RESUMEN
The discovery of RNA interference has led to the development of short interfering RNA (siRNA) screening, which has been widely used to study biological pathways. Here, we describe the development and validation of a system suitable to identify cellular genes involved in interferon A2 (IFNA2) promoter activation and interleukin (IL)-8 secretion downstream of MyD88. Forty genes were identified. Five genes were selected for further study. One gene, protein kinase, DNA-activated catalytic polypeptide (PRKDC), was confirmed to play a role in MyD88-induced IFNA2 activation and IL-8 secretion.
Asunto(s)
Proteína Quinasa Activada por ADN/metabolismo , Interferón-alfa/genética , Interleucina-8/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , Proteínas Nucleares/metabolismo , Supervivencia Celular , Proteína Quinasa Activada por ADN/genética , Biblioteca de Genes , Células HEK293 , Humanos , Proteínas Nucleares/genética , Regiones Promotoras Genéticas/genética , ARN Interferente Pequeño/genética , Regulación hacia Arriba/genéticaRESUMEN
The paramyxovirus Sendai (SV), is a well-established inducer of IFN-alphabeta gene expression. In this study we show that SV induces IFN-alphabeta gene expression normally in cells from mice with targeted deletions of the Toll-IL-1 resistance domain containing adapters MyD88, Mal, Toll/IL-1R domain-containing adaptor inducing IFN-beta (TRIF), and TRIF-related adaptor molecule TLR3, or the E3 ubiquitin ligase, TNFR-associated factor 6. This TLR-independent induction of IFN-alphabeta after SV infection is replication dependent and mediated by the RNA helicase, retinoic acid-inducible gene-I (RIG-I) and not the related family member, melanoma differentiation-associated gene 5. Furthermore, we characterize a RIG-I-like RNA helicase, Lgp2. In contrast to RIG-I or melanoma differentiation-associated gene 5, Lgp2 lacks signaling caspase recruitment and activation domains. Overexpression of Lgp2 inhibits SV and Newcastle disease virus signaling to IFN-stimulated regulatory element- and NF-kappaB-dependent pathways. Importantly, Lgp2 does not prevent TLR3 signaling. Like RIG-I, Lgp2 binds double-stranded, but not single-stranded, RNA. Quantitative PCR analysis demonstrates that Lgp2 is present in unstimulated cells at a lower level than RIG-I, although both helicases are induced to similar levels after virus infection. We propose that Lgp2 acts as a negative feedback regulator of antiviral signaling by sequestering dsRNA from RIG-I.
Asunto(s)
ARN Helicasas/fisiología , Receptores Toll-Like/fisiología , Transactivadores/fisiología , Replicación Viral/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Antígenos de Diferenciación/genética , Línea Celular , Humanos , Proteínas de Transporte de Membrana/genética , Ratones , Ratones Noqueados , Proteínas de la Mielina/genética , Proteínas Proteolipídicas Asociadas a Mielina y Linfocito , Factor 88 de Diferenciación Mieloide , Proteolípidos/genética , ARN Bicatenario/metabolismo , Receptores Inmunológicos/genética , Virus Sendai/fisiología , Transducción de Señal/fisiología , Factor 6 Asociado a Receptor de TNF/genética , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/metabolismo , Transactivadores/genéticaRESUMEN
Interferon regulatory factors (IRFs) are critical components of virus-induced immune activation and type I interferon regulation. IRF3 and IRF7 are activated in response to a variety of viruses or after engagement of Toll-like receptor (TLR) 3 and TLR4 by double-stranded RNA and lipopolysaccharide, respectively. The activation of IRF5, is much more restricted. Here we show that in contrast to IRF3 and IRF7, IRF5 is not a target of the TLR3 signaling pathway but is activated by TLR7 or TLR8 signaling. We also demonstrate that MyD88, interleukin 1 receptor-associated kinase 1, and tumor necrosis factor receptor-associated factor 6 are required for the activation of IRF5 and IRF7 in the TLR7 signaling pathway. Moreover, ectopic expression of IRF5 enabled type I interferon production in response to TLR7 signaling, whereas knockdown of IRF5 by small interfering RNA reduced type I interferon induction in response to the TLR7 ligand, R-848. IRF5 and IRF7, therefore, emerge from these studies as critical mediators of TLR7 signaling.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Glicoproteínas de Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Factores de Transcripción/fisiología , Proteínas Adaptadoras Transductoras de Señales , Antígenos de Diferenciación/metabolismo , Bioensayo , Línea Celular , Relación Dosis-Respuesta a Droga , Electroporación , Genes Reporteros , Glutatión Transferasa/metabolismo , Humanos , Factor 3 Regulador del Interferón , Factor 7 Regulador del Interferón , Factores Reguladores del Interferón , Interferón Tipo I/metabolismo , Ligandos , Lipopolisacáridos/metabolismo , Microscopía Confocal , Modelos Biológicos , Factor 88 de Diferenciación Mieloide , Fosforilación , Interferencia de ARN , ARN Bicatenario/metabolismo , ARN Interferente Pequeño/metabolismo , Receptores Inmunológicos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Receptor Toll-Like 3 , Receptor Toll-Like 4 , Receptor Toll-Like 7 , Receptor Toll-Like 8 , Receptores Toll-Like , Factores de Transcripción/metabolismo , TransfecciónRESUMEN
Microbial DNA sequences containing unmethylated CpG dinucleotides activate Toll-like receptor 9 (TLR9). We have found that TLR9 is localized to the endoplasmic reticulum (ER) of dendritic cells (DCs) and macrophages. Because there is no precedent for immune receptor signaling in the ER, we investigated how TLR9 is activated. We show that CpG DNA binds directly to TLR9 in ligand-binding studies. CpG DNA moves into early endosomes and is subsequently transported to a tubular lysosomal compartment. Concurrent with the movement of CpG DNA in cells, TLR9 redistributes from the ER to CpG DNA-containing structures, which also accumulate MyD88. Our data indicate a previously unknown mechanism of cellular activation involving the recruitment of TLR9 from the ER to sites of CpG DNA uptake, where signal transduction is initiated.
