Structure of Human Enterovirus 70 and Its Inhibition by Capsid-Binding Compounds

Figures

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  FIG 1. Virion structure of EV70. (A) Molecular surface of the EV70 virion colored according to capsid proteins. VP1 subunits are shown in blue, VP2 subunits are in green, and VP3 subunits are in red. The positions of selected icosahedral symmetry axes are indicated with an oval for 2-fold, a triangle for 3-fold, and a pentagon for 5-fold. An asymmetric unit is outlined with a triangle. The white arrow indicates the position of an opening into a selected VP1 pocket. Bar, 10 nm. (B and C) Cartoon representations of the protomer of capsid proteins in two orientations related by 90° rotation along the x axis and 90° rotation along the y axis. The coloring of the subunits is the same as that described above for panel A, and the VP4 subunit is shown in yellow. The inset of panel C shows the differences between the BC and DE loops of VP1 and the C termini of VP3 (red arrow) of EV70 (dark colors), EV-D68 strain Fermon (light colors), and EV-D68 strain MO (magenta).

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  FIG 2. Conservation of pocket-forming residues among enteroviruses. (A) Cartoon representation of the EV70 protomer colored according to sequence conservation among 30 selected enteroviruses (for details, see Fig. S8 in the supplemental material) (85). The position of the VP1 pocket with pleconaril is highlighted, represented as a green semitransparent molecular surface. The arrows indicate the BC and DE loops of VP1, which are part of antigenic sites in enteroviruses. (B) Detail of the VP1 pocket with side chains of residues shown in a stick representation. (C) Conservation of pocket-forming residues of VP1 and VP3 (the asterisk denotes a residue contributing to a pocket in a neighboring protomer). The table shows the conservation of amino acid residues among 30 selected enteroviruses. The consensus of residues at the levels of 70% to 100% is indicated. Capital letters indicate consensus amino acid residues, and lowercase letters indicate classes of residues with specific physicochemical properties (86).

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  FIG 3. The pocket in VP1 of EV70 and its interactions with capsid-binding inhibitors. (A to D) Molecular surface representations of capsid proteins clipped to show the VP1 pocket of EV70. (A and B) The pocket is partially blocked by Met224 of VP1 (in cyan) in the virion (A) and completely collapsed in the altered particle (B). (C and D) The binding of WIN51711 (C) and pleconaril (D) induces the expansion of the pocket. The inhibitors are shown in a stick representation. The VP1 subunit is shown in blue, VP3 is in red, VP3 from a neighboring protomer is in orange, and inhibitors are in magenta. (E) Cryo-EM maps showing inhibitors bound in the VP1 pocket of EV70. The inhibitors are shown in a stick representation. (F and G) Stick representations of the interactions of the side chains of residues with WIN51711 (F) and pleconaril (G). The inhibitors are shown as semitransparent molecular surfaces. The inhibitors interact with the same residues.

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  FIG 4. Structural comparison of the virion, altered particle, and empty particle of EV70. (A to C) Molecular surfaces rainbow-colored according to the distance from the particle center of the virion (A), altered particle (B), and empty particle (C). Altered and empty particles contain pores on the 2-fold axes of their symmetry. (D to F) Central slices of cryo-EM maps of the virion (D), altered particle (E), and empty particle (F). The insets show electron micrographs of the corresponding particles. The positions of selected icosahedral symmetry axes are indicated in panel D. (G to I) Cartoon representations of capsid proteins around the 2-fold symmetry axes of the virion (G), altered particle (H), and empty particle (I). The formation of a pore around the 2-fold symmetry axis in altered (H) and empty (I) particles is enabled by the reduction in interpentamer contacts and the loss of structure of parts of VP2 subunits. The sizes of the pores are shown in panels H and I. The arrow in panel H indicates the externalization of the N-terminal part of VP1 through a pore above the 2-fold symmetry axis in altered particles. VP1 subunits are shown in blue, VP2 subunits in green, and VP3 subunits…

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  FIG 5. Effects of capsid-binding inhibitors on the infectivity and particle stability of EV70. (A) The reduction in the infectivity of EV70 was assayed on rPE1 cells using a plaque assay. Sigmoidal dose-response curves were fitted to the experimental data and EC50 values of the inhibitors were estimated. (B) Denaturation curves from thermal stability assays performed on native EV70 and EV70 mixed with capsid-binding inhibitors (15-fold molar excess of the inhibitor per binding site). As the temperature increased, the structure of the particle relaxed, and the SYBR green present in the solution interacted with genomic RNA, which caused the increase in fluorescence. EV70 particles in the presence of capsid-binding inhibitors exhibit higher capsid stability and, thus, a higher particle denaturation temperature (Tm).