RESUMEN
The helicase RIG-I initiates an antiviral immune response after recognition of pathogenic RNA. TRIM25, an E3 ubiquitin ligase, mediates K63-linked ubiquitination of RIG-I, which is crucial for RIG-I downstream signaling and the antiviral innate immune response. The components and mode of the RIG-I-initiated innate signaling remain to be fully understood. Here we identify a novel long noncoding RNA (Lnczc3h7a) that binds to TRIM25 and promotes RIG-I-mediated antiviral innate immune responses. Depletion of Lnczc3h7a impairs RIG-I signaling and the antiviral innate response to RNA viruses in vitro and in vivo. Mechanistically, Lnczc3h7a binds to both TRIM25 and activated RIG-I, serving as a molecular scaffold for stabilization of the RIG-I-TRIM25 complex at the early stage of viral infection. Lnczc3h7a facilitates TRIM25-mediated K63-linked ubiquitination of RIG-I and thus promotes downstream signaling transduction. Our findings reveal that host RNAs can enhance the response of innate immune sensors to foreign RNAs, ensuring effective antiviral defense.
Asunto(s)
Proteína 58 DEAD Box/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Inmunidad Innata/genética , ARN Largo no Codificante/genética , Factores de Transcripción/genética , Animales , Línea Celular , Humanos , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/virología , Ratones , Modelos Biológicos , Interferencia de ARN , Virus ARN/inmunología , Transducción de Señal , Virosis/genética , Virosis/inmunología , Virosis/metabolismo , Virosis/virologíaRESUMEN
Much attention has focused on commensal bacteria in health and disease, but the role of commensal viruses is understudied. Although metagenomic analysis shows that the intestine of healthy humans and animals harbors various commensal viruses and the dysbiosis of these viruses can be associated with inflammatory diseases, there is still a lack of causal data and underlying mechanisms to understand the physiological role of commensal viruses in intestinal homeostasis. In the present study, we show that commensal viruses are essential for the homeostasis of intestinal intraepithelial lymphocytes (IELs). Mechanistically, the cytosolic viral RNA-sensing receptor RIG-I in antigen-presenting cells can recognize commensal viruses and maintain IELs via a type I interferon-independent, but MAVS-IRF1-IL-15 axis-dependent, manner. The recovery of IELs by interleukin-15 administration reverses the susceptibility of commensal virus-depleted mice to dextran sulfate sodium-induced colitis. Collectively, our results indicate that commensal viruses maintain the IELs and consequently sustain intestinal homeostasis via noncanonical RIG-I signaling.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Infecciones por Caliciviridae/inmunología , Colitis/inmunología , Proteína 58 DEAD Box/metabolismo , Intestinos/inmunología , Linfocitos Intraepiteliales/inmunología , Norovirus/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Infecciones por Caliciviridae/virología , Células Cultivadas , Colitis/inducido químicamente , Colitis/virología , Proteína 58 DEAD Box/genética , Sulfato de Dextran , Susceptibilidad a Enfermedades , Homeostasis , Factor 1 Regulador del Interferón/genética , Factor 1 Regulador del Interferón/metabolismo , Interleucina-15/metabolismo , Intestinos/virología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal , Simbiosis/inmunologíaRESUMEN
RIG-I is essential for host defense against viral pathogens, as it triggers the release of type I interferons upon encounter with viral RNA molecules. In this study, we show that RIG-I is rapidly and efficiently activated by small quantities of incoming viral RNA and that it relies exclusively on the constitutively expressed resident pool of RIG-I receptors for a strong antiviral response. Live-cell imaging of RIG-I following stimulation with viral or synthetic dsRNA reveals that RIG-I signaling occurs without mass aggregation at the mitochondrial membrane. By contrast, interferon-induced RIG-I protein becomes embedded in cytosolic aggregates that are functionally unrelated to signaling. These findings suggest that endogenous RIG-I efficiently recognizes viral RNA and rapidly relays an antiviral signal to MAVS via a transient signaling complex and that cellular aggregates of RIG-I have a function that is distinct from signaling.
Asunto(s)
Interferón Tipo I , Transducción de Señal , Transducción de Señal/genética , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/metabolismo , Antivirales/farmacología , Interferón Tipo I/genética , ARN Bicatenario/genética , ARN Viral/genética , Inmunidad InnataRESUMEN
Faithful maintenance of immune homeostasis relies on the capacity of the cellular immune surveillance machinery to recognize "nonself", such as the presence of pathogenic RNA. Several families of pattern-recognition receptors exist that detect immunostimulatory RNA and then induce cytokine-mediated antiviral and proinflammatory responses. Here, we review the distinct features of bona fide RNA sensors, Toll-like receptors and retinoic-acid inducible gene-I (RIG-I)-like receptors in particular, with a focus on their functional specificity imposed by cell-type-dependent expression, subcellular localization, and ligand preference. Furthermore, we highlight recent advances on the roles of nucleotide-binding oligomerization domain (NOD)-like receptors and DEAD-box or DEAH-box RNA helicases in an orchestrated RNA-sensing network and also discuss the relevance of RNA sensor polymorphisms in human disease.
Asunto(s)
Inmunidad Innata/inmunología , ARN Viral/inmunología , Receptores de Reconocimiento de Patrones/inmunología , Citocinas/inmunología , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/inmunología , Proteína 58 DEAD Box/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/inmunología , ARN Helicasas DEAD-box/metabolismo , Humanos , Interferón Tipo I/inmunología , Proteínas NLR/genética , Proteínas NLR/inmunología , Proteínas NLR/metabolismo , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/metabolismo , Transducción de Señal/inmunología , Receptores Toll-Like/metabolismoRESUMEN
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.
Asunto(s)
Proteína 58 DEAD Box/metabolismo , Inmunidad Innata , Helicasa Inducida por Interferón IFIH1/metabolismo , Receptores Inmunológicos/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Microscopía por Crioelectrón , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/ultraestructura , Epítopos , Evolución Molecular , Células HEK293 , Humanos , Helicasa Inducida por Interferón IFIH1/genética , Helicasa Inducida por Interferón IFIH1/ultraestructura , Modelos Moleculares , Filogenia , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Receptores Inmunológicos/genética , Receptores Inmunológicos/ultraestructura , Proteínas de Motivos Tripartitos/genética , Proteínas de Motivos Tripartitos/ultraestructura , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/ultraestructuraRESUMEN
Mitochondrial morphology shifts rapidly to manage cellular metabolism, organelle integrity, and cell fate. It remains unknown whether innate nucleic acid sensing, the central and general mechanisms of monitoring both microbial invasion and cellular damage, can reprogram and govern mitochondrial dynamics and function. Here, we unexpectedly observed that upon activation of RIG-I-like receptor (RLR)-MAVS signaling, TBK1 directly phosphorylated DRP1/DNM1L, which disabled DRP1, preventing its high-order oligomerization and mitochondrial fragmentation function. The TBK1-DRP1 axis was essential for assembly of large MAVS aggregates and healthy antiviral immunity and underlay nutrient-triggered mitochondrial dynamics and cell fate determination. Knockin (KI) strategies mimicking TBK1-DRP1 signaling produced dominant-negative phenotypes reminiscent of human DRP1 inborn mutations, while interrupting the TBK1-DRP1 connection compromised antiviral responses. Thus, our findings establish an unrecognized function of innate immunity governing both morphology and physiology of a major organelle, identify a lacking loop during innate RNA sensing, and report an elegant mechanism of shaping mitochondrial dynamics.
Asunto(s)
Dinaminas/metabolismo , Mitocondrias/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , ARN/metabolismo , Pez Cebra/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/metabolismo , Dinaminas/genética , Células HCT116 , Células HEK293 , Humanos , Masculino , Ratones , Ratones Transgénicos , Mutación , Proteínas Serina-Treonina Quinasas/genética , ARN/genética , Transducción de Señal/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Circular RNAs (circRNAs) are a class of single-stranded RNAs with a contiguous structure that have enhanced stability and a lack of end motifs necessary for interaction with various cellular proteins. Here, we show that unmodified exogenous circRNA is able to bypass cellular RNA sensors and thereby avoid provoking an immune response in RIG-I and Toll-like receptor (TLR) competent cells and in mice. The immunogenicity and protein expression stability of circRNA preparations are found to be dependent on purity, with small amounts of contaminating linear RNA leading to robust cellular immune responses. Unmodified circRNA is less immunogenic than unmodified linear mRNA in vitro, in part due to the evasion of TLR sensing. Finally, we provide the first demonstration to our knowledge of exogenous circRNA delivery and translation in vivo, and we show that circRNA translation is extended in adipose tissue in comparison to unmodified and uridine-modified linear mRNAs.
Asunto(s)
Proteína 58 DEAD Box/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN/genética , Animales , Proteína 58 DEAD Box/inmunología , Regulación de la Expresión Génica , Redes Reguladoras de Genes/genética , Inmunidad Innata/genética , Ratones , MicroARNs/genética , ARN Circular , Receptores Toll-Like/genética , Receptores Toll-Like/inmunología , Uridina/genética , Vacunas Sintéticas/genéticaRESUMEN
The innate immune receptor RIG-I provides a first line of defense against viral infections. Viral RNAs are recognized by RIG-I's C-terminal domain (CTD), but the RNA must engage the helicase domain to release the signaling CARD (Caspase Activation and Recruitment Domain) domains from their autoinhibitory CARD2:Hel2i interactions. Because the helicase itself lacks RNA specificity, mechanisms to proofread RNAs entering the helicase domain must exist. Although such mechanisms would be crucial in preventing aberrant immune responses by non-specific RNAs, they remain largely uncharacterized to date. This study reveals a previously unknown proofreading mechanism through which RIG-I ensures that the helicase engages RNAs explicitly recognized by the CTD. A crucial part of this mechanism involves the intrinsically disordered CARDs-Helicase Linker (CHL), which connects the CARDs to the helicase subdomain Hel1. CHL uses its negatively charged regions to antagonize incoming RNAs electrostatically. In addition to this RNA gating function, CHL is essential for stabilization of the CARD2:Hel2i interface. Overall, we uncover that the CHL and CARD2:Hel2i interface work together to establish a tunable gating mechanism that allows CTD-chosen RNAs to bind the helicase domain, while at the same time blocking non-specific RNAs. These findings also indicate that CHL could represent a novel target for RIG-I-based therapeutics.
Asunto(s)
ARN Helicasas DEAD-box , ARN Bicatenario , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , ADN Helicasas/metabolismo , Inmunidad Innata , Estructura Terciaria de Proteína , ARN Viral/genéticaRESUMEN
Nucleic acid sensors survey subcellular compartments for atypical or mislocalized RNA or DNA, ultimately triggering innate immune responses. Retinoic acid-inducible gene-I (RIG-I) is part of the family of cytoplasmic RNA receptors that can detect viruses. A growing literature demonstrates that mammalian RNA polymerase III (Pol III) transcribes certain viral or cellular DNA sequences into immunostimulatory RIG-I ligands, which elicits antiviral or inflammatory responses. Dysregulation of the Pol III-RIG-I sensing axis can lead to human diseases including severe viral infection outcomes, autoimmunity, and tumor progression. Here, we summarize the newly emerging role of viral and host-derived Pol III transcripts in immunity and also highlight recent advances in understanding how mammalian cells prevent unwanted immune activation by these RNAs to maintain homeostasis.
Asunto(s)
Antivirales , ARN Polimerasa III , Animales , Humanos , Proteína 58 DEAD Box/genética , Inmunidad Innata , Inflamación , ARN , ARN Viral , MamíferosRESUMEN
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.
Asunto(s)
ARN Helicasas DEAD-box , ARN Helicasas , Humanos , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/metabolismo , ARN Helicasas DEAD-box/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Helicasa Inducida por Interferón IFIH1/metabolismo , Unión Proteica/fisiología , Receptores Inmunológicos/genética , ARN Helicasas/metabolismo , ARN Bicatenario , ARN Viral/metabolismoRESUMEN
Transmissible gastroenteritis virus (TGEV)-induced enteritis is characterized by watery diarrhea, vomiting, and dehydration, and has high mortality in newborn piglets, resulting in significant economic losses in the pig industry worldwide. Conventional cell lines have been used for many years to investigate inflammation induced by TGEV, but these cell lines may not mimic the actual intestinal environment, making it difficult to obtain accurate results. In this study, apical-out porcine intestinal organoids were employed to study TEGV-induced inflammation. We found that apical-out organoids were susceptible to TGEV infection, and the expression of representative inflammatory cytokines was significantly upregulated upon TGEV infection. In addition, retinoic acid-inducible gene I (RIG-I) and the nuclear factor-kappa B (NF-κB) pathway were responsible for the expression of inflammatory cytokines induced by TGEV infection. We also discovered that the transcription factor hypoxia-inducible factor-1α (HIF-1α) positively regulated TGEV-induced inflammation by activating glycolysis in apical-out organoids, and pig experiments identified the same molecular mechanism as the ex vivo results. Collectively, we unveiled that the inflammatory responses induced by TGEV were modulated via the RIG-I/NF-κB/HIF-1α/glycolysis axis ex vivo and in vivo. This study provides novel insights into TGEV-induced enteritis and verifies intestinal organoids as a reliable model for investigating virus-induced inflammation. IMPORTANCE: Intestinal organoids are a newly developed culture system for investigating immune responses to virus infection. This culture model better represents the physiological environment compared with well-established cell lines. In this study, we discovered that inflammatory responses induced by TGEV infection were regulated by the RIG-I/NF-κB/HIF-1α/glycolysis axis in apical-out porcine organoids and in pigs. Our findings contribute to understanding the mechanism of intestinal inflammation upon viral infection and highlight apical-out organoids as a physiological model to mimic virus-induced inflammation.
Asunto(s)
Gastroenteritis Porcina Transmisible , Glucólisis , Inflamación , Organoides , Virus de la Gastroenteritis Transmisible , Animales , Citocinas/metabolismo , Proteína 58 DEAD Box/metabolismo , Proteína 58 DEAD Box/genética , Gastroenteritis Porcina Transmisible/virología , Gastroenteritis Porcina Transmisible/metabolismo , Gastroenteritis Porcina Transmisible/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Inflamación/metabolismo , Inflamación/virología , Intestinos/virología , Intestinos/patología , FN-kappa B/metabolismo , Organoides/virología , Organoides/metabolismo , Organoides/patología , Transducción de Señal , Porcinos , Virus de la Gastroenteritis Transmisible/fisiologíaRESUMEN
Singleton-Merten syndrome (SMS) is a rare immunogenetic disorder affecting multiple systems, characterized by dental dysplasia, aortic calcification, glaucoma, skeletal abnormalities, and psoriasis. Glaucoma, a key feature of both classical and atypical SMS, remains poorly understood in terms of its molecular mechanism caused by DDX58 mutation. This study presented a novel DDX58 variant (c.1649A>C [p.Asp550Ala]) in a family with childhood glaucoma. Functional analysis showed that DDX58 variant caused an increase in IFN-stimulated gene expression and high IFN-ß-based type-I IFN. As the trabecular meshwork (TM) is responsible for controlling intraocular pressure (IOP), we examine the effect of IFN-ß on TM cells. Our study is the first to demonstrate that IFN-ß significantly reduced TM cell viability and function by activating autophagy. In addition, anterior chamber injection of IFN-ß remarkably increased IOP level in mice, which can be attenuated by treatments with autophagy inhibitor chloroquine. To uncover the specific mechanism underlying IFN-ß-induced autophagy in TM cells, we performed microarray analysis in IFN-ß-treated and DDX58 p.Asp550Ala TM cells. It showed that RSAD2 is necessary for IFN-ß-induced autophagy. Knockdown of RSAD2 by siRNA significantly decreased autophagy flux induced by IFN-ß. Our findings suggest that DDX58 mutation leads to the overproduction of IFN-ß, which elevates IOP by modulating autophagy through RSAD2 in TM cells.
Asunto(s)
Autofagia , Proteína 58 DEAD Box , Glaucoma , Presión Intraocular , Malla Trabecular , Animales , Femenino , Humanos , Masculino , Ratones , Enfermedades de la Aorta , Autofagia/efectos de los fármacos , Proteína 58 DEAD Box/metabolismo , Proteína 58 DEAD Box/genética , Hipoplasia del Esmalte Dental , Glaucoma/patología , Glaucoma/metabolismo , Glaucoma/genética , Pérdida Auditiva Sensorineural/genética , Pérdida Auditiva Sensorineural/patología , Pérdida Auditiva Sensorineural/metabolismo , Interferón beta/metabolismo , Presión Intraocular/genética , Metacarpo/anomalías , Ratones Endogámicos C57BL , Enfermedades Musculares , Mutación , Odontodisplasia , Atrofia Óptica/genética , Atrofia Óptica/metabolismo , Atrofia Óptica/patología , Osteoporosis , Linaje , Receptores Inmunológicos , Malla Trabecular/metabolismo , Malla Trabecular/efectos de los fármacos , Calcificación VascularRESUMEN
Type I interferons (IFNs) exhibit strong antiviral activity and induce the expression of antiviral proteins. Since excessive expression of type I IFNs is harmful to the host, their expression should be turned off at the appropriate time. In this study, we find that post-translational modification of LGP2, a member of the RIG-I-like receptor family, modulates antiviral innate immune responses. The LGP2 protein undergoes K63-linked polyubiquitination in response to cytoplasmic double-stranded RNAs or viral infection. Our mass spectrometry analysis reveals the K residues ubiquitinated by the Riplet ubiquitin ligase. LGP2 ubiquitination occurs with a delay compared to RIG-I ubiquitination. Interestingly, ubiquitination-defective LGP2 mutations increase the expression of type I IFN at a late phase, whereas the mutant proteins attenuate other antiviral proteins, such as SP100, PML, and ANKRD1. Our data indicate that delayed polyubiquitination of LGP2 fine-tunes RIG-I-dependent antiviral innate immune responses at a late phase of viral infection.
Asunto(s)
Proteína 58 DEAD Box , ARN Helicasas DEAD-box , Inmunidad Innata , Interferón Tipo I , Virosis , Humanos , Antivirales , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/metabolismo , ARN Helicasas DEAD-box/genética , Interferón Tipo I/genética , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
Most SARS-CoV-2 proteins are translated from subgenomic RNAs (sgRNAs). While the majority of these sgRNAs are monocistronic, some viral mRNAs encode more than one protein. One example is the ORF3a sgRNA that also encodes ORF3c, an enigmatic 41-amino-acid peptide. Here, we show that ORF3c is expressed in SARS-CoV-2-infected cells and suppresses RIG-I- and MDA5-mediated IFN-ß induction. ORF3c interacts with the signaling adaptor MAVS, induces its C-terminal cleavage, and inhibits the interaction of RIG-I with MAVS. The immunosuppressive activity of ORF3c is conserved among members of the subgenus sarbecovirus, including SARS-CoV and coronaviruses isolated from bats. Notably, however, the SARS-CoV-2 delta and kappa variants harbor premature stop codons in ORF3c, demonstrating that this reading frame is not essential for efficient viral replication in vivo and is likely compensated by other viral proteins. In agreement with this, disruption of ORF3c does not significantly affect SARS-CoV-2 replication in CaCo-2, CaLu-3, or Rhinolophus alcyone cells. In summary, we here identify ORF3c as an immune evasion factor of SARS-CoV-2 that suppresses innate sensing in infected cells.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Células CACO-2 , COVID-19/genética , Transducción de Señal , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/metabolismo , Inmunidad Innata/genéticaRESUMEN
Tetherin (BST2/CD317) is an interferon-inducible antiviral factor known for its ability to block the release of enveloped viruses from infected cells. Yet its role in type I interferon (IFN) signaling remains poorly defined. Here, we demonstrate that Tetherin is a negative regulator of RIG-I like receptor (RLR)-mediated type I IFN signaling by targeting MAVS. The induction of Tetherin by type I IFN accelerates MAVS degradation via ubiquitin-dependent selective autophagy in human cells. Moreover, Tetherin recruits E3 ubiquitin ligase MARCH8 to catalyze K27-linked ubiquitin chains on MAVS at lysine 7, which serves as a recognition signal for NDP52-dependent autophagic degradation. Taken together, our findings reveal a negative feedback loop of RLR signaling generated by Tetherin-MARCH8-MAVS-NDP52 axis and provide insights into a better understanding of the crosstalk between selective autophagy and optimal deactivation of type I IFN signaling.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antígenos CD/metabolismo , Autofagia/fisiología , Interferón Tipo I/metabolismo , Proteínas Nucleares/metabolismo , Transducción de Señal/fisiología , Células A549 , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Antígenos CD/genética , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/metabolismo , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Células HeLa , Humanos , Interferón Tipo I/genética , Ratones , Proteínas Nucleares/genética , Células RAW 264.7 , Receptores Inmunológicos , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/fisiologíaRESUMEN
Circular RNAs (circRNAs) are single-stranded RNAs that are joined head to tail with largely unknown functions. Here we show that transfection of purified in vitro generated circRNA into mammalian cells led to potent induction of innate immunity genes and confers protection against viral infection. The nucleic acid sensor RIG-I is necessary to sense foreign circRNA, and RIG-I and foreign circRNA co-aggregate in cytoplasmic foci. CircRNA activation of innate immunity is independent of a 5' triphosphate, double-stranded RNA structure, or the primary sequence of the foreign circRNA. Instead, self-nonself discrimination depends on the intron that programs the circRNA. Use of a human intron to express a foreign circRNA sequence abrogates immune activation, and mature human circRNA is associated with diverse RNA binding proteins reflecting its endogenous splicing and biogenesis. These results reveal innate immune sensing of circRNA and highlight introns-the predominant output of mammalian transcription-as arbiters of self-nonself identity.
Asunto(s)
Virus de la Encefalitis Equina Venezolana/inmunología , Encefalomielitis Equina Venezolana/prevención & control , Tolerancia Inmunológica , Inmunidad Innata , Intrones , Procesamiento Postranscripcional del ARN , Proteínas de Unión al ARN/inmunología , ARN/genética , ARN/inmunología , Animales , Secuencia de Bases , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/inmunología , Proteína 58 DEAD Box/metabolismo , Virus de la Encefalitis Equina Venezolana/genética , Virus de la Encefalitis Equina Venezolana/metabolismo , Encefalomielitis Equina Venezolana/genética , Encefalomielitis Equina Venezolana/inmunología , Encefalomielitis Equina Venezolana/metabolismo , Células HEK293 , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Tolerancia Inmunológica/genética , Inmunidad Innata/genética , Ratones , Conformación de Ácido Nucleico , Unión Proteica , Células RAW 264.7 , ARN/biosíntesis , ARN/química , ARN Circular , ARN Mensajero/genética , ARN Mensajero/inmunología , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Receptores Inmunológicos , Empalmosomas/inmunología , Empalmosomas/metabolismo , TransfecciónRESUMEN
Mitochondrial antiviral signaling platform protein (MAVS) acts as a central hub for RIG-I receptor proximal signal propagation. However, key components in the assembly of the MAVS mitochondrial platform that promote RIG-I mitochondrial localization and optimal activation are still largely undefined. Employing pooled RNAi and yeast two-hybrid screenings, we report that the mitochondrial adaptor protein tripartite motif (TRIM)14 provides a docking platform for the assembly of the mitochondrial signaling complex required for maximal activation of RIG-I-mediated signaling, consisting of WHIP and protein phosphatase PPP6C. Following viral infection, the ubiquitin-binding domain in WHIP bridges RIG-I with MAVS by binding to polyUb chains of RIG-I at lysine 164. The ATPase domain in WHIP contributes to stabilization of the RIG-I-dsRNA interaction. Moreover, phosphatase PPP6C is responsible for RIG-I dephosphorylation. Together, our findings define the WHIP-TRIM14-PPP6C mitochondrial signalosome required for RIG-I-mediated innate antiviral immunity.
Asunto(s)
Proteínas Portadoras/inmunología , Proteína 58 DEAD Box/inmunología , Proteínas de Unión al ADN/inmunología , Inmunidad Innata , Mitocondrias/inmunología , Proteínas Mitocondriales/inmunología , Complejos Multiproteicos/inmunología , Fosfoproteínas Fosfatasas/inmunología , Transducción de Señal/inmunología , ATPasas Asociadas con Actividades Celulares Diversas , Animales , Proteínas Portadoras/genética , Línea Celular Tumoral , Chlorocebus aethiops , Proteína 58 DEAD Box/genética , Proteínas de Unión al ADN/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Mitocondrias/genética , Proteínas Mitocondriales/genética , Complejos Multiproteicos/genética , Fosfoproteínas Fosfatasas/genética , Receptores Inmunológicos , Transducción de Señal/genética , Proteínas de Motivos Tripartitos , Células Vero , Virosis/genética , Virosis/inmunología , Virus/genética , Virus/inmunologíaRESUMEN
The innate immune receptor RIG-I recognizes 5'-triphosphate double-stranded RNAs (5' PPP dsRNA) as pathogenic RNAs. Such RNA-ends are present in viral genomes and replication intermediates, and they activate the RIG-I signaling pathway to produce a potent interferon response essential for viral clearance. Endogenous mRNAs cap the 5' PPP-end with m7G and methylate the 2'-O-ribose to evade RIG-I, preventing aberrant immune responses deleterious to the cell. Recent studies have identified RNAs in cells capped with metabolites such as NAD+, FAD and dephosphoCoA. Whether RIG-I recognizes these metabolite-capped RNAs has not been investigated. Here, we describe a strategy to make metabolite-capped RNAs free from 5' PPP dsRNA contamination, using in vitro transcription initiated with metabolites. Mechanistic studies show that metabolite-capped RNAs have a high affinity for RIG-I, stimulating the ATPase activity at comparable levels to 5' PPP dsRNA. Cellular signaling assays show that the metabolite-capped RNAs potently stimulate the innate antiviral immune response. This demonstrates that RIG-I can tolerate diphosphate-linked, capped RNAs with bulky groups at the 5' RNA end. This novel class of RNAs that stimulate RIG-I signaling may have cellular roles in activating the interferon response and may be exploited with proper functionalities for RIG-I-related RNA therapeutics.
Asunto(s)
ARN Helicasas DEAD-box , ARN Bicatenario , Proteína 58 DEAD Box/genética , ARN Helicasas DEAD-box/metabolismo , Inmunidad Innata , Interferones/genética , Ligandos , Caperuzas de ARN , ARN Viral/genética , ARN Viral/metabolismo , Transducción de Señal , HumanosRESUMEN
RIG-I (retinoic acid inducible gene-I) can sense subtle differences between endogenous and viral RNA in the cytoplasm, triggering an anti-viral immune response through induction of type I interferons (IFN) and other inflammatory mediators. Multiple crystal and cryo-EM structures of RIG-I suggested a mechanism in which the C-terminal domain (CTD) is responsible for the recognition of viral RNA with a 5'-triphoshate modification, while the CARD domains serve as a trigger for downstream signaling, leading to the induction of type I IFN. However, to date contradicting conclusions have been reached around the role of ATP in the mechanism of the CARD domains ejection from RIG-I's autoinhibited state. Here we present an application of NMR spectroscopy to investigate changes induced by the binding of 5'-triphosphate and 5'-OH dsRNA, both in the presence and absence of nucleotides, to full length RIG-I with all its methionine residues selectively labeled (Met-[ϵ-13CH3]). With this approach we were able to identify residues on the CTD, helicase domain, and CARDs that served as probes to sense RNA-induced conformational changes in those respective regions. Our results were analyzed in the context of either agonistic or antagonistic RNAs, by and large supporting a mechanism proposed by the Pyle Lab in which CARD release is primarily dependent on the RNA binding event.
Asunto(s)
Transactivadores , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/metabolismo , Interferón Tipo I/genética , Estructura Terciaria de Proteína , ARN Bicatenario , ARN Viral/genética , ARN Viral/metabolismo , Transducción de Señal , Transactivadores/metabolismoRESUMEN
RIG-I is a cytosolic receptor of viral RNA essential for the immune response to numerous RNA viruses. Accordingly, RIG-I must sensitively detect viral RNA yet tolerate abundant self-RNA species. The basic binding cleft and an aromatic amino acid of the RIG-I C-terminal domain(CTD) mediate high-affinity recognition of 5'triphosphorylated and 5'base-paired RNA(dsRNA). Here, we found that, while 5'unmodified hydroxyl(OH)-dsRNA demonstrated residual activation potential, 5'-monophosphate(5'p)-termini, present on most cellular RNAs, prevented RIG-I activation. Determination of CTD/dsRNA co-crystal structures and mutant activation studies revealed that the evolutionarily conserved I875 within the CTD sterically inhibits 5'p-dsRNA binding. RIG-I(I875A) was activated by both synthetic 5'p-dsRNA and endogenous long dsRNA within the polyA-rich fraction of total cellular RNA. RIG-I(I875A) specifically interacted with long, polyA-bearing, mitochondrial(mt) RNA, and depletion of mtRNA from total RNA abolished its activation. Altogether, our study demonstrates that avoidance of 5'p-RNA recognition is crucial to prevent mtRNA-triggered RIG-I-mediated autoinflammation.