Polyprotein processing and intermolecular interactions within the viral replication complex spatially and temporally...

Norovirus infections are a major cause of acute viral gastroenteritis and a significant burden on global human health. A vital process for norovirus replication is the processing of the nonstructural polyprotein by a viral protease into the viral components required to form the viral replication complex. This cleavage occurs at different rates, resulting in the accumulation of stable precursor forms. Here, we characterized how precursor forms of the norovirus protease accumulate during infection. Using stable forms of the protease precursors, we demonstrated that all of them are proteolytically active in vitro, but that when expressed in cells, their activities are determined by both substrate and protease localization. Although all precursors could cleave a replication complex-associated substrate, only a subset of precursors lacking the NS4 protein were capable of efficiently cleaving a cytoplasmic substrate. By mapping the full range of protein-protein interactions among murine and human norovirus proteins with the LUMIER assay, we uncovered conserved interactions between replication complex members that modify the localization of a protease precursor subset. Finally, we demonstrate that fusion to the membrane-bound replication complex components permits efficient cleavage of a fused substrate when active polyprotein-derived protease is provided in trans These findings offer a model for how norovirus can regulate the timing of substrate cleavage throughout the replication cycle. Because the norovirus protease represents a key target in antiviral therapies, an improved understanding of its function and regulation, as well as identification of interactions among the other nonstructural proteins, offers new avenues for antiviral drug design.