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

• 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
• 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

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.

项目摘要 轮状病毒（RVS）是严重胃肠炎的最常见原因，并使腹泻脱水 宠物和幼儿在全球。尽管多种疫苗的管理很广泛，但RV仍然是 高度重要的人类病原体，每年导致215,000多人死亡。对 RV-host相互作用极大地阻碍了改善的疫苗和治疗干预措施的发展。 在初步研究中，Ding博士进行了无偏基因组CRISPR-CAS9屏幕 以及高通量的RV-host蛋​​白质组学相互作用组映射，以弥合知识的差距并确定 基质抗原2（Stag2）是RV感染所需的新型宿主因子。人类和动物的感染力 在Stag2 - / - 细胞中，RV显着降低。 Stag2是核粘着蛋白复合物的组成部分 它如何促进细胞质RV复制令人困惑和有趣。在此应用程序中，使用一组 丁博士将测试RV劫持stag2/粘连的假设 复合物可阻止DNA损伤并抑制IFN诱导，从而增强病毒感染。在K99期间 阶段，丁博士将仔细检查介导对RV感染的抗性性的Stag2 - / - 细胞中的信号通路 （AIM 1; K99）。丁博士将进一步生成一种新的鼠标模型，该模型特别缺乏肠道中的stag2 上皮（AIM 2; K99），并采用新型的人类肠癌（AIM 2; r00）来研究Stag2 缺乏会影响生理上有效的环境中的RV感染。最后，丁博士将跟进他的职业 声音发现，粘蛋白复合物的几个亚基在物理上与RV非结构蛋白结合。 他将使用新的重新确定体内RV复制和发病机理的这种相互作用的结果 经文遗传学系统（AIM 3; R00）。总的来说，这些关于RV-粘蛋白相互作用的研究将构成 抗病毒抑制剂合理设计的科学基础（通过瞬时靶向Stag2）和疫苗候选 日期（RV突变体无粘蛋白相互作用），将在未来的R01应用中进一步追求。 Ding博士在博士的直接指导下制定了一项全面的职业发展计划。 斯坦福大学的哈里·格林伯格（Harry Greenberg Sional（Lab Management和Grantsship）技能，以实现他成功执行的短期目标 拟议的项目并建立独立性。享有声望的咨询委员会（Ann Arvin博士，彼得 斯坦福大学的杰克逊（Jackson）和扬·卡莱特（Jan Carette），与一支优秀的外部顾问和合作团队结合 演说家将在基因组筛查，蛋白质组学和小鼠遗传学方面提供必要的指导，以补充 Ding博士在病毒学和免疫学方面的良好背景。部门的模范环境 斯坦福大学医学院的微生物学和免疫学，将使丁博士能够从中充分受益 奖励并实现他的长期职业目标，以领导世界一流的实验室研究肠道病毒 CIAL专注于病毒发病机理和与宿主先天免疫系统的相互作用。