Norovirus VPg Binds RNA through a Conserved N-Terminal K/R Basic Patch.

Alice M Mcsweeney,Vivienne L Young,Vernon K Ward

doi:10.3390/v13071282

Abstract

The viral protein genome-linked (VPg) of noroviruses is a multi-functional protein that participates in essential roles during the viral replication cycle. Predictive analyses indicate that murine norovirus (MNV) VPg contains a disordered N-terminal region with RNA binding potential. VPg proteins were expressed with an N-terminal spidroin fusion protein in insect cells and the interaction with RNA investigated by electrophoretic mobility shift assays (EMSA) against a series of RNA probes (pentaprobes) representing all possible five nucleotide combinations. MNV VPg and human norovirus (HuNV) VPg proteins were directly bound to RNA in a non-specific manner. To identify amino acids involved in binding to RNA, all basic (K/R) residues in the first 12 amino acids of MNV VPg were mutated to alanine. Removal of the K/R amino acids eliminated RNA binding and is consistent with a K/R basic patch RNA binding motif within the disordered N-terminal region of norovirus VPgs. Finally, we show that mutation of the K/R basic patch required for RNA binding eliminates the ability of MNV VPg to induce a G0/G1 cell cycle arrest.

Highlights

An alignment with the RNA binding motif of PVA viral protein genome-linked (VPg) identified that the 20 N-terminal amino acids of murine norovirus (MNV) VPg have some conservation with several basic and glycine residues (Figure 2c), suggesting that this region may have RNA
Bioinformatic analysis indicates that MNV VPg contains regions of disorder, consistent with Nuclear magnetic resonance (NMR) data and disorder predictions of other norovirus VPg proteins [10,40]
VPg proteins used in this study were expressed as a fusion with the NT* protein

Summary

Introduction

Human noroviruses (HuNV) are a significant cause of gastroenteritis and account for approximately one-fifth of acute gastroenteritis across all age groups [1]. Recent studies have demonstrated HuNV replication in a continuous human B cell line, stem cell derived human enteroids or zebrafish larvae, significantly increasing our ability to directly study. HuNV [2,3,4]. Implementation of these systems within the laboratory can be complex and there is considerable variation in replication between HuNV strains [5]. Murine norovirus (MNV) remains a commonly utilised model virus for studying the viral lifecycle. MNV replicates in laboratory cell culture lines and retains a similar genomic layout to

Methods

Results

Conclusion