RESUMEN
Cellular messenger RNA (mRNA) of higher eukaryotes and many viral RNAs are methylated at the N-7 and 2'-O positions of the 5' guanosine cap by specific nuclear and cytoplasmic methyltransferases (MTases), respectively. Whereas N-7 methylation is essential for RNA translation and stability, the function of 2'-O methylation has remained uncertain since its discovery 35 years ago. Here we show that a West Nile virus (WNV) mutant (E218A) that lacks 2'-O MTase activity was attenuated in wild-type primary cells and mice but was pathogenic in the absence of type I interferon (IFN) signalling. 2'-O methylation of viral RNA did not affect IFN induction in WNV-infected fibroblasts but instead modulated the antiviral effects of IFN-induced proteins with tetratricopeptide repeats (IFIT), which are interferon-stimulated genes (ISGs) implicated in regulation of protein translation. Poxvirus and coronavirus mutants that lacked 2'-O MTase activity similarly showed enhanced sensitivity to the antiviral actions of IFN and, specifically, IFIT proteins. Our results demonstrate that the 2'-O methylation of the 5' cap of viral RNA functions to subvert innate host antiviral responses through escape of IFIT-mediated suppression, and suggest an evolutionary explanation for 2'-O methylation of cellular mRNA: to distinguish self from non-self RNA. Differential methylation of cytoplasmic RNA probably serves as an example for pattern recognition and restriction of propagation of foreign viral RNA in host cells.
Asunto(s)
Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/inmunología , Interferones/inmunología , Proteínas/metabolismo , Caperuzas de ARN/metabolismo , ARN Viral/metabolismo , Células 3T3 , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas Reguladoras de la Apoptosis , Proteínas Portadoras/genética , Células Cultivadas , Coronavirus/enzimología , Coronavirus/genética , Coronavirus/inmunología , Coronavirus/fisiología , Fibroblastos , Regulación de la Expresión Génica/genética , Humanos , Inmunidad Innata/genética , Interferones/deficiencia , Interferones/genética , Metilación , Metiltransferasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Genéticos , Modelos Inmunológicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Poxviridae/enzimología , Poxviridae/genética , Poxviridae/inmunología , Poxviridae/fisiología , Biosíntesis de Proteínas/inmunología , Proteínas/genética , Caperuzas de ARN/genética , Caperuzas de ARN/inmunología , ARN Viral/genética , ARN Viral/inmunología , Proteínas de Unión al ARN , Receptor de Interferón alfa y beta/deficiencia , Receptor de Interferón alfa y beta/genética , Tasa de Supervivencia , Replicación Viral , Virus del Nilo Occidental/enzimología , Virus del Nilo Occidental/genética , Virus del Nilo Occidental/inmunología , Virus del Nilo Occidental/fisiologíaAsunto(s)
Virus de la Bronquitis Infecciosa/metabolismo , Proteínas del Envoltorio Viral/fisiología , Animales , Chlorocebus aethiops , ADN Complementario/metabolismo , Modelos Biológicos , Estructura Terciaria de Proteína , Factores de Tiempo , Células Vero , Proteínas del Envoltorio Viral/química , Replicación ViralRESUMEN
Coronavirus spike (S) proteins are responsible for binding and fusion with target cells and thus play an essential role in virus infection. Recently, we identified a dilysine endoplasmic reticulum (ER) retrieval signal and a tyrosine-based endocytosis signal in the cytoplasmic tail of the S protein of infectious bronchitis virus (IBV). Here, an infectious cDNA clone of IBV was used to address the importance of the S protein trafficking signals to virus infection. We constructed infectious cDNA clones lacking the ER retrieval signal, the endocytosis signal, or both. The virus lacking the ER retrieval signal was viable. However, this virus had a growth defect at late times postinfection and produced larger plaques than IBV. Further analysis confirmed that the mutant S protein trafficked though the secretory pathway faster than wild-type S protein. A more dramatic phenotype was obtained when the endocytosis signal was mutated. Recombinant viruses lacking the endocytosis signal (in combination with a mutated dilysine signal or alone) could not be recovered, even though transient syncytia were formed in transfected cells. Our results suggest that the endocytosis signal of IBV S is essential for productive virus infection.