RESUMEN
In response to stress, cells induce ribonucleoprotein aggregates, termed stress granules (SGs). SGs are transient loci containing translation-stalled mRNA, which is eventually degraded or recycled for translation. Infection of some viruses, including influenza A virus with a deletion of nonstructural protein 1 (IAVΔNS1), induces SG-like protein aggregates. Previously, we showed that IAVΔNS1-induced SGs are required for efficient induction of type I interferon (IFN). Here, we investigated SG formation by different viruses using green fluorescent protein (GFP)-tagged Ras-Gap SH3 domain binding protein 1 (GFP-G3BP1) as an SG probe. HeLa cells stably expressing GFP-G3BP1 were infected with different viruses, and GFP fluorescence was monitored live with time-lapse microscopy. SG formations by different viruses was classified into 4 different patterns: no SG formation, stable SG formation, transient SG formation, and alternate SG formation. We focused on encephalomyocarditis virus (EMCV) infection, which exhibited transient SG formation. We found that EMCV disrupts SGs by cleavage of G3BP1 at late stages of infection (>8 h) through a mechanism similar to that used by poliovirus. Expression of a G3BP1 mutant that is resistant to the cleavage conferred persistent formation of SGs as well as an enhanced induction of IFN and other cytokines at late stages of infection. Additionally, knockdown of endogenous G3BP1 blocked SG formation with an attenuated induction of IFN and potentiated viral replication. Taken together, our findings suggest a critical role of SGs as an antiviral platform and shed light on one of the mechanisms by which a virus interferes with host stress and subsequent antiviral responses.
Asunto(s)
Virus de la Encefalomiocarditis/inmunología , Virus de la Encefalomiocarditis/patogenicidad , Ribonucleoproteínas/metabolismo , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Citocinas/genética , ADN Helicasas , Virus ADN/patogenicidad , Virus de la Encefalomiocarditis/fisiología , Expresión Génica , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Innata/genética , Interferones/genética , Mutación , Proteínas de Unión a Poli-ADP-Ribosa , ARN Helicasas , Proteínas con Motivos de Reconocimiento de ARN , Virus ARN/patogenicidad , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteínas/inmunología , Estrés Fisiológico , Replicación ViralRESUMEN
In virus-infected cells, RIG-I-like receptor (RLR) recognizes cytoplasmic viral RNA and triggers innate immune responses including production of type I and III interferon (IFN) and the subsequent expression of IFN-inducible genes. Interferon-beta promoter stimulator 1 (IPS-1, also known as MAVS, VISA and Cardif) is a downstream molecule of RLR and is expressed on the outer membrane of mitochondria. While it is known that the location of IPS-1 is essential to its function, its underlying mechanism is unknown. Our aim in this study was to delineate the function of mitochondria so as to identify more precisely its role in innate immunity. In doing so we discovered that viral infection as well as transfection with 5'ppp-RNA resulted in the redistribution of IPS-1 to form speckle-like aggregates in cells. We further found that Mitofusin 1 (MFN1), a key regulator of mitochondrial fusion and a protein associated with IPS-1 on the outer membrane of mitochondria, positively regulates RLR-mediated innate antiviral responses. Conversely, specific knockdown of MFN1 abrogates both the virus-induced redistribution of IPS-1 and IFN production. Our study suggests that mitochondria participate in the segregation of IPS-1 through their fusion processes.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , GTP Fosfohidrolasas/inmunología , Proteínas de Transporte de Membrana/inmunología , Proteínas Mitocondriales/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Línea Celular , Humanos , Inmunidad Innata , Ratones , Mitocondrias/inmunología , Mitocondrias/patología , Proteínas de Transporte de Membrana Mitocondrial , ARN ViralRESUMEN
Activation of antiviral innate immunity is triggered by cellular pattern recognition receptors. Retinoic acid inducible gene-I (RIG-I)-like receptors (RLRs) detect viral non-self RNA in cytoplasm of virus-infected cells and play a critical role in the clearance of the invaded viruses through production of antiviral cytokines. Among the three known RLRs, RIG-I and melanoma differentiation-associated gene 5 recognize distinct non-self signatures of viral RNA and activate antiviral signaling. Recent reports have clearly described the molecular machinery underlying the activation of RLRs and interactions with the downstream adaptor, mitochondrial antiviral signaling protein (MAVS). RLRs and MAVS are thought to form large multimeric filaments around cytoplasmic organelles depending on the presence of Lys63-linked ubiquitin chains. Furthermore, RLRs have been shown to localize to stress-induced ribonucleoprotein aggregate known as stress granules and utilize them as a platform for recognition/activation of signaling. In this review, we will focus on the current understanding of RLR-mediated signal activation and the interactions with stress-induced RNA granules.
Asunto(s)
Gránulos Citoplasmáticos/inmunología , Infecciones por Virus ADN/inmunología , Inmunidad Innata , Infecciones por Virus ARN/inmunología , ARN Viral/inmunología , Receptores de Reconocimiento de Patrones/inmunología , Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Gránulos Citoplasmáticos/virología , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/inmunología , ARN Helicasas DEAD-box/metabolismo , Humanos , Helicasa Inducida por Interferón IFIH1 , Ratones , Poliubiquitina/metabolismo , ARN Helicasas/inmunología , ARN Helicasas/metabolismo , Receptores Inmunológicos , Receptores de Reconocimiento de Patrones/metabolismo , Ribonucleoproteínas/metabolismo , Transducción de Señal , Estrés Fisiológico/inmunologíaRESUMEN
In higher vertebrates, recognition of the non-self signature of invading viruses by genome-encoded pattern recognition receptors initiates antiviral innate immunity. Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) detect viral RNA as a non-self pattern in the cytoplasm and activate downstream signaling. Detection of viral RNA also activates stress responses resulting in stress granule-like aggregates, which facilitate RLR-mediated antiviral immunity. Among the three RLR family members RIG-I and melanoma differentiation-associated gene 5 (MDA5) recognize distinct viral RNA species with differential molecular machinery and activate signaling through mitochondrial antiviral signaling (MAVS, also known as IPS-1/VISA/Cardif), which leads to the expression of cytokines including type I and III interferons (IFNs) to restrict viral propagation. In this review, we summarize recent knowledge regarding RNA recognition and signal transduction by RLRs and MAVS/IPS-1.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , ARN Viral/metabolismo , Transducción de Señal , Animales , Humanos , Inmunidad Innata , Fosforilación , Unión Proteica , ARN Viral/inmunología , Estrés Fisiológico , Ubiquitina/metabolismoRESUMEN
Retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) function as cytoplasmic sensors for viral RNA to initiate antiviral responses including type I interferon (IFN) production. It has been unclear how RIG-I encounters and senses viral RNA. To address this issue, we examined intracellular localization of RIG-I in response to viral infection using newly generated anti-RIG-I antibody. Immunohistochemical analysis revealed that RLRs localized in virus-induced granules containing stress granule (SG) markers together with viral RNA and antiviral proteins. Because of similarity in morphology and components, we termed these aggregates antiviral stress granules (avSGs). Influenza A virus (IAV) deficient in non-structural protein 1 (NS1) efficiently generated avSGs as well as IFN, however IAV encoding NS1 produced little. Inhibition of avSGs formation by removal of either the SG component or double-stranded RNA (dsRNA)-dependent protein kinase (PKR) resulted in diminished IFN production and concomitant enhancement of viral replication. Furthermore, we observed that transfection of dsRNA resulted in IFN production in an avSGs-dependent manner. These results strongly suggest that the avSG is the locus for non-self RNA sensing and the orchestration of multiple proteins is critical in the triggering of antiviral responses.