The MNV-1 protease-polymerase precursor cleaves a novel site in the NS1-2 protein.

Human noroviruses (HuNVs) are members of the Caliciviridae family and are a significant cause of gastroenteritis worldwide. Cell culture models are challenging for studying the intracellular replication of HuNV, with murine norovirus (MNV) routinely used to study norovirus replication. Norovirus genomes contain a non-structural (NS) polyprotein that is processed by the viral protease, generating precursor and mature proteins during replication. We identified a putative viral protease cleavage site at E66/G67 in the MNV-1 NS1-2 protein by sequence analysis that was cleaved by the protease-polymerase (ProPol) precursor but not Pro in vitro and during MNV-1 replication, with the cleavage site confirmed by MS. The P4-P4' LHAE66/G67PLA cleavage site of MNV-1 is conserved in some MNV strains, while other strains contain a predicted caspase cleavage site (LHAD66/G67PHA) at this position, indicating that cleavage of NS1 is widely conserved in MNV. Mutation of the ProPol cleavage site in MNV-1 NS1-2 to the caspase motif reduced viral protein expression and replication in Huh7CD300lf and BV2 cells, while removal of the site via an E66A mutation caused a 100-fold reduction in viral yield in the naturally permissive BV-2 cell line. This study demonstrates that the protease function of the MNV-1 ProPol precursor has an important role during replication that is distinct to the mature protease and that cleavage of NS1-2 by ProPol at E66/G67 is important for MNV-1 replication.