Genetic Analysis of Resistance to Viral Infection

抗病毒感染的遗传分析

• 批准号: 8338920
• 负责人: BRUCE A BEUTLER
• 金额: $ 272.54万
• 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 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8338920
• 关键词: Adaptor Protein Complex 3; Adaptor Signaling Protein; Address; Affect; Age; Alleles; Antiviral Agents; Antiviral Response; Area; Autophagocytosis; Biochemical; Bunyavirus Infections; CRSP3 gene; Categories; Cell physiology; Cells; Cellular Stress; Cessation of life; Child; Communication; Communities; Competence; Complex; Computer Simulation; Cryopreservation; Cytomegalovirus; Cytomegalovirus Infections; DNA; Dendritic Cells; Derivation procedure; Drosophila genus; Elements; Embryo; Endosomes; Extended Family; Fibroblasts; Funding; Gene Expression; Gene Targeting; Generations; Genes; Genetic; Genetic Materials; Genetic Techniques; Genomics; Goals; Grant; Gray unit of radiation dose; HIV; Health; Herpesviridae Infections; Homologous Gene; Host Defense; Host resistance; Human; I-kappa B Proteins; Image; Immune; Immune response; Immune system; Immunity; Individual; Infection; Insecta; Institutes; Interferon Type I; Interferons; Investigation; Knock-out; Knockout Mice; Learning; Life; Lipopolysaccharides; Mammalian Cell; Mammals; Maps; Measures; Mediating; Medical center; Medicine; Membrane Protein Traffic; Memory; Methods; Microbe; Modeling; Molecular; Molecular Target; Movement; Murid herpesvirus 1; Mus; Mutagenesis; Mutation; Natural Immunity; Natural Killer Cells; Natural regeneration; Nucleic Acids; Organism; Participant; Pathway interactions; Pharmaceutical Preparations; Physiology; Pluripotent Stem Cells; Population; Post-Transcriptional Regulation; Post-Translational Protein Processing; Predisposition; Principal Investigator; Process; Productivity; Proliferating; Proteins; RNA; RNA Interference; Reaction; Reagent; Recording of previous events; Regulation; Research; Resistance; Resistance to infection; Resolution; Rift Valley fever virus; Risk; Role; Screening procedure; Signal Pathway; Signal Transduction; Small RNA; Smallpox; Speed; Sting Injury; Structural Models; Systemic Lupus Erythematosus; TLR7 gene; Talents; Technology; Therapeutic; Time; Toll-like receptors; Transcriptional Regulation; Transmembrane Transport; Universities; Ursidae Family; Vaccines; Viral; Viral Genes; Viral Pathogenesis; Virus; Virus Diseases; Work; Writing; Zinc Fingers; base; combat; coping; design; expression cloning; fly; gene induction; gene interaction; genetic analysis; genome sequencing; human disease; human mortality; induced pluripotent stem cell; member; mutant; new technology; pathogen; positional cloning; pressure; prevent; programs; receptor; resistance mechanism; response; sensor; simulation; small molecule; sound; stem cell technology; transcription factor; viral DNA; viral RNA

DESCRIPTION (provided by applicant): The present application extends a successful multifaceted investigation of host resistance to viral infection. The strengths of our approach include: 1) an unbiased component based on mutagenesis combined with a hypothesis-driven component; 2) the study of distantly related organisms (mice and Drosophila) to appreciate which elements of defense are conserved; 3) the embrace of new and powerful methods to support our efforts. In our work to date, we have collectively identified new sensors (e.g., LGP2; DICER-2) necessary for activation of antiviral defenses, and delineated pathways of response to viruses, both at a biochemical level and in terms of communication between cells. We have identified previously unknown molecular participants in host defense. Among these are channel proteins (SLC15A4; KCNJ8/SUR2), transcription factors (IKB;AKIRIN2), proteins concerned with membrane trafficking or organellar mobility (AP3B1; STING; TR1M56; ATG9A; UNC93B), cell stress (SLFN2), post-translational modification (TRIM56; TR1M23), and endosome physiology (SLC15A4). Some of these proteins are members of extended families and may open the way to broad new models of host defense. Others highlight the importance of intermediary steps in host defense (e.g., the movement of molecules within cells) in a way that has not been considered before. Each participating group (Dallas, Osaka, and Strasbourg) has its special talents, and these have been combined to take us beyond genetics per se, incorporating new technologies that will accelerate the discovery of essential elements of the host defense apparatus. We recognize that it is not enough to possess a list of parts to understand how a machine operates. As new proteins are shown to be essential for particular aspects of host defense, we will establish how they interact with one another and/or other proteins to support resistance; how they catalyze particular reactions within cells, and how they drive or suppress the expression of genes in what we see as a highly dynamic process. We view the continuation of this POl as an opportunity to build upon an approach with established productivity: one that has generated new molecules, concepts, and reagents for use by the scientific community as a whole. The POl has been, and will continue to be, highly collaborative in the exchange of methods, genetic materials, and most importantly, ideas, ultimately derived from genetics. PUBLIC HEALTH RELEVANCE: Historically, viruses have been responsible for more death among humans than any other single cause, and still constitute a serious threat to mankind. We will use strong genetic techniques to study both host resistance mechanisms (innate immunity to viral infection), and the essential requirements that viruses have in order to cause infection. Basic scientific discovery in these areas will pave the way to the design of effective anti-viral drugs and vaccines, and has the highest relevance to human health. Project 1: Forward genetic analysis of Mammalian Resistance to Viral Infection Project 1 Leader (PL): Bruce Beutler DESCRIPTION (provided by applicant): We will extend our successful forward genetic analysis of susceptibility to mouse cytomegalovirus (MCMV) infection, and institute a new screen for cell-autonomous resistance to Rift Valley Fever Virus (RVFV). These studies will build upon technological advances that permit us to find mutations faster than ever before, using massively parallel sequencing, both by itself and in combination with inducible pluripotent stem cell (IPS) technology. We have found that it is possible to screen mutagenized MEFs from reprogrammable C57BL/6J mice for resistance to RVFV ex vivo, to identify resistant clones, to use these clones to regenerate live mice, and at the same time, detect most of the mutations in these cells by sequencing genomic DNA. We have determined, based on in silico simulations, that compound heterozygous null alleles at almost all loci will result from mutagenesis on the scale we contemplate. As such, we hope to enrich our understanding of what it takes to combat a model herpesvirus infection with strong relevance to a human disease (HCMV infection), and also, to determine the critical host proteins necessary for a Category A pathogen (RVFV) to proliferate in mammalian cells. This work, pursued in depth, will inform us of molecular targets for the treatment of bunyavirus infections in general. In a third, circumscribed specific aim, we will analyze a new mutation, identified in screening that abolishes the responses of plasmacytoid dendritic cells (PDCs) to nucleic acids. This mutation, called feeble, affects a channel protein that acts in conjunction with the adaptor protein 3 (AP3) complex to condition endosomes in PDCs (but not other cells), making them competent to signal via TLRs. Based on our studies to date, we infer the existence of an ARF1-->AP3 -->SIc45a-->TLR7/9 pathway essential for the support of type I interferon gene induction in PDCs. The further elucidation of this pathway will deepen understanding of PDC function, and the role of PDC-derived interferon both in infection and in pathological settings such as systemic lupus erythematosus.

描述(由申请人提供)：本申请扩展了对宿主对病毒感染的抵抗力的成功的多方面调查。我们方法的优势包括：1)基于突变的无偏见组件与假说驱动的组件相结合；2)研究远亲生物(小鼠和果蝇)，以了解哪些防御元件是保守的；3)采用新的强大的方法来支持我们的努力。到目前为止，在我们的工作中，我们已经集体确定了激活抗病毒防御所需的新传感器(例如，LGP2；DICER-2)，并描绘了在生化水平和细胞间通信方面对病毒做出反应的途径。我们已经确定了以前未知的分子参与者 在宿主防御中。其中包括通道蛋白(SLC15A4；KCNJ8/SUR2)、转录因子(IKB；AKIRIN2)、与膜运输或细胞器移动有关的蛋白(AP3B1；STING；TR1M56；ATG9A；UNC93B)、细胞应激(SLFN2)、翻译后修饰(TRIM56；TR1M23)和内体生理学(SLC15A4)。其中一些蛋白质是大家庭的成员，可能会为更广泛的新寄主防御模式开辟道路。其他人则强调了宿主防御的中间步骤(例如，分子在细胞内的运动)的重要性，这是以前从未考虑过的。每个参与小组(达拉斯、大阪和斯特拉斯堡)都有自己的特殊才能，这些天赋结合在一起，使我们超越了遗传学本身，结合了新技术，将加速发现宿主防御机构的基本要素。我们认识到，仅仅拥有一份部件清单是不够的，以了解机器是如何运行的。随着新的蛋白质被证明对宿主防御的特定方面至关重要，我们将确定它们如何相互作用和/或其他蛋白质来支持抗性；它们如何在细胞内催化特定的反应，以及它们如何在我们认为是一个高度动态的过程中驱动或抑制基因的表达。我们认为，这一POL的延续是一个机会，可以在现有生产力的基础上建立一种方法：一种产生了供整个科学界使用的新分子、概念和试剂的方法。波兰人一直是，并将继续在交流方法、遗传物质，以及最重要的、最终源于遗传学的想法方面进行高度合作。 公共卫生相关性：从历史上看，病毒在人类中造成的死亡比任何其他单一原因都要多，并且仍然对人类构成严重威胁。我们将使用强大的基因技术来研究宿主抵抗机制(对病毒感染的先天免疫)，以及病毒引起感染的基本要求。这些领域的基础科学发现将为设计有效的抗病毒药物和疫苗铺平道路，与人类健康具有最高的相关性。 项目1：哺乳动物对病毒感染抵抗力的正向遗传分析 项目1负责人(PL)：Bruce Beutler 描述(申请人提供)：我们将扩展我们成功的对小鼠巨细胞病毒(MCMV)感染易感性的正向遗传分析，并建立一种新的细胞自主抵抗裂谷热病毒(RVFV)的筛查。这些研究将建立在技术进步的基础上，使我们能够比以往任何时候都更快地发现突变，使用大规模并行测序，既可以单独进行，也可以与可诱导的多能干细胞(IPS)技术相结合。我们已经发现，从可重编程的C57BL/6J小鼠中筛选诱变的MEF体外对RVFV的抗性是可能的，识别抗性克隆，使用这些克隆再生活小鼠，同时通过测序基因组DNA来检测这些细胞中的大多数突变。在电子计算机模拟的基础上，我们已经确定，几乎所有基因座上的复合杂合零等位基因都将导致我们所考虑的规模的突变。因此，我们希望丰富我们对如何对抗与人类疾病(HCMV感染)密切相关的模型疱疹病毒感染的理解，并确定A类病原体(RVFV)在哺乳动物细胞中增殖所需的关键宿主蛋白。这项深入开展的工作将使我们了解治疗布尼亚病毒感染的一般分子靶点。在第三个有限制的特定目标中，我们将分析一种新的突变，该突变是在筛查中发现的，它取消了浆细胞样树突状细胞(PDC)对核酸的反应。这种突变被称为弱突变，它影响一种通道蛋白，该通道蛋白与适配器蛋白3(AP3)复合体一起作用，调节PDCs(但不是其他细胞)中的内小体，使它们能够通过TLR发出信号。根据我们迄今的研究，我们推测存在一条ARF1--&gt；AP3--&gt；SIc45a--&gt；TLR7/9通路，对于支持PDCs中I型干扰素基因的诱导是必不可少的。对这一途径的进一步阐明将加深对PDC功能的理解，以及PDC衍生的干扰素在感染和系统性红斑狼疮等病理环境中的作用。