PROJECT SUMMARY: Project 2 The focus of Project 2 of this Program Project is to achieve a fully permissive replication system and detailed molecular understanding of host and viral functions that control human norovirus (HuNoV) replication. For almost 50 years, the most significant barrier to HuNoV research was the lack of an efficient in vitro cell cultivation system. We recently succeeded in establishing a robust and reproducible cultivation system for many HuNoV strains in human stem cell-derived intestinal enteroids (HIEs) that overcomes the longstanding hurdle to cultivation. Strain-specific differences in replication were discovered and the system is being used to study virus neutralization, antiviral activity and HuNoV biology. The goal of Project 2 is to continue to understand and overcome newly recognized remaining barriers in the cultivation of all HuNoVs in HIEs. We will address this goal through two aims that outline studies to (i) continue to improve and simplify the cultivation system by understanding virus entry into cells and (ii) identify the cellular receptor(s) for HuNoVs (Aim 1), and (iii) understand the molecular mechanisms by which norovirus replication induces and regulates cellular innate responses and how these cellular responses restrict viral replication and spread (Aim 2). Therapeutics and host molecules associated with disease and virus clearance (Project 1), and structural insight into viral-host interactions (Project 3) will be tested for their effects on HuNoV replication. Overall, these studies are designed to lead to a fully permissive, simple and affordable replication system, gain new molecular understanding of host and viral functions that control virus replication and provide new insight about HuNoV biology. The program project brings together a highly collaborative group of investigators with diverse skills and talents, and substantial record of working together. As in the previous funding period, the interactions among each project and each of the cores will be extensive such that the activities of each project will be enhanced considerably over what could be accomplished if the projects were pursued independently.