RESUMO
The innate immune sensor retinoic acid-inducible gene I (RIG-I) detects cytosolic viral RNA and requires a conformational change caused by both ATP and RNA binding to induce an active signaling state and to trigger an immune response. Previously, we showed that ATP hydrolysis removes RIG-I from lower-affinity self-RNAs (
Assuntos
Adenosina Trifosfatases/química , Trifosfato de Adenosina/química , Doenças da Aorta/genética , Proteína DEAD-box 58/química , Hipoplasia do Esmalte Dentário/genética , Metacarpo/anormalidades , Doenças Musculares/genética , Odontodisplasia/genética , Osteoporose/genética , Calcificação Vascular/genética , Proteínas AAA/química , Proteínas AAA/genética , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/metabolismo , Doenças da Aorta/enzimologia , Doenças da Aorta/patologia , Citosol/virologia , Proteína DEAD-box 58/genética , Hipoplasia do Esmalte Dentário/enzimologia , Hipoplasia do Esmalte Dentário/patologia , Humanos , Hidrólise , Imunidade Inata/genética , Metacarpo/enzimologia , Metacarpo/patologia , Doenças Musculares/enzimologia , Doenças Musculares/patologia , Mutação , Odontodisplasia/enzimologia , Odontodisplasia/patologia , Osteoporose/enzimologia , Osteoporose/patologia , Ligação Proteica , Conformação Proteica , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/genética , RNA Viral/química , RNA Viral/genética , Receptores Imunológicos , Calcificação Vascular/enzimologia , Calcificação Vascular/patologiaRESUMO
The sensor RIG-I detects double-stranded RNA derived from RNA viruses. Although RIG-I is also known to have a role in the antiviral response to DNA viruses, physiological RNA species recognized by RIG-I during infection with a DNA virus are largely unknown. Using next-generation RNA sequencing (RNAseq), we found that host-derived RNAs, most prominently 5S ribosomal RNA pseudogene 141 (RNA5SP141), bound to RIG-I during infection with herpes simplex virus 1 (HSV-1). Infection with HSV-1 induced relocalization of RNA5SP141 from the nucleus to the cytoplasm, and virus-induced shutoff of host protein synthesis downregulated the abundance of RNA5SP141-interacting proteins, which allowed RNA5SP141 to bind RIG-I and induce the expression of type I interferons. Silencing of RNA5SP141 strongly dampened the antiviral response to HSV-1 and the related virus Epstein-Barr virus (EBV), as well as influenza A virus (IAV). Our findings reveal that antiviral immunity can be triggered by host RNAs that are unshielded following depletion of their respective binding proteins by the virus.
Assuntos
Proteína DEAD-box 58/imunologia , Herpesvirus Humano 1/imunologia , Imunidade/imunologia , RNA Ribossômico 5S/imunologia , Animais , Células Cultivadas , Chlorocebus aethiops , Proteína DEAD-box 58/metabolismo , Expressão Gênica/imunologia , Células HEK293 , Herpesvirus Humano 1/fisiologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Camundongos Knockout , Pseudogenes/genética , Transporte de RNA/imunologia , RNA Ribossômico 5S/genética , RNA Ribossômico 5S/metabolismo , Receptores Imunológicos , Células VeroRESUMO
Cytosolic DNA arising from intracellular pathogens triggers a powerful innate immune response. It is sensed by cyclic GMP-AMP synthase (cGAS), which elicits the production of type I interferons by generating the second messenger 2'3'-cyclic-GMP-AMP (cGAMP). Endogenous nuclear or mitochondrial DNA can also be sensed by cGAS under certain conditions, resulting in sterile inflammation. The cGAS dimer binds two DNA ligands shorter than 20 base pairs side-by-side, but 20-base-pair DNA fails to activate cGAS in vivo and is a poor activator in vitro. Here we show that cGAS is activated in a strongly DNA length-dependent manner both in vitro and in human cells. We also show that cGAS dimers form ladder-like networks with DNA, leading to cooperative sensing of DNA length: assembly of the pioneering cGAS dimer between two DNA molecules is ineffective; but, once formed, it prearranges the flanking DNA to promote binding of subsequent cGAS dimers. Remarkably, bacterial and mitochondrial nucleoid proteins HU and mitochondrial transcription factor A (TFAM), as well as high-mobility group box 1 protein (HMGB1), can strongly stimulate long DNA sensing by cGAS. U-turns and bends in DNA induced by these proteins pre-structure DNA to nucleate cGAS dimers. Our results suggest a nucleation-cooperativity-based mechanism for sensitive detection of mitochondrial DNA and pathogen genomes, and identify HMGB/TFAM proteins as DNA-structuring host factors. They provide an explanation for the peculiar cGAS dimer structure and suggest that cGAS preferentially binds incomplete nucleoid-like structures or bent DNA.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/química , DNA/metabolismo , Proteínas HMGB/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas Mitocondriais/metabolismo , Nucleotidiltransferases/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Feminino , Humanos , Camundongos , Modelos Moleculares , Conformação de Ácido Nucleico , Nucleotídeos Cíclicos/metabolismo , Nucleotidiltransferases/química , Multimerização ProteicaRESUMO
RIG-I-like receptors (RLRs) are cytosolic innate immune sensors that detect pathogenic RNA and induce a systemic antiviral response. During the last decade, many studies focused on their molecular characterization and the identification of RNA agonists. Therefore, it became more and more clear that RLR activation needs to be carefully regulated, because constitutive signaling or detection of endogenous RNA through loss of specificity is detrimental. Here, we review the current understanding of RLR activation and selectivity. We specifically focus upon recent findings on the function of the helicase domain in discriminating between different RNAs, and whose malfunctioning causes serious autoimmune diseases.
Assuntos
Doenças Autoimunes/imunologia , Proteína DEAD-box 58/imunologia , Imunidade Inata , RNA/imunologia , Animais , HumanosRESUMO
RIG-I-like receptors detect cytosolic viral RNA and activate an antiviral innate immune response. A new study employs the one STrEP-purification technique and next generation sequencing to characterize physiological ligands in an infected cell. The view of all three RLRs bound to viral RNAs shows specialization, collaboration and new binding sites.
Assuntos
Proteína DEAD-box 58/imunologia , Imunidade Inata/imunologia , Helicase IFIH1 Induzida por Interferon/imunologia , Infecções por Vírus de RNA/imunologia , Linhagem Celular , Células HEK293 , Humanos , Infecções por Vírus de RNA/diagnóstico , RNA Viral/genética , Receptores Imunológicos , Transdução de Sinais/imunologiaRESUMO
The cytosolic antiviral innate immune sensor RIG-I distinguishes 5' tri- or diphosphate containing viral double-stranded (ds) RNA from self-RNA by an incompletely understood mechanism that involves ATP hydrolysis by RIG-I's RNA translocase domain. Recently discovered mutations in ATPase motifs can lead to the multi-system disorder Singleton-Merten Syndrome (SMS) and increased interferon levels, suggesting misregulated signaling by RIG-I. Here we report that SMS mutations phenocopy a mutation that allows ATP binding but prevents hydrolysis. ATPase deficient RIG-I constitutively signals through endogenous RNA and co-purifies with self-RNA even from virus infected cells. Biochemical studies and cryo-electron microscopy identify a 60S ribosomal expansion segment as a dominant self-RNA that is stably bound by ATPase deficient RIG-I. ATP hydrolysis displaces wild-type RIG-I from this self-RNA but not from 5' triphosphate dsRNA. Our results indicate that ATP-hydrolysis prevents recognition of self-RNA and suggest that SMS mutations lead to unintentional signaling through prolonged RNA binding.
Assuntos
Trifosfato de Adenosina/metabolismo , RNA Helicases DEAD-box/metabolismo , RNA Viral/metabolismo , Linhagem Celular , Proteína DEAD-box 58 , Humanos , Hidrólise , Receptores Imunológicos , Especificidade por SubstratoRESUMO
RIG-I-like receptors (RLRs: RIG-I, MDA5 and LGP2) play a major role in the innate immune response against viral infections and detect patterns on viral RNA molecules that are typically absent from host RNA. Upon RNA binding, RLRs trigger a complex downstream signaling cascade resulting in the expression of type I interferons and proinflammatory cytokines. In the past decade extensive efforts were made to elucidate the nature of putative RLR ligands. In vitro and transfection studies identified 5'-triphosphate containing blunt-ended double-strand RNAs as potent RIG-I inducers and these findings were confirmed by next-generation sequencing of RIG-I associated RNAs from virus-infected cells. The nature of RNA ligands of MDA5 is less clear. Several studies suggest that double-stranded RNAs are the preferred agonists for the protein. However, the exact nature of physiological MDA5 ligands from virus-infected cells needs to be elucidated. In this work, we combine a crosslinking technique with next-generation sequencing in order to shed light on MDA5-associated RNAs from human cells infected with measles virus. Our findings suggest that RIG-I and MDA5 associate with AU-rich RNA species originating from the mRNA of the measles virus L gene. Corresponding sequences are poorer activators of ATP-hydrolysis by MDA5 in vitro, suggesting that they result in more stable MDA5 filaments. These data provide a possible model of how AU-rich sequences could activate type I interferon signaling.