Norovirus translation requires an interaction between the C Terminus of the genome-linked viral protein VPg and eukaryotic translation initiation factor 4G.

Viruses have evolved a variety of mechanisms to usurp the host cell translation machinery to enable translation of the viral genome in the presence of high levels of cellular mRNAs. Noroviruses, a major cause of gastroenteritis in man, have evolved a mechanism that relies on the interaction of translation initiation factors with the virus-encoded VPg protein covalently linked to the 5' end of the viral RNA. To further characterize this novel mechanism of translation initiation, we have used proteomics to identify the components of the norovirus translation initiation factor complex. This approach revealed that VPg binds directly to the eIF4F complex, with a high affinity interaction occurring between VPg and eIF4G. Mutational analyses indicated that the C-terminal region of VPg is important for the VPg-eIF4G interaction; viruses with mutations that alter or disrupt this interaction are debilitated or non-viable. Our results shed new light on the unusual mechanisms of protein-directed translation initiation.This work was supported by funding from the BBSRC (BB/I012303/1) and the Wellcome Trust (WT097997MA) to IG, funding from BBSRC to LR and NL (BB/I01232X/1), as well as to SC (BB/J001708/1). IG is a Wellcome Senior Fellow.This is the final published version. It's also available on the publisher's website at: http://www.jbc.org/content/early/2014/06/13/jbc.M114.550657.abstrac

Structure of a Murine Norovirus NS6 Protease-Product Complex Revealed by Adventitious Crystallisation

Murine noroviruses have emerged as a valuable tool for investigating the molecular basis of infection and pathogenesis of the closely related human noroviruses, which are the major cause of non-bacterial gastroenteritis. The replication of noroviruses relies on the proteolytic processing of a large polyprotein precursor into six non-structural proteins (NS1–2, NS3, NS4, NS5, NS6pro, NS7pol) by the virally-encoded NS6 protease. We report here the crystal structure of MNV NS6pro, which has been determined to a resolution of 1.6 Å. Adventitiously, the crystal contacts are mediated in part by the binding of the C-terminus of NS6pro within the peptide-binding cleft of a neighbouring molecule. This insertion occurs for both molecules in the asymmetric unit of the crystal in a manner that is consistent with physiologically-relevant binding, thereby providing two independent views of a protease-peptide complex. Since the NS6pro C-terminus is formed in vivo by NS6pro processing, these crystal contacts replicate the protease-product complex that is formed immediately following cleavage of the peptide bond at the NS6-NS7 junction. The observed mode of binding of the C-terminal product peptide yields new insights into the structural basis of NS6pro specificity

Sequence conservation of polyprotein junction in MNV that are cleaved by NS6^pro.

<p>(A) The five cleavage junctions of MNV CW1 polyprotein (NCBI accession number YP_720001) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038723#pone.0038723-Sosnovtsev1" target="_blank">[14]</a>. (B) Weblogo of polyprotein cleavage junctions cleaved by MNV NS6^pro. This Weblogo was generated using 39 MNV polyprotein sequences and the Weblogo sequence logo generator <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038723#pone.0038723-Crooks1" target="_blank">[44]</a>. The height of the letter in each case is indicative of the degree of conservation. The Genbank accession numbers of the other sequences used to prepare the alignment are ABU55618, ABU55627, ABU55615, ABU55621, ABU55612, ABU55624, AEE10026, ABU55600, AEY83582, AEE10023, ABU55606, AEE10020, ABU55609, AEE10017, ABB02416, AEE10002, ACJ72215, AEE09999, ABU55591, AEE10005, ABU55570, AEE10008, ABU55585, AEE10014, ABU55579, AEE10011, ABU55576, ABU55597, ABU55573, ABU55603, ABU55582, ABU55594, ABU55588, ABU55567, ACS70958, ACJ72218, ABS29272, ABS29274.</p

Comparative analysis of protease-peptide interactions for the P6–P1 residues in MNV and SV NS6^pro and CAV 3C^pro.

<p>The N-terminal and C-terminal β-barrel domains of each protease are coloured green and orange respectively. (A) Binding of residues P5–P1 (C-terminus of NS6^pro), shown as sticks colour-coded by atom type (Carbon – light-blue; Oxygen – red; Nitrogen – blue), within the peptide binding grove of MNV NS6^pro. Selected side-chains from the protease are also shown as sticks. Hydrogen bonds and salt-bridges mentioned in the text are indicated by black dashed lines; all such bonds shown are ≤3.1 Å. (B) Same view as in A but showing the surface of MNV NS6^pro. (C) Binding of residues P5–P1 from a peptide-like inhibitor to SV (a human norovirus) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038723#pone.0038723-Hussey1" target="_blank">[23]</a>. Water molecules involved in the protease-peptide interaction are shown as red spheres. (D) Same view as in B but showing the surface of SV NS6^pro. (E) The refined σ-weighted 2F_o-F_c map electron density (where F_o and F_c are the observed and calculated structure factors respectively) for an A-chain C-terminal peptide, shown at 1.5 σ. (F) The interaction between residues P6–P1 of a peptide ‘product’ and CAV 3C^pro<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038723#pone.0038723-Lu1" target="_blank">[30]</a>.</p

Cloning and C139A mutagenic primers used in the course of the study.

<p>Restriction sites used in cloning are underlined. Mutations introduced using QuikChange mutagenesis are in boldface.</p

Structural comparison of the MNV NS6 protease with human norovirus NS6^pro and foot-and-mouth disease virus 3C^pro.

<p>(A) Cartoon representation of the MNV NS6^pro structure. The N and C-terminal domains are coloured green and orange respectively. The side-chains of the amino acids that make up the catalytic triad, A139 (mutated from Cys), H30 and D54, are shown as sticks. A disordered loop formed by residues 124–130 (residues 124–131 in chain B) is indicated as a dashed line. The peptide bound to NS6^pro is not shown in this representation. (B) Overlay of HuNV NS6 protease structures from Chiba (PDB-ID: 1WQS), Norwalk (PDB-ID: 2FYQ) and Southampton (PDB-ID: 2IPH) viruses <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038723#pone.0038723-Nakamura1" target="_blank">[19]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038723#pone.0038723-Zeitler1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038723#pone.0038723-Hussey1" target="_blank">[23]</a>. Excluding the variable C-termini, the root mean square deviations of the backbone atoms of Chiba, Norwalk and Southampton virus NS6^pro from MNV NS6^pro are 0.62, 0.43 and 0.41 respectively. The disordered C-terminus of the Chibavirus protease is shown as a dashed line. (C) Structure of FMDV 3C^pro (PDB-ID: 2J92) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038723#pone.0038723-Sweeney1" target="_blank">[26]</a>, coloured as in panel (A).</p

Variations in the crystal packing of the MNV NS6^pro A and B chains in the asymmetric unit.

<p>(A) Crystal packing of A and B chains of MNV NS6^pro. The A and B chains of one asymmetric unit are shown along with the neighbouring molecules (labelled A' and B') into which they insert their C-termini. (B) This panel shows the same molecules that are depicted in panel A (with the same colouring) but in this case the A and B chains within the asymmetric unit are superposed; this reveals the very different contacts that they make with their closest neighbour in the crystal. (C) Here the A' and B' chains from panel A are now superposed in order to show the similarity of the conformations of the bound C-termini (shown as sticks) from the A and B chains respectively. Colour-coding is the same as panel A.</p

Crystallographic data collection and model refinement statistics for MNV NS6^pro.

1<p>Values for highest resolution shell given in parentheses.</p>2<p>R_merge  = 100 ×Σ_hkl|I_j(hkl) − j(hkl)>|/Σ_hklΣ_jI(hkl), where I_j(hkl) and j(hkl)> are the intensity of measurement j and the mean intensity for the reflection with indices hkl, respectively.</p>3<p>R_work  = 100 ×Σ_hkl||F_obs| − |F_calc||/Σ_hkl|F_obs|.</p>4<p>R_free is the R_model calculated using a randomly selected 5% sample of reflection data that were omitted from the refinement.</p>5<p>RMS, root-mean-square; deviations are from the ideal geometry defined by the Engh and Huber parameters <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038723#pone.0038723-Engh1" target="_blank">[45]</a>.</p

A Conserved Interaction between a C-Terminal Motif in Norovirus VPg and the HEAT-1 Domain of eIF4G Is Essential for Translation Initiation

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 in vitro 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.ISSN:1553-7374ISSN:1553-736