PROJECT SUMMARY –Project 3 The major goal of Project 3 is to provide a detailed structural understanding of various aspects human norovirus (HuNoV) biology to synergistically complement the studies in Projects 1 and 2, and aid in the design and development of antiviral strategies. HuNoVs are the leading cause of epidemic acute gastroenteritis. These viruses show enormous genotypic diversity and based on this they are classified into several genogroups and genotypes. Susceptibility to HuNoVs is determined by genetically controlled expression of histo-blood group antigen (HBGA), which is also critical for their attachment to host cells. In the previous grant periods, from extensive structural studies, we have shown how the sequence changes alter the HBGA binding specificity and how they impact the antigenic landscape to drive the evolution of these viruses. Studies in Projects 1 during the current grant period, have further amplified this notion by identifying several HBGA blocking human monoclonal antibodies (mAbs) of various isotypes with an unexpected degree of breadth and neutralization activity using the human intestinal enteroid (HIE) system developed in Project 2. Our studies in Project 3 during this period have shown while the blockade mechanism of a GI.1-specific mAb is by directly blocking the HBGA binding site in GI.1 HuNoV, with a cross-reactive GII-specific mAb, the blockade/neutralization mechanism is different and potentially unique. The goal of AIM 1 is to further our understanding of potentially varying mechanisms of blockade/neutralization of HuNoVs by human mAbs using X-ray crystallography, cryo-EM, and other complementary biophysical techniques. The emphasis in our following two AIMs is on the non-structural proteins of HuNoV that regulate virus replication to provide a structural framework for design and optimization of novel antiviral compounds that are being discovered through high-throughput screening procedures (Projects 1 and 2). AIM 2 is designed to provide a structural understanding of how GII.4 protease with its unique features interacts with the substrates, how the newly discovered small molecules inhibit GI and GII.4 HuNoV proteases and GII.4 polymerase, and what is the mechanism by which some small molecules discovered during screening similar to VPg enhance polymerase activity. The focus of AIM 3 is on the p41 protein encoded by G1.1 HuNoV. p41, with its membrane-anchoring domain, NTPase and helicase activities, plays a critical role in virus replication. In the previous grant period, we determined the first crystal structure of p41 lacking the membrane anchoring domain. Although it provided structural insight into the NTPase activity, it was not sufficient to provide a complete mechanistic understanding of the potentially membrane-associated helicase, RNA remodeling/RNA chaperone activity of p41. Based on the exciting preliminary data, studies in AIM 3 will focus on determining the structure of the soluble and nan...