MNV VPg 104–124 interacts with the HEAT-1 domains of eIF4GI, eIF4GII and DAP5 with low micromolar affinity.

<p>(A-B) FITC-labelled peptides– MNV VPg(104–124) and MNV VPg(108–124)–were used in fluorescence anisotropy binding assays with unlabelled HEAT-1 domains of eIF4GI (748–993), eIF4GII (745–1003) and DAP5 (61–323) in order to measure the affinity of the interaction. The normalised change in fluorescence anisotropy (relative to a no-protein control), ΔFA, is plotted against protein concentration. Error bars in ΔFP indicate the standard deviation of 5 (eIF4GI) or 10 (eIF4GII) independent measurements. The solid lines indicate the fit to a single-site binding model. (A) Comparison of the binding of MNV VPg(104–124) and MNV VPg(108–124) to eIF4GI and eIF4GII HEAT-1 domains. (B) Binding of MNV VPg(104–124) to the HEAT-1 domain of DAP5. The fit is calculated with the fluorescence anisotropy data from three independent experiments (all included in the graph). (C) ITC experiments in which unlabelled eIF4GII (745–1003) was titrated into MNV VPg(1–124). Top panel: raw data obtained for a representative experiment from 20 injections (firstly with a volume of 0.5 μL followed by 19 injections of 2 μL of eIF4GII HEAT-1). Bottom panel: the integrated data with a best-fit curve for the representative experiment generated for a single-site binding model using the Origin software package.