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.

项目摘要/摘要 该建议描述了四年的职业发展计划和罗伯特博士的研究策略 果园从博士后研究员过渡到一个独立的学术教师职位，调查主人 - 病原体相互作用。奖项的指导阶段（K99）将在续下完成 华盛顿大学病理学和免疫学系Herbert'Skip'Virgin博士的指导 医学院。该提案的总体研究目标是确定分子机制 诺如病毒辅助因子进入病毒。 候选人：我对理解基本机制的兴趣很长 复杂的宿主 - 病原体相互作用。我从得克萨斯A＆M大学毕业 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课程 西南医学院。对于我在尼尔·阿尔托（Neal Alto）博士实验室中的博士学位论文，我描述了细菌 毒力蛋白篡夺宿主细胞骨架机制和工程师的致病回路 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金 Skip Virgin博士的指导。在维珍博士实验室的奖学金期间，我的目标是使我 通过剖析宿主 - 病原体信号网络的经验，具有定义体内相关性的能力 动物模型中的宿主病原体相互作用。为此，我的研究项目集中在 了解由于其坚固耐用的鼠（MNOV）复制和向量的理解的分子机制 体外和体内系统。具体来说，我们最近完成了一个主机的全基因组CRISPR屏幕 MNOV复制所需的基因。我们发现MNOV结合了蛋白质受体CD300LF， 在体外和体内都是必要的，以进行MNOV复制，并且在足够的人类细胞中表达时 打破MNOV复制的物种障碍。此外，我们的工作描述了一个小说，身份不明的辅助因子 有效的MNOV与细胞结合所需的血清。这项工作是研究建议的基础 在这里概述。我计划将其余的奖学金集中在获得专业技能和科学方面 过渡到终身位置所需的见解。 职业发展计划：在我作为博士后研究员的最后一年中，我将重点关注一个重要的 努力（15％）为成功挑战我的专业技能，这是成功的 独立调查员。我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 Michael Diamond博士和Thaddeus Stappenbeck博士将评估我克服的进度 我 科学进步。此外，我将参加华盛顿内外的特定研讨会 大学以加强我的培训和准备过渡到独立的准备。最后，我已经开发了 时间表的特定里程碑将指导自己，我的导师维尔京博士和我的职业咨询 委员会为我准备成功竞争在结束时成功争夺独立的RO1赠款的目标 这四年的建议。 研究项目：诺如病毒鼠（MNOV）是了解人类的重要模型 诺病毒（HNOVS），用于阐明病毒，宿主的微生物群和宿主之间的复合互动 免疫系统。 HNOV培养系统的最新进展已发现促进病毒的辅助因子 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 测试新的假设，即诺如病毒tropism是由受体和辅因子的组合决定的 互动。我们的初步数据表明，胆固醇衍生的胆汁酸之间未开发的联系， MNOV受体CD300LF和宿主在促进病毒入口的宿主衍生的神经酰胺脂质物种。重要的是，是 很好地确定，MNOV和HNOV在非允许细胞中复制的无能是由于 病毒进入中的缺陷。我们将以组合方式探讨这些组件中每个组件的相互作用。 此外，我们将直接测试MNOV建立和维持小鼠持续感染的能力 当这些途径使用遗传和药理方法扰动时。我期待我们的 结果将揭示诺如病毒进入的基本原则，并提供建立强大和的见解 可重现的体外HNOV复制系统。更广泛地说我们的假设，如果证明是真的， 建立一种新颖的方法来为当前无法培养的新方法生成体外培养系统 病毒。该项目的最初发现将帮助我过渡到独立的学术职位研究 诺夫病毒及其宿主之间的相互作用。该项目的完成不仅会提供新颖 洞悉诺如病毒生物学，但是竞争性RO1应用的框架。