The focus of Project 2 of this Program Project is to continue to pursue approaches to cultivated human caliciviruses and to understand the molecular basis of restriction of virus gene expression, replication and growth in cell systems. Although the clinical and economic significance of the human noroviruses as the most important cause of global foodborne and waterborne illness is now recognized, factors affecting the persistence and transmission of viable virus in the environment and the underlying mechanisms of pathogenesis and immunity are poorly understood. This lack of understanding is largely due to the lack of available in vitro cultivation or animal models of infection. Our long-term goal is to exploit fundamental discoveries made in the previous project period that can measure in vitro infectivity and new in vitro cell culture systems for gastrointestinal epithelium. In Specific Aim 1, we will improve and develop efficient in vitro methods for cultivation of Norwalk virus and other noroviruses. Studies in this aim will determine if the restriction of viral spread in RNA-transfected cells is due to the lack of a co-receptor. These studies will also exploit new ideas to obtain and maintain primary human intestinal epithelial cells in culture. In Specific Aim 2, we propose to understand the molecular mechanisms by which norovirus protein expression regulates cellular innate responses as well as whether these cellular responses regulate viral replication and spread. In Specific Aim 3, we propose to use gene expression and replication systems to dissect the mechanisms by which VP1, VP2, and VPg interact with each other and with genomic RNA for RNA encapsidation. The results from these studies will generate new knowledge about the replication of noroviruses and will complement and allow functional assessment of methods of virus detection as well as of antiviral that are developed in Projects 1 and 3.