Gene Silencing as an Antiviral Defense in Animal Cells

描述(申请人提供)：RNA病毒包括重要的人类病原体，如丙型肝炎病毒、西尼罗河病毒和登革热病毒，并显示出与DNA病毒不同的遗传和免疫学特性。在植物和无脊椎动物中，RNA病毒的复制触发病毒来源的小干扰RNAs(SiRNAs)的依赖的生物发生，通过RNA干扰(RNAi)指导特异性的抗病毒免疫。然而，没有直接证据支持在哺乳动物中siRNA途径具有类似的抗病毒作用。利用果蝇的鸡舍病毒感染果蝇，我们已经获得了基于RNA的抗病毒免疫的关键机制见解，这已经成为研究RNAi和天然免疫的有力模型。我们最近报道了在培养的果蝇细胞中产生一种新型的病毒衍生的小RNA，称为PIWI相互作用RNAs(PiRNAs)，它是在果蝇和哺乳动物中独立于Dester产生的，用于沉默生殖系中的转座元件。进一步的遗传学研究表明，在果蝇中，piRNA途径具有一种新的抗病毒功能，可以抑制病毒垂直传播到后代。AI-1将确定piRNA导向的抗病毒防御的遗传要求，并研究病毒piRNAs的种群、生物发生和活性。目的2选择一种节肢动物传播的编码RNAi病毒抑制因子(VSR)的哺乳动物RNA病毒，验证在病毒RNA复制的早期阶段在哺乳动物细胞中诱导抗病毒RNAi的假说。我们发现，该病毒VSR缺陷突变体的感染在哺乳动物细胞中得到有效抑制，并与病毒siRNAs的产生有关，该病毒siRNAs的产生与果蝇对鸡舍病毒感染的病毒siRNAs的关键特性相同。目的2研究病毒siRNAs在哺乳动物细胞中的生物发生，并确定检测到的病毒siRNAs是否直接在哺乳动物宿主细胞中产生特异性的抗病毒防御。这些研究可能会开辟病毒piRNAs功能研究的新领域，并建立一个哺乳动物系统来研究病毒来源的小RNA的生物发生和抗病毒功能。

DESCRIPTION (provided by applicant): RNA viruses include important human pathogens such as hepatitis C, West Nile and dengue viruses and exhibit distinct genetic and immunological properties from DNA viruses. In plants and invertebrates, replication of RNA viruses triggers Dicer-dependent biogenesis of virus-derived small interfering RNAs (siRNAs) to guide specific antiviral immunity by RNA interference (RNAi). However, direct evidence supporting a similar antiviral role for the siRNA pathway in mammals is not available. We have obtained key mechanistic insights to the RNA-based antiviral immunity using Flock house virus infection of the fruit fly Drosophila melanogaster, which has been a powerful model for studies on both RNAi and innate immunity. We have recently reported production of a novel class of virus-derived small RNAs in cultured Drosophila cells known as PIWI- interacting RNAs (piRNAs), which are produced independently of Dicer in flies and mammals to silence transposable elements in the germline. Further genetic studies indicate a novel antiviral function for the piRNA pathway in the fruit fly that inhibits virus vertical transmission to the progeny. Ai 1 will determine the genetic requirements of the piRNA-directed antiviral defense and investigate the population, biogenesis and activity of viral piRNAs. Aim 2 chose an arthropod-borne mammalian RNA virus that encodes a viral suppressor of RNAi (VSR) to examine the hypothesis that antiviral RNAi is induced in the mammalian cells during the early stages of viral RNA replication. We found that infection of a VSR-deficient mutant of the virus was effectively suppressed in mammalian cells and was associated with production of viral siRNAs that share key properties with the viral siRNAs produced in the fruit fly in response to Flock house virus infection. Aim 2 will investigate the biogenesis of viral siRNAs in mammalian cells and determine if the detected viral siRNAs direct specific antiviral defense in the mammalian host cells. These studies will likely open up a new area of research on the function of viral piRNAs and establish a mammalian system to investigate the biogenesis and antiviral function of virus-derived small RNAs.