Mechanism of Rotavirus Entry

轮状病毒侵入机制

• 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
• 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

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，每年造成20多万儿童死亡。目前的RV疫苗在流行国家效力有限，而且没有直接的抗病毒药物可用。我们的总体目标是更好地了解人类rv进入人小肠上皮细胞（IECs）的机制。RV外衣壳刺突蛋白VP4的头部区域（VP8*）负责病毒的进入。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级CRISPR/Cas9基因敲除筛选，并确定了几种跨膜宿主蛋白作为推测的附着因子。在Aim 1中，我们将使用CRISPR/Cas9基因编辑和经典吸附试验，从功能上检查这些候选物在人RV进入HT-29细胞和原代人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，鉴定人类RV进入过程中VP8*相互作用的细胞伙伴。通过宿主基因筛选、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制定对策，以阻止感染和预防腹泻疾病。