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.

项目总结/摘要 本建议书描述了罗伯特博士四年的职业发展计划和研究策略 果园从博士后过渡到一个独立的学术教师职位调查主机- 病原体相互作用该奖项的辅导阶段（K99）将在继续 华盛顿大学病理学和免疫学系赫伯特博士的指导 医学院。该提案的总体研究目标是确定 诺如病毒辅因子。 候选人：我一直对理解潜在的分子机制感兴趣 复杂的宿主-病原体相互作用。我以最优异的成绩毕业于德克萨斯农工大学， 微生物学我随后加入了德克萨斯大学的分子微生物学研究生课程 西南医学院。在我的博士论文中，我描述了细菌是如何 毒力蛋白篡夺宿主细胞骨架机制，并在宿主细胞内设计致病信号通路。 真核宿主细胞质的复杂环境。然后，我开始了博士后奖学金， 斯基普·维珍博士的指导在维珍博士的实验室工作期间，我的目标是将我的 具有解剖宿主-病原体信号传导网络的经验，能够定义 宿主-病原体相互作用的动物模型。为此，我的研究项目一直专注于 了解小鼠诺如病毒（MNoV）复制和嗜性的分子机制， 体外和体内系统。具体来说，我们最近完成了一项针对宿主的全基因组CRISPR筛选， MNoV复制所需的基因。我们发现MNoV结合蛋白质受体CD 300 lf， 是MNoV复制在体外和体内所必需的，并且当在人细胞中表达时足以 打破了MNoV复制的物种屏障。此外，我们的工作描述了一个新的，身份不明的辅因子， MNoV与细胞有效结合所需的血清。这项工作是研究方案的基础 在这里概述。我计划把剩下的奖学金集中在获得专业技能和科学知识上。 这是过渡到终身职位所必需的洞察力。 职业发展计划：在我作为博士后研究员的最后一年，我将专注于一个重要的 努力（15%）发展对我来说具有挑战性的专业技能，这是成功所必需的 独立调查员。我召集了一个职业顾问委员会成员包括戴维·弗里蒙特博士 博士迈克尔·戴蒙德和撒迪厄斯·斯塔彭贝克博士将评估我克服 在科学写作和数据展示，指导和实验室管理方面的缺陷，沿着我的 科学进步。此外，我将参加华盛顿内外的特定研讨会 大学加强我的训练和准备过渡到独立。最后，我开发了一个 时间轴与具体的里程碑，将指导我自己，我的导师博士。 委员会为我准备我的目标，成功地竞争一个独立的RO 1赠款年底 这四年的提案。 研究项目：鼠诺如病毒（MNoV）是了解人类免疫系统的重要模型。 诺如病毒（HNoV），并阐明病毒，宿主的微生物群，和病毒之间的复杂相互作用。 免疫系统HNoV培养系统的最新进展已经揭示了促进病毒增殖的辅因子。 通过目前未知的机制在体外复制。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 检验诺如病毒嗜性是由受体和辅因子的组合决定的新假设 交互.我们的初步数据表明胆固醇衍生的胆汁酸， MNoV受体CD 300 lf和宿主衍生的神经酰胺脂质物质促进病毒进入。重要的是 已经充分确定，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一个的相互作用。 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。 当这些途径被遗传和药理学方法干扰时。我预计我们的 结果将揭示诺如病毒进入的基本原理，并为建立一个强大的 可重复的体外HNoV复制系统。更广泛地说，我们的假设，如果被证明是正确的， 建立一种新的方法来产生体外培养系统的潜力， 病毒这个项目的初步结果将帮助我过渡到一个独立的学术立场，研究 诺如病毒与宿主之间的相互作用该项目的完成不仅将提供新颖的 深入了解诺如病毒生物学，但竞争性RO 1应用的框架。