Identification and Characterization of Norovirus Cofactors for Entry

诺如病毒进入辅因子的鉴定和表征

• 批准号: 9980887
• 负责人: Robert C. Orchard
• 金额: $ 24.9万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-03 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9980887
• 关键词: Advisory Committees; Amino Acids; Anabolism; Animal Model; Antiviral Agents; Award; Bile Acids; Bile fluid; Binding; Biological; Biology; Blood Group Antigens; CRISPR screen; Cell membrane; Cell surface; Cells; Ceramides; Cholesterol; Complex; Cytoplasm; Data; Defect; Dependence; Development Plans; Diamond; Engineering; Environment; Faculty; Fellowship; Foundations; Gastroenteritis; Genes; Genetic; Goals; Grant; Human; Immune system; Immunology; In Vitro; Infection; Integration Host Factors; Intestines; Investigation; Laboratories; Ligands; Link; Lipids; Mentors; Mentorship; Microbiology; Modeling; Molecular; Multienzyme Complexes; Mus; Norovirus; Pathogenicity; Pathology; Pathway interactions; Pharmacology; Phase; Phospholipids; Physiological; Positioning Attribute; Postdoctoral Fellow; Preparation; Process; Proteins; Reproducibility; Research; Research Personnel; Research Project Grants; Research Proposals; Role; Serum; Signal Transduction; System; Testing; Texas; Therapeutic; TimeLine; Training; Tropism; Universities; Vaccines; Viral; Virulence; Virus; Virus Diseases; Virus Receptors; Virus Replication; Washington; Work; Writing; career; career development; cell type; chronic infection; cofactor; combinatorial; experience; experimental study; host microbiota; in vivo; in vivo Model; insight; interest; medical schools; novel; novel strategies; pathogen; prevent; programs; receptor; receptor binding; skills; small molecule; structured data; tenure track; therapy design; whole genome

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.

项目概要/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策略 果园从博士后研究员过渡到独立学术教职职位调查主机 - 病原体相互作用。该奖项（K99）的指导阶段将在持续的指导下完成 华盛顿大学病理学与免疫学系 Herbert `Skip' Virgin 博士指导 医学院。该提案的总体研究目标是确定分子机制 病毒进入后诺如病毒辅因子。 候选人：我长期以来对了解其背后的分子机制很感兴趣 复杂的宿主-病原体相互作用。我以优异成绩从德克萨斯农工大学毕业并获得学位 在微生物学中。随后我加入了德克萨斯大学分子微生物学研究生项目 西南医学院。在 Neal Alto 博士实验室的博士论文中，我描述了细菌如何 毒力蛋白篡夺宿主细胞骨架机制并改造宿主细胞内的致病信号通路 真核宿主细胞的细胞质环境复杂。然后我开始了博士后奖学金 Skip Virgin 博士的指导。在维珍博士实验室的研究期间，我的目标是结合我的 具有解剖宿主-病原体信号网络的经验以及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，我的研究项目主要集中在 了解鼠诺如病毒 (MNoV) 复制和向性的分子机制，因为它具有强大的功能 体外和体内系统。具体来说，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 结合蛋白质受体 CD300lf， 在体外和体内对于 MNoV 复制都是必需的，并且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颖的、未鉴定的辅助因子 MNoV 与细胞有效结合所需的血清。这项工作是研究计划的基础 此处概述。我计划将剩余的研究金重点放在获得专业技能和科学知识上 过渡到终身职位所需的洞察力。 职业发展计划：在博士后的最后一年，我将重点关注一个重要的领域 付出大量努力（15%）来发展对我来说具有挑战性的成功所必需的专业技能 独立调查员。我组建了一个职业咨询委员会，由 Daved Fremont 博士组成， Michael Diamond 博士和 Thaddeus Stappenbeck 博士将评估我在克服困难方面的进展 我在科学写作和数据呈现、指导和实验室管理方面的缺陷 科学进步。此外，我将参加华盛顿境内外的特定研讨会 大学加强我的训练并为过渡到独立做好准备。最后，我开发了一个 包含具体里程碑的时间表，将指导我自己、我的导师 Virgin 博士和我的职业咨询 委员会帮助我为我的目标做好准备，即在年底成功竞争独立 RO1 拨款 这项为期四年的提案。 研究项目：鼠诺如病毒（MNoV）是了解人类的重要模型 诺如病毒 (HNoV) 并阐明病毒、宿主微生物群和宿主之间复杂的相互作用 免疫系统。 HNoV 培养系统的最新进展发现了促进病毒传播的辅助因子 通过目前未知的机制进行体外复制。在此，我们将继续调查此事 MNoV 辅助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 检验诺如病毒向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的胆汁酸之间存在未经探索的联系， MNoV 受体 CD300lf 和宿主衍生的神经酰胺脂质物种促进病毒进入。重要的是，它是 众所周知，MNoV 和 HNoV 无法在不允许的细胞中复制是由于 病毒进入的缺陷。我们将以组合方式探索每个组件的相互作用。 此外，我们将直接测试 MNoV 在小鼠体内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途径时。我预计我们的 结果将揭示诺如病毒进入的基本原理，并为建立强大和可靠的病毒库提供见解。 可重复的体外 HNoV 复制系统。更广泛地说，如果我们的假设被证明是正确的， 建立一种新方法来产生目前无法培养的体外培养系统的潜力 病毒。该项目的初步发现将帮助我过渡到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提供新颖的 对诺如病毒生物学的见解，但也是具有竞争力的 RO1 应用的框架。