Gene Silencing as an Antiviral Defense in Animal Cells

描述（由申请人提供）：拟议研究的长期目标是阐明宿主先天免疫反应和病毒反防御反应的机制。RNAi是指由小RNA引导的基因调控机制，从植物、无脊椎动物到人类都高度保守。在2002年，我们使用果蝇细胞培养模型提供了RNAi在动物中天然抗病毒作用的第一个直接证据。随后，我们发现正链RNA病毒复制也触发了蚊子和线虫的RNAi免疫，并建立了第一个由RNAi的dsRNA-siRNA途径介导的新病毒免疫的分子框架。最近的研究表明，RNAi在哺乳动物对病毒的反应中也起着重要作用。作为一种反防御，包括感染人类的病毒在内的病毒编码能够抑制RNAi的蛋白质，称为RNAi的病毒抑制子（VSR）。鸡舍病毒B2蛋白在防止病毒dsRNA切割成siRNA中的VSR活性对于感染是必需的，因为B2表达在RNAi缺陷型果蝇突变体的感染中变得不稳定。然而，在病毒感染期间RNAi抑制中的这种特定作用尚未在任何其他动物VSR中建立。目标1将确定是否需要主动RNAi抑制脊髓灰质炎样板球麻痹病毒的感染，并测试病毒在RNAi缺陷突变果蝇中的连续传代将导致其VSR基因内功能丧失突变的积累，因为消除了病毒免疫力。小分子RNA决定了RNAi机制的特异性，其来源揭示了RNAi诱导剂的身份。果蝇在不同的遗传途径中产生小干扰RNA（siRNA）、微小RNA（miRNA）和重复相关siRNA（rasiRNA）。目的2将克隆，测序，并调查病毒衍生的siRNA在感染果蝇中的生物起源和抗病毒活性，确定miRNA和/或rasiRNA通路是否也参与病毒免疫，并表征在全基因组筛选中鉴定的免疫新组分。此外，已经鉴定了靶向RNAi途径中的不同步骤的多种VSR。相反，很少有人知道，如果以及如何宿主生物体调节病毒VSR活动。目的3将测试的假设，丝氨酸磷酸化和赖氨酸泛素化，我们检测到的B2蛋白在感染的果蝇细胞中表达的代表一种新的宿主防御策略，以破坏VSR的磷酸化依赖的蛋白酶体途径。果蝇已成为阐明人类先天免疫和RNAi分子机制的有力模型。通过对果蝇对模型正链RNA病毒感染的免疫反应和病毒的反防御策略的研究，可能会为病毒与哺乳动物宿主之间的分子相互作用提供机理上的见解。

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to elucidate the mechanism of host innate immune responses and viral counter-defense responses. RNAi refers to the gene regulatory mechanism guided by small RNAs and is highly conserved from plants, invertebrates to humans. In 2002 we provided the first direct evidence for a natural antiviral role of RNAi in animals using a Drosophila cell culture model. Subsequently we showed that positive-strand RNA virus replication also triggers the RNAi immunity in mosquitoes and nematodes, and established the first molecular framework for the new viral immunity mediated by the dsRNA-siRNA pathway of RNAi in adult Drosophila. Recent studies from others indicate that RNAi also plays an important role in mammalian responses to viruses. As a counter-defense, viruses including those infecting humans encode proteins capable of RNAi suppression, referred to as viral suppressors of RNAi (VSR). The VSR activity of flock house virus B2 protein in preventing viral dsRNA from dicing into siRNAs is essential for infection because B2 expression becomes dispensable in infection of RNAi-defective Drosophila mutants. However, such a specific role in RNAi suppression during virus infection has not been established for any other animal VSR. Aim 1 will determine if active RNAi suppression is required for infection by the polio-like cricket paralysis virus, and test the hypothesis that serial passage of viruses in RNAi-defective mutant flies would result in the accumulation of loss-of-function mutations within their VSR genes because of elimination of the viral immunity. Small RNAs determine the specificity of RNAi mechanism and their origin reveals the identity of RNAi inducer. Drosophila produces small-interfering RNAs (siRNA), microRNAs (miRNA) and repeat- associated siRNAs (rasiRNA) in distinct genetic pathways. Aim 2 will clone, sequence, and investigate the biogenesis and antiviral activities of virus-derived siRNAs in infected Drosophila, determine if miRNA and/or rasiRNA pathways also participate in the viral immunity, and characterize new components of the immunity identified in a genome-wide screen. Furthermore, diverse VSRs that target distinct steps in the RNAi pathway have been identified. In contrast, little is known about if and how host organisms regulate the viral VSR activities. Aim 3 will test the hypothesis that serine phosphorylation and lysine ubiquitination we detected in the B2 protein expressed in infected Drosophila cells represent a new host defense strategy to destroy VSR in a phosphorylation-dependent proteasome pathway. Fruit fly has been a powerful model for elucidating the molecular mechanisms of both innate immunity and RNAi in humans. It is likely that the proposed studies on Drosophila immune responses to infection by model positive-strand RNA viruses and the viral counter- defensive strategies will provide mechanistic insights into the molecular interactions between viruses and mammalian hosts.