RESUMEN
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.
Asunto(s)
Gránulos Citoplasmáticos/inmunología , ARN Helicasas DEAD-box/inmunología , Inmunidad Innata/inmunología , Virus de la Influenza A/inmunología , Infecciones por Orthomyxoviridae/inmunología , ARN Viral/metabolismo , eIF-2 Quinasa/inmunología , Animales , Anticuerpos Antivirales/inmunología , Chlorocebus aethiops , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/metabolismo , Fibroblastos , Células HeLa , Humanos , Inmunohistoquímica , Interferón Tipo I/inmunología , Ratones , Ratones Noqueados , Células Vero , eIF-2 Quinasa/metabolismoRESUMEN
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
Viral infections trigger innate immune responses, including the production of type I interferons (IFN-alpha and -beta) and other proinflammatory cytokines. Novel antiviral cytokines IFN-lambda1, IFN-lambda2, and IFN-lambda3 are classified as type III IFNs and have evolved independently of type I IFNs. Type III IFN genes are regulated at the level of transcription and induced by viral infection. Although the regulatory mechanism of type I IFNs is well elucidated, the expression mechanism of IFN-lambdas is not well understood. Here, we analyzed the mechanism by which IFN-lambda gene expression is induced by viral infections. Loss- and gain-of-function experiments revealed the involvement of RIG-I (retinoic acid-inducible gene I), IPS-1, TBK1, and interferon regulatory factor-3, key regulators of the virus-induced activation of type I IFN genes. Consistent with this, a search for the cis-regulatory element of the human ifnlambda1 revealed a cluster of interferon regulatory factor-binding sites and a NF-kappaB-binding site. Functional analysis demonstrated that all of these sites are essential for gene activation by the virus. These results strongly suggest that types I and III IFN genes are regulated by a common mechanism.
Asunto(s)
Citocinas/genética , Regulación de la Expresión Génica , Interferón Tipo I/genética , Interleucinas/genética , Virosis/inmunología , Proteínas Adaptadoras Transductoras de Señales/fisiología , Animales , Sitios de Unión , Células Cultivadas , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/fisiología , Humanos , Factor 3 Regulador del Interferón/fisiología , Interferones , Ratones , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Receptores Inmunológicos , Transducción de Señal , Sitio de Iniciación de la Transcripción , Activación TranscripcionalRESUMEN
The taste buds of bovine fungiform papillae were studied by light and electron microscopy using both histological and immunohistochemical methods. The taste buds existed in the epithelium of the apical region of the papillae. By electron microscopy, two types of taste cells, namely type I and type II cells, could be classified according to the presence of dense-cored vesicles, the cytoplasmic density and the cell shape. Type I cells were thin, had an electron-dense cytoplasm containing dense-cored vesicles, and possessed long thick apical processes in the taste pore. Type II cells were thick, had an electron-lucent cytoplasm containing many electron-lucent vesicles, rather than dense-cored vesicles, and possessed microvilli in the taste pore. Immunohistochemical staining with an antiserum against gustducin was investigated by both light and electron microscopy using the avidin-biotin complex (ABC) method. Some, but not all, of the type II cells exhibited gustducin immunoreactivity, whereas none of the type I cells showed any immunoreactivity.