Mechanism of Rotavirus Entry

轮状病毒侵入机制

基本信息

批准号：
10592070
负责人：
Siyuan Ding
金额：
$ 23.33万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-16 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10592070
关键词：
Adsorption Affinity Chromatography Age Amino Acids Animal Model Antigens Antiviral Agents Apical B-Lymphocytes Binding Binding Sites Biological Assay Biotin Blood Group Antigens CRISPR screen CRISPR/Cas technology Capsid Cell Line Cell Separation Cells Cessation of life Chemicals Child Clinical Co-Immunoprecipitations Colon Complex Country Crystallography Data Dependence Diarrhea Distant EXTL3 gene Enterocytes Enteroendocrine Cell Epidemiology Epithelial Cells Epitopes Escape Mutant Etiology Event Flow Cytometry Gastroenteritis Genes Genetic Screening HT29 Cells Head Heparitin Sulfate Human Incubated Infant Infection Integral Membrane Protein Integration Host Factors Intestines Knock-out Knowledge Label Ligation Lymphocyte Maps Mass Spectrum Analysis Mediating Membrane Membrane Proteins Molecular Monkeys Monoclonal Antibodies Morbidity - disease rate Norovirus Organoids Outcome Persons Polysaccharides Preventive vaccine Proteins Proteomics Recombinants Reporter Role Rotavirus Rotavirus Infections Rotavirus Vaccines Route Sialic Acids Side Small Intestines Structural Protein Surface System Therapeutic Transmission Electron Microscopy Tropism Vaccines Viral Virion cyanine dye 5 data modeling design diarrheal disease genome-wide high throughput screening human monoclonal antibodies improved in vivo in vivo Model infancy innovation intestinal epithelium kidney epithelial cell monolayer mortality mouse model neutralizing monoclonal antibodies novel therapeutics novel vaccines rational design receptor reverse genetics suckling tool

项目摘要

Rotavirus (RV) is a major etiological cause of severe gastroenteritis and diarrhea worldwide, resulting in the death of more than 200,000 children each year. Current RV vaccines have limited efficacy in endemic countries and no direct antivirals are available. Our overall objective is to better understand the mechanism by which human RVs enter human small bowel intestinal epithelial cells (IECs). The head region (VP8*) of the RV outer capsid spike protein VP4 is responsible for viral entry. However, the identities of host factors that interact with VP8* and their roles in human RV entry is unclear. In preliminary studies, we conducted a flow cytometry-based genome-scale CRISPR/Cas9 knockout screen and identified several transmembrane host proteins as putative attachment factors. In Aim 1, we will use CRISPR/Cas9 gene editing and classic adsorption assays to functionally examine the role of these candidates in human RV entry into HT-29 cells and primary human IECs. In Aim 2, we will employ chemical labeling and proteomic approaches and identify the VP8*-interacting cellular partners during human RV entry. Using host genetic screens, RV reverse genetics, and human intestinal organoid cultures, we expect that our study will elucidate the molecular mechanisms by which host molecules facilitate the early replication of human RVs. We ultimately aim to harness that knowledge to develop countermeasures to impede infection and protect against diarrheal diseases.

轮状病毒 (RV) 是全世界严重胃肠炎和腹泻的主要病因，每年导致超过 200,000 名儿童死亡。目前的 RV 疫苗在流行国家的功效有限，并且没有直接的抗病毒药物。我们的总体目标是更好地了解人类 RV 进入人类小肠肠上皮细胞 (IEC) 的机制。 RV 外衣壳刺突蛋白 VP4 的头部区域 (VP8*) 负责病毒进入。然而，与 VP8* 相互作用的宿主因子的身份及其在人类 RV 进入中的作用尚不清楚。在初步研究中，我们进行了基于流式细胞术的基因组规模 CRISPR/Cas9 敲除筛选，并鉴定了几种跨膜宿主蛋白作为假定的附着因子。在目标 1 中，我们将使用 CRISPR/Cas9 基因编辑和经典吸附测定来功能性检查这些候选物在人类 RV 进入 HT-29 细胞和原代人类 IEC 中的作用。在目标 2 中，我们将采用化学标记和蛋白质组学方法，并在人类 RV 进入过程中识别 VP8* 相互作用的细胞伙伴。使用宿主遗传筛选、RV 反向遗传学和人类肠道类器官培养，我们期望我们的研究将阐明宿主分子促进人类 RV 早期复制的分子机制。我们的最终目标是利用这些知识来制定预防感染和预防腹泻疾病的对策。