ArticleLiterature Review

LGP2 synergy with MDA5 in RLR-mediated RNA recognition and antiviral signaling

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Abstract

Mammalian cells have the ability to recognize virus infection and mount a powerful antiviral response. Pattern recognition receptor proteins detect molecular signatures of virus infection and activate antiviral signaling. The RIG-I-like receptor (RLR) proteins are expressed in the cytoplasm of nearly all cells and specifically recognize virus-derived RNA species as a molecular feature discriminating the pathogen from the host. The RLR family is composed of three homologous proteins, RIG-I, MDA5, and LGP2. All RLRs have the ability to detect virus-derived dsRNA and hydrolyze ATP, but display individual differences in enzymatic activity, intrinsic ability to recognize RNA, and mechanisms of activation. Emerging evidence suggests that MDA5 and RIG-I utilize distinct mechanisms to form oligomeric complexes along dsRNA. Aligning of their signaling domains creates a platform capable of propagating and amplifying antiviral signaling responses. LGP2 with intact ATP hydrolysis is critical for the MDA5-mediated antiviral response, but LGP2 lacks the domains essential for activation of antiviral signaling, leaving the role of LGP2 in antiviral signaling unclear. Recent studies revealed a mechanistic basis of synergy between LGP2 and MDA5 leading to enhanced antiviral signaling. This review briefly summarizes the RLR system, and focuses on the relationship between LGP2 and MDA5, describing in detail how these two proteins work together to detect foreign RNA and generate a fully functional antiviral response. Copyright © 2015 Elsevier Ltd. All rights reserved.

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... Later on, using the LGP2 gene KO mice, the LGP2 was suggested to positively regulate RIG-I and MDA5 signaling (15,16). Further studies tried to reconcile the controversial regulatory roles of LGP2 by proposing that LGP2 is a concentration dependent biphasic switch: at low concentration when cells are in steady state, LGP2 enhances MDA5 activation to help fight infection, whereas at high concentration in late infection stages, LGP2 exerts a general inhibition to RIG-I and MDA5 signaling to resolve the inflammation (17,18). Nevertheless, the exact role of LGP2 in the regulation of RIG-I/MDA5 and in antiviral signaling still remains an open question (8,19). ...
... Importantly, we found porcine LGP2 positively regulates porcine MDA5 as well as RIG-I activity. Positive regulation of MDA5 activation by physiological low concentration of LGP2 was widely accepted (15,45,46) and the biochemical and molecular mechanism recently became clear (17,18,43,47): MDA5 binds dsRNA weakly and the filament formation on long dsRNA chain is slow and unstable. On the contrary, LGP2 binds dsRNA with the highest affinity among three RLRs, regardless of 5'-PPP and RNA length, but not forming filaments (18,40,48). ...
... Positive regulation of MDA5 activation by physiological low concentration of LGP2 was widely accepted (15,45,46) and the biochemical and molecular mechanism recently became clear (17,18,43,47): MDA5 binds dsRNA weakly and the filament formation on long dsRNA chain is slow and unstable. On the contrary, LGP2 binds dsRNA with the highest affinity among three RLRs, regardless of 5'-PPP and RNA length, but not forming filaments (18,40,48). By utilizing its ATP hydrolysis activity, LGP2 is able to not only increase initial binding of dsRNA by MDA5, but also stabilize the shorter MDA5 filaments on dsRNA which are signaling active, thus promoting MDA5 activity. ...
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The RLRs play critical roles in sensing and fighting viral infections especially RNA virus infections. Despite the extensive studies on RLRs in humans and mice, there is a lack of systemic investigation of livestock animal RLRs. In this study, we characterized the porcine RLR members RIG-I, MDA5 and LGP2. Compared with their human counterparts, porcine RIG-I and MDA5 exhibited similar signaling activity to distinct dsRNA and viruses, via similar and cooperative recognitions. Porcine LGP2, without signaling activity, was found to positively regulate porcine RIG-I and MDA5 in transfected porcine alveolar macrophages (PAMs), gene knockout PAMs and PK-15 cells. Mechanistically, LGP2 interacts with RIG-I and MDA5 upon cell activation, and promotes the binding of dsRNA ligand by MDA5 as well as RIG-I. Accordingly, porcine LGP2 exerted broad antiviral functions. Intriguingly, we found that porcine LGP2 mutants with defects in ATPase and/or dsRNA binding present constitutive activity which are likely through RIG-I and MDA5. Our work provided significant insights into porcine innate immunity, species specificity and immune biology.
... Instead, MDA5 rather shows an open and flexible and thus uninhibited conformation. This entails downstream signaling upon overexpression of MDA5 in the absence of an RNA ligand [26][27][28]. Due to the lack of CARDs LGP2 cannot directly initiate downstream signaling via MAVS. But it seems to function as modulator of MDA5 signaling. ...
... But it seems to function as modulator of MDA5 signaling. At low protein levels, LGP2 accelerates and stabilizes MDA5-RNA interaction, whereas high levels of LGP2 lead to MDA5 inhibition [27,29,30]. ...
Article
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Cells have developed different strategies to cope with viral infections. Key to initiating a defense response against viruses is the ability to distinguish foreign molecules from their own. One central mechanism is the perception of foreign nucleic acids by host proteins which, in turn, initiate an efficient immune response. Nucleic acid sensing pattern recognition receptors have evolved, each targeting specific features to discriminate viral from host RNA. These are complemented by several RNA-binding proteins that assist in sensing of foreign RNAs. There is increasing evidence that the interferon-inducible ADP-ribosyltransferases (ARTs; PARP9—PARP15) contribute to immune defense and attenuation of viruses. However, their activation, subsequent targets, and precise mechanisms of interference with viruses and their propagation are still largely unknown. Best known for its antiviral activities and its role as RNA sensor is PARP13. In addition, PARP9 has been recently described as sensor for viral RNA. Here we will discuss recent findings suggesting that some PARPs function in antiviral innate immunity. We expand on these findings and integrate this information into a concept that outlines how the different PARPs might function as sensors of foreign RNA. We speculate about possible consequences of RNA binding with regard to the catalytic activities of PARPs, substrate specificity and signaling, which together result in antiviral activities.
... Despite its lack of signaling domains, LGP2 is consistently observed to play an important role in modulating signaling by MDA5 and RIG-I. 14,17,153,197,198,199,200 Crystal structures of chicken LGP2 with dsRNA show that the helicase domain of LGP2 resembles that of MDA5, although the CTD of LGP2 caps the ends of dsRNA in a fashion similar to that of RIG-I. 38,192,193 LGP2 displays high RNA binding affinity and the isolated CTD of LGP2 has a comparatively higher RNA binding affinity than the CTD of RIG-I. ...
... 103,195,204 LGP2 may facilitate MDA5/RNA interactions or regulate MDA5 filament assembly, presumably through heterocomplex formation, potentially resulting in alternative MDA5 filaments that are somehow active for signaling. 195,197,200,203 Taken together, the functional diversity of RLRs may be linked to their inherent differences in RNA recognition, binding, and signaling mechanism. ...
Article
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RIG‐I is our first line of defense against RNA viruses, serving as a pattern recognition receptor that identifies molecular features common among dsRNA and ssRNA viral pathogens. RIG‐I is maintained in an inactive conformation as it samples the cellular space for pathogenic RNAs. Upon encounter with the triphosphorylated terminus of blunt‐ended viral RNA duplexes, the receptor changes conformation and releases a pair of signaling domains (CARDs) that are selectively modified and interact with an adapter protein (MAVS), thereby triggering a signaling cascade that stimulates transcription of interferons. Here, we describe the structural determinants for specific RIG‐I activation by viral RNA, and we describe the strategies by which RIG‐I remains inactivated in the presence of host RNAs. From the initial RNA triggering event to the final stages of interferon expression, we describe the experimental evidence underpinning our working knowledge of RIG‐I signaling. We draw parallels with behavior of related proteins MDA5 and LGP2, describing evolutionary implications of their collective surveillance of the cell. We conclude by describing the cell biology and immunological investigations that will be needed to accurately describe the role of RIG‐I in innate immunity and to provide the necessary foundation for pharmacological manipulation of this important receptor.
... On the other hand, MDA5 mainly recognizes positive-stranded RNA viruses, such as picornaviruses and arteriviruses [40]. Despite the fact that LGP2 does not contain the N-terminal CARD, it still can bind varied dsRNAs irrespective of 5 -PPP or RNA length [47]. ...
... LGP2 does not possess signaling CARD domains and its role in antiviral response induction is unclear. Strong activation of LGP2 expression occurs upon viral infection, poly(I:C) stimulation and IFNα treatment [47]. It is intriguing that in various experiments, LGP2 receptor has been shown to function as a positive or negative regulator of RLR-mediated signaling. ...
Article
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Recognition of the non-self signature of invading pathogens is a crucial step for the initiation of the innate immune mechanisms of the host. The host response to viral and bacterial infection involves sets of pattern recognition receptors (PRRs), which bind evolutionarily conserved pathogen structures, known as pathogen-associated molecular patterns (PAMPs). Recent advances in the identification of different types of PRRs in teleost fish revealed a number of cytosolic sensors for recognition of viral and bacterial nucleic acids. These are DExD/H-box RNA helicases including a group of well-characterized retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) and non-RLR DExD/H-box RNA helicases (e.g., DDX1, DDX3, DHX9, DDX21, DHX36 and DDX41) both involved in recognition of viral RNAs. Another group of PRRs includes cytosolic DNA sensors (CDSs), such as cGAS and LSm14A involved in recognition of viral and intracellular bacterial dsDNAs. Moreover, dsRNA-sensing protein kinase R (PKR), which has a role in antiviral immune responses in higher vertebrates, has been identified in fish. Additionally, fish possess a novel PKR-like protein kinase containing Z-DNA binding domain, known as PKZ. Here, we review the current knowledge concerning cytosolic sensors for recognition of viral and bacterial nucleic acids in teleosts.
... The DDX58 (DExD/H-box helicase 58) gene encodes a cytosolic receptor called RIG-I (retinoic acid-inducible gene I) and belongs to the RIG-I-like receptor (RLR) family (1). DDX58, MDA5 (melanoma differentiation Factor 5) and LGP2 (laboratory of genetics and physiology 2) are three central members of the RLR family (2). ...
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Introduction Triple-negative breast cancer (TNBC) is characterized by its aggressive nature and absence of specific therapeutic targets, necessitating the reliance on chemotherapy as the primary treatment modality. However, the drug resistance poses a significant challenge in the management of TNBC. In this study, we investigated the role of DDX58 (DExD/H-box helicase 58), also known as RIG-I, in TNBC chemoresistance. Methods The relationship between DDX58 expression and breast cancer prognosis was investigated by online clinical databases and confirmed by immunohistochemistry analysis. DDX58 was knockout by CRISPR-Cas9 system (DDX58-KO), knockdown by DDX58-siRNA (DDX58-KD), and stably over expressed (DDX58-OE) by lentivirus. Western blotting, immunofluorescence and qPCR were used for related molecules detection. Apoptosis was analyzed through flow cytometry (Annexin V/7AAD apoptosis assay) and Caspase 3/7 activity assay. Results Patients with lower expression of DDX58 led to lower rate of pathological complete response (pCR) and worse prognosis by online databases and hospital clinical data. DDX58-KD cells showed multiple chemo-drugs resistance (paclitaxel, doxorubicin, 5-fluorouracil) in TNBC cell lines. Similarly, DDX58-KO cells also showed multiple chemo-drugs resistance in a dosage-dependent manner. In the CDX model, tumours in the DDX58-KO group had a 25% reduction in the tumour growth inhibition rate (IR) compared to wild-type (WT) group after doxorubicin (Dox) treatment. The depletion of DDX58 inhibited proliferation and promoted the migration and invasion in MDA-MB-231 cells. The findings of our research indicated that DDX58-KO cells exhibit a reduction in Dox-induced apoptosis both in vivo and in vitro. Mechanistically, Dox treatment leads to a significant increase in the expression of double-stranded RNAs (dsRNAs) and activates the DDX58-Type I interferon (IFN) signaling pathway, ultimately promoting apoptosis in TNBC cells. Discussion In the process of TNBC chemotherapy, the deficiency of DDX58 can inhibit Dox-induced apoptosis, revealing a new pathway of chemotherapy resistance, and providing a possibility for developing personalized treatment strategies based on DDX58 expression levels.
... Activated RIG-I and MDA5 interact with mitochondrial antiviral-signaling protein (MAVS); when stimulated by MDA5 or RIG-I CARD, MAVS oligomerizes and further recruits multiple tumor necrosis factor (TNF) receptor-associated factor (TRAF) proteins (such as TRAF2, TRAF3, and TRAF6), which are necessary for the activation of TANKbinding kinase 1 (TBK1) and inhibitor of nuclear factor kappa-B kinase ε (IKKε). Ultimately, these kinases phosphorylate interferon regulatory factor 3/7 (IRF3/7), which in turn leads to the production of IFN-I and interferon-stimulated genes (ISGs) [66][67][68][69]. ...
Article
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Immune signal transduction is crucial to the body’s defense against viral infection. Recognition of pathogen-associated molecular patterns by pattern recognition receptors (PRRs) activates the transcription of interferon regulators and nuclear factor-κB (NF-κB); this promotes the release of interferons and inflammatory factors. Efficient regulation of type I interferon and NF-κB signaling by members of the mitogen-activated protein (MAP) kinase kinase kinase (MAP3K) family plays an important role in antiviral immunity. Elucidating the specific roles of MAP3K activation during viral infection is essential to develop effective antiviral therapies. In this review, we outline the specific regulatory mechanisms of MAP3Ks in antiviral immunity and discuss the feasibility of targeting MAP3Ks for the treatment of virus-induced diseases.
... Although LGP2 itself is inactive in signaling, it has a stronger ability to bind with dsRNA and enables to regulate RIG-I and MDA5 activity [8]. In spite of the controversial role of LGP2 in the regulation of RIG-I and MDA5 signaling [5,[9][10][11], our recent work showed that porcine LGP2 is able to positively regulate RIG-I and MDA5 signaling by promoting dsRNA binding and exerts an antiviral role against several porcine viruses [12]. ...
Article
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The RIG-I-like receptors (RLRs) play critical roles in sensing and combating viral infections, particularly RNA virus infections. However, there is a dearth of research on livestock RLRs due to a lack of specific antibodies. In this study, we purified porcine RLR proteins and developed monoclonal antibodies (mAbs) against porcine RLR members RIG-I, MDA5 and LGP2, for which one, one and two hybridomas were obtained, respectively. The porcine RIG-I and MDA5 mAbs each targeted the regions beyond the N-terminal CARDs domains, whereas the two LGP2 mAbs were both directed to the N-terminal helicase ATP binding domain in the Western blotting. In addition, all of the porcine RLR mAbs recognized the corresponding cytoplasmic RLR proteins in the immunofluorescence and immunochemistry assays. Importantly, both RIG-I and MDA5 mAbs are porcine specific, without demonstrating any cross-reactions with the human counterparts. As for the two LGP2 mAbs, one is porcine specific, whereas another one reacts with both porcine and human LGP2. Thus, our study not only provides useful tools for porcine RLR antiviral signaling research, but also reveals the porcine species specificity, giving significant insights into porcine innate immunity and immune biology.
... Chronic high-fat diet downregulated the protein expression of CSAD, ST3GAL5, and LPIN2 by about 4-fold, 6-fold, and 10-fold, respectively. In addition, DHX58 is a RIG-I-like receptor involved in cytoplasmic RNA recognition and antiviral signaling (Bruns & Horvath, 2015). DHX58 protein expression is more than 3-fold upregulated by a high-fat diet. ...
Article
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Abstract Non-alcoholic fatty liver disease (NAFLD) is now considered to be the most common liver disease worldwide, caused by fat deposition in hepatocytes. High-fat diet is considered to be a major lifestyle factor predisposing to NAFLD. However, the effect of different cycles of high-fat diets on changes in NAFLD-related gene and protein expression is unclear. In this study, NAFLD mouse models were established by feeding C57BL/6 male mice a high-fat diet for 16 and 38 weeks. The transcriptome and proteome of mouse liver were profiled by RNA sequencing and high-resolution mass spectrometry, respectively. The results show that accumulation of liver lipids was observed at 38 weeks of treatment on a high-fat diet. At the same time, the expression profiles of 1329 genes and 802 proteins involved in NAFLD were changed, with a total of 234 genes and 37 proteins significantly changing by more than twice. These differentially expressed genes and proteins were significantly enriched in fatty acid metabolism and organic acid biosynthesis. 18 genes and their corresponding protein overlaps were identified using Venn diagrams, and most of them were regulated by high-fat diet in an aging-dependent manner. All in all, our study is valuable for understanding the high-fat diet on the developmental process of NAFLD.
... Fixation sur l'ARN afin d'inhiber sa traduction par compétition Abbas et al., 2013;Pichlmair et al., 2011 3. Les récepteurs qui activent la réponse interféron Les récepteurs induisant une réponse antivirale lors de la reconnaissance d'ARNdb appartiennent à la famille des récepteurs de reconnaissance de motifs moléculaires (pattern (Civril et al., 2013;Hornung et al., 2009 Goubau et al., 2014;Marq et al., 2011;Poeck et al., 2010 MDA5/LGP2 ARNdb Bruns & Horvath, 2015;Pichlmair et al., 2009;del Toro Duany et al., 2015 Ces récepteurs ont tous pour finalité l'activation d'une réponse antivirale consistant en la production d'interféron, de cytokines pro-inflammatoires et la stimulation de gènes induits par l'interféron (IFN-stimulated genes -ISG) ( Figure 9). ...
Thesis
Tous les organismes sont en permanence soumis à des attaques par des agents pathogènes dont le matériel génétique est perçu comme étranger par la cellule. Pour se protéger de ces dangers potentiels, différents mécanismes inhibiteurs permettant de percevoir et d’agir directement sur cette information génétique exogène ont été mis en place au cours de l’évolution dans les différents règnes du vivant. L’interférence par ARN et la réponse interféron appartiennent toutes deux à ces mécanismes de défense. Les virus, parasites intracellulaires obligatoires, sont l’une des sources majeures de matériel génétique étranger et produisent la principale forme d’acide nucléique reconnue par l’interférence par ARN et la réponse interféron : l’ARN double brin. Mon projet a pour vocation l’identification et à la caractérisation des différents facteurs pouvant être impliqués dans la réponse cellulaire à l’ARN double brin dans des cellules somatiques de mammifères.
... It has been reported that porcine LGP2 positively regulated porcine RIG-I and MDA5 in activated PAMs by interacting with RIG-I and MDA5, promoting dsRNA binding to MDA5 as well as RIG-I (35). It was demonstrated that LGP2 functioned in viral sensing through cooperation with MDA5 and subsequent MDA5 antiviral innate immune responses in response to many viruses, such as Tembusu virus, MVA, and avian influenza viruses (26,(36)(37)(38). In our study, we are interested in the preference of LGP2 for the interaction with RIG-I or MDA5 following PRRSV infection. ...
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LGP2, a member of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), shows higher-affinity binding to RNA and work synergism with RIG-I or MDA5. However, LGP2 has divergent responses to different viruses, which remains controversial in antiviral immune responses. Here, we present the detailed process of LGP2 in positively regulating the anti-PRRSV response. Upon PRRSV infection, LGP2 was prone to bind to MDA5 and enhanced MDA5 signaling, manifesting the enrichment of viral RNA on MDA5 and the activation of downstream IRF3 and NF-κB, which results in increased proinflammatory cytokines and type I interferon expression, ultimately inhibiting PRRSV at the early stage of infection. Moreover, PRRSV Nsp1 and Nsp2 interacted with LGP2 via ubiquitin-proteasome pathways, thus blocking LGP2-mediated immune response. This research helps us understand the host recognition and innate antiviral response to PRRSV infection by neglected pattern recognition receptors, which sheds light on the detailed mechanism of virus-host interaction.
... This interaction is also present in two other K63-linked E3s (TRAF6 and RNF125) to negatively regulate innate immune signaling [24,25]. However, accumulating evidence has shown that LGP2 can positively regulate MDA5-mediated antiviral signaling by synergistically sensing viral dsRNA [26,27]. However, it is unclear whether LGP2 affects the activation of MDA5 during the ubiquitylation process. ...
Article
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The outbreaks caused by RNA and DNA viruses, such as SARS-CoV-2 and monkeypox, pose serious threats to human health. The RLR and cGAS-STING pathways contain major cytoplasmic sensors and signaling transduction axes for host innate antiviral immunity. In physiological and virus-induced pathological states, the activation and inactivation of these signal axes are tightly controlled, especially post-translational modifications (PTMs). E3 ubiquitin ligases (E3s) are the direct manipulator of ubiquitin codons and determine the type and modification type of substrate proteins. Therefore, members of the E3s family are involved in balancing the host’s innate antiviral immune responses, and their functions have been extensively studied over recent decades. In this study, we overviewed the mechanisms of different members of three E3s families that mediate the RLR and cGAS-STING axes and analyzed them as potential molecular targets for the prevention and treatment of virus-related diseases.
... Furthermore, 3A has recently been shown to play a critical role in FMDV replication by interacting with swine annexin A1 (ANXA1) [78]. This member of the annexin family is known to be an important modulator of the immune response and particularly of the IFN pathway, since it is able to interact with MAVS, NEMO, IRF3 or TBK1 [162][163][164]. ...
Article
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Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals with a significant socioeconomic impact. One of the issues related to this disease is the ability of its etiological agent, foot-and-mouth disease virus (FMDV), to persist in the organism of its hosts via underlying mechanisms that remain to be elucidated. The establishment of a virus–host equilibrium via protein–protein interactions could contribute to explaining these phenomena. FMDV has indeed developed numerous strategies to evade the immune response, especially the type I interferon response. Viral proteins target this innate antiviral response at different levels, ranging from blocking the detection of viral RNAs to inhibiting the expression of ISGs. The large diversity of impacts of these interactions must be considered in the light of the in vitro models that have been used to demonstrate them, some being sometimes far from biological systems. In this review, we have therefore listed the interactions between FMDV and the interferon response as exhaustively as possible, focusing on both their biological effect and the study models used.
... MAVS is embedded in the mitochondrial-associated membranes (MAMs) and in oxidative organelles (peroxisomes) (27). Canonically, MAVS forms a scaffold at the MAM with TNFR-associated factor 2 (TRAF2), TRAF5, and TRAF6 to activate the inhibitor of IkB kinase (IKK) family and TBK1 (32). The inhibitor of IKK family kinases and TBK1 phosphorylate NF-kB, IRF3, and IRF7, which allows for their translocation to the nucleus and transcription of IFN, antiviral genes, proapoptotic genes, and immune cell programming genes (27). ...
Article
Retinoic acid-inducible gene I-like receptors (RLRs) are cytosolic RNA sensors critical for initiation of antiviral immunity. Activation of RLRs following RNA recognition leads to production of antiviral genes and IFNs for induction of broad antiviral immunity. Although the RLRs are ubiquitously expressed, much of our understanding of these molecules comes from their study in epithelial cells and fibroblasts. However, RLR activation is critical for induction of immune function and long-term protective immunity. Recent work has focused on the roles of RLRs in immune cells and their contribution to programming of effective immune responses. This new understanding of RLR function in immune cells and immune programming has led to the development of vaccines and therapeutics targeting the RLRs. This review covers recent advances in our understanding of the contribution of RLRs to immune cell function during infection and the emerging RLR-targeting strategies for induction of immunity against cancer and viral infection.
... These results also suggest that the LGP2's stimulatory pattern in mammalian cells under low concentrations of poly(I:C) is indeed similar to that in fish cells by titration of LGP2 alone without poly(I:C) ( Figure 5F). That is, if the stimulatory potential of ectopically-expressed LGP2s in fish cells requires pre-conjugation to trace dsRNA produced by plasmid transfection, as does human LGP2 in mammalian cells (15)(16)(17)47), it is easy to understand that fish and human LGP2s actually display a same regulatory effect on stimulating IFN response, in fish cells by overexpression alone, and in mammalian cells by combined transfection of poly(I:C) at low concentrations ( Figure 5). ...
Article
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Retinoic acid inducible gene-I (RIG-I)-like receptors (RLRs) are viral RNA sensors that regulate host interferon (IFN)-mediated antiviral signaling. LGP2 (laboratory genetics and physiology 2) lacks the N-terminal caspase activation and recruitment domains (CARDs) responsible for signaling transduction in the other two RLR proteins, RIG-I and melanoma differentiation associated gene-5 (MDA5). How LGP2 regulates IFN signaling is controversial, and inconsistent results have often been obtained in overexpression assays when performed in fish cells and mammalian cells. Here we report that the differential sensitivity of fish cells and mammalian cells to poly(I:C) transfection conceals the function conservation of zebrafish and human LGP2. In fish cells, overexpression of zebrafish or human LGP2 initially activates IFN signaling in a dose-dependent manner, followed by inhibition at a critical threshold of LGP2 expression. A similar trend exists for LGP2-dependent IFN induction in response to stimulation by low and high concentrations of poly(I:C). In contrast, overexpression of zebrafish or human LGP2 alone in mammalian cells does not activate IFN signaling, but co-stimulation with very low or very high concentrations of poly(I:C) shows LGP2-dependent enhancement or inhibition of IFN signaling, respectively. Titration assays show that LGP2 promotes MDA5 signaling in mammalian cells mainly under low concentration of poly(I:C) and inhibits RIG-I/MDA5 signaling mainly under high concentration of poly(I:C). Our results suggest that fish and human LGP2s switch regulatory roles from a positive one to a negative one in increasing concentrations of poly(I:C)-triggered IFN response.
... After secreted in an autocrine and paracrine manner, type I IFNs specifically bind to the IFN-α/β receptor (IFNAR), which activates the intracellular Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, especially STAT1 and STAT2, causing downstream signal transduction [83] [84]. Once phosphorylated, STAT1 and STAT2 rapidly bind to IRF9 and constitute the interferon-stimulated gene factor 3 (ISGF3) transcriptional complex 11. ...
Article
Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), which are pivotal sensors of RNA virus invasions, mediate the transcriptional induction of genes encoding type I interferons (IFNs) and proinflammatory cytokines, successfully establishing host antiviral immune response. A few excellent reviews have elaborated on the structural biology of RLRs and the antiviral mechanisms of RLR activation. In this review, we give a basic understanding of RLR biology and summarize recent findings of how RLR signaling cascade is strictly controlled by host regulatory mechanisms, which include RLR-interacting proteins, post-translational modifications and microRNAs (miRNAs). Furthermore, we pay particular attention to the relationship between RLRs and diseases, especially how RLRs participate in SARS-CoV-2, malaria or bacterial infections, how single-nucleotide polymorphisms (SNPs) or mutations in RLRs and antibodies against RLRs lead to autoinflammatory diseases and autoimmune diseases, and how RLRs are involved in anti-tumor immunity. These findings will provide insights and guidance for antiviral and immunomodulatory therapies targeting RLRs.
... Retinoic acid-inducible gene I-like receptors (RLRs) family is composed of retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5) and LGP2. RLR are HIV-1 (128) cytoplasmic sensors expressed in nearly all cell types that upon virus-derived RNA binding, interact with the mitochondrial antiviral signaling protein or MAVS (also known as IPS-1) to induce an antiviral environment (see (137,138) for review). RLRs can be activated by a broad spectrum of viruses including ssRNA(+) Flavivirirus (WNV; HVC; Zika virus) or Coronavirus (SARS coronavirus); ssRNA(-) Filovirus (EBOV, Marburg virus); ssRNA (RT) retrovirus (HIV) but also viral RNA derived from dsDNA viruses such as Herpervirus (HSV-1, Epstein-Barr) (138) Hence, there is an increasing interest to explore RIG-I activation as a therapeutic approach. ...
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The development of antiviral drugs, has provided enormous achievements in our recent history in the fight against viral infections. To date, most of the approved antiviral drugs target virus-encoded proteins to achieve direct antiviral activity. Nonetheless, the inherent idiosyncrasy of viral mutations during their replication cycle, enable many viruses to adapt to the new barriers, becoming resistant to therapies, therefore, representing an ever-present menace and prompting the scientific community towards the development of novel therapeutic strategies. Taking advantage of the increasing knowledge of virus-host cell interactions, the targeting of cellular factors or pathways essential for virus survival turns into an alternative strategy to intervene in almost every step of viral replication cycle. Since host factors are evolutionary conserved, viral evasion to host-directed therapies (HDT) would impose a higher genetic barrier to the emergence of resistant strains. Thus, targeting host factors has long been considered an alternative strategy to overcome viral resistance. Nevertheless, targeting host factors or pathways potentially hints undesired off targets effects, and therefore, a critical risk-benefit evaluation is required. The present review discusses the current state-of-the-art on the identification of viral host dependency factors (HDF) and the workflow required for the development of HDT as antivirals. Then, we focus on the feasibility of using a specific class of host factors, those involved in innate immune modulation, as broad-spectrum antiviral therapeutic strategies. Finally, a brief summary of major roadblocks derived from targeting host cellular proteins and putative future strategies to overcome its major limitations is proposed.
... However, as it lacks a CARD, LGP2 negatively regulates RIG-I-mediated recognition of viral dsRNA, reduces the production of IFNs and inflammatory factors, and ultimately inhibits the antiviral innate immune response [130]. However, LGP2 facilitates the antiviral response mediated by MDA5 [131][132][133]. ...
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The TANK-binding kinase 1 (TBK1) is a serine/threonine kinase belonging to the non-canonical inhibitor of nuclear factor-κB (IκB) kinase (IKK) family. TBK1 can be activated by pathogen-associated molecular patterns (PAMPs), inflammatory cytokines, and oncogenic kinases, including activated K-RAS/N-RAS mutants. TBK1 primarily mediates IRF3/7 activation and NF-κB signaling to regulate inflammatory cytokine production and the activation of innate immunity. TBK1 is also involved in the regulation of several other cellular activities, including autophagy, mitochondrial metabolism, and cellular proliferation. Although TBK1 mutations have not been reported in human cancers, aberrant TBK1 activation has been implicated in the oncogenesis of several types of cancer, including leukemia and solid tumors with KRAS -activating mutations. As such, TBK1 has been proposed to be a feasible target for pharmacological treatment of these types of cancer. Studies suggest that TBK1 inhibition suppresses cancer development not only by directly suppressing the proliferation and survival of cancer cells but also by activating antitumor T-cell immunity. Several small molecule inhibitors of TBK1 have been identified and interrogated. However, to this point, only momelotinib (MMB)/CYT387 has been evaluated as a cancer therapy in clinical trials, while amlexanox (AMX) has been evaluated clinically for treatment of type II diabetes, nonalcoholic fatty liver disease, and obesity. In this review, we summarize advances in research into TBK1 signaling pathways and regulation, as well as recent studies on TBK1 in cancer pathogenesis. We also discuss the potential molecular mechanisms of targeting TBK1 for cancer treatment. We hope that our effort can help to stimulate the development of novel strategies for targeting TBK1 signaling in future approaches to cancer therapy.
... In some cases, the RLR-mediated IFN induction is reduced in the cells overexpressed with LGP2 protein. In contrast, in response to various RNA virus infections, the production of IFNβ is dramatically decreased in mice lacking LGP2, suggesting a positive role in RLR-dependent signaling (Bruns and Horvath, 2015). Consistent with MDA5, LGP2 is also targeted by V proteins of PIV5, HPIV2, MuV, SeV, NiV, and HeV. ...
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For efficient replication, viruses have developed multiple strategies to evade host antiviral innate immunity. Paramyxoviruses are a large family of enveloped RNA viruses that comprises diverse human and animal pathogens which jeopardize global public health and the economy. The accessory proteins expressed from the P gene by RNA editing or overlapping open reading frames (ORFs) are major viral immune evasion factors antagonizing type I interferon (IFN-I) production and other antiviral innate immune responses. However, the antagonistic mechanisms against antiviral innate immunity by accessory proteins differ among viruses. Here, we summarize the current understandings of immune evasion mechanisms by paramyxovirus accessory proteins, specifically how accessory proteins directly or indirectly target the adaptors in the antiviral innate immune signaling pathway to facilitate virus replication. Additionally, some cellular responses, which are also involved in viral replication, will be briefly summarized.
... From Bowzard et al. 2011 While LPG2 is considered as an RLR and displays the highest RNA binding affinity among them, the absence of a CARD domain prevents it from activating downstream antiviral signal (Wu and Chen 2014). It has been suggested it acts as a regulator of RIG-I and MDA5 signaling, specifically of MDA5 sensing (Bruns and Horvath 2015). ...
Thesis
The identification of cellular proteins that interfere with virus replication is a key challenge in virology. Amongst them, finding those engaged in long-term virus-host interaction and co-evolution is of particular interest. In the host, such selective pressures induce diverse genetic innovations, such as site-specific positive selection, gene copy number variation, recombination, etc. Under the hypothesis that genetic innovations in innate immunity may particularly occur in viral interacting proteins, we developed a pipeline for retrieving orthologous sequences, aligning them and reconstructing their phylogeny, followed by the detection of genetic innovations. This streamlined procedure uniquely allows for the detection of paralogous genes, recombination breakpoints, and signatures of positive selection with several widely-used methods. We validated this evolutionary and predictive pipeline on genes with known selection profiles. Furthermore, we screened two datasets of candidate genes. The first one was composed of 56 genes which knock-downs impact the interferon response to viral infection. The second one was composed of 60 genes upregulated in macrophages resistant to HIV infection. We found numerous genes presenting important marks of genetic conflict, thus potentially encoding for novel Viral Interacting Proteins. Two of these candidates are undergoing functional characterization for their role in the HIV replicative cycle, and others are pending further investigation. Overall, we designed a complete and highly-flexible pipeline, available to the public, that can screen large datasets and allow researchers to rank candidate genes in order to prioritize their wet-lab experiments.
... The RLRs group has three members: melanoma differentiation-associated protein-5 (MDA5), retinoic acid-inducible gene-I (RIG-I), and laboratory of genetics and physiology-2 (LGP2) (Yoneyama et al., 2015;Lee et al., 2019b). MDA5 and RIG-I are equipped with two structural motifs, caspase activation and recruitment domains (CARDs) that promote downstream signaling, whereas LGP2 lacks CARD domains and thus downstream signaling, instead it enhances MDA5's function (Bruns et al., 2014;Tan et al., 2018;Bruns and Horvath, 2015). Upon sensing viral RNA, RLHs transmit the signal to mitochondrial antiviral-signaling protein (MAVS) via CARD-CARD domains interactions (Jiang, 2018). ...
Article
DEAD/H-box RNA helicases make the prominent family of helicases super family-2 which take part in almost all RNA-related processes, from initiation of transcription to RNA decay pathways. In addition to these RNA-related activities, in recent years a certain number of these helicases are reported to play important roles in anti-viral immunity through various ways. Along with RLHs, endosomal TLRs, and cytosolic DNA receptors, many RNA helicases including DDX3, DHX9, DDX6, DDX41, DHX33, DDX60, DHX36 and DDX1-DDX21-DHX36 complex act as viral nucleic acid sensors or co-sensors. These helicases mostly follow RLHs-MAVS and STING mediated signaling cascades to trigger induction of type-I interferons and pro-inflammatory cytokines. Many of them also function as downstream adaptor molecules (DDX3), segments of stress and processing bodies (DDX3 and DDX6) or negative regulators (DDX19, DDX24, DDX25, DDX39A and DDX46). On the contrary, many studies indicated that several DEAD/H-box helicases such as DDX1, DDX3, DDX6, DDX24, and DHX9 could be exploited by viruses to evade innate immune responses, suggesting that these helicases seem to have a dual function as anti-viral innate immune mediators and viral replication cofactors. In this review, we summarized the current knowledge on several representative DEAD/H-box helicases, with an emphasis on their functions in innate immunity responses, involved in their anti-viral and pro-viral roles.
... LGP2 plays a critical role in regulating MDA5-and RIG-I-mediated antiviral responses [40,41]. DDX39A was identified as a binding partner of LGP2 by a yeast two-hybrid system screen and further confirmed by coimmunoprecipitation assay. ...
Article
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DExD/H-box helicases play critical roles in multiple cellular processes, including transcription, cellular RNA metabolism, translation, and infections. Several seminal studies over the past decades have delineated the distinct functions of DExD/H-box helicases in regulating antiviral innate immune signaling pathways, including Toll-like receptors, retinoic acid-inducible gene I-like receptors, cyclic GMP-AMP synthase-the stimulator of interferon gene, and NOD-like receptors signaling pathways. Besides the prominent regulatory roles, there is increasing attention on their functions as nucleic acid sensors involved in antiviral innate immunity. Here we summarize the complex regulatory roles of DExD/H-box helicases in antiviral innate immunity. A better understanding of the underlying molecular mechanisms of DExD/H-box helicases’ regulatory roles is vital for developing new therapeutics targeting DExD/H-box helicases and their mediated signaling transduction in viral infectious diseases.
... Both of these domains are able to detect and bind RNA; moreover, they both possess caspase activation and recruitment domains (CARDs), which mediate signal transduction and lead to type I IFN gene expression [43]. LGP2 lacks CARDs, thus it initiates signaling by regulating RIG-I and MDA5 [44]. The regulatory C-terminal domain of LGP2 binds to the dsRNA binding protein PACT, and this complex inhibits the RIG-I-dependent response and activates the MDA5-dependent response. ...
Article
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Nucleic acid-based therapeutics have gained increased attention during recent decades because of their wide range of application prospects. Immunostimulatory nucleic acids represent a promising class of potential drugs for the treatment of tumoral and viral diseases due to their low toxicity and stimulation of the body's own innate immunity by acting on the natural mechanisms of its activation. The repertoire of nucleic acids that directly interact with the components of the immune system is expanding with the improvement of both analytical methods and methods for the synthesis of nucleic acids and their derivatives. Despite the obvious progress in this area, the problem of delivering therapeutic acids to target cells as well as the unresolved issue of achieving a specific therapeutic effect based on activating the mechanism of interferon and anti-inflammatory cytokine synthesis. Minimizing the undesirable effects of excessive secretion of inflammatory cytokines remains an unsolved task. This review examines recent data on the types of immunostimulatory nucleic acids, the receptors interacting with them, and the mechanisms of immunity activation under the action of these molecules. Finally, data on immunostimulatory nucleic acids in ongoing and completed clinical trials will be summarized.
... Unlike the previous two RLRs, LGP2 lacks the caspase recruitment domains (CARDs) necessary for downstream signaling. Instead, recent data suggest that it has a synergistic effect with MDA5 [35,36] (Figure 1A). Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) make up another family of cytoplasmic PRRs involved in viral recognition, with one of their most well-known roles being inflammasome activation. ...
Article
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The ability of dendritic cells (DCs) to sense viral pathogens and orchestrate a proper immune response makes them one of the key players in antiviral immunity. Different DC subsets have complementing functions during viral infections, some specialize in antigen presentation and cross-presentation and others in the production of cytokines with antiviral activity, such as type I interferons. In this review, we summarize the latest updates concerning the role of DCs in viral infections, with particular focus on the complex interplay between DC subsets and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Despite being initiated by a vast array of immune receptors, DC-mediated antiviral responses often converge towards the same endpoint, that is the production of proinflammatory cytokines and the activation of an adaptive immune response. Nonetheless, the inherent migratory properties of DCs make them a double-edged sword and often viral recognition by DCs results in further viral dissemination. Here we illustrate these various aspects of the antiviral functions of DCs and also provide a brief overview of novel antiviral vaccination strategies based on DCs targeting.
... 124 LGP2 is also critical in the antiviral response mediated by MDA5. 125 LGP2 exhibits a concentration-dependent conversion between MDA5specific enhancement and interference. 126 The latest research revealed a mechanistic basis for LGP2-mediated regulation of MDA5 antiviral innate immune responses. ...
Article
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Pattern recognition receptors (PRRs) are a class of receptors that can directly recognize the specific molecular structures on the surface of pathogens, apoptotic host cells, and damaged senescent cells. PRRs bridge nonspecific immunity and specific immunity. Through the recognition and binding of ligands, PRRs can produce nonspecific anti-infection, antitumor, and other immunoprotective effects. Most PRRs in the innate immune system of vertebrates can be classified into the following five types based on protein domain homology: Toll-like receptors (TLRs), nucleotide oligomerization domain (NOD)-like receptors (NLRs), retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), C-type lectin receptors (CLRs), and absent in melanoma-2 (AIM2)-like receptors (ALRs). PRRs are basically composed of ligand recognition domains, intermediate domains, and effector domains. PRRs recognize and bind their respective ligands and recruit adaptor molecules with the same structure through their effector domains, initiating downstream signaling pathways to exert effects. In recent years, the increased researches on the recognition and binding of PRRs and their ligands have greatly promoted the understanding of different PRRs signaling pathways and provided ideas for the treatment of immune-related diseases and even tumors. This review describes in detail the history, the structural characteristics, ligand recognition mechanism, the signaling pathway, the related disease, new drugs in clinical trials and clinical therapy of different types of PRRs, and discusses the significance of the research on pattern recognition mechanism for the treatment of PRR-related diseases.
... LGP2 synergy with MDA5 to generate antiviral immune response during RLR-dependent dsRNA recognition [51]. ...
Chapter
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The COVID-19 pandemic that started in December 2019 in Wuhan city, China has created chaos all over the world with over 185 million infection cases and 4 million deaths world-wide. The pathogen behind COVID-19 has been identified as severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) that is more close to the previous SARS-CoV responsible for SARS epidemic 2002–2003. Although, SARS-CoV-2 also differs from SARS-CoV in many aspects as indicated by genetic studies. For example, SARS-CoV does not have a furin binding domain or site, whereas its presence in SARS-CoV-2 spike (S) protein increases its potential for infectivity. The horseshoe bats (Rhinolphus species) from China are considered as primary animal reservoirs for SARS-CoV and SARS-CoV-2. However, along with CoVs, bats also harbor many other viral pathogens (Ebola, Nipah, and Hendra viruses) without having serious infections. The bat physiology plays a crucial role in harboring these viruses along with adaptations to longevity and slow aging process. The immune system plays a crucial role in the clearance or establishment of the infection. Present chapter discusses different immunological aspects (innate immune response comprising the virus recognizing pattern recognition receptors (PRRs), type 1 interferon production, pro- and anti-inflammatory immune response, and adaptive immune response) that help bats to control viral infection without getting a severe infection as compared to other mammals, including humans.
... RIG-I signaling is negatively regulated by LGP2 which does not contain a CARD domain. However, LGP2 initiates RNA binding to MDA5 and thus LGP2 usually has a positive effect on MDA5-mediated dsRNA sensing (Bruns & Horvath 2015). Unique features of viral RNA (usually not found in mature RNA molecules of the host) are recognized by MDA5 and RIG-I (discussed in detail in the section below). ...
... Additionally, RIG-I and LGP2 have a repressor domain (RD) within the C-terminal regulatory domain (28,30). As LGP2 lacks CARD domains, it is unable to initiate antiviral signaling and instead likely acts as a concentration dependent biphasic switch in mammals, positively regulating MDA5 at low concentrations and negatively regulating RIG-I and MDA5 at high concentrations (33)(34)(35). Some RLRs with sequences most similar to RIG-I have been identified in the N. vectensis genome (9). ...
Article
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Pattern recognition receptors (PRRs) are evolutionarily ancient and crucial components of innate immunity, recognizing danger-associated molecular patterns (DAMPs) and activating host defenses. Basal non-bilaterian animals such as cnidarians must rely solely on innate immunity to defend themselves from pathogens. By investigating cnidarian PRR repertoires we can gain insight into the evolution of innate immunity in these basal animals. Here we utilize the increasing amount of available genomic resources within Cnidaria to survey the PRR repertoires and downstream immune pathway completeness within 15 cnidarian species spanning two major cnidarian clades, Anthozoa and Medusozoa. Overall, we find that anthozoans possess prototypical PRRs, while medusozoans appear to lack these immune proteins. Additionally, anthozoans consistently had higher numbers of PRRs across all four classes relative to medusozoans, a trend largely driven by expansions in NOD-like receptors and C-type lectins. Symbiotic, sessile, and colonial cnidarians also have expanded PRR repertoires relative to their non-symbiotic, mobile, and solitary counterparts. Interestingly, cnidarians seem to lack key components of mammalian innate immune pathways, though similar to PRR numbers, anthozoans possess more complete immune pathways than medusozoans. Together, our data indicate that anthozoans have greater immune specificity than medusozoans, which we hypothesize to be due to life history traits common within Anthozoa. Overall, this investigation reveals important insights into the evolution of innate immune proteins within these basal animals.
... Generally, the activation of TBK1 by IPS-1 is a complex process, which requires recruiting the preassociation complex TBK1-TRAFs (TRA2/3/6) to approach IPS-1 for TBK1 autophosphorylation (9,10). Activated TBK1 leads phosphorylation and nuclear translocation of IRF3 and NF-kB, which can induce type I interferon (IFN-I) production (11)(12)(13)(14). ...
Article
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Swine acute diarrhea syndrome coronavirus (SADS-CoV), first discovered in 2017, is a porcine enteric coronavirus that can cause acute diarrhea syndrome (SADS) in piglets. Here, we studied the role of SADS-CoV nucleocapsid (N) protein in innate immunity. Our results showed that SADS-CoV N protein could inhibit type I interferon (IFN) production mediated by Sendai virus (Sev) and could block the phosphorylation and nuclear translocation of interferon regulatory factor 3 (IRF3). Simultaneously, the IFN-β promoter activity mediated by TANK binding kinase 1 (TBK1) or its upstream molecules in the RLRs signal pathway was inhibited by SADS-CoV N protein. Further investigations revealed that SADS-CoV N protein could counteract interaction between TNF receptor-associated factor 3 (TRAF3) and TBK1, which led to reduced TBK1 activation and IFN-β production. Our study is the first report of the interaction between SADS-CoV N protein and the host antiviral innate immune responses, and the mechanism utilized by SADS-CoV N protein provides a new insight of coronaviruses evading host antiviral innate immunity.
... The cellular response against picornavirus infection largely relies on the early detection of the dsRNA intermediates generated during infection by MDA5 (Feng et al., 2012), while LGP2 promotes the viral RNA-MDA5 interaction (Bruns & Horvath, 2015). Interestingly, MDA5 pulldown carried out with FMDV-infected cell lysates retrieved FMDV viral RNA (Rodriguez Pulido et al., 2020). ...
Article
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RNA viruses have developed specialized mechanisms to subvert host RNA‐binding proteins (RBPs) favoring their own gene expression. The Leader (L) protein of foot‐and‐mouth disease virus, a member of the Picornaviridae family, is a papain‐like cysteine protease that self‐cleaves from the polyprotein. Early in infection, the L protease cleaves the translation initiation factors eIF4GI and eIF4GII, inducing the shutdown of cap‐dependent translation. However, the cleavage sites on the viral polyprotein, eIF4GI, and eIF4GII differ in sequence, challenging the definition of a consensus site for L targets. Identification of Gemin5 and Daxx proteolytic products in infected cells unveiled a motif centered on the RKAR sequence. The RBP Gemin5 is a member of the survival of motor neurons complex, a ribosome interacting protein, and a translation downregulator. Likewise, the Fas‐ligand Daxx is a multifunctional adaptor that plays key roles in transcription control, apoptosis, and innate immune antiviral response. Remarkably, the cleavage site on the RNA helicases MDA5 and LGP2, two relevant immune sensors of the retinoic acid‐inducible gene‐I (RIG‐I)‐like receptors family, resembles the L target site of Gemin5 and Daxx, and similar cleavage sites have been reported in ISG15 and TBK1, two proteins involved in type I interferon response and signaling pathway, respectively. In this review we dissect the features of the L cleavage sites in essential RBPs, eventually helping in the discovery of novel L targets. This article is categorized under: • RNA in Disease and Development > RNA in Disease • Translation > Translation Regulation Abstract Overview of RNA‐binding proteins modifications operating in cells during RNA viral infections.
... LGP2 does not directly activate MAVS and the downstream pathway. Although some studies suggest that LGP2 has a role in modulating RIG-I/MDA5 functions (Bruns and Horvath, 2015;Venkataraman et al., 2007), other studies suggest a cell typeand ligand-dependent phenotype of LGP2 deletion (Satoh et al., 2010;Suthar et al., 2012), raising questions regarding the true functions of LGP2. Nevertheless, given the structural similarity of TRIM65:MDA5 and RIPLET:RIG-I binding, we speculated that a related TRIM protein might bind LGP2 in a similar manner. ...
Article
RNA helicases and E3 ubiquitin ligases mediate many critical functions in cells, but their actions have largely been studied in distinct biological contexts. Here, we uncover evolutionarily conserved rules of engagement between RNA helicases and tripartite motif (TRIM) E3 ligases that lead to their functional coordination in vertebrate innate immunity. Using cryoelectron microscopy and biochemistry, we show that RIG-I-like receptors (RLRs), viral RNA receptors with helicase domains, interact with their cognate TRIM/TRIM-like E3 ligases through similar epitopes in the helicase domains. Their interactions are avidity driven, restricting the actions of TRIM/TRIM-like proteins and consequent immune activation to RLR multimers. Mass spectrometry and phylogeny-guided biochemical analyses further reveal that similar rules of engagement may apply to diverse RNA helicases and TRIM/TRIM-like proteins. Our analyses suggest not only conserved substrates for TRIM proteins but also, unexpectedly, deep evolutionary connections between TRIM proteins and RNA helicases, linking ubiquitin and RNA biology throughout animal evolution.
Article
Retinoic acid inducible gene I (RIG-I)-like receptors (RLRs), including RIG-I, MDA5 and LGP2, recognize viral RNA to mount an antiviral interferon (IFN) response RLRs share three different protein domains: C-terminal domain, DExD/H box RNA helicase domain, and an N-terminal domain with two tandem repeats (CARDs). LGP2 lacks tandem CARD and is not able to induce an IFN response. However, LGP2 positively enhances MDA5 and negatively regulates RIG-I signaling. In this study, we determined the LGP2 alternative transcripts in humans to further comprehend the mechanism of its regulation, their evolutionary origin, and the isoforms functionallity. The results showed new eight alternative transcripts in the samples tested. The presence of these transcripts demonstrated that the main mechanisms for the regulation of LGP2 expression are both by insertion of introns and by the loss of exons. The phylogenetic analysis of the comparison between sequences from exon 1 to exon 3 of humans and those previously described in non-human primates showed three well-differentiated groups (lineages) originating from gorillas, suggesting that the transspecies evolution has been maintained for 10 million years. The corresponding protein models (isoforms) were also established, obtaining four isoforms: one complete and three others lacking the C-terminal domain or this domain and the partial or total He2 Helicase domain, which would compromise the functionality of LGP2. In conclusion, this is the first study that elucidate the large genomic organization and complex transcriptional regulation of human LGP2, its pattern of sequence generation, and a mode of evolutionary inheritance across species.
Preprint
RIG-I-like receptors (RLRs) detect cytosolic viral RNAs and initiate innate immune response. MDA5 is an RLR that recognizes viral long double-stranded RNA (dsRNA). The central helicase and C-terminal domains of MDA5 form a ring-like clamp around dsRNA, and its activation requires the oligomerization of N-terminal caspase activation and recruitment domains (CARDs). Another RLR, LGP2, lacks CARDs but plays essential roles in MDA5 signaling. However, the mechanisms underlying MDA5 CARDs assembly and the collaborative mechanics between MDA5 and LGP2 remain largely unresolved. Here we found the MDA5 ring-like clamp operates as an ATP-hydrolysis-driven motor, with the self-assembly of its CARDs strictly suppressed by the protein one-dimensional (1D) translocation along dsRNA. LGP2 recognizes a mobile MDA5 and inhibits the motor's movement, thereby allowing MDA5 CARDs to assemble into oligomers that can further interact with downstream components. These findings highlight how RLR sensors orchestrate higher-order assemblies through 1D movement to direct immune response.
Article
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Background: Evolutionarily, immune response is a complex mechanism that protects the host from internal and external threats. Pattern-recognition receptors (PRRs) recognize MAMPs, PAMPs, and DAMPs to initiate a protective pro-inflammatory immune response. PRRs are expressed on the cell membranes by TLR1, 2, 4, and 6 and in the cytosolic organelles by TLR3, 7, 8, and 9, NLRs, ALRs, and cGLRs. We know their downstream signaling pathways controlling immunoregulatory and pro-inflammatory immune response. However, the impact of PRRs on metabolic control of immune cells to control their pro- and anti-inflammatory activity has not been discussed extensively. Summary: Immune cell metabolism or immunometabolism critically determines immune cells’ pro-inflammatory phenotype and function. The current article discusses immunometabolic reprogramming (IR) upon activation of different PRRs, such as TLRs, NLRs, cGLRs, and RLRs. The duration and type of PRR activated, species studied, and location of immune cells to specific organ are critical factors to determine the IR-induced immune response. Key Message: The work herein describes IR upon TLR, NLR, cGLR, and RLR activation. Understanding IR upon activating different PRRs is critical for designing better immune cell-specific immunotherapeutics and immunomodulators targeting inflammation and inflammatory diseases.
Article
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The RIG-I family helicases, comprising RIG-I, MDA5 and LGP2, are cytoplasmic RNA sensors that trigger an antiviral immune response by specifically recognizing foreign RNAs. While LGP2 lacks the signaling domain necessary for immune activation, it plays a vital role in regulating the RIG-I/MDA5 signaling pathway. In this study, we investigate the mechanisms underlying this regulation by examining the oligomeric state, RNA binding specificity, and translocation activity of human LGP2 and the impact of ATPase activity. We show that LGP2, like RIG-I, prefers binding blunt-ended double-stranded (ds) RNAs over internal dsRNA regions or RNA overhangs and associates with blunt-ends faster than with overhangs. Unlike RIG-I, a 5′-triphosphate (5′ppp), Cap0, or Cap1 RNA-end does not influence LGP2’s RNA binding affinity. LGP2 hydrolyzes ATP in the presence of RNA but at a 5–10 fold slower rate than RIG-I. Nevertheless, LGP2 uses its ATPase activity to translocate and displace biotin-streptavidin interactions. This activity is significantly hindered by a methylated RNA patch, particularly on the 3′-strand, suggesting a 3′-strand tracking mechanism like RIG-I. The preference of LGP2 for blunt-end RNA binding, its insensitivity to Cap0/Cap1 modification, and its translocation/protein displacement ability have substantial implications for how LGP2 regulates the RNA sensing process by MDA5/RIG-I.
Chapter
Current vaccine development is highly focused on safer vaccine strategies such as subunit vaccines containing purified parts of the pathogen. However, subunit vaccines suffer from low immunogenicity, often requiring the inclusion of an adjuvant to augment immune responses. Adjuvants boost vaccine efficacy by acting as agonists for pattern-recognition receptors (PRRs), which are present on the cell membrane and in different intracellular compartments of immune cells. The mechanistic insights into PRR activation led to the discovery of several adjuvant molecules that stimulate the immune system by diverse mechanisms. The physicochemical properties associated with these adjuvants and their ability to reach intracellular compartments of immune cells limit their usage. Nanoparticulate delivery systems aid adjuvants in overcoming these issues by enabling efficient encapsulation and precisely delivering them to intracellular compartments while safeguarding them from harsh in vivo conditions. Of note, nanoparticles possess self-adjuvant properties due to their pathogen-mimicking nature. Specifically, nano-adjuvant systems have been widely studied in the development of vaccines against infectious diseases, including the recent coronavirus disease 19 (COVID-19). This chapter focuses on the adjuvants and nano-adjuvant strategies studied for various infectious diseases.
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During RNA viral infection, RIG-I-like receptors (RLRs) recognize the intracellular pathogenic RNA species derived from viral replication and activate antiviral innate immune response by stimulating type 1 interferon expression. Three RLR members, namely, RIG-I, MDA5, and LGP2 are homologous and belong to a subgroup of superfamily 2 Helicase/ATPase that is preferably activated by double-stranded RNA. RLRs are significantly different in gene architecture, RNA ligand preference, activation, and molecular functions. As switchable macromolecular sensors, RLRs' activities are tightly regulated by RNA ligands, ATP, posttranslational modifications, and cellular cofactors. We provide a comprehensive review of the structure and function of the RLRs and summarize the molecular understanding of sensing and signaling events during the RLR activation process. The key roles RLR signaling play in both anti-infection and immune disease conditions highlight the therapeutic potential in targeting this important molecular pathway.
Article
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Retinoic acid inducible gene (RIG)-I-like receptors (RLRs), including RIG-I, melanoma differentiation associated-5 (MDA5), and laboratory of genetics and physiology 2 (LGP2), play pivotal roles in viral RNA sensing to initiate antiviral interferon (IFN) responses. We previously reported that an RNA-silencing regulator, transactivation response RNA-binding protein (TRBP), up-regulates MDA5/LGP2-mediated IFN responses through interaction with LGP2. Here, we aimed to investigate the mechanism underlying the TRBP-mediated up-regulation of IFN response. Data indicated that phosphomimetic TRBP showed a modest effect, whereas the nonphosphorylated form exhibited hyperactivity in enhancing Cardiovirus-triggered IFN responses. These results suggest that encephalomyocarditis virus (EMCV) attenuates the TRBP-mediated IFN response via TRBP phosphorylation, since EMCV infection activates the kinase responsible for TRBP phosphorylation for virus replication. Furthermore, we found that TRBP-mediated up-regulation of IFN response required the ATP hydrolysis and RNA binding of LGP2. TRBP enhanced RNA-dependent ATP hydrolysis by LGP2 but not that by RIG-I or MDA5. Nonphosphorylated TRBP exhibited higher levels of activity than phosphomimetic TRBP did, suggesting its possible involvement in the mechanism underlying the up-regulation of IFN response. TRBP activated the ATP hydrolysis of LGP2 and RIG-I, but not that of MDA5, in the absence of RNA. Collectively, we showed that TRBP differentially regulated RLR-mediated ATP hydrolysis. Further elucidation of the mechanism underlying the regulation of ATP hydrolysis leading to IFN response and self- and non-self-RNA discrimination could advance the development of effective therapeutic agents against autoimmune diseases.
Article
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DEAD/H-box helicases are implicated in virtually every aspect of RNA metabolism, including transcription, pre-mRNA splicing, ribosomes biogenesis, nuclear export, translation initiation, RNA degradation, and mRNA editing. Most of these helicases are upregulated in various cancers and mutations in some of them are associated with several malignancies. Lately, synthetic lethality (SL) and synthetic dosage lethality (SDL) approaches, where genetic interactions of cancer-related genes are exploited as therapeutic targets, are emerging as a leading area of cancer research. Several DEAD/H-box helicases, including DDX3, DDX9 (Dbp9), DDX10 (Dbp4), DDX11 (ChlR1), and DDX41 (Sacy-1), have been subjected to SL analyses in humans and different model organisms. It remains to be explored whether SDL can be utilized to identity druggable targets in DEAD/H-box helicase overexpressing cancers. In this review, we analyze gene expression data of a subset of DEAD/H-box helicases in multiple cancer types and discuss how their SL/SDL interactions can be used for therapeutic purposes. We also summarize the latest developments in clinical applications, apart from discussing some of the challenges in drug discovery in the context of targeting DEAD/H-box helicases.
Article
Type I interferon (IFN) is considered as a bridge between innate and adaptive immunity. Proper activation or inhibition of type I IFN signaling is essential for host defense against pathogen invasion, tumor cell proliferation, and overactive immune responses. Due to intricate and diverse chemical structures, natural products and their derivatives have become an invaluable source inspiring innovative drug discovery. In addition, some natural products have been applied in clinical practice for infection, cancer, and autoimmunity over thousands of years and their promising curative effects and safety have been well-accepted. However, whether these natural products are primarily targeting type I IFN signaling and specific molecular targets involved are not fully elucidated. In the current review, we thoroughly summarize recent advances in the pharmacology researches of natural products for their type I IFN activity, including both agonism/activation and antagonism/inhibition, and their potential application as therapies. Furthermore, the source and chemical nature of natural products with type I IFN activity are highlighted and their specific molecular targets in the type I IFN pathway and mode of action are classified. In conclusion, natural products possessing type I IFN activity represent promising therapeutic strategies and have a bright prospect in the treatment of infection, cancer, and autoimmune diseases.
Article
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Background & aims: RIG-I-like receptors, including RIG-I, MDA5 and LGP2, sense viral RNA to induce the antiviral interferon (IFN) response. LGP2, unable to activate the IFN response itself, modulates RIG-I and MDA5 signaling. Hepatitis D virus (HDV), a small RNA virus causing the most severe form of viral hepatitis, is sensed by MDA5. The mechanism underlying IFN induction and its effect on HDV replication is unclear. Here, we aimed to unveil the role of LGP2 and clinically relevant variants thereof in these processes. Methods: RLRs were depleted in HDV susceptible HepaRGNTCP cells and primary human hepatocytes. Cells were reconstituted to express different LGP2 versions. HDV and IFN markers were quantified in a time-resolved manner. Interaction studies between LGP2, MDA5 and RNA were performed by pull-down assays. Results: LGP2 is essential for the MDA5-mediated IFN response induced upon HDV infection. This induction requires both RNA binding and ATPase activities of LGP2. The IFN response only moderately reduced HDV replication in resting cells but profoundly suppressed cell division-mediated HDV spread. An LGP2 variant (Q425R), predominating in Africans who develop less severe chronic hepatitis D, mediated detectably higher basal and faster HDV-induced IFN response as well as stronger HDV suppression. Mechanistically, LGP2 RNA binding was a prerequisite for the formation of stable MDA5-RNA complexes. MDA5 binding to RNA was enhanced by the Q425R LGP2 variant. Conclusions: LGP2 is essential to mount an antiviral IFN response induced by HDV and stabilizes MDA5-RNA interaction required for downstream signaling. The natural Q425R LGP2 is a gain-of-function variant and might contribute to an attenuated course of hepatitis D. Lay summary: Hepatitis D virus (HDV) is the causative pathogen of chronic hepatitis D, a severe form of viral hepatitis. Upon infection, the human immune system senses HDV and mounts an antiviral interferon (IFN) response. However, the mechanism of IFN response induction and its effect on HDV replication are not well understood. Taking advantage of relevant cell culture models, gene inactivation and overexpression techniques, we demonstrate that the immune sensor LGP2 is essential for recognizing HDV in infected cells to activate an IFN response that represses HDV replication. Moreover, we found that a natural genetic variant of LGP2 reported to predominate in Sub-Saharan African individuals can accelerate the IFN response induced by HDV. Genetic determinants, possibly including LGP2, might contribute to slower disease progression of hepatitis D in this population. This work helps to understand the interaction between the human IFN system and HDV, and the potential role of natural LGP2 variants for infection outcome in different populations.
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In mammals, LGP2 is the enigmatic RLR family member, being initially believed as an inhibitor of RLR-triggered IFN response but subsequently as an activator of MDA5 signaling and an inhibitor of RIG-I signaling. The contradiction happens to fish LGP2. Here, we generate a lgp2 loss-of-function (lgp2lof/lof) zebrafish mutant, which is highly susceptible to SVCV infection, displaying an initially decreased activation of IFN response and a following increased one. Mechanistically, zebrafish LGP2 functions as the essential activator of IFN response dependently of MDA5 at the early stage of viral infection, but as a negative regulator by impairing mRNA levels of tbk1 and ikki at the late stage of viral infection. The function switch of LGP2 is related to cellular IFN production during viral infection. Our data demonstrate that zebrafish LGP2 is a key homeostatic regulator of IFN response and thus essential for zebrafish survival against SVCV infection.
Article
Mitochondria are traditionally known as the powerhouse of the cell, but their functions extend far beyond energy production. They are vital in cellular and organismal pathways that direct metabolism, stress responses, immunity, and cellular fate. To accomplish these tasks, mitochondria have established networks of both intra- and extracellular communication. Intracellularly, these communication routes comprise direct contacts between mitochondria and other subcellular components as well as indirect vesicle transport of ions, metabolites, and other intracellular messengers. Extracellularly, mitochondria can induce stress responses or other cellular changes that secrete mitochondrial cytokine (mitokine) factors that can travel between tissues as well as respond to immune challenges from extracellular sources. Here we provide a current perspective on the major routes of communication for mitochondrial signaling, including their mechanisms and physiological impact. We also review the major diseases and age-related disorders associated with defects in these signaling pathways. An understanding of how mitochondrial signaling controls cellular homeostasis will bring greater insight into how dysfunctional mitochondria affect health in disease and aging. Expected final online publication date for the Annual Review of Cell and Developmental Biology Volume 38 is October 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
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The past decade has provided critical information about the cytoplasmic innate immune sensing pathway of cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). These discoveries have broadened our understanding of the interconnectedness of the cGAS-STING pathway with autophagy, programmed cell death, Rig-I-like receptor (RLR) signaling, DNA independent interferon induction, and how this pathway responds to RNA virus infection. These advances highlight how multiple families of RNA viruses are restricted by and in turn have mechanisms to inhibit cGAS-STING dependent type-I interferon (IFN-I) induction. Here we review recent discoveries of how and why the cGAS-STING pathway responds to infection with RNA viruses, novel findings of RNA viral antagonism of the cGAS-STING innate immune sensing pathway, and attempt to provide context for a shift in thinking as to how critical this DNA sensing pathway is for the restriction of a wide range of RNA viruses.
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Sensing of pathogen-associated molecular patterns including viral RNA by innate immunity represents the first line of defense against viral infection. In addition to RIG-I-like receptors and NOD-like receptors, several other RNA sensors are known to mediate innate antiviral response in the cytoplasm. Double-stranded RNA-binding protein PACT interacts with prototypic RNA sensor RIG-I to facilitate its recognition of viral RNA and induction of host interferon response, but variations of this theme are seen when the functions of RNA sensors are modulated by other RNA-binding proteins to impinge on antiviral defense, proinflammatory cytokine production and cell death programs. Their discrete and coordinated actions are crucial to protect the host from infection. In this review, we will focus on cytoplasmic RNA sensors with an emphasis on their interplay with RNA-binding partners. Classical sensors such as RIG-I will be briefly reviewed. More attention will be brought to the new insights on how RNA-binding partners of RNA sensors modulate innate RNA sensing and how viruses perturb the functions of RNA-binding partners.
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Mitochondrial antiviral signaling (MAVS) protein is required for innate immune responses against RNA viruses. In virus-infected cells MAVS forms prion-like aggregates to activate antiviral signaling cascades, but the underlying structural mechanism is unknown. Here we report cryo-electron microscopic structures of the helical filaments formed by both the N-terminal caspase activation and recruitment domain (CARD) of MAVS and a truncated MAVS lacking part of the proline-rich region and the C-terminal transmembrane domain. Both structures are left-handed three-stranded helical filaments, revealing specific interfaces between individual CARD subunits that are dictated by electrostatic interactions between neighboring strands and hydrophobic interactions within each strand. Point mutations at multiple locations of these two interfaces impaired filament formation and antiviral signaling. Super-resolution imaging of virus-infected cells revealed rod-shaped MAVS clusters on mitochondria. These results elucidate the structural mechanism of MAVS polymerization, and explain how an α-helical domain uses distinct chemical interactions to form self-perpetuating filaments.
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RNA virus infections are detected by the RIG-I family of receptors, which induce type-I interferons through the mitochondrial protein MAVS. MAVS forms large prion-like polymers that activate the cytosolic kinases IKK and TBK1, which in turn activate NF-κB and IRF3, respectively, to induce interferons. Here we show that MAVS polymers recruit several TRAF proteins, including TRAF2, TRAF5, and TRAF6, through distinct TRAF-binding motifs. Mutations of these motifs that disrupted MAVS binding to TRAFs abrogated its ability to activate IRF3. IRF3 activation was also abolished in cells lacking TRAF2, 5, and 6. These TRAF proteins promoted ubiquitination reactions that recruited NEMO to the MAVS signaling complex, leading to the activation of IKK and TBK1. These results delineate the mechanism of MAVS signaling and reveal that TRAF2, 5, and 6, which are normally associated with NF-κB activation, also play a crucial role in IRF3 activation in antiviral immune responses.
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Mammalian cells possess mechanisms to detect and defend themselves from invading viruses. In the cytosol, the RIG-I-like receptors (RLRs), RIG-I (retinoic acid-inducible gene I; encoded by DDX58) and MDA5 (melanoma differentiation-associated gene 5; encoded by IFIH1) sense atypical RNAs associated with virus infection. Detection triggers a signalling cascade via the adaptor MAVS that culminates in the production of type I interferons (IFN-α and β; hereafter IFN), which are key antiviral cytokines. RIG-I and MDA5 are activated by distinct viral RNA structures and much evidence indicates that RIG-I responds to RNAs bearing a triphosphate (ppp) moiety in conjunction with a blunt-ended, base-paired region at the 5'-end (reviewed in refs 1, 2, 3). Here we show that RIG-I also mediates antiviral responses to RNAs bearing 5'-diphosphates (5'pp). Genomes from mammalian reoviruses with 5'pp termini, 5'pp-RNA isolated from yeast L-A virus, and base-paired 5'pp-RNAs made by in vitro transcription or chemical synthesis, all bind to RIG-I and serve as RIG-I agonists. Furthermore, a RIG-I-dependent response to 5'pp-RNA is essential for controlling reovirus infection in cultured cells and in mice. Thus, the minimal determinant for RIG-I recognition is a base-paired RNA with 5'pp. Such RNAs are found in some viruses but not in uninfected cells, indicating that recognition of 5'pp-RNA, like that of 5'ppp-RNA, acts as a powerful means of self/non-self discrimination by the innate immune system.
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Mammalian cells have the ability to recognize virus infection and mount a powerful antiviral transcriptional response that provides an initial barrier to replication and impacts both innate and adaptive immune responses. Retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) proteins mediate intracellular virus recognition and are activated by viral RNA ligands to induce antiviral signal transduction. While the mechanisms of RIG-I regulation are already well understood, less is known about the more enigmatic melanoma differentiation-associated 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2). Emerging evidence suggests that these two RLRs are intimately associated as both accomplices and antagonists of antiviral signal transduction.
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The type I interferon system is integral to human antiviral immunity. However, inappropriate stimulation or defective negative regulation of this system can lead to inflammatory disease. We sought to determine the molecular basis of genetically uncharacterized cases of the type I interferonopathy Aicardi-Goutières syndrome and of other undefined neurological and immunological phenotypes also demonstrating an upregulated type I interferon response. We found that heterozygous mutations in the cytosolic double-stranded RNA receptor gene IFIH1 (also called MDA5) cause a spectrum of neuroimmunological features consistently associated with an enhanced interferon state. Cellular and biochemical assays indicate that these mutations confer gain of function such that mutant IFIH1 binds RNA more avidly, leading to increased baseline and ligand-induced interferon signaling. Our results demonstrate that aberrant sensing of nucleic acids can cause immune upregulation.
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Significance An undesirable outcome of radiotherapy (ionizing radiation, IR) of cancer is the emergence of radioresistant cells. We report that Laboratory of Genetics and Physiology 2 (LGP2), a resident RIG-I (retinoic acid inducible gene I)–like receptor protein, can induce radioresistance. IR induces interferon and stimulates accumulation of LGP2. In turn, LGP2 shuts off the synthesis of interferon and blocks its cytotoxic effects. Ectopic expression of LGP2 enhances resistance to IR, whereas depletion enhances cytotoxic effects of IR. Here we show that LGP2 is associated with radioresistance in numerous diverse cancer cell lines. Examination of available databases links expression of LGP2 with poor prognosis in cancer patients.
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Type I interferons (IFNs) activate intracellular antimicrobial programmes and influence the development of innate and adaptive immune responses. Canonical type I IFN signalling activates the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway, leading to transcription of IFN-stimulated genes (ISGs). Host, pathogen and environmental factors regulate the responses of cells to this signalling pathway and thus calibrate host defences while limiting tissue damage and preventing autoimmunity. Here, we summarize the signalling and epigenetic mechanisms that regulate type I IFN-induced STAT activation and ISG transcription and translation. These regulatory mechanisms determine the biological outcomes of type I IFN responses and whether pathogens are cleared effectively or chronic infection or autoimmune disease ensues.
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STAT1 and STAT2 proteins are key mediators of type I and type III interferon (IFN) signaling, and are essential components of the cellular antiviral response and adaptive immunity. They associate with IFN regulatory factor 9 (IRF9) to form a heterotrimeric transcription factor complex known as ISGF3. The regulation of IFN-stimulated gene (ISG) expression has served as a model of JAK-STAT signaling and mammalian transcriptional regulation, but to date has primarily been analyzed at the single gene level. While many aspects of ISGF3-mediated gene regulation are thought to be common features applicable to several ISGs, there are also many reports of distinct cases of non-canonical STAT1 or STAT2 signaling events and distinct patterns of co-regulators that contribute to gene-specific transcription. Recent genome-wide studies have begun to uncover a more complete profile of ISG regulation, moving toward a genome-wide understanding of general mechanisms that underlie gene-specific behaviors.
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Transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor κB (NFκB) are activated by external stimuli, including virus infection, to translocate to the nucleus and bind genomic targets important for immunity and inflammation. To investigate RNA polymerase II (Pol II) recruitment and elongation in the human antiviral gene regulatory network, a comprehensive genome-wide analysis was conducted during the initial phase of virus infection. Results reveal extensive integration of IRF3 and NFκB with Pol II and associated machinery and implicate partners for antiviral transcription. Analysis indicates that both de novo polymerase recruitment and stimulated release of paused polymerase work together to control virus-induced gene activation. In addition to known messenger-RNA-encoding loci, IRF3 and NFκB stimulate transcription at regions not previously associated with antiviral transcription, including abundant unannotated loci that encode novel virus-inducible RNAs (nviRNAs). These nviRNAs are widely induced by virus infections in diverse cell types and represent a previously overlooked cellular response to virus infection.
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Paramyxovirus V proteins bind to MDA5 (melanoma differentiation-associated gene 5) and LGP2 (laboratory of genetics and physiology gene 2) but not RIG-I (retinoic acid-inducible gene I). The results demonstrate MDA5 R806 is essential for inhibition by diverse V proteins. Complementary substitution for the analogous RIG-I L714 confers V protein recognition. The analogous LGP2 R455 is required for recognition by measles V protein, but not other V proteins. These findings indicate that paramyxoviruses use a single amino acid to distinguish MDA5 from RIG-I and have evolved distinct contact sites for LGP2 interference.
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Laboratory of genetics and physiology 2 (LGP2) is a member of the RIG-I-like receptor family of cytoplasmic pattern recognition receptors that detect molecular signatures of virus infection and initiate antiviral signal transduction cascades. The ATP hydrolysis activity of LGP2 is essential for antiviral signaling, but it has been unclear how the enzymatic properties of LGP2 regulate its biological response. Quantitative analysis of the dsRNA binding and enzymatic activities of LGP2 revealed high dsRNA-independent ATP hydrolysis activity. Biochemical assays and single-molecule analysis of LGP2 and mutant variants that dissociate basal from dsRNA-stimulated ATP hydrolysis demonstrate that LGP2 utilizes basal ATP hydrolysis to enhance and diversify its RNA recognition capacity, enabling the protein to associate with intrinsically poor substrates. This property is required for LGP2 to synergize with another RIG-I-like receptor, MDA5, to potentiate IFNβ transcription in vivo during infection with encephalomyocarditis virus or transfection with poly(I:C). These results demonstrate previously unrecognized properties of LGP2 ATP hydrolysis and RNA interaction and provide a mechanistic basis for a positive regulatory role for LGP2 in antiviral signaling.
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RIG-I and MDA5 are cytosolic RNA sensors that play a critical role in innate antiviral responses. Major advances have been made in identifying RIG-I ligands, but our knowledge of the ligands for MDA5 remains restricted to data from transfection experiments mostly using poly(I:C), a synthetic dsRNA mimic. Here, we dissected the IFN-α/β-stimulatory activity of different viral RNA species produced during picornavirus infection, both by RNA transfection and in infected cells in which specific steps of viral RNA replication were inhibited. Our results show that the incoming genomic plus-strand RNA does not activate MDA5, but minus-strand RNA synthesis and production of the 7.5 kbp replicative form trigger a strong IFN-α/β response. IFN-α/β production does not rely on plus-strand RNA synthesis and thus generation of the partially double-stranded replicative intermediate. This study reports MDA5 activation by a natural RNA ligand under physiological conditions.
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The viral sensor MDA5 distinguishes between cellular and viral dsRNAs by length-dependent recognition in the range of ∼0.5-7 kb. The ability to discriminate dsRNA length at this scale sets MDA5 apart from other dsRNA receptors of the immune system. We have shown previously that MDA5 forms filaments along dsRNA that disassemble upon ATP hydrolysis. Here, we demonstrate that filament formation alone is insufficient to explain its length specificity, because the intrinsic affinity of MDA5 for dsRNA depends only moderately on dsRNA length. Instead, MDA5 uses a combination of end disassembly and slow nucleation kinetics to "discard" short dsRNA rapidly and to suppress rebinding. In contrast, filaments on long dsRNA cycle between partial end disassembly and elongation, bypassing nucleation steps. MDA5 further uses this repetitive cycle of assembly and disassembly processes to repair filament discontinuities, which often are present because of multiple, internal nucleation events, and to generate longer, continuous filaments that more accurately reflect the length of the underlying dsRNA scaffold. Because the length of the continuous filament determines the stability of the MDA5-dsRNA interaction, the mechanism proposed here provides an explanation for how MDA5 uses filament assembly and disassembly dynamics to discriminate between self vs. nonself dsRNA.
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Melanoma differentiation-associated protein 5 (MDA5) detects viral dsRNA in the cytoplasm. On binding of RNA, MDA5 forms polymers, which trigger assembly of the signaling adaptor mitochondrial antiviral-signaling protein (MAVS) into its active fibril form. The molecular mechanism of MDA5 signaling is not well understood, however. Here we show that MDA5 forms helical filaments on dsRNA and report the 3D structure of the filaments using electron microscopy (EM) and image reconstruction. MDA5 assembles into a polar, single-start helix around the RNA. Fitting of an MDA5 homology model into the structure suggests a key role for the MDA5 C-terminal domain in cooperative filament assembly. Our study supports a signal transduction mechanism in which the helical array of MDA5 within filaments nucleates the assembly of MAVS fibrils. We conclude that MDA5 is a polymerization-dependent signaling platform that uses the amyloid-like self-propagating properties of MAVS to amplify signaling.
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Melanoma differentiation-associated gene-5 (MDA5) detects viral double-stranded RNA in the cytoplasm. RNA binding induces MDA5 to activate the signalling adaptor MAVS through interactions between the caspase recruitment domains (CARDs) of the two proteins. The molecular mechanism of MDA5 signalling is not well understood. Here, we show that MDA5 cooperatively binds short RNA ligands as a dimer with a 16-18-basepair footprint. A crystal structure of the MDA5 helicase-insert domain demonstrates an evolutionary relationship with the archaeal Hef helicases. In X-ray solution structures, the CARDs in unliganded MDA5 are flexible, and RNA binds on one side of an asymmetric MDA5 dimer, bridging the two subunits. On longer RNA, full-length and CARD-deleted MDA5 constructs assemble into ATP-sensitive filaments. We propose a signalling model in which the CARDs on MDA5-RNA filaments nucleate the assembly of MAVS filaments with the same polymeric geometry.
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MDA5, an RIG-I-like helicase, is a conserved cytoplasmic viral RNA sensor, which recognizes dsRNA from a wide-range of viruses in a length-dependent manner. It has been proposed that MDA5 forms higher-order structures upon viral dsRNA recognition or during antiviral signaling, however the organization and nature of this proposed oligomeric state is unknown. We report here that MDA5 cooperatively assembles into a filamentous oligomer composed of a repeating segmental arrangement of MDA5 dimers along the length of dsRNA. Binding of MDA5 to dsRNA stimulates its ATP hydrolysis activity with little coordination between neighboring molecules within a filament. Individual ATP hydrolysis in turn renders an intrinsic kinetic instability to the MDA5 filament, triggering dissociation of MDA5 from dsRNA at a rate inversely proportional to the filament length. These results suggest a previously unrecognized role of ATP hydrolysis in control of filament assembly and disassembly processes, thereby autoregulating the interaction of MDA5 with dsRNA, and provides a potential basis for dsRNA length-dependent antiviral signaling.
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RIG‐I detects cytosolic viral dsRNA and initiates antiviral innate immune responses. Four recent reports of crystal structures that enlighten how RIG‐I is activated and recognizes RNA are discussed.
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RIG-I is a key innate immune pattern-recognition receptor that triggers interferon expression upon detection of intracellular 5'triphosphate double-stranded RNA (5'ppp-dsRNA) of viral origin. RIG-I comprises N-terminal caspase activation and recruitment domains (CARDs), a DECH helicase, and a C-terminal domain (CTD). We present crystal structures of the ligand-free, autorepressed, and RNA-bound, activated states of RIG-I. Inactive RIG-I has an open conformation with the CARDs sequestered by a helical domain inserted between the two helicase moieties. ATP and dsRNA binding induce a major rearrangement to a closed conformation in which the helicase and CTD bind the blunt end 5'ppp-dsRNA with perfect complementarity but incompatibly with continued CARD binding. We propose that after initial binding of 5'ppp-dsRNA to the flexibly linked CTD, co-operative tight binding of ATP and RNA to the helicase domain liberates the CARDs for downstream signaling. These findings significantly advance our molecular understanding of the activation of innate immune signaling helicases.
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RIG-I detects cytosolic viral dsRNA with 5' triphosphates (5'-ppp-dsRNA), thereby initiating an antiviral innate immune response. Here we report the crystal structure of superfamily 2 (SF2) ATPase domain of RIG-I in complex with a nucleotide analogue. RIG-I SF2 comprises two RecA-like domains 1A and 2A and a helical insertion domain 2B, which together form a 'C'-shaped structure. Domains 1A and 2A are maintained in a 'signal-off' state with an inactive ATP hydrolysis site by an intriguing helical arm. By mutational analysis, we show surface motifs that are critical for dsRNA-stimulated ATPase activity, indicating that dsRNA induces a structural movement that brings domains 1A and 2A/B together to form an active ATPase site. The structure also indicates that the regulatory domain is close to the end of the helical arm, where it is well positioned to recruit 5'-ppp-dsRNA to the SF2 domain. Overall, our results indicate that the activation of RIG-I occurs through an RNA- and ATP-driven structural switch in the SF2 domain.
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Retinoic-acid-inducible gene-I (RIG-I; also known as DDX58) is a cytoplasmic pathogen recognition receptor that recognizes pathogen-associated molecular pattern (PAMP) motifs to differentiate between viral and cellular RNAs. RIG-I is activated by blunt-ended double-stranded (ds)RNA with or without a 5'-triphosphate (ppp), by single-stranded RNA marked by a 5'-ppp and by polyuridine sequences. Upon binding to such PAMP motifs, RIG-I initiates a signalling cascade that induces innate immune defences and inflammatory cytokines to establish an antiviral state. The RIG-I pathway is highly regulated and aberrant signalling leads to apoptosis, altered cell differentiation, inflammation, autoimmune diseases and cancer. The helicase and repressor domains (RD) of RIG-I recognize dsRNA and 5'-ppp RNA to activate the two amino-terminal caspase recruitment domains (CARDs) for signalling. Here, to understand the synergy between the helicase and the RD for RNA binding, and the contribution of ATP hydrolysis to RIG-I activation, we determined the structure of human RIG-I helicase-RD in complex with dsRNA and an ATP analogue. The helicase-RD organizes into a ring around dsRNA, capping one end, while contacting both strands using previously uncharacterized motifs to recognize dsRNA. Small-angle X-ray scattering, limited proteolysis and differential scanning fluorimetry indicate that RIG-I is in an extended and flexible conformation that compacts upon binding RNA. These results provide a detailed view of the role of helicase in dsRNA recognition, the synergy between the RD and the helicase for RNA binding and the organization of full-length RIG-I bound to dsRNA, and provide evidence of a conformational change upon RNA binding. The RIG-I helicase-RD structure is consistent with dsRNA translocation without unwinding and cooperative binding to RNA. The structure yields unprecedented insight into innate immunity and has a broader impact on other areas of biology, including RNA interference and DNA repair, which utilize homologous helicase domains within DICER and FANCM.
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In response to viral infection, RIG-I-like RNA helicases bind to viral RNA and activate the mitochondrial protein MAVS, which in turn activates the transcription factors IRF3 and NF-κB to induce type I interferons. [corrected] We have previously shown that RIG-I binds to unanchored lysine-63 (K63) polyubiquitin chains and that this binding is important for MAVS activation; however, the mechanism underlying MAVS activation is not understood. Here, we show that viral infection induces the formation of very large MAVS aggregates, which potently activate IRF3 in the cytosol. We find that a fraction of recombinant MAVS protein forms fibrils that are capable of activating IRF3. Remarkably, the MAVS fibrils behave like prions and effectively convert endogenous MAVS into functional aggregates. We also show that, in the presence of K63 ubiquitin chains, RIG-I catalyzes the conversion of MAVS on the mitochondrial membrane to prion-like aggregates. These results suggest that a prion-like conformational switch of MAVS activates and propagates the antiviral signaling cascade.
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RNA helicases of the DEAD box family are present in all eukaryotic cells and in many bacteria and Archaea. These highly conserved enzymes are required for RNA metabolism from transcription to degradation and are therefore important players in gene expression. DEAD box proteins use ATP to unwind short duplex RNA in an unusual fashion and remodel RNA-protein complexes, but they can also function as ATP-dependent RNA clamps to provide nucleation centres that establish larger RNA-protein complexes. Structural, mechanistic and molecular biological studies have started to reveal how these conserved proteins can perform such diverse functions and how accessory proteins have a central role in their regulation.
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On detecting viral RNAs, the RNA helicase retinoic acid-inducible gene I (RIG-I) activates the interferon regulatory factor 3 (IRF3) signalling pathway to induce type I interferon (IFN) gene transcription. How this antiviral signalling pathway might be negatively regulated is poorly understood. Microarray and bioinformatic analysis indicated that the expression of RIG-I and that of the tumour suppressor CYLD (cylindromatosis), a deubiquitinating enzyme that removes Lys 63-linked polyubiquitin chains, are closely correlated, suggesting a functional association between the two molecules. Ectopic expression of CYLD inhibits the IRF3 signalling pathway and IFN production triggered by RIG-I; conversely, CYLD knockdown enhances the response. CYLD removes polyubiquitin chains from RIG-I as well as from TANK binding kinase 1 (TBK1), the kinase that phosphorylates IRF3, coincident with an inhibition of the IRF3 signalling pathway. Furthermore, CYLD protein level is reduced in the presence of tumour necrosis factor and viral infection, concomitant with enhanced IFN production. These findings show that CYLD is a negative regulator of RIG-I-mediated innate antiviral response.
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Increased IFN-α signaling is a heritable risk factor for systemic lupus erythematosus (SLE). IFN induced with helicase C domain 1 (IFIH1) is a cytoplasmic dsRNA sensor that activates IFN-α pathway signaling. We studied the impact of the autoimmune-disease-associated IFIH1 rs1990760 (A946T) single nucleotide polymorphism upon IFN-α signaling in SLE patients in vivo. We studied 563 SLE patients (278 African-American, 179 European-American, and 106 Hispanic-American). Logistic regression models were used to detect genetic associations with autoantibody traits, and multiple linear regression was used to analyze IFN-α-induced gene expression in PBMCs in the context of serum IFN-α in the same blood sample. We found that the rs1990760 T allele was associated with anti-dsDNA Abs across all of the studied ancestral backgrounds (meta-analysis odds ratio = 1.34, p = 0.026). This allele also was associated with lower serum IFN-α levels in subjects who had anti-dsDNA Abs (p = 0.0026). When we studied simultaneous serum and PBMC samples from SLE patients, we found that the IFIH1 rs1990760 T allele was associated with increased IFN-induced gene expression in PBMCs in response to a given amount of serum IFN-α in anti-dsDNA-positive patients. This effect was independent of the STAT4 genotype, which modulates sensitivity to IFN-α in a similar way. Thus, the IFIH1 rs1990760 T allele was associated with dsDNA Abs, and in patients with anti-dsDNA Abs this risk allele increased sensitivity to IFN-α signaling. These studies suggest a role for the IFIH1 risk allele in SLE in vivo.
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Cytoplasmic pattern recognition receptors detect non-self RNAs during virus infections and initiate antiviral signaling. One receptor, MDA5, possesses essential signaling domains, but weak RNA binding. A second receptor, LGP2, rapidly detects diverse dsRNA species, but lacks signaling domains. Accumulating evidence suggests LGP2 and MDA5 work together to detect viral RNA and generate a complete antiviral response, but the basis for their cooperation has been elusive. Experiments presented here address this gap in antiviral signaling, revealing that LGP2 assists MDA5-RNA interactions leading to enhanced MDA5-mediated antiviral signaling. LGP2 increases the initial rate of MDA5-RNA interaction and regulates MDA5 filament assembly, resulting in the formation of more numerous, shorter MDA5 filaments that are shown to generate equivalent or greater signaling activity in vivo than the longer filaments containing only MDA5. These findings provide a mechanism for LGP2 coactivation of MDA5 and a biological context for MDA5-RNA filaments in antiviral responses.
Article
Virus-encoded molecular signatures, such as cytosolic double-stranded or otherwise biochemically distinct RNA species, trigger cellular antiviral signaling. Cytoplasmic proteins recognize these non-self RNAs and activate signal transduction pathways that drive the expression of virus-induced genes, including the primary antiviral cytokine, IFNβ, and diverse direct and indirect antiviral effectors 0005, 0010, 0015 and 0020. One important group of cytosolic RNA sensors known as the RIG-I like receptors (RLRs) is comprised of three proteins that are similar in structure and function. The RLR proteins, RIG-I, MDA5, and LGP2, share the ability to recognize nucleic acid signatures produced by virus infections and activate antiviral signaling. Emerging evidence indicates that RNA detection by RLRs culminates in the assembly of dynamic multimeric ribonucleoprotein (RNP) complexes. These RNPs can act as signaling platforms that are capable of propagating and amplifying antiviral signaling responses. Despite their common domain structures and similar abilities to induce antiviral responses, the RLRs differ in their enzymatic properties, their intrinsic abilities to recognize RNA, and their ability to assemble into filamentous complexes. This molecular specialization has enabled the RLRs to recognize and respond to diverse virus infections, and to mediate both unique and overlapping functions in immune regulation 0025 and 0030.
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A new study shows that gain-of-function mutations in IFIH1, which encodes the cytosolic double-stranded RNA sensor MDA5, lead to upregulated type I interferon responses. Individuals with these mutations exhibit phenotypes consistent with autoimmune diseases, including Aicardi-Goutières syndrome and systemic lupus erythematosus.
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Ubiquitin (Ub) has important roles in a wide range of intracellular signalling pathways. In the conventional view, ubiquitin alters the signalling activity of the target protein through covalent modification, but accumulating evidence points to the emerging role of non-covalent interaction between ubiquitin and the target. In the innate immune signalling pathway of a viral RNA sensor, RIG-I, both covalent and non-covalent interactions with K63-linked ubiquitin chains (K63-Ubn) were shown to occur in its signalling domain, a tandem caspase activation and recruitment domain (hereafter referred to as 2CARD). Non-covalent binding of K63-Ubn to 2CARD induces its tetramer formation, a requirement for downstream signal activation. Here we report the crystal structure of the tetramer of human RIG-I 2CARD bound by three chains of K63-Ub2. 2CARD assembles into a helical tetramer resembling a 'lock-washer', in which the tetrameric surface serves as a signalling platform for recruitment and activation of the downstream signalling molecule, MAVS. Ubiquitin chains are bound along the outer rim of the helical trajectory, bridging adjacent subunits of 2CARD and stabilizing the 2CARD tetramer. The combination of structural and functional analyses reveals that binding avidity dictates the K63-linkage and chain-length specificity of 2CARD, and that covalent ubiquitin conjugation of 2CARD further stabilizes the Ub-2CARD interaction and thus the 2CARD tetramer. Our work provides unique insights into the novel types of ubiquitin-mediated signal-activation mechanism, and previously unexpected synergism between the covalent and non-covalent ubiquitin interaction modes.
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MDA5 is an essential intracellular sensor for several viruses, including picornaviruses, and elicits antiviral interferon (IFN) responses by recognizing viral dsRNAs. MDA5 has been implicated in autoimmunity. However, the mechanisms of how MDA5 contributes to autoimmunity remain unclear. Here we provide direct evidence that dysregulation of MDA5 caused autoimmune disorders. We established a mutant mouse line bearing MDA5 mutation by ENU mutagenesis, which spontaneously developed lupus-like autoimmune symptoms without viral infection. Inflammation was dependent on an adaptor molecule, MAVS indicating the importance of MDA5-signaling. In addition, intercrossing the mutant mice with type I IFN receptor-deficient mice ameliorated clinical manifestations. This MDA5 mutant could activate signaling in the absence of its ligand but was paradoxically defective for ligand- and virus-induced signaling, suggesting that the mutation induces a conformational change in MDA5. These findings provide insight into the association between disorders of the innate immune system and autoimmunity.
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Cytokine signaling is mediated by the combinatorial usage of seven STAT proteins that form homo- or heterodimers involved in the regulation of specific transcriptional programs. Among STATs, STAT2 is classically known to dimerize with STAT1 and together with IRF9 forms the ISGF3 transcription factor complex that has long been considered a hallmark of activation by type I and type III interferons. However, accumulating evidence reveal distinct facets of STAT2 and IRF9 activity mediated by the segregation in alternative STAT1-independent complexes/pathways that are thought to trigger different transcriptional programs. The goal of this review is to summarize our current knowledge of the stimuli, regulatory mechanisms, and function of these alternative pathways.
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Host cell invasion is monitored by a series of pattern recognition receptors (PRRs) that activate the innate immune machinery upon detection of a cognate pathogen associated molecular pattern (PAMP). The RIG-I like receptor (RLR) family of PRRs includes three proteins — RIG-I, MDA5, and LGP2 — responsible for the detection of intracellular pathogenic RNA. All RLR proteins are built around an ATPase core homologous to those found in canonical Superfamily 2 (SF2) RNA helicases, which has been modified through the addition of novel accessory domains to recognize duplex RNA. This review focuses on the structural bases for pathogen-specific dsRNA binding and ATPase activation in RLRs, differential RNA recognition by RLR family members, and implications for other duplex RNA activated ATPases, such as Dicer.
Article
RIG-I-like receptors (RLRs) are nucleic acid sensors that activate antiviral innate immune response. These molecules recognize diverse non-self RNA substrates and are antagonized by several viral inhibitors. We performed an evolutionary analysis of RLR genes (RIG-I, MDA5, and LGP2) in mammals. Results indicated that purifying selection had a dominant role in driving the evolution of RLRs. However, application of maximum-likelihood analyses identified several positions that evolved adaptively. Positively selected sites are located in all domains of MDA5 and RIG-I, whereas in LGP2 they are confined to the helicase domain. In both MDA5 and RIG-I, the linkers separating the CARD and helicase domains represented preferential targets of positive selection. Independent selective events in RIG-I and LGP2 targeted the corresponding site (Asp421 and Asp179, respectively) within a protruding α-helix that grips the V-shaped structure formed by the pincer. Most of the positively selected sites in MDA5 are in regions unique to this RLR, including a characteristic insertion within the helicase domain. Additional selected sites are located at the contact interface between MDA5 monomers, in spatial proximity to a positively selected human polymorphism (Arg843His), and immediately external to the parainfluenza virus 5 V protein (PIV5) binding region. Structural analyses suggested that the positively selected His834 residue is involved in PIV5 binding. Data herein suggest that RLRs have been engaged in host-virus genetic conflict leading to diversifying selection, and indicate parallel evolution at the same site in RIG-I and LGP2, a position likely to be of central importance in antiviral responses.
Article
Mitochondria have emerged as critical platforms for antiviral innate immune signaling. This is due in large part to the mitochondrial localization of the innate immune signaling adaptor mitochondrial antiviral signaling protein (MAVS), which coordinates signals received from two independent cytosolic pathogen recognition receptors (PRRs) to induce antiviral genes. The existence of a shared adaptor for two central PRRs presents an ideal target by which the host cell can prevent cellular damage induced by uncontrolled inflammation through alteration of MAVS expression and/or signaling. In this review, we focus on the MAVS regulome and review the cellular factors that regulate MAVS by (1) protein-protein interactions, (2) alterations in mitochondrial dynamics, and/or (3) post-translational modifications.
Article
Retinoic acid-inducible gene 1 (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) are paralogous receptors for viral double-stranded RNA (dsRNA) with divergent specificity. We have previously shown that MDA5 forms filaments upon viral dsRNA recognition and that this filament formation is essential for interferon signal activation. Here, we show that while RIG-I binds to a dsRNA end as a monomer in the absence of ATP, it assembles in the presence of ATP into a filament that propagates from the dsRNA end to the interior. Furthermore, RIG-I filaments directly stimulate mitochondrial antiviral signaling (MAVS) filament formation without any cofactor, such as polyubiquitin chains, and forced juxtaposition of the isolated signaling domain of RIG-I, as it would be in the filament, is sufficient to activate interferon signaling. Our findings thus define filamentous architecture as a common yet versatile molecular platform for divergent viral RNA detection and proximity-induced signal activation by RIG-I and MDA5.
Article
Initiating the immune response to invading pathogens, the innate immune system is constituted of immune receptors (pattern recognition receptors, PRR) that sense microbe-associated molecular patterns (MAMPs). Detection of pathogens triggers intracellular defense mechanisms, such as the secretion of cytokines or chemokines to alarm neighboring cells and attract or activate immune cells. The innate immune response to viruses is mostly based on PRRs that detect the unusual structure, modification or location of viral nucleic acids. Most of the highly pathogenic and emerging viruses are RNA genome-based viruses, which can give rise to zoonotic and epidemic diseases or cause viral hemorrhagic fever. As viral RNA is located in the same compartment as host RNA, PRRs in the cytosol have to discriminate between viral and endogenous RNA by virtue of their structure or modification. This challenging task is taken on by the homologous cytosolic DExD/H-box family helicases RIG-I and MDA5, which control the innate immune response to most RNA viruses. This review focuses on the molecular basis for RIG-I like receptor (RLR) activation by synthetic and natural ligands and will discuss controversial ligand definitions.
Article
The cytosolic pathogen sensor RIG-I is activated by RNAs with exposed 5'-triphosphate (5'-ppp) and terminal double-stranded structures, such as those that are generated during viral infection. RIG-I has been shown to translocate on dsRNA in an ATP-dependent manner. However, the precise role of the ATPase activity in RIG-I activation remains unclear. Using in vitro-transcribed Sendai virus defective interfering RNA as a model ligand, we show that RIG-I oligomerizes on 5'-ppp dsRNA in an ATP hydrolysis-dependent and dsRNA length-dependent manner, which correlates with the strength of type-I interferon (IFN-I) activation. These results establish a clear role for the ligand-induced ATPase activity of RIG-I in the stimulation of the IFN response.
Article
Cells are equipped with mechanisms that allow them to rapidly detect and respond to viruses. These defense mechanisms rely partly on receptors that monitor the cytosol for the presence of atypical nucleic acids associated with virus infection. RIG-I-like receptors detect RNA molecules that are absent from the uninfected host. DNA receptors alert the cell to the abnormal presence of that nucleic acid in the cytosol. Signaling by RNA and DNA receptors results in the induction of restriction factors that prevent virus replication and establish cell-intrinsic antiviral immunity. In light of these formidable obstacles, viruses have evolved mechanisms of evasion, masking nucleic acid structures recognized by the host, sequestering themselves away from the cytosol or targeting host sensors, and signaling adaptors for deactivation or degradation. Here, we detail recent advances in the molecular understanding of cytosolic nucleic acid detection and its evasion by viruses.