Genetic and Biochemical Interrogation of Rotavirus-Cohesin Interaction

轮状病毒-粘连蛋白相互作用的遗传和生化研究

基本信息

批准号：
10046745
负责人：
Siyuan Ding
金额：
$ 24.9万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-07-27 至 2021-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10046745
关键词：
Accounting Advisory Committees Affect Affinity Chromatography Animals Antigens Antiviral Agents Arvin Autoimmunity Award Binding Biochemical Biological Models CRISPR screen Cell Line Cells Cessation of life Chemicals Chikungunya virus Child Chromosomal Instability Clustered Regularly Interspaced Short Palindromic Repeats Complement Complex Coupled DNA DNA Damage Dependence Development Development Plans Diarrhea Embryo Enteral Environment Epithelial Cells Future Gamma-H2AX Gastroenteritis Gastrointestinal Diseases Genetic Genetic Transcription Genomic Instability Genomics Goals Head Histones Human Immunology Incidence Infant Infection Innate Immune System Integration Host Factors Interferon Activation Interferon Type I Interferon-beta Interferons Intestines Knock-out Knowledge Laboratory Study Lead Mass Spectrum Analysis Mediating Mentorship Microbiology Mitosis Molecular Mucosal Immunity Mus Natural Immunity Nonstructural Protein Norovirus Nuclear Organoids Outcome Pathogenesis Pathway interactions Pattern Pattern recognition receptor Phase Physiological Production Proteins Proteomics Public Health RNA Viruses Receptor Signaling Regulation Reporting Resistance Role Rotavirus Rotavirus Infections Signal Pathway Signal Transduction Sister Chromatid Small Interfering RNA Stimulator of Interferon Genes System Testing Therapeutic Therapeutic Intervention Training Universities Vaccines Viral Pathogenesis Virulent Virus Virus Diseases Virus Replication Vocation Work burden of illness career career development cohesin cohesion conditional knockout design follow-up genome-wide histidylproline human pathogen improved in vivo influenzavirus inhibitor/antagonist insight interdisciplinary approach intestinal epithelium medical schools member mimetics mortality mouse genetics mouse model mutant new therapeutic target novel pathogen prevent response reverse genetics skills transcription factor vaccine candidate villin virology

项目摘要

Project Summary Rotaviruses (RVs) are the most common cause of severe gastroenteritis and dehydrating diarrhea in in- fants and young children worldwide. Despite the wide administration of several vaccines, RVs still remain a highly significant human pathogen, leading to over 215,000 deaths annually. The inadequate understanding of RV-host interaction greatly impedes the development of improved vaccines and therapeutic interventions. In the preliminary studies, Dr. Ding has performed an unbiased genome-wide CRISPR-Cas9 screen and a high-throughput RV-host proteomic interactome mapping, to bridge this gap in knowledge and identified stromal antigen 2 (STAG2) as a novel host factor required for RV infection. The infectivity of human and animal RVs was significantly reduced in STAG2-/- cells. STAG2 is an integral member of the nuclear cohesin complex and how it promotes cytoplasmic RV replication is perplexing and intriguing. In this application, using a set of powerful and tractable model systems, Dr. Ding will test the hypothesis that RV hijacks the STAG2/cohesin complex to block DNA damage and inhibit IFN induction, thereby enhancing virus infection. During the K99 phase, Dr. Ding will scrutinize the signaling pathways in STAG2-/- cells that mediate resistance to RV infection (Aim 1; K99). Dr. Ding will further generate a new mouse model that specifically lacks Stag2 in the intestinal epithelium (Aim 2; K99) and employ the novel human intestinal organoids (Aim 2; R00) to study how STAG2 deficiency affects RV infection in a physiologically valid environment. Finally, Dr. Ding will follow up on his pro- vocative finding that several subunits of the cohesin complex physically bind to an RV non-structural protein. He will determine the outcome of such interaction for RV replication and pathogenesis in vivo, using a new re- verse genetics system (Aim 3; R00). Collectively, these studies on RV-cohesin interaction will constitute the scientific basis for the rational design of antiviral inhibitors (by transiently targeting STAG2) and vaccine candi- dates (RV mutants incapable of cohesin interaction), which will be further pursued in future R01 applications. Dr. Ding has developed a comprehensive career development plan, under the direct mentorship of Dr. Harry Greenberg at Stanford University, to perfect his training in experimental (human organoids) and profes- sional (lab management and grantsmanship) skills to achieve his short-term goals of successfully carrying out the proposed project and establishing independence. A prestigious advisory committee (Drs. Ann Arvin, Peter Jackson and Jan Carette at Stanford), in combination with a team of excellent external consultants and collab- orators, will provide essential guidance in genomic screens, proteomics, and mouse genetics, to complement Dr. Ding's strong background in virology and immunology. The exemplary environment in the Department of Microbiology and Immunology at the Stanford School of Medicine, will enable Dr. Ding to fully benefit from this Award and attain his long-term career goal to lead a world-class laboratory studying enteric viruses with a spe- cial focus on the viral pathogenesis and interaction with the host intestinal innate immune system.

项目概要轮状病毒（RV）是严重胃肠炎和脱水性腹泻的最常见原因。全世界的粉丝和幼儿。尽管多种疫苗被广泛使用，RV 仍然是一种高度重要的人类病原体，每年导致超过 215,000 人死亡。认识不足 RV-宿主相互作用极大地阻碍了改进疫苗和治疗干预措施的开发。在初步研究中，丁博士进行了无偏见的全基因组CRISPR-Cas9筛选以及高通量 RV 宿主蛋白质组相互作用组图谱，以弥合这一知识差距并确定基质抗原 2 (STAG2) 作为 RV 感染所需的新型宿主因子。人类和动物的传染性 STAG2-/- 细胞中的 RV 显着减少。 STAG2 是核粘连蛋白复合体的重要成员它如何促进细胞质 RV 复制令人困惑且有趣。在此应用中，使用一组强大且易于处理的模型系统，丁博士将测试 RV 劫持 STAG2/cohesin 的假设复合物可阻断 DNA 损伤并抑制 IFN 诱导，从而增强病毒感染。 K99期间在这一阶段，丁博士将仔细研究 STAG2-/- 细胞中介导 RV 感染抵抗力的信号通路（目标 1；K99）。丁博士将进一步生成肠道中特异缺乏Stag2的新小鼠模型上皮细胞（目标 2；K99）并利用新型人类肠道类器官（目标 2；R00）来研究 STAG2 缺乏会影响生理有效环境中的 RV 感染。最后，丁博士将跟进他的亲呼唤发现粘连蛋白复合物的几个亚基与 RV 非结构蛋白物理结合。他将使用一种新的方法来确定 RV 复制和体内发病机制的这种相互作用的结果。反向遗传学系统（目标 3；R00）。总的来说，这些关于 RV-粘连蛋白相互作用的研究将构成合理设计抗病毒抑制剂（通过暂时靶向 STAG2）和候选疫苗的科学依据日期（RV 突变体无法进行粘连蛋白相互作用），这将在未来的 R01 应用中进一步研究。丁博士在丁博士的直接指导下制定了全面的职业发展计划。斯坦福大学的哈里·格林伯格，为了完善他在实验（人类类器官）和专业方面的培训 sional（实验室管理和资助）技能，以实现成功实施的短期目标拟议的项目并建立独立性。享有盛誉的顾问委员会（Ann Arvin 博士、Peter 斯坦福大学的 Jackson 和 Jan Carette），与优秀的外部顾问团队和合作者一起演说家将在基因组筛选、蛋白质组学和小鼠遗传学方面提供必要的指导，以补充丁博士在病毒学和免疫学方面拥有深厚的背景。系内模范环境斯坦福大学医学院微生物学和免疫学专业，将使丁博士充分受益于此获奖并实现他的长期职业目标，领导一个世界一流的实验室，专门研究肠道病毒重点关注病毒发病机制以及与宿主肠道先天免疫系统的相互作用。