Crystal structures of murine norovirus-1 RNA-dependent RNA polymerase

Abstract

Norovirus is one of the leading agents of gastroenteritis and is a major public health concern. In this study, the crystal structures of recombinant RNA-dependent RNA polymerase (RdRp) from murine norovirus-1 (MNV-1) and its complex with 5-fluorouracil (5FU) were determined at 2.5 Å resolution. Crystals with C2 symmetry revealed a dimer with half a dimer in the asymmetrical unit, and the protein exists predominantly as a monomer in solution, in equilibrium with a smaller population of dimers, trimers and hexamers. MNV-1 RdRp exhibited polymerization activity with a right-hand fold typical of polynucleotide polymerases. The metal ion modelled in close proximity to the active site was found to be coordinated tetrahedrally to the carboxyl groups of aspartate clusters. The orientation of 5FU observed in three molecules in the asymmetrical unit was found to be slightly different, but it was stabilized by a network of favourable interactions with the conserved active-site residues Arg185, Asp245, Asp346, Asp347 and Arg395. The information gained on the structural and functional features of MNV-1 RdRp will be helpful in understanding replication of norovirus and in designing novel therapeutic agents against this important pathogen.

Structure-Based Inhibition of Norovirus RNA-Dependent RNA Polymerases

“…As already described by Lee et al, MNV RdRp shows a propensity to oligomerize with increasing concentrations that does not seem biochemically relevant. 11 In fact, in gel-filtration experiments, where the …”

“…11 The protein crystal structure was refined to a final crystallographic R-factor of 18.6% and R free of 22.7%, for the 29.0-to 2.0-Å resolution data set ( Table 2). Structural superposition of the three independent protein C α backbones yielded r.m.s.d.…”

“…The three-dimensional structure of MNV RdRp was solved by the molecular replacement method using the program MOLREP 42 and a search model based on the hNV RNA-CTP complex structure (PDB ID 3BSO 9 ) since the structure of RdRp MNV 11 was not available when we started this work. The three independent molecules in the crystal asymmetric unit (A, B and C) were subjected to rigid-body refinement and subsequently refined using REFMAC5.…”

Nonnucleoside Inhibitors of Norovirus RNA Polymerase: Scaffolds for Rational Drug Design

“…One key target for NoV antivirals is the viral polymerase (RdRp) because of its essential role in viral replication and the lack of homologous human enzymes. A number of studies have used recombinant NoV RdRp to characterize its biochemical properties in vitro, and the X-ray crystal structures of RdRps from human GI and GII and mouse GV NoVs have been solved (16)(17)(18).…”

Anti-norovirus therapeutics: a patent review (2010-2015)

“…The mechanism of action of nucleoside analogs involves conversion of the inactive nucleosides into their phosphate or diphosphate forms, which are the actual inhibitory species. Consequently, the norovirus RdRp is an attractive target that is well-suited to the development of norovirus-specific therapeutics due to (a) the vital role RdRp plays in the norovirus replication cycle, (b) the availability of high resolution X-ray crystal structures of free and ligand-bound enzyme complexes [72–75] and (c) the absence of a human homolog which diminishes the likelihood of off-target effects. Several novel nucleoside inhibitors have been shown to inhibit norovirus via the inhibition of RdRp (Figure 4) [76–77].…”