The Mechanisms of IFITM-Mediated Restriction

描述（由申请人提供）：候选人。我的近期目标是发展我成为一名成功的独立调查员所必需的技能和专业知识。我的长期目标是帮助全面了解病毒感染的先天免疫控制，并利用这些知识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，获得博士学位。2008年毕业于哈佛大学。我的博士本论文主要研究了甲型流感病毒（IAV）和SARS冠状病毒进入细胞过程中的酶促调控。这些研究使我能够与Stephen Elledge博士一起开发一种RNAi屏幕，用于调节IAV复制的因素。该筛选鉴定了干扰素诱导跨膜（IFITM）蛋白家族，其对干扰素介导的几种致病性病毒（包括IAV、登革热病毒和西尼罗河病毒）的控制至关重要。对这些重要蛋白质的活性和机制的研究是我未来几年研究计划和科学目标的基础。 环境和职业发展计划。我的发展计划集中在三个方向，这将最能加强我作为一个成功的独立调查员的能力。(1)我将加强我在先天免疫方面的知识背景和对我的科学目标很重要的新技术方面的经验。为此，我将访问新英格兰灵长类动物研究中心（NEPRC）、哈佛医学院、新英格兰地区卓越/生物防御和新发传染病中心（NERCE/BEID）以及哈佛和麻省理工学院布罗德研究所的许多研讨会、课程和其他资源。(2)我将进一步积累进行独立动物研究所需的经验。在这方面，我将依靠国家环境审查委员会提供的特殊专门知识和正式培训。(3)我将提高我的沟通，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，通过参加多个本科生和研究生培训计划，并通过我的导师和NEPRC科学领导的指导下的直接经验来发展。 研究计划。我们以前的研究表明，IFITM蛋白是IAV和其他几种人类病原体免疫控制的关键介质。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这些蛋白质的活性和机制。这些研究分为三个具体目标。目的1的特点，这些蛋白质的生物化学和功能，使用一系列已建立的方法。我们将确定其差异限制活性的分子决定因素，确定限制的病毒范围，并在体外描述已知的人类IFITM多态性的表型。目的2试图了解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来定位限制位点，并使用基于亲和力和shRNA的方法来识别IFITM介导的限制的细胞靶点和辅因子。目的3评估IFITM蛋白对IAV控制的体内贡献，使用两组敲除小鼠，缺乏单独的Ifitm 3基因或五个Ifitm基因。在存在和不存在I型和II型干扰素的情况下，用感染性IAV攻击这些小鼠，监测免疫应答和临床症状，并在死后表征病理学差异。目标1和目标2的一部分将在本建议书的辅导（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。我最近对IFITM蛋白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中，我可以追求我的科学和职业发展目标。 项目叙述 干扰素诱导的跨膜蛋白（IFITM）是重要的人类病原体的内在和先天控制的独特和关键的效应子。这项研究的目标是全面了解这些蛋白质的体外和体内活性，重点是它们的潜在细胞机制。

DESCRIPTION (provided by applicant): 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. PROJECT NARRATIVE Interferon-inducible transmembrane (IFITM) proteins are unique and critical effectors of the intrinsic and innate control of important human pathogens. The goal of this proposed research is to comprehensively understand the in vitro and in vivo activities of these proteins, with focus on their underlying cellular mechanisms.