RESUMEN
Intrinsic immune responses autonomously inhibit viral replication and spread. One pathway that restricts viral infection in plants and insects is RNA interference (RNAi), which targets and degrades viral RNA to limit infection. To identify additional genes involved in intrinsic antiviral immunity, we screened Drosophila cells for modulators of viral infection using an RNAi library. We identified Ars2 as a key component of Drosophila antiviral immunity. Loss of Ars2 in cells, or in flies, increases susceptibility to RNA viruses. Consistent with its antiviral properties, we found that Ars2 physically interacts with Dcr-2, modulates its activity in vitro, and is required for siRNA-mediated silencing. Furthermore, we show that Ars2 plays an essential role in miRNA-mediated silencing, interacting with the Microprocessor and stabilizing pri-miRNAs. The identification of Ars2 as a player in these small RNA pathways provides new insight into the biogenesis of small RNAs that may be extended to other systems.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/genética , Drosophila/inmunología , Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Interferencia de ARN , Vesiculovirus/inmunología , Animales , Drosophila/virología , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , MicroARNs/genética , ARN Bicatenario/metabolismo , ARN Interferente Pequeño/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Vesiculovirus/genéticaRESUMEN
Intrinsic innate immune mechanisms are the first line of defense against pathogens and exist to control infection autonomously in infected cells. Here, we showed that autophagy, an intrinsic mechanism that can degrade cytoplasmic components, played a direct antiviral role against the mammalian viral pathogen vesicular stomatitis virus (VSV) in the model organism Drosophila. We found that the surface glycoprotein, VSV-G, was likely the pathogen-associated molecular pattern (PAMP) that initiated this cell-autonomous response. Once activated, autophagy decreased viral replication, and repression of autophagy led to increased viral replication and pathogenesis in cells and animals. Lastly, we showed that the antiviral response was controlled by the phosphatidylinositol 3-kinase (PI3K)-Akt-signaling pathway, which normally regulates autophagy in response to nutrient availability. Altogether, these data uncover an intrinsic antiviral program that links viral recognition to the evolutionarily conserved nutrient-signaling and autophagy pathways.
Asunto(s)
Autofagia/inmunología , Drosophila/inmunología , Vesiculovirus/fisiología , Animales , Células Cultivadas , Drosophila/virología , Técnica del Anticuerpo Fluorescente , Vesiculovirus/inmunologíaRESUMEN
A common application of fluorescent proteins is to label whole cells, but many RFPs are cytotoxic when used with standard high-level expression systems. We engineered a rapidly maturing tetrameric fluorescent protein called DsRed-Express2 that has minimal cytotoxicity. DsRed-Express2 exhibits strong and stable expression in bacterial and mammalian cells, and it outperforms other available RFPs with regard to photostability and phototoxicity.