The Mechanisms of IFITM-Mediated Restriction

项目总结 候选人。我的近期目标是培养成为一名成功人士所必需的技能和专业知识。 独立调查员。我的长期目标是为全面理解 对病毒感染的免疫控制，并利用这一知识帮助创造针对病毒疾病的新疗法。 为了实现这些目标，我将利用我作为一名训练有素的临床医生和一名成熟的研究科学家的背景。我 2000年在台湾大学获得医学博士学位，2004年完成临床培训， 2008年在哈佛大学获得博士学位。我的博士论文关注的是酶 甲型流感病毒(IAV)和SARS冠状病毒进入过程的监管。这些研究让我 与Stephen Elledge博士一起开发RNAi筛查，以了解调节IAV复制的因素。此屏幕 发现干扰素诱导的跨膜蛋白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。对中国传统文化的研究 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。 环境与职业发展计划。我的发展计划集中在三个方向上，这三个方向将是最 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 与生俱来的免疫力和对我的科学目标很重要的新技术的经验。为此，我将 访问我在新英格兰灵长类动物协会的许多研讨会、课程和其他资源 研究中心、哈佛医学院、新英格兰地区中心 卓越/生物防御和新发传染病(NERCE/BEID)和哈佛大学博德研究所 还有麻省理工学院。(2)我将进一步积累进行独立动物研究所需的经验。在这里，我会 依靠非国家反兴奋剂机构提供的特殊专门知识和正规培训。(3)我会提高我的能力 沟通、手稿准备、拨款、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，通过参与多项 本科和研究生培训计划，以及在我导师的指导下的直接经验 以及全国科学研究委员会的科学领导层。 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预防感染。我们的总体目标是全面了解活动和 这些蛋白质的机制。这些研究被组织成三个具体目标。目标1的特点是 这些蛋白质的生化和功能，使用一系列既定的方法。我们将确定 它们不同限制活性的分子决定因素，决定限制的病毒范围，以及 在体外描述已知的人类IFITM多态的表型。目标2试图理解 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来定位 限制，以及基于亲和力和shRNA的方法来识别IFITM的细胞靶点和辅助因子- 居间限制。目的3评估IFITM蛋白在体内对控制IAV的贡献，使用两种 一组基因敲除的小鼠，缺乏单独的IFITM3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型干扰素的情况下监测传染性IAV的免疫反应 和临床症状，并以死后病理差异为特征。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的其余部分和目标3的所有部分 将在独立(R00)阶段完成。 总结。我的目标是发展自己的事业，成为一名天生免疫方面的独立调查员。我最近的研究 IFITM蛋白质的研究将是这些努力的最初重点。我处于一个高度支持和 智力丰富的环境，我可以在其中追求我的科学和职业发展目标。

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.