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特性。在植物和无脊椎动物中，RNA病毒的复制触发病毒衍生的小干扰RNA（siRNA）的Dicer依赖性生物合成，以通过RNA干扰（RNAi）引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。我们已经获得了关键的机制的见解RNA为基础的抗病毒免疫使用Flock房子病毒感染的果蝇，这一直是一个强大的模型研究RNAi和先天免疫。我们最近报道了在培养的果蝇细胞中产生一类新的病毒衍生的小RNA，称为PIWI相互作用RNA（piRNA），其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，果蝇中的皮尔纳途径具有新的抗病毒功能，可抑制病毒垂直传播到后代。Ai 1将确定piRNA指导的抗病毒防御的遗传要求，并研究病毒piRNA的群体、生物发生和活性。目的2选择一种编码RNA干扰抑制因子（VSR）的节肢动物RNA病毒，验证在病毒RNA复制的早期阶段，抗病毒RNA干扰在哺乳动物细胞中被诱导的假说。我们发现，病毒的VSR缺陷突变体的感染在哺乳动物细胞中被有效抑制，并且与病毒SiRNA的产生有关，这些病毒SiRNA与果蝇响应Flock house病毒感染而产生的病毒SiRNA具有相同的关键特性。目的2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是否指导哺乳动物宿主细胞的特异性抗病毒防御。这些研究将可能开辟病毒piRNA功能研究的新领域，并建立一个研究病毒衍生小RNA的生物起源和抗病毒功能的哺乳动物系统。

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.