Genetic and Biochemical Interrogation of Rotavirus-Cohesin Interaction

项目摘要 轮状病毒(RV)是引起严重胃肠炎和脱水腹泻的最常见原因。 世界各地的胖子和小孩子。尽管已经广泛接种了几种疫苗，但房车仍然是一种 高度重要的人类病原体，每年导致超过215,000人死亡。对……的认识不足 轮状病毒与宿主的相互作用极大地阻碍了改进疫苗和治疗干预措施的发展。 在初步研究中，丁博士进行了无偏见的全基因组CRISPR-Cas9筛查 以及高通量的RV-宿主蛋白质组相互作用组图，以弥合这一知识差距并识别 基质抗原2(STAG2)是轮状病毒感染所必需的新宿主因子。人和动物的传染性 STAG2-/-细胞的RVS显著减少。STAG2是核粘连蛋白复合体的一个重要成员 它如何促进细胞质RV复制是令人困惑和耐人寻味的。在此应用程序中，使用一组 强大且易于处理的模型系统，丁博士将检验房车劫持STAG2/粘附素的假设 复合体可阻断DNA损伤，抑制干扰素诱导，从而增强病毒感染。在K99期间 在这一阶段，丁博士将仔细研究STAG2-/-细胞中介导对RV感染的抵抗的信号通路 (目标1；K99)。丁博士将进一步创造一种新的小鼠模型，这种模型在肠道中特别缺乏STAG2 上皮细胞(Aim 2；K99)，并使用新的人类肠道器官(Aim 2；R00)来研究STAG2如何 缺乏会在生理上有效的环境中影响轮状病毒感染。最后，丁医生将跟进他的亲身- 开创性的发现，粘附素复合体的几个亚基在物理上与RV的非结构蛋白结合。 他将使用一种新的Re-Re来确定这种相互作用对RV复制和体内致病作用的结果。 诗歌遗传学系统(目标3；R00)。总的来说，这些关于轮状病毒-粘附素相互作用的研究将构成 为合理设计抗病毒抑制剂(瞬时靶向STAG2)和疫苗糖提供科学依据。 DATES(不能与粘附素相互作用的RV突变体)，这将在未来的R01应用中进一步研究。 在丁博士的直接指导下，丁博士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(人类有机化合物)和教授方面的培训- (实验室管理和资质)技能，以实现他成功实施的短期目标 提议的项目和建立独立性。享有盛誉的咨询委员会(安·阿文、彼得博士 杰克逊和Jan Carette在斯坦福大学)，与优秀的外部顾问和合作团队相结合- 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有很强的背景。该部的模范环境 斯坦福医学院的微生物学和免疫学，将使丁博士充分受益于这一点 获奖并实现了长期的职业目标，领导一个世界级的实验室研究肠道病毒，并致力于- 关注病毒的发病机制以及与宿主肠道天然免疫系统的相互作用。

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.