling independent of the classic TLR and retinoic acid-inducible gene-I-like receptor (RLR) pathways. We generated a stable A549 human epithelial cell line with inducible expression of the hepatitis C virus protease NS3/4A, which efficiently cleaves TRIF and IFN-β promoter stimulator 1, adaptors for TLR3 and the RLRs, respectively. Cells expressing NS3/4A and TLR3/MyD88/IFN-β promoter stimulator 1(-/-) mouse embryonic fibroblasts completely lacked antiviral activity to extracellular dsRNA relative to control cells, suggesting that SR-As do not possess signaling capacity independent of TLR3 or the RLRs. Previous studies implicated PI3K signaling in SR-A-mediated activities and in downstream production of type I IFN. We found that SR-A-mediated dsRNA internalization occurs independent of PI3K activation, whereas downstream signaling leading to IFN production was partially dependent on PI3K activity. Overall, these findings suggest that SR-A-mediated dsRNA internalization is independent of innate antiviral signaling.

ID: 82: The Innate Immune Sensor, LGP2, Interferes with TRAF Proteins to Prevent NFkB Activation

The detection of RNA virus infections triggers the production type I interferon (IFN), and is a major component of the powerful antiviral system that directly interferes with virus replication and contributes to both innate and adaptive immune responses. Virus replication intermediates such as double stranded RNA (dsRNA) or RNAs with distinct non-self modifications are recognized in the cytoplasm by pattern recognition receptors including RIG-I, MDA5, and LGP2. These innate sensors recognize non-self RNAs via their C-terminal domains, and RIG-I and MDA5 use N-terminal CARDs to induce the activation of the downstream signaling scaffold, MAVS. MAVS coordinates the assembly of signaling machinery including TRAFs and IKK-family kinases that activate IRF3 and NFkB transcription factors. Available evidence suggests LGP2 acts as a concentration dependent switch to regulate RLR antiviral signaling. For positive actions, LGP2 uses its unique catalytic activity to enhance MDA5 RNA recognition, dsRNA filament assembly, and enable more efficient activation of MAVS and downstream signal transduction. The mechanism of LGP2 negative regulation have been investigated, and results indicate that LGP2-mediated inhibition occurs downstream of RIG-I, MDA5, and MAVS. LGP2 inhibition does not require ATPase activity or RNA binding. Instead, results indicate that LGP2 targets TRAF signaling proteins, disengaging their ubiquitin ligase activity, and potently preventing NFkB activation in MAVS dependent and independent systems. Identification of TRAF proteins as inhibitory targets not only resolves the dual roles of LGP2 in antiviral signaling regulation, but also expands the potential impact of LGP2 negative regulation beyond innate antiviral signaling.

Trim21 regulates Nmi-IFI35 complex-mediated inhibition of innate antiviral response

In this study, using an immunoprecipitation coupled with mass spectrometry approach, we have identified the E3 ubiquitin ligase Trim21 as an interacting partner of IFI35 and Nmi. We found that this interaction leads to K63-linked ubiquitination on K22 residue of Nmi, but not IFI35. Using domain deletion analysis, we found that the interaction is mediated via the coiled-coil domain of Nmi and the carboxyl-terminal SPRY domain of Trim21. Furthermore, we show that depletion of Trim21 leads to significantly reduced interaction of Nmi with IFI35, which results in the abrogation of the negative regulatory function of the Nmi-IFI35 complex on innate antiviral signaling. Thus, Trim21 appears to be a critical regulator of the functions of the Nmi-IFI35 complex. Overall, the results presented here uncover a new mechanism of regulation of the Nmi-IFI35 complex by Trim21, which may have implications for various autoimmune diseases associated uncontrolled antiviral signaling.

Aryl Hydrocarbon Receptor Interacting Protein Targets IRF7 to Suppress Antiviral Signaling and the Induction of Type I Interferon

The transcription factor IRF7 (interferon regulatory factor 7) is a key regulator of type I interferon and plays essential roles in restricting virus infection and spread. IRF7 activation is tightly regulated to prevent excessive inflammation and autoimmunity; however, how IRF7 is suppressed by negative regulators remains poorly understood. Here, we have identified AIP (aryl hydrocarbon receptor interacting protein) as a new binding partner of IRF7. The interaction between AIP and IRF7 is enhanced upon virus infection, and AIP potently inhibits IRF7-induced type I IFN (IFNα/β) production. Overexpression of AIP blocks virus-induced activation of IFN, whereas knockdown of AIP by siRNA potentiates virally activated IFN production. Consistently, AIP-deficient murine embryonic fibroblasts are highly resistant to virus infection because of increased production of IFNα/β. AIP inhibits IRF7 function by antagonizing the nuclear localization of IRF7. Together, our study identifies AIP as a novel inhibitor of IRF7 and a negative regulator of innate antiviral signaling.

Regulation of cellular innate antiviral signaling by ubiquitin modification.

Host pattern-recognition receptors (PRRs) recognize pathogen-associated molecular patterns generated by invading viruses and initiate a series of signaling cascades that lead to the activation of interferon-regulatory factor 3 (IRF3) and nuclear factor-κB (NF-κB) and subsequent induction of type I interferons (IFNs). Posttranslational modification of proteins by ubiquitin plays an essential role in mediating or regulating the virus-triggered PRRs-mediated signaling. Deubiquitination is the reversible process of ubiquitination and its role in regulating PRRs-mediated signaling has recently been explored. In this review, we first summarize the ubiquitination events in PRRs-mediated signaling that is triggered by viral nucleic acid and then focus on host and viral deubiquitinating enzymes-mediated regulation of virus-triggered signaling that modulates the activation of IRF3 and NF-κB and subsequent induction of type I IFNs.

Innate Antiviral Host Defense Attenuates TGF-β Function through IRF3-Mediated Suppression of Smad Signaling

TGF-β signaling is essential in many processes, including immune surveillance, and its dysregulation controls various diseases, including cancer, fibrosis, and inflammation. Studying the innate host defense, which functions in most cell types, we found that RLR signaling represses TGF-β responses. This regulation is mediated by activated IRF3, using a dual mechanism of IRF3-directed suppression. Activated IRF3 interacts with Smad3, thus inhibiting TGF-β-induced Smad3 activation and, in the nucleus, disrupts functional Smad3 transcription complexes by competing with coregulators. Consequently, IRF3 activation by innate antiviral signaling represses TGF-β-induced growth inhibition, gene regulation and epithelial-mesenchymal transition, and the generation of Treg effector lymphocytes from naive CD4(+) lymphocytes. Conversely, silencing IRF3 expression enhances epithelial-mesenchymal transition, TGF-β-induced Treg cell differentiation upon virus infection, and Treg cell generation invivo. Wepresent a mechanism of regulation of TGF-β signaling by the antiviral defense, with evidence for its role in immune tolerance and cancer cell behavior.

Asthma is associated with multiple alterations in anti-viral innate signalling pathways.

BackgroundHuman rhinovirus (HRV) infection is a major trigger for asthma exacerbations. Anti-viral immunity appears to be abnormal in asthma, with immune dysfunction reported in both airway structural cells and migratory, bone marrow derived cells. Though decreased capacity to produce anti-viral interferons (IFNs) has been reported in asthma, a detailed analysis of the molecular events involved has not been undertaken.ObjectiveTo compare the molecular pathway controlling type I IFN synthesis in HRV-stimulated peripheral blood mononuclear cells (PBMC) from asthmatic and healthy subjects.MethodsPBMC from 22 allergic asthmatics and 20 healthy donors were cultured with HRV for 24 hours. Multiple components of the Toll-like receptor (TLR), IFN regulatory and NFκβ pathways were compared at the mRNA and protein level.ResultsMultiple deficiencies in the innate immune response to HRV were identified in asthma, with significantly lower expression of IFNα, IFNβ and interferon stimulated genes than in healthy subjects. This was accompanied by reduced expression of intra-cellular signalling molecules including interferon regulatory factors (IRF1, IRF7), NF-κB family members (p50, p52, p65 and IκKα) and STAT1, and by reduced responsiveness to TLR7/TLR8 activation. These observations could not be attributed to alterations in the numbers of dendritic cell (DC) subsets in asthma or baseline expression of the viral RNA sensing receptors TLR7/TLR8. In healthy subjects, blocking the activity of type-I IFN or depleting plasmacytoid DC recapitulated many of the abnormalities observed in asthma.ConclusionsMultiple abnormalities in innate anti-viral signalling pathways were identified in asthma, with deficiencies in both IFN-dependent and IFN-independent molecules identified.

A Novel Mitochondrial MAVS/Caspase-8 Platform Links RNA Virus–Induced Innate Antiviral Signaling to Bax/Bak-Independent Apoptosis

Semliki Forest virus (SFV) requires RNA replication and Bax/Bak for efficient apoptosis induction. However, cells lacking Bax/Bak continue to die in a caspase-dependent manner. In this study, we show in both mouse and human cells that this Bax/Bak-independent pathway involves dsRNA-induced innate immune signaling via mitochondrial antiviral signaling (MAVS) and caspase-8. Bax/Bak-deficient or Bcl-2- or Bcl-xL-overexpressing cells lacking MAVS or caspase-8 expression are resistant to SFV-induced apoptosis. The signaling pathway triggered by SFV does neither involve death receptors nor the classical MAVS effectors TNFR-associated factor-2, IRF-3/7, or IFN-β but the physical interaction of MAVS with caspase-8 on mitochondria in a FADD-independent manner. Consistently, caspase-8 and -3 activation are reduced in MAVS-deficient cells. Thus, after RNA virus infection MAVS does not only elicit a type I antiviral response but also recruits caspase-8 to mitochondria to mediate caspase-3 activation and apoptosis in a Bax/Bak-independent manner.

The ORF4b-encoded accessory proteins of Middle East respiratory syndrome coronavirus and two related bat coronaviruses localize to the nucleus and inhibit innate immune signalling

The recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV), a betacoronavirus, is associated with severe pneumonia and renal failure. The environmental origin of MERS-CoV is as yet unknown; however, its genome sequence is closely related to those of two bat coronaviruses, named BtCoV-HKU4 and BtCoV-HKU5, which were derived from Chinese bat samples. A hallmark of highly pathogenic respiratory viruses is their ability to evade the innate immune response of the host. CoV accessory proteins, for example those from severe acute respiratory syndrome CoV (SARS-CoV), have been shown to block innate antiviral signalling pathways. MERS-CoV, similar to SARS-CoV, has been shown to inhibit type I IFN induction in a variety of cell types in vitro. We therefore hypothesized that MERS-CoV and the phylogenetically related BtCoV-HKU4 and BtCoV-HKU5 may encode proteins with similar capabilities. In this study, we have demonstrated that the ORF4b-encoded accessory protein (p4b) of MERS-CoV, BtCoV-HKU4 and BtCoV-HKU5 may indeed facilitate innate immune evasion by inhibiting the type I IFN and NF-κB signalling pathways. We also analysed the subcellular localization of p4b from MERS-CoV, BtCoV-HKU4 and BtCoV-HKU5 and demonstrated that all are localized to the nucleus.

The expanding role of NLRs in antiviral immunity.

Nucleotide oligomerization and binding domain (NOD)-like receptors (NLRs) are a major constituent of the cytosolic innate immune-sensing machinery and participate in a wide array of pathways including nuclear factor κB (NF-κB), mitogen-activated protein kinase (MAPK), inflammasome, and type I interferon (IFN) signaling. NLRs have known roles in autoimmune, autoinflammatory, and infectious diseases. With respect to virus infection, NLRP3 is the most extensively studied NLR, including mechanisms of activation and inhibition. Furthermore, the importance of NLRP3 in both innate and adaptive immunity has been demonstrated. In comparison to NLRP3, the roles of other NLRs during virus infection are only just emerging. NLRC2 is an important activator of innate antiviral signaling and was recently found to mitigate inflammation during virus infection through autophagy. Finally, functions for NLRX1 in immune modulation and reactive oxygen species production require further examination and the importance of NLRC5 as a transactivator of major histocompatibility complex (MHC) class I and antigen presentation is currently developing. In this review, we discuss current knowledge pertaining to viruses and NLRs as well as areas of potential research, which will help advance the study of NLR biology during virus infection.

Negative Regulation of RIG-I-Mediated Innate Antiviral Signaling by SEC14L1

Retinoic acid-inducible gene I (RIG-I) is a key sensor for recognizing nucleic acids derived from RNA viruses and triggers beta interferon (IFN-β) production. Because of its important role in antiviral innate immunity, the activity of RIG-I must be tightly controlled. Here, we used yeast two-hybrid screening to identify a SEC14 family member, SEC14L1, as a RIG-I-associated negative regulator. Transfected SEC14L1 interacted with RIG-I, and endogenous SEC14L1 associated with RIG-I in a viral infection-inducible manner. Overexpression of SEC14L1 inhibited transcriptional activity of the IFN-β promoter induced by RIG-I but not TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3). Knockdown of endogenous SEC14L1 in both HEK293T cells and HT1080 cells potentiated RIG-I and Sendai virus-triggered IFN-β production as well as attenuated the replication of Newcastle disease virus. SEC14L1 interacted with the N-terminal domain of RIG-I (RIG-I caspase activation and recruitment domain [RIG-I-CARD]) and competed with VISA/MAVS/IPS-1/Cardif for RIG-I-CARD binding. Domain mapping further indicated that the PRELI-MSF1 and CRAL-TRIO domains but not the GOLD domain of SEC14L1 are required for interaction and inhibitory function. These findings suggest that SEC14L1 functions as a novel negative regulator of RIG-I-mediated antiviral signaling by preventing RIG-I interaction with the downstream effector.

RING Finger Protein 11 Targets TBK1/IKKi Kinases to Inhibit Antiviral Signaling

A key feature of the innate antiviral immune response is a rapid nonspecific response to virus infection largely mediated by the induction and extracellular secretion of type I interferons (IFNs) that restrict virus replication. Cytoplasmic sensors such as RIG-I recognize viral RNA and trigger antiviral signaling pathways that upregulate IFN transcription. However, it remains largely unknown how antiviral signaling is negatively regulated to maintain homeostasis after the elimination of virus. In this report, we have identified the RING domain-containing protein RING finger 11 (RNF11) as a novel negative regulator of innate antiviral signaling. Overexpression of RNF11 downregulated IFN-β expression and enhanced viral replication whereas siRNA-mediated knockdown of RNF11 suppressed viral replication. RNF11 interacted with the noncanonical IKK kinases TBK1/IKKi and attenuated their Lys63-linked polyubiquitination by blocking interactions with the E3 ligase TRAF3. The inhibitory function of RNF11 was dependent on the ubiquitin-binding adaptor molecule TAX1BP1 which was required for RNF11 to target TBK1/IKKi. Collectively, these results indicate that RNF11 functions together with TAX1BP1 to restrict antiviral signaling and IFN-β production.

The NLRP4-DTX4 axis: a key suppressor of TBK1 and innate antiviral signaling

The NLRP4-DTX4 axis: a key suppressor of TBK1 and innate antiviral signaling