Human Norovirus NS7 Protein Activates Canonical Inflammasome Pathways via NLRP6 in Intestinal Epithelial Cells

Abstract Human norovirus (HNoV) is a leading cause of acute gastroenteritis, yet the mechanisms by which it interfaces with host innate immunity remain elusive. Here, we demonstrate that the HNoV NS7 protein, an RNA-dependent RNA polymerase, acts as a direct activator of canonical inflammasomes. Using reconstituted cell systems and human intestinal enteroids (HIEs), we found that NS7 interacts with both NLRP3 and NLRP6, promoting ASC speck formation, caspase-1 cleavage, and secretion of IL-1β and IL-18. HNoV infection of HIEs recapitulated these events, including gasdermin D processing and robust IL-18 release. Importantly, CRISPR/Cas9-mediated NLRP6 deficiency abrogated inflammasome activation and markedly enhanced viral replication, underscoring the essential role of NLRP6 in epithelial antiviral defense. These findings identify NS7 as a novel inflammasome activator and establish NLRP6 as a key determinant of innate immune control of HNoV. Our study highlights inflammasome signaling as a potential therapeutic target for norovirus infection. Author summary In this study, we used human intestinal organoid models to explore how norovirus infection triggers a specific immune response known as inflammasome, which helps protect the gut from viral invaders. We focused on a viral protein called NS7 and discovered that it directly activates two types of inflammasome sensors, NLRP3 and NLRP6. We found that NLRP6, which is abundant in the gut lining, is especially important for detecting norovirus and launching an immune response. When we removed NLRP6 from intestinal cells, the virus was able to replicate more easily, and normal immune activation was lost. Our results reveal that norovirus uses its NS7 protein to interact with the body’s immune machinery in the intestine, and that NLRP6 plays a key role in controlling infection. This work highlights a new way in which the gut senses and responds to norovirus and may help guide future efforts to develop treatments that target these immune pathways.