Fig. 6: Three CVA9 VP1 amino acid residues contribute to the recruitment of Subtilisin A to CVA9 capsid and are structurally compatible to enzymatic cleavage.

a SDS-PAGE analysis of CVA9 capsid protein integrity following the digestion of purified native virus in solution with Subtilisin A. The two arrows point the proteolytic viral fragments observed after 15 min digestion with Subtilisin A. b Schematic representation of the disintegration of CVA9 viral capsid following the predictive attack of VP1 on the 5-fold vertex and subsequent use of each independent viral protein as substrate. c Deductive inference process used to identify amino acid residues involved in CVA9 inactivation by Subtilisin A. d Surface representation of the two interfaces (in purple) predicted by AF2-M to interact with Subtilisin A during the early stages of CVA9 inactivation. e Mass spectrometry identification of the six amino acid residues for which the C-terminal peptide bond was cleaved following in-gel digestion of denatured VP1 with Subtilisin A. f Site-directed docking of Subtilisin A on three of six amino acid residues deduced during the two first steps of the selection process. Protein/peptide docking have been performed using HADDOCK 2.4 and interatomic contacts (IC) for each output have been determined using Prodigy. Surface views of Subtilisin A contact points with each VP1 regions are shown to the right of each docking structure. Pink surface regions correspond to the catalytic triad of the enzyme. g Point mutations of VP1 applied in silico and used to predict the significance of each amino acid residue in Subtilisin A recruitment on viral capsid. On the left of the dotted line, the sequence corresponds to the DE loop and on the right, the five first amino acid of the C-terminal end of VP1. h Influence of CVA9 VP1 point mutations on the prediction of Subtilisin A recruitment to viral capsid. Subtilisin A recruitments on CVA9 protomer were predicted with AF2-M using 6 simulation cycles. To the right of each prediction (rank 1 to 5), the squares represent the response of both DE loop and C-terminal end sequence trajectories. The absence of a square indicates that the corresponding trajectory event was not observed for any of the analysis.