TY - JOUR T1 - A conserved interaction between a C-terminal motif in Norovirus VPg and the HEAT-1 domain of eIF4G is essential for translation initiation JF - bioRxiv DO - 10.1101/024349 SP - 024349 AU - Eoin N. Leen AU - Frédéric Sorgeloos AU - Samantha Correia AU - Yasmin Chaudhry AU - Fabien Cannac AU - Chiara pastore AU - Yingqi Xu AU - Stephen C. Graham AU - Stephen J. Matthews AU - Ian G. Goodfellow AU - Stephen Curry Y1 - 2015/01/01 UR - http://biorxiv.org/content/early/2015/08/10/024349.abstract N2 - Translation initiation is a critical early step in the replication cycle of the positive-sense, single-stranded RNA genome of noroviruses, a major cause of gastroenteritis in humans. Norovirus RNA, which has neither a 5′ m7G cap nor an internal ribosome entry site (IRES), adopts an unusual mechanism to initiate protein synthesis that relies on interactions between the VPg protein covalently attached to the 5′-end of the viral RNA and eukaryotic initiation factors (eIFs) in the host cell.For murine norovirus (MNV) we previously showed that VPg binds to the middle fragment of eIF4G (4GM; residues 652-1132). Here we have used pull-down assays, fluorescence anisotropy, and isothermal titration calorimetry (ITC) to demonstrate that a stretch of ∼20 amino acids at the C terminus of MNV VPg mediates direct and specific binding to the HEAT-1 domain within the 4GM fragment of eIF4G. Our analysis further reveals that the MNV C-terminus binds to eIF4G HEAT-1 via a motif that is conserved in all known noroviruses. Fine mutagenic mapping suggests that the MNV VPg C terminus may interact with eIF4G in a helical conformation. NMR spectroscopy was used to define the VPg binding site on eIF4G HEAT-1, which was confirmed by mutagenesis and binding assays. We have found that this site is non-overlapping with the binding site for eIF4A on eIF4G HEAT-1 by demonstrating that norovirus VPg can form ternary VPg-eIF4G-eIF4A complexes. The functional significance of the VPg-eIF4G interaction was shown by the ability of fusion proteins containing the C-terminal peptide of MNV VPg to inhibit translation of norovirus RNA but not cap- or IRES-dependent translation. These observations define important structural details of a functional interaction between norovirus VPg and eIF4G and reveal a binding interface that might be exploited as a target for antiviral therapy. ER -