Fig. 4
sGAG analogues stimulate EV-D68-947 uncoating. a Structure of the EV-D68-947 native virion. Density map (low passed to 14 Å resolution) colored by radial distance to the particle center (Å). A pentangle, oval, and two triangles indicate five-, two-, and three-fold axes and delimit an icosahedral asymmetric unit. A rectangle outlines the limit of the close-up view in b. b Sialic acid-binding site in EV-D68-947. Shown are residues (yellow) within 4 Å from any atom of sialic acid (green). Black dashed lines indicate polar interactions. Residues are numbered by adding 1000 (VP1), 2000 (VP2), or 3000 (VP3) to their positions. c Receptor analogues induce conformational changes of EV-D68-947 to eject the pocket factor. Left panel: a biological capsid protomer is colored blue (VP1), green (VP2), red (VP3), and orange (the VP1 hydrophobic pocket). A rectangle delimits the three close-up views of the pocket (right panels): superimposed structures of native virus and virus with receptor analogues. VP1 Ile217 moves such that it would clash with a pocket factor if it were present. d EV-D68-947 particles were incubated for 1 h at 33 °C with receptor analogues. Full and empty particles in cryo-EM images were counted. e Structure of EV-D68-947 empty capsids formed in the absence or presence of LMWH. VP2 residues 53–60, 98–100, and 240–242 and VP3 residues 148–150, which line the hole around the two-fold axis, are less ordered in emptied particles formed after LMWH treatment than in those formed without ligand. Color scale indicates radial distance to the particle center (Å). Scale bar: 35 Å. f Hypothetical model summarizing uncoating cues required by EV-D68 strains. Bar sizes are not based on quantitative measurements. g Infectivity of EV-A71 before (passage 0) and after (passage 5) five passages in H1-HeLa PLA2G16KO cells, as determined by end-point dilution on WT and PLA2G16KO H1-HeLa cells. TCID50: median tissue culture infective dose. Error bars represent mean ± s.e.m. of two biological replicates. h EV-A71 was incubated at 50 °C for the indicated times, after which the amount of intact virus was determined by end-point dilution on HeLa-R19 cells. Two biological replicates are shown
