Caspase-mediated cleavage of murine norovirus NS1/2 potentiates apoptosis and is required for persistent infection of intestinal epithelial cells

Human norovirus (HNoV) is the leading cause of acute gastroenteritis and is spread by fecal shedding that can often persist for weeks to months after the resolution of symptoms. Elimination of persistent viral reservoirs has the potential to prevent outbreaks. Similar to HNoV, murine norovirus (MNV) is spread by persistent shedding in the feces and provides a tractable model to study molecular mechanisms of enteric persistence. Previous studies have identified non-structural protein 1 (NS1) from the persistent MNV strain CR6 as critical for persistent infection in intestinal epithelial cells (IECs), but its mechanism of action remains unclear. We now find that the function of CR6 NS1 is regulated by apoptotic caspase cleavage. Following induction of apoptosis in infected cells, caspases cleave the precursor NS1/2 protein, and this cleavage is prevented by mutation of caspase target motifs. These mutations profoundly compromise CR6 infection of IECs and persistence in the intestine. Conversely, NS1/2 cleavage is not required for acute replication in extra-intestinal tissues or in cultured myeloid cells, indicating an IEC-specific role. Intriguingly, we find that caspase cleavage of NS1/2 reciprocally promotes caspase activity, potentiates cell death, and amplifies spread among cultured IEC monolayers. Together, these data indicate that the function of CR6 NS1 is regulated by apoptotic caspases, and suggest that apoptotic cell death enables epithelial spread and persistent shedding. Author Summary Human Norovirus infection is highly contagious and the most common cause of acute gastroenteritis. Norovirus can be persistently shed after resolution of symptoms, perpetuating or initiating new outbreaks. Murine norovirus (MNV) is also persistently shed, enabling study of host and viral determinants of norovirus pathogenesis. We previously identified a critical role for MNV non-structural protein 1 (NS1), in persistence. Herein we find that regulation of NS1 by host apoptotic caspases is required for infection of intestinal epithelial cells, but not for extra-intestinal spread. Additionally, we demonstrate that NS1 reciprocally promotes cell death and spread among epithelial cells. These data identify regulation of NS1 by host proteases and suggest that apoptotic death is a determinant of epithelial spread and persistence.