Gene Silencing as an Antiviral Defense in Animal Cells

描述（由申请人提供）：拟建研究的长期目标是阐明宿主先天免疫反应和病毒对抗防御反应的机制。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。2002年，我们利用果蝇细胞培养模型首次提供了RNAi在动物体内天然抗病毒作用的直接证据。随后，我们发现正链RNA病毒复制也能触发蚊子和线虫的RNAi免疫，并建立了RNAi dsRNA-siRNA通路介导的成虫新型病毒免疫的第一个分子框架。最近其他人的研究表明，RNAi在哺乳动物对病毒的反应中也起着重要作用。作为一种对抗防御，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的病毒抑制因子（VSR）。禽舍病毒B2蛋白在阻止病毒dsRNA切割成sirna中的VSR活性对于感染至关重要，因为在rnai缺陷的果蝇突变体感染中B2表达变得必不可少。然而，在病毒感染期间，这种特定的RNAi抑制作用尚未在任何其他动物VSR中得到证实。目的1将确定脊髓灰质炎样蟋蟀麻痹病毒的感染是否需要活性RNAi抑制，并验证病毒在RNAi缺陷突变果蝇中的连续传代将导致其VSR基因中由于病毒免疫的消除而积累功能丧失突变的假设。小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）。Aim 2将克隆、测序并研究受感染果蝇中病毒衍生sirna的生物发生和抗病毒活性，确定miRNA和/或rasiRNA途径是否也参与病毒免疫，并表征在全基因组筛选中鉴定出的免疫新组分。此外，已经确定了针对RNAi途径中不同步骤的多种VSRs。相比之下，宿主生物是否以及如何调节病毒的VSR活动却知之甚少。目的3将验证我们在受感染果蝇细胞中表达的B2蛋白中检测到的丝氨酸磷酸化和赖氨酸泛素化是一种新的宿主防御策略，可以在磷酸化依赖的蛋白酶体途径中破坏VSR。果蝇已成为阐明人类先天免疫和RNAi分子机制的有力模型。研究果蝇对模型正链RNA病毒感染的免疫反应和病毒的反防御策略，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制上的见解。

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.