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.