Gene Silencing as an Antiviral Defense in Animal Cells

描述（由申请人提供）：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，并表现出与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dicer依赖性病毒衍生的小干扰RNA （sirna）的生物发生，通过RNA干扰（RNAi）指导特异性抗病毒免疫。然而，没有直接证据支持siRNA途径在哺乳动物中具有类似的抗病毒作用。我们利用果蝇的Flock house病毒感染获得了基于rna的抗病毒免疫机制的关键见解，这已经成为RNAi和先天免疫研究的有力模型。我们最近报道了在培养的果蝇细胞中产生的一类新的病毒衍生小rna，称为PIWI-相互作用rna (piRNAs)，它在果蝇和哺乳动物中独立于Dicer产生，以沉默种系中的转座因子。进一步的遗传研究表明，果蝇piRNA通路具有一种新的抗病毒功能，可以抑制病毒向后代的垂直传播。Ai 1将确定pirna定向抗病毒防御的遗传需求，并研究病毒pirna的种群、生物发生和活性。目的2选择一种节肢动物传播的哺乳动物RNA病毒，该病毒编码RNAi病毒抑制因子（VSR），以检验在病毒RNA复制的早期阶段在哺乳动物细胞中诱导抗病毒RNAi的假设。我们发现，在哺乳动物细胞中，病毒的vsr缺陷突变体的感染被有效抑制，并且与病毒sirna的产生有关，这些sirna与果蝇响应Flock house病毒感染时产生的病毒sirna具有相同的关键特性。目的2将研究病毒sirna在哺乳动物细胞中的生物发生，并确定检测到的病毒sirna是否直接在哺乳动物宿主细胞中进行特异性抗病毒防御。这些研究将有可能为病毒pirna功能的研究开辟一个新的领域，并建立一个哺乳动物系统来研究病毒源性小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.