Mechanism of Rotavirus Entry

轮状病毒(RV)是世界范围内引起严重胃肠炎和腹泻的主要病原，每年导致20多万儿童死亡。目前的轮状病毒疫苗在流行国家的效力有限，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轮状病毒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规模的CRISPR/Cas9基因敲除筛选，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试验从功能上研究这些候选基因在人RV进入HT-29细胞和原代人IECs中的作用。在目标2中，我们将使用化学标记和蛋白质组学方法，并鉴定在人类RV进入过程中与VP8*相互作用的细胞伙伴。利用宿主遗传筛选、轮状病毒反向遗传学和人类肠道器官培养，我们预计我们的研究将阐明宿主分子促进人类轮状病毒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，以防止感染和预防腹泻疾病。

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.