Molecular analysis of nodaviral suppression of RNA interference

术语“RNA干扰”（RNAi）是指在几乎所有的哺乳动物中都是可操作的基因失活途径。 真核细胞它开始于长dsRNA被切割成21-25个核苷酸（nt）的片段（siRNA）， RNA酶III样核酸酶Dicer，并且通常导致携带RNA的mRNA的降解或失活。 与siRNA序列互补。RNA沉默的自然作用与 调节基因表达的内源性RNA（miRNAs）的加工和防御机制 对抗转座子和病毒。反过来，病毒通过编码蛋白质， 抑制RNA沉默。已经鉴定了十几种病毒编码的抑制蛋白， 大多数是（+）-正义RNA病毒。该提案概述了研究昆虫如何 Nodavirus Flock House病毒抑制受感染宿主中的RNAi。FHV编码一种强大的RNAi抑制剂 称为B2。B2是一种小RNA结合蛋白，其特异性地但不依赖于序列地结合长链RNA。 和短的dsRNA。它抑制不同物种的RNAi，包括昆虫，植物，哺乳动物和蠕虫。的 蛋白质的生物化学和结构特征，但其确切的作用机制，特别是 在FHV感染周期中，仍然未知。建议的项目有三个具体目标：（1） 鉴定在FHV感染者中诱导和抑制RNAi关键的病毒和宿主决定因子 果蝇细胞;（2）测试B2在FHV感染期间抑制RNAi的机制可能性， (3)建立反式显性B2变异体，以开发FHV作为昆虫中外源基因表达的载体。 根据本提案中提供的初步数据，我们认为我们处于独特的地位， 描绘导致抑制RNA干扰的分子事件，从而提供对RNA干扰的见解 RNAi途径的调控。从长远来看，我们希望将FHV/B2系统作为一个重要的 研究果蝇中RNA沉默的自然作用的工具，并开发新的方法， 转基因在多种昆虫中的表达。

The term "RNA interference" (RNAi) refers to a gene inactivation pathway that is operational in almost all eukaryotic cells. It begins with cleavage of long dsRNAs into 21-25 nucleotide (nt) segments (siRNA) by the RNaselll-like nuclease Dicer and typically leads to degradation or inactivation of mRNAs that bear sequence complementarity to the siRNAs. The natural role of RNA silencing has been linked to the processing of endogenous RNAs that regulate gene expression (miRNAs) and a defense mechanism against transposons and viruses. Viruses in turn have developed counter-defenses by encoding proteins that suppress RNA silencing. More than a dozen virus-encoded suppressor proteins have been identified, most of them in (+)-sense RNA viruses. This proposal outlines experiments to investigate how the insect nodavirus Flock House virus suppresses RNAi in infected hosts. FHV encodes a powerful RNAi inhibitor called B2. B2 is a small RNA binding protein that specifically, but sequence independently, binds to long and short dsRNAs. It inhibits RNAi in diverse species including insects, plants, mammals and worms. The protein has been characterized biochemically and structurally but its exact mechanism of action, particularly during the FHV infection cycle, remains unknown. The proposed project has three specific aims: (1) to identify viral and host determinants critical for induction and suppression of RNAi in FHV-infected Drosophila cells; (2) to test mechanistic possibilities by which B2 suppresses RNAi during FHV infection and (3) to create trans-dominant B2 variants to develop FHV as a vector for foreign gene expression in insects. Based on the preliminary data presented in this proposal we believe that we are in a unique position to delineate the molecular events that lead to suppression of RNAi and thereby provide insights into the regulation of the RNAi pathway. In the longterm, we hope to develop the FHV/B2 system as an important tool to study the natural role of RNA silencing in Drosophila and to develop novel approaches for sustained transgene expression in a variety of insects.