Genetic analysis of the Drosophila Response to Viral Infection

果蝇对病毒感染反应的遗传分析

• 批准号: 8678819
• 负责人: Jules A. Hoffmann
• 金额: $ 35.64万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-15 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8678819
• 关键词: Affinity; Animal Model; Anti-Bacterial Agents; Antifungal Agents; Antiviral Agents; Antiviral Response; Arboviruses; Avidin; Biotin; Boxing; Cell Communication; Cells; Cohort Studies; Collaborations; Complex; Cytotoxic T-Lymphocytes; DNA Resequencing; Dicer Pathway; Drosophila genus; Drosophila melanogaster; Genes; Genetic Screening; Genome; Goals; High-Throughput Nucleotide Sequencing; Host Defense; Host Defense Mechanism; Human; Image; Imaging Techniques; Immune response; Immunity; Insect Vectors; Instruction; Japanese American; Knowledge; Laboratories; Lead; Life; Light; Location; Mammals; Methods; Molecular; Molecular Biology; Mus; Mutagenesis; Mutate; Mutation; Natural Immunity; Pathway interactions; Phylogeny; Play; Postdoctoral Fellow; Proteomics; RNA Interference; RNA Viruses; Reading; Resistance; Role; Signal Transduction; Small RNA; Students; Testing; Toll-like receptors; Viral; Viral Load result; Virus; Virus Diseases; base; cost effective; deep sequencing; fighting; fly; genetic analysis; genome sequencing; helicase; human DICER1 protein; mutant; neutralizing antibody; novel; novel strategies; programs; protein complex; receptor; recombinant virus; research study; response; tandem mass spectrometry; viral RNA; working group

The objective of this project is to analyze host-defense mechanisms against viral infection in the model organism Drosophila melanogaster, and to do so in the light of concurrent studies of viral infection in mice, carried out by our colleagues in La Jolla and in Osaka. By studying the response of Drosophila to viral infection we will uncover evolutionarily conserved mechanisms of innate immunity that, together with the programs developed in the other participating laboratories, will help to unravel the essential aspects of the antiviral response in mammals. The project has two specific aims, which are based on the original results obtained by the laboratory in the past five years. The picture emerging from these studies is that of a complex, multi-faceted program of defense, essentially based on RNA interference and an inducible response, on which our knowledge is still fragmentary. In this new application, we aim to produce an integrative view of the sensing, signaling and effector mechanisms leading to resistance to virus infection in drosophila. The first aim is to conduct unbiased forward genetic screens to identify mutations that impair the antiviral response. The readout for the screens will be the viral load (using GFP-expressing recombinant viruses), and the survival to viral infection. We propose to take advantage of the technological breakthrough of SOLiD high throughput sequencing to rapidly identify the mutated genes in a cost effective manner by whole genome resequencing. The second aim is to characterize the role of three evolutionarily conserved pathways, namely RNAi, Toll and Imd, in antiviral defense in drosophila. The experimental strategy will be to identify by a powerful method combining the unparalleled affinity of avidin for biotin and the exquisite sensitivity of nanoLC-tandem mass spectrometry the molecules interacting with selected components of the RNA interference, Imd and Toll pathways. The composition of these complexes will be compared in cells either uninfected or infected by viruses replicating in different cytoplasmic locations. The function of the identified molecules will be characterized in cells and flies. In addition, we will study by live imaging in infected cells the interaction of the canonical molecules of the RNAi pathway (Dicer-2, R2D2 and AGO2) with viral RNAs.

该项目的目的是分析模型中针对病毒感染的宿主防御机制 黑腹果蝇，并根据小鼠病毒感染的同时研究， 由我们在拉霍亚和大阪的同事进行。通过研究果蝇对病毒的反应 感染，我们将揭示先天免疫的进化保守机制，连同 其他参与实验室开发的计划将有助于阐明该研究的基本方面 哺乳动物的抗病毒反应。 该项目有两个具体目标，这些目标基于实验室在 过去五年。这些研究显示出一个复杂的、多方面的计划 防御，主要基于 RNA 干扰和诱导反应，对此我们的知识仍然未知 零碎的。在这个新应用中，我们的目标是产生传感、信号和信号的综合视图。 导致果蝇抵抗病毒感染的效应机制。第一个目标是进行 无偏见的正向遗传筛查，以识别损害抗病毒反应的突变。读数为 筛选将是病毒载量（使用表达 GFP 的重组病毒）和病毒的存活率 感染。我们建议利用SOLiD高通量的技术突破 通过全基因组重测序以经济高效的方式快速识别突变基因。 第二个目标是表征三个进化保守途径的作用，即 RNAi、Toll 和 Imd，研究果蝇的抗病毒防御。实验策略将是通过强大的 该方法结合了亲和素对生物素无与伦比的亲和力和 nanoLC-tandem 的精湛灵敏度 质谱分析与 RNA 干扰、Imd 和选定成分相互作用的分子 收费通道。这些复合物的组成将在未感染或感染的细胞中进行比较 病毒在不同的细胞质位置复制。已识别分子的功能将是 以细胞和果蝇为特征。此外，我们将通过受感染细胞的实时成像来研究 RNAi 途径的经典分子（Dicer-2、R2D2 和 AGO2）与病毒 RNA。