Identification and Characterization of Norovirus Cofactors for Entry

项目摘要/摘要 该提案描述了罗伯特博士的四年职业发展计划和研究战略。 Orchard将从博士后过渡到独立的学术教师职位，调查主持人- 病原体相互作用。该奖项的指导阶段(K99)将在继续 赫伯特·斯基普·维珍博士在华盛顿大学病理和免疫学系的指导 医学院。该提案的总体研究目标是确定糖尿病的分子机制。 诺沃克病毒在病毒进入时起辅助作用。 应聘者：我对了解其背后的分子机制有着长期的兴趣 复杂的寄主-病原体相互作用。我以优异的成绩毕业于德克萨斯农工大学，获得学位 微生物学。后来我加入了德克萨斯大学的分子微生物学研究生项目 西南医学院。在尼尔·阿尔托博士实验室的博士论文中，我描述了细菌 毒力蛋白侵占宿主的细胞骨架机制，并设计致病信号通路 真核宿主细胞细胞质的复杂环境。然后，我开始了博士后研究，在 斯基普·维珍博士的指导。在我在维珍博士的实验室做研究员期间，我的目标是让我的 解剖宿主-病原体信号网络的经验与他定义体内相关性的能力 动物模型中宿主与病原体的相互作用。为此，我的研究项目一直集中在 了解小鼠诺如病毒(MNoV)复制和趋向性的分子机制 体外和体内系统。具体地说，我们最近完成了宿主的全基因组CRISPR筛选 MNoV复制所需的基因。我们发现MNoV与蛋白质受体CD300lf结合， 在体外和体内都是MNoV复制所必需的，当在人类细胞中表达时足以 打破MNOV复制的物种障碍。此外，我们的工作描述了一种新的、未确定的辅因子 MNoV与细胞有效结合所需的血清。这项工作是提出研究方案的基础 概述如下。我计划将我剩下的奖学金集中在获得专业技能和科学知识上。 过渡到终身教职所需的洞察力。 职业发展计划：在我博士后的最后一年，我将专注于一项重要的 努力培养对我来说是成功所必需的专业技能(15%) 独立调查人员。我已经组建了一个由戴维德·弗里蒙特博士组成的职业咨询委员会， 迈克尔·戴蒙德博士和撒迪厄斯·斯塔彭贝克博士将评估我在克服 在科学写作和数据展示、指导和实验室管理方面的不足以及我的 科学进步。此外，我还将参加华盛顿内外的特定研讨会 大学加强了我向独立过渡的培训和准备。最后，我开发了一个 有具体里程碑的时间表，将指导我自己、我的导师维珍博士和我的职业建议 委员会帮助我为我的目标做好准备，我的目标是在年底成功竞争一项独立的RO1拨款 这份为期四年的提案。 研究项目：小鼠诺如病毒(MNoV)是了解人类的重要模型 诺如病毒(HNov)，以及阐明病毒、宿主微生物区系和 免疫系统。人类新城疫病毒培养系统的最新进展揭示了促进病毒传播的辅因子 通过目前未知的机制进行体外复制。在这里，我们将继续调查 MNoV辅因子和受体之间的相互作用。更具体地说，概述的实验将直接 验证诺如病毒嗜性是由受体和辅因子的组合决定的新假设 互动。我们的初步数据表明，胆固醇衍生胆汁酸之间存在一种未知的联系， MNoV受体CD300lf和宿主来源的神经酰胺脂类物质在促进病毒进入中的作用。重要的是，它是 众所周知，MNoV和hNoV都不能在非允许细胞中复制是由于 病毒进入的缺陷。我们将以组合的方式探索这些组件中的每个组件的相互作用。 此外，我们将直接测试MNOV在小鼠中建立和维持持续感染的能力 当使用遗传和药理学方法扰乱这些通路时。我期待着我们的 研究结果将揭示诺沃克病毒进入的基本原理，并为建立健全和 体外可重复性的hNoV复制系统。更广泛地说，如果我们的假设被证明是真的，那么 建立一种新的方法来产生目前无法培养的植物的体外培养系统的可能性 病毒。这个项目的初步发现将帮助我过渡到一个独立的学术职位学习 诺如病毒与其宿主之间的相互作用。该项目的完成不仅将为 对诺沃克病毒生物学的见解，但具有竞争力的RO1应用程序的框架。

Project Summary/Abstract This proposal describes a four year career development plan and a research strategy for Dr. Robert Orchard to transition from a postdoctoral fellow to an independent academic faculty position investigating host- pathogen interactions. The mentored phase of the award (K99) will be completed under the continued guidance of Dr. Herbert `Skip' Virgin in the Department of Pathology and Immunology at Washington University School of Medicine. The overall research goal of the proposal is to determine molecular mechanisms of norovirus cofactors upon viral entry. Candidate: I have a long standing interest in understanding the molecular mechanisms underlying complex host-pathogen interactions. I graduated summa cum laude from Texas A&M University with a degree in Microbiology. I subsequently joined the Molecular Microbiology Graduate Program at University of Texas Southwestern Medical School. For my doctoral thesis in Dr. Neal Alto's laboratory, I described how bacterial virulence proteins usurp the host cytoskeletal machinery and engineer pathogenic-signaling circuits within the complex environment of the cytoplasm of eukaryotic host cells. I then began a postdoctoral fellowship under the mentorship of Dr. Skip Virgin. During my fellowship in Dr. Virgin's lab, it has been my goal to couple my experiences with dissecting host-pathogen signaling networks with his ability to define the in vivo relevance of host-pathogen interactions in animal models. To this end, my research project has been focused on understanding the molecular mechanisms of murine norovirus (MNoV) replication and tropism due to its robust in vitro and in vivo systems. Specifically, we recently completed a whole-genome CRISPR screen for host genes required for MNoV replication. We discovered that MNoV binds a proteinaceous receptor, CD300lf, that is necessary both in vitro and in vivo for MNoV replication and when expressed in human cells sufficient to break the species barrier of MNoV replication. Additionally, our work described a novel, unidentified cofactor in serum required for efficient MNoV binding to cells. This work is the foundation for the research proposal outlined here. I plan to focus the remainder of my fellowship on obtaining professional skills and scientific insight necessary to transition to a tenure-track position. Career Development Plan: During my final year as a postdoctoral fellow, I will focus a significant amount of effort (15%) to developing the professional skills challenging to me that are necessary for successful independent investigators. I have assembled a career advisory committee composed of Dr. Daved Fremont, Dr. Michael Diamond, and Dr. Thaddeus Stappenbeck that will evaluate my progress in overcoming deficiencies in scientific writing and data presentation, mentoring, and laboratory management along with my scientific progress. Additionally, I will attend specific seminars both within and outside of Washington University to enhance my training and preparation for transition to independence. Lastly, I have developed a timeline with specific milestones that will guide myself, my mentor Dr. Virgin, and my career advisory committee in preparing me for my goal of successfully competing for an independent RO1 grant at the end of this four year proposal. Research Project: Murine norovirus (MNoV) is an important model for understanding human noroviruses (HNoVs) and for elucidating complex interactions between viruses, the host's microbiota, and the immune system. Recent advances in HNoV culture systems have uncovered cofactors that promote viral replication in vitro through currently unknown mechanisms. Here, we will continue our investigations into the interactions between MNoV cofactors and receptors. More specifically, the experiments outlined will directly test the novel hypothesis that norovirus tropism is determined by the combination of receptor and cofactor interactions. Our preliminary data suggests an unexplored connection between cholesterol derived bile acids, the MNoV receptor CD300lf, and host derived ceramide lipid species in promoting viral entry. Importantly, it is well established that the inability of both MNoV and HNoV to replicate in non-permissive cells are due to defects in viral entry. We will explore the interactions of each of these components in a combinatorial fashion. Furthermore, we will directly test the ability of MNoV to establish and maintain a persistent infection in mice when these pathways are perturbed using genetic and pharmacological approaches. I anticipate that our results will reveal fundamental principles of norovirus entry and provide insights into establishing a robust and reproducible in vitro HNoV replication system. More broadly speaking our hypothesis, if proven true, has the potential for establishing a novel approach for generating in vitro culture systems for currently uncultivable viruses. The initial findings of this project will help me transition to an independent academic position studying the interaction between noroviruses and their hosts. The completion of this project will not only provide novel insights into norovirus biology but the framework for a competitive RO1 application.