Fig_4.tif (2.11 MB)

The C-termini of Noroviral VPg proteins contain a conserved sequence motif that binds to eIF4G HEAT-1.

figure

posted on 2016-01-18, 14:36 authored by Eoin N. Leen, Frédéric Sorgeloos, Samantha Correia, Yasmin Chaudhry, Fabien Cannac, Chiara Pastore, Yingqi Xu, Stephen C. Graham, Stephen J. Matthews, Ian G. Goodfellow, Stephen Curry

(A) Top: Amino acid sequence alignments of representative sequences of the C-termini of all 6 genogroups of Norovirus (GI-GIV). Second from top: Alignment of the C-termini of Human Astrovirus 4 (HuAST4) VPg with MNV (GV) VPg. Third from top: Alignment of the C-termini of FCV VPg with MNV (GV) VPg. Bottom: Alignment of the C terminus of MNV (GV) VPg with Rice Yellow Mottle Virus (RYMV) VPg. The representative strains used in the alignments are GI—Hu/GI/Norwalk/1968/US, (NCBI accession AAC64602), GII—Lordsdale virus Hu/GII/Lordsdale/1993/UK (NCBI accession P54634), GIII—Bo/GIII/B309/2003/BEL (NCBI accession ACJ04905.1), GIV—Hu/GIV.1/LakeMacquarie/NSW268O (NCBI accession AFJ21375), GV—Mu/NoV/GV/MNV1/2002/USA (NCBI accession ABU55564.1), GVI—dog/GVI.1/HKU_Ca035F/2007/HKG (NCBI accession FJ692501), FCV—F9 strain (NCBI accession P27409.1), HuAst4—Human astrovirus 4 (NCBI accession Q3ZN07), Rice yellow mottle virus isolate CI4 (NCBI accession NC_001575). Sequence alignments were performed by ClustalW [6] and BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). ^#Denotes sequences that were used in pull-down assays (see panel B). (B) The indicated VPg sequences in panel A were fused to the C terminus of GST for use as prey in cobalt affinity pull-down assays in which His-tagged eIF4GI HEAT-1 was used as bait, and analysed by SDS PAGE. Lanes 1–3: input samples of some of the proteins used in pull-down experiments (black labels); lanes 4–11: eluted proteins (red labels; bands at ~27 kDa indicate GST-VPg C-terminal constructs that bound to His-eIF4G HEAT-1); lanes 12–19 (blue labels)–protein mixes used in the pull-down experiments. (C, D) Mutational analysis of the C-terminal sequences of MNV VPg were performed using GST-MNV VPg C-terminal fusions in the same way as the experiment presented in panel B. Labelling is colour-coded as in panel B. (E) Quantification of the pull-down results shown in panels C and D. (F) Helical wheel representation of MNV VPg 102–124 generated using http://heliquest.ipmc.cnrs.fr/ [44]. Residues mutated for the pull-down assays are indicated by bold-face labels and colour-coded by the effect of the substitution on binding to eIF4G HEAT-1: red–no binding; black—binding at or near wild-type levels.