Function and mechanism of the mammalian RNA interference response to virus infection

人类进化出了多种防御系统来对抗病毒病原体。这项建议旨在 RNA干扰(RNAi)介导的哺乳动物抗病毒免疫新机制的研究 在成年小鼠模型中的途径。先天免疫和获得性免疫机制已被广泛研究 特色化的。先天免疫系统在检测到病毒核酸后被激活，并被 主要由I型干扰素(干扰素)调节。适应性免疫系统在T和B的控制下 引导抗原特异性和持久的抗病毒免疫的淋巴细胞。真菌、植物、昆虫和 线虫宿主，病毒dsRNA由Dester处理成小干扰RNA(SiRNAs)，以指导有效的 以及通过保守的RNAi途径进行的特异性抗病毒防御。最近的研究表明，产生了 小鼠胚胎干细胞、分化的仓鼠、猴和人中丰富的病毒来源的siRNAs 细胞或乳鼠感染来自三个家族的四种正链或负链RNA病毒。 此外，三种病毒已经进化出不同的RNAi病毒抑制物(VSR)，具有相同的活性 抑制病毒siRNAs的生物发生和ArgAerte2的RNA切片活性具有抗病毒作用 老鼠和人类的细胞。这些发现使我们假设存在一种新的哺乳动物抗病毒药物 RNAi途径引导的免疫机制。然而，关于是否有很多争论 干扰素对抗病毒RNAi的反应是抑制的。目前还不清楚哺乳动物的病毒siRNA是否 从体内的反应可以指导特定的，Argavite2介导的靶向RNA的切割。vbl.使用 诺达村病毒作为正链RNA病毒的模型，我们将研究其在 在成年小鼠体内诱导和抑制哺乳动物抗病毒RNAi反应 干扰素系统有缺陷。拟议中的研究将首次揭示其性质、丰度 以及哺乳动物病毒siRNA在感染成年小鼠中的持久性，并表明哺乳动物是否 在体内感染期间，VSR抑制抗病毒RNAi和/或干扰素反应。我们将描述 两种方法制备的哺乳动物病毒siRNA在成年小鼠主动siRNA反应中的抗病毒活性 独立的方法。此外，我们将在成年小鼠身上开发一种条件基因敲除系统。 为了确定核心RNAi途径基因DICER和ArgAerte2在体内的抗病毒功能，它具有 由于哺乳动物在动物发育中的重要作用，到目前为止在哺乳动物身上还不可能做到这一点。调查结果： 拟议的研究将为新的哺乳动物抗病毒反应提供一个机制框架 体内和成年小鼠模型的抗病毒RNAi，从而促进对siRNA反应的研究 由医学上重要的RNA病毒病原体诱导，这些病原体是对人类健康的主要威胁。

A multitude of human defense systems have evolved to combat viral pathogens. This proposal aims to characterize a new mammalian antiviral immunity mechanism mediated by the RNA interference (RNAi) pathway in an adult mouse model. The innate and adaptive immunity mechanisms have been extensively characterized. The innate immune system is activated upon the detection of viral nucleic acids and is primarily regulated by type I interferons (IFN). The adaptive immune system is under the control of T and B lymphocytes that direct an antigen-specific and long-lasting antiviral immunity. In fungal, plant, insect and nematode hosts, viral dsRNA is processed by Dicer into small interfering RNAs (siRNAs) to direct a potent and specific antiviral defense by the conserved RNAi pathway. Recent studies have revealed production of abundant virus-derived siRNAs in mouse embryonic stem cells, differentiated hamster, monkey and human cells or suckling mice after infection with four positive- or negative-strand RNA viruses from three families. Moreover, three viruses have evolved distinct viral suppressors of RNAi (VSRs) with the same activity to inhibit the biogenesis of viral siRNAs and the RNA slicing activity of Argonaute2 is antiviral in cultured mouse and human cells. These findings lead us to hypothesize that there is a new mammalian antiviral immunity mechanism directed by the RNAi pathway. However, there has been much debate about whether the IFN response is inhibitory to antiviral RNAi. It also remains unknown whether mammalian viral siRNAs from an in vivo response can guide specific, Argonaute2-mediated cleavages of the targeted RNAs. Using Nodamura virus as a model for positive-strand RNA viruses, we will investigate the mechanisms in the induction and suppression of mammalian antiviral RNAi response in adult mice that are either active or defective in the IFN system. The proposed studies will reveal for the first time the properties, abundance and persistence of mammalian viral siRNAs in the infected adult mouse and show whether mammalian VSRs suppress antiviral RNAi and/or the IFN response during in vivo infection. We will characterize the antiviral activity of mammalian viral siRNAs made during active siRNA response of adult mice by two independent approaches. Furthermore, we will develop a conditional gene knockout system in adult mice for defining the in vivo antiviral function of the core RNAi pathway genes Dicer and Argonaute2, which has not been possible thus far in mammals due to their essential roles in animal development. Results from the proposed studies will provide both a mechanistic framework for the new mammalian antiviral response in vivo and an adult mouse model for antiviral RNAi, thereby facilitating investigations on the siRNA response induced by medically important RNA virus pathogens that are a major threat to human health.