Convergent evolution in the mechanisms of ACBD3 recruitment to picornavirus replication sites
Fig 5
Analysis of the dimerization interface of the GOLD: EVD68 3A complexes.
a, Overall fold of the heterotetramer composed of two GOLD: 3A complexes (upper panel) and a detailed view of the 3A dimerization interface (lower panel). The ACBD3 GOLD domains are depicted in grey, the EVD68 3A proteins in green and violet. The hydrophobic core of the dimerization interface is highlighted in red and the additional salt-bridge formed by D24 and K41 in yellow. b, Elution profiles of the GB1-fused wild-type 3A protein (blue curve) and its LVVY mutant (red curve) in size-exclusion chromatography, monitored by the absorbance at 280 nm. c, Elution profiles of the uncomplexed ACBD3 GOLD domain (green curve), GOLD domain fused to the wild-type 3A protein (blue curve) and its LVVY mutant (red curve) in size-exclusion chromatography, monitored by the absorbance at 280 nm. d-e, SAXS analysis of the wild-type GOLD-3A fusion protein (d) and its LVVY mutant (e). In the upper panel, SAXS intensity profiles for three protein concentrations are shown in green, blue, and orange. Structural models (detailed in S7 Fig, panel a) of the dimeric wild-type GOLD-3A fusion protein and its monomeric LVVY mutant yield the scattering curves shown as black solid and dashed lines (d) or vice versa (e). In the bottom panel, the Guinier plots with the curves colored as in the upper panel are shown. The region of the Guinier approximation valid for globular proteins is shaded in grey. Rg, radius of gyration. f-g, FRET analysis of the wild-type GOLD-3A fusion protein and its LVVY mutant. mAmetrine- and mPlum-fusion proteins were transiently co-expressed in HeLa cells and the FRET intensity was determined by flow cytometry. The data are visualized as the FRET signal against a wide range of the acceptor signal from one representative experiment (f) or as the mean FRET signal ± SEM at a fixed acceptor signal (marked by a dashed line in (f)) from three independent experiments (g). h, Model of the GOLD: 3A heterotetramer on the lipid bilayer. The ACBD3 GOLD domains are depicted in grey, the EVD68 3A proteins in yellow and red. i, Viral subgenomic replicon assay. U-87 MG and HaCaT cells were transfected with the T7-amplified EVD68 subgenomic replicon wild-type RNA or its mutants as indicated, and the percentage of cells with the reporter mCherry fluorescence above background was determined by flow cytometry. The viral polymerase-lacking mutant (Δ3D) was used as a negative control. The data are presented as means ± SEMs from 2 independent experiments.