Abstract Noroviruses (NoVs) are a leading cause of gastroenteritis world-wide, yet host factors that restrict NoV replication are not well understood. Here, we use a CRISPR activation (CRISPRa) genome-wide screening to identify host genes that can inhibit murine norovirus (MNoV) replication in either mouse or human cells. Our screens identified with high confidence 57 genes that can inhibit MNoV infection when overexpressed. A significant number of these genes are in interferon and immune regulation signaling networks, but surprising, the majority of the genes identified are not associated with innate or adaptive immunity nor with any antiviral activity. Confirmatory studies of eight of the genes in validate the initial screening data. Mechanistic studies on TRIM7 demonstrated a conserved role of the molecule in mouse and human cells in restricting MNoV in a step of infection after viral entry. Furthermore, we demonstrate that two isoforms of TRIM7 have differential antiviral activity. Taken together these data provide a resource for understanding norovirus biology and demonstrate a robust methodology for identifying new antiviral molecules across cell types and species. Author Summary Norovirus is one of the leading causes of foodborne illness world-wide. Despite its prevalence, our understanding of norovirus biology is limited due to the difficulty in growing human norovirus in vitro and a lack of an animal model. Murine norovirus (MNoV) is a model norovirus system because MNoV replicates robustly in cell culture and in mice. To identify host genes that can restrict norovirus replication when overexpressed we performed genome-wide CRISPR activation (CRISPRa) screens to induce gene overexpression at the native locus through recruitment of transcriptional activators to individual gene promoters. We found 57 genes could block murine norovirus replication in either mouse or human cells. Several of these genes are associated with classical immune signaling pathways, while many of the molecules we identified have not been previously associated with antiviral activity. Our data is a resource for those studying norovirus and we provide a robust approach to identify novel antiviral genes.

Identification of anti-norovirus genes in mouse and human cells using genome-wide CRISPR activation screening