Norovirus (NoV) is a non-enveloped single-stranded RNA virus and a leading cause of acute gastroenteritis worldwide. NoV has a major impact on human health, and represents a significant economic burden, in both developed and developing countries. Antivirals targeting the NoV RNA-dependent RNA polymerase (RdRp) are divided into two classes, nucleoside inhibitors (NI) and non-nucleoside inhibitors (NNI). Previous work showed that our previously developed NoV NNI, NIC02, displayed half-maximal inhibitory concentrations (IC50) in the low micromolar range across RdRps from a range of members of the Caliciviridae family. The aim of this study was to determine the RdRp binding site and mode of action of NIC02.
The NoV RdRp sequence (GII.4 Den Haag 2006b variant) was amplified, cloned into pET26b, expressed in Escherichia coli and purified using metal affinity and size-exclusion chromatography. Determination of the NoV RdRp crystal structure in complex with NIC02, at a resolution of 3.4Å, indicated that several residues within motif B potentially interact with NIC02, including V302, P303 and S306. To confirm this interaction, RdRp mutants V302E, V302F, P303S, P303G, S306A and S306Y were generated and fluorescent-based RdRp assays were used to determine if any changes conferred NIC02 resistance by measuring NIC02 IC50 values. The P303G mutant displayed a 3-fold increase in IC50 (28.31 µM) and the P303S mutant had a 1.5-fold increase in IC50 (16.63 µM), compared to wild-type enzyme (IC50 9.96 µM). In conclusion, the resistance observed with RdRp mutants together with the crystallography studies, suggests that motif B interacts with NIC02. This could represent a new NNI RdRp binding site and may explain the broad spectrum activity seen with this inhibitor.