Abstract
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 E(66)/G(67) 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' LHAE(66)/G(67)PLA cleavage site of MNV-1 is conserved in some MNV strains, while other strains contain a predicted caspase cleavage site (LHAD(66)/G(67)PHA) 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 E(66)A 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 E(66)/G(67) is important for MNV-1 replication.