The Mechanisms of IFITM-Mediated Restriction

IFITM 介导的限制机制

• 批准号: 8544970
• 负责人: I-Chueh Huang
• 金额: $ 24万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8544970
• 关键词: Affinity; Animals; Autophagocytosis; Biochemical; Biological Response Modifiers; Cellular Membrane; Clinical; Collaborations; Communication; Data; Dengue Virus; Development Plans; Doctor of Medicine; Doctor of Philosophy; Dominant-Negative Mutation; Ebola virus; Emerging Communicable Diseases; Environment; Family; Filovirus; Flavivirus; Foundations; Frankfurt-Marburg Syndrome Virus; Genes; Genetic Polymorphism; Glycoproteins; Goals; Grant; Human; IFITM1 gene; Immune; Immune response; In Vitro; Infection; Infection prevention; Influenza A virus; Institutes; Integral Membrane Protein; Integration Host Factors; Interferons; Knockout Mice; Knowledge; Laboratories; Leadership; Libraries; Life; Manuscripts; Measures; Mediating; Mediator of activation protein; Mentors; Methods; Molecular; Monitor; Mus; Natural Immunity; New England; Orthologous Gene; Pathway interactions; Phase; Phenotype; Preparation; Primates; Process; Protein Family; Protein S; Proteins; RNA Interference; Regulation; Research; Research Personnel; Research Proposals; Resources; Role; SARS coronavirus; Scientist; Site; Staging; Symptoms; Taiwan; Training; Training Programs; Universities; Variant; Viral; Virus; Virus Diseases; Virus Replication; West Nile virus; Writing; adaptive immunity; base; biodefense; career; career development; cellular imaging; cellular targeting; cofactor; experience; improved; in vivo; medical schools; new technology; novel; overexpression; particle; pathogen; programs; skills; small hairpin RNA; trafficking

PROJECT SUMMARY Candidate. My immediate goal is to develop the skills and expertise essential for me to become a successful independent investigator. My long-term goal is to contribute to a comprehensive understanding of the innate immune control of viral infections, and to use this knowledge to help create novel therapies for viral diseases. To achieve these goals, I will use my background as a trained clinician and a developed research scientist. I acquired my M.D. degree from National Taiwan University in 2000, finished my clinical training in 2004, and received my Ph.D. degree from Harvard University in 2008. My Ph.D. dissertation focused the enzymatic regulation of the entry processes of influenza A virus (IAV) and SARS coronavirus. These studies allowed me to develop an RNAi screen with Dr. Stephen Elledge for factors that modulate IAV replication. This screen identified a family of interferon-inducible transmembrane (IFITM) proteins critical to the interferon-mediated control of several pathogenic viruses, including IAV, dengue virus and West Nile virus. The study of the activities and mechanisms of these important proteins is the basis of my research proposal and my scientific goals in the next several years. Environment and Career Development Plan. My development plan focuses on three directions that will most strengthen my abilities as a successful independent investigator. (1) I will enhance my intellectual background in innate immunity and my experience in novel technologies important to my scientific goals. To do so, I will access the many seminars, classes and other resources available to me at the New England Primate Research Center (NEPRC), Harvard Medical School, the New England Regional Center of Excellence/Biodefense and Emerging Infectious Diseases (NERCE/BEID), and the Broad Institute of Harvard and MIT. (2) I will further develop experience necessary for conducting independent animal studies. Here I will rely on the exceptional expertise and formal training provided by the NEPRC. (3) I will improve my communication, manuscript preparation, grant-writing, lab management, and mentoring skills. These skills will be developed through formal classes provided by Harvard University, by participation in the multiple undergraduate and graduate training programs, and through direct experience with the guidance of my mentor and the scientific leadership at the NEPRC. Research Program. Our previous studies indicate that the IFITM proteins are critical mediators of the immune control of IAV and several other human pathogens. They are unique as viral restriction factors because they prevent infection by inhibiting viral entry. Our overall goal is to understand comprehensively the activities and mechanisms of these proteins. These studies are organized into three specific aims. Aim 1 characterizes these proteins biochemically and functionally, using a range of established methods. We will identify the molecular determinants of their differential restriction activities, determine the range of viruses restricted, and describe in vitro the phenotypes of known human IFITM polymorphisms. Aim 2 seeks to understand the mechanism by which IFITM proteins restrict viral entry. Here we will use live-cell imaging to localize the site of restriction, and affinity- and shRNA-based approaches to identify cellular targets and cofactors of IFITM- mediated restriction. Aim 3 evaluates the in vivo contribution of IFITM proteins to the control of IAV, using two sets of knockout mice, lacking either the Ifitm3 gene alone or five Ifitm genes. These mice will be challenged with infectious IAV in the presence and absence of type I and II interferons, monitored for immune responses and clinical symptoms, and characterized post-mortem for pathological differences. Aim 1 and part of Aim 2 will be accomplished during the mentored (K99) phase of this proposal. The remainder of Aim 2 and all of Aim 3 will be accomplished during the independent (R00) phase. Summary. My goal is to develop a career as an independent investigator in innate immunity. My recent studies of the IFITM proteins will be the initial focus of these efforts. I am situated in a highly supportive and intellectually rich environment in which I can pursue my scientific and career development goals.

项目摘要 候选人我的近期目标是发展技能和专业知识，使我成为一个成功的 独立调查员我的长期目标是为全面了解先天性 病毒感染的免疫控制，并利用这些知识来帮助创造病毒性疾病的新疗法。 为了实现这些目标，我将利用我的背景作为一个训练有素的临床医生和发达的研究科学家。我 获得了医学博士学位2000年毕业于国立台湾大学，2004年完成临床培训， 获得了博士学位2008年毕业于哈佛大学。我的博士本论文重点研究了酶促 调节甲型流感病毒（IAV）和SARS冠状病毒的进入过程。这些研究让我 与Stephen Elledge博士一起开发一种RNAi筛选，以寻找调节IAV复制的因子。此屏幕 鉴定了一个干扰素诱导的跨膜蛋白家族（IFITM），该家族对干扰素介导的 控制几种致病病毒，包括IAV、登革热病毒和西尼罗河病毒。的研究 这些重要蛋白质的活性和机制是我的研究计划和我的科学研究的基础。 未来几年的目标。 环境和职业发展计划。我的发展计划集中在三个方向， 加强我作为一名成功的独立调查员的能力(1)我会加强我的知识背景 先天免疫和我在新技术方面的经验对我的科学目标很重要。为此，我将 访问许多研讨会，课程和其他资源提供给我在新英格兰灵长类 研究中心（NEPRC），哈佛医学院，新英格兰区域中心， 卓越/生物防御和新兴传染病（NERCE/BEID）和哈佛布罗德研究所 和麻省理工学院(2)我将进一步积累进行独立动物研究所需的经验。下面笔者就 依靠NEPRC提供的特殊专业知识和正式培训。(3)我会提高我的 沟通，手稿准备，赠款写作，实验室管理和指导技能。这些技能将 通过哈佛大学提供的正式课程， 本科生和研究生的培训计划，并通过直接的经验与指导我的导师 以及NEPRC的科学领导层。 研究计划。我们以前的研究表明IFITM蛋白是免疫调节的关键介质， 控制IAV和其他几种人类病原体。它们作为病毒限制因子是独特的，因为它们 通过抑制病毒进入来防止感染。我们的总体目标是全面了解这些活动， 这些蛋白质的机制。这些研究分为三个具体目标。目标1描述了 这些蛋白质的生物化学和功能，使用一系列既定的方法。我们将确定 它们的不同限制活性的分子决定因素，决定限制的病毒范围，和 描述了已知人IFITM多态性的体外表型。目标2旨在了解 IFITM蛋白限制病毒进入的机制。在这里，我们将使用活细胞成像定位的网站， 限制，以及基于亲和力和shRNA的方法来鉴定IFITM的细胞靶点和辅因子。 介导的限制。目的3评价IFITM蛋白对IAV控制的体内贡献，使用两种方法， 一组敲除小鼠，缺乏单独的Ifitm 3基因或五个Ifitm基因。这些老鼠将受到挑战 在存在和不存在I型和II型干扰素的情况下感染IAV，监测免疫应答 和临床症状，并以病理差异为特征。目标1和目标2的一部分将 在本建议书的指导（K99）阶段完成。目标2的剩余部分和目标3的全部 将在独立（R 00）阶段完成。 摘要我的目标是发展一个职业生涯作为一个独立的调查员在先天免疫。我最近的研究 IFITM蛋白质的研究将是这些工作的最初重点。我处于一个高度支持和 智力丰富的环境中，我可以追求我的科学和职业发展目标。