Cryo-EM structures reveal two distinct conformational states in a picornavirus cell entry intermediate

Fig 1. Anti-VP1 antibody trapped expanded particle. (A) Top row: The 3D classes from the dataset, with undecorated 160S particles (left, blue border), the anti-VP1 mAb decorated 135S-like particle (middle, orange border) and the mAb decorated empty particle (right, yellow border). Second row: Radially colored isosurface representations of the refined particles are shown at a high contour, where the holes at the 2-fold are clearly observed. Third row, isosurface representations of the mAb-decorated 135S (center) and 80S particles (right) contoured at a lower level showing the diffuse density attributable to the bottom half of the Fab part of the mAb. Bar indicates the radial range that each color corresponds to. (B) Density for an asymmetric focus class calculated from the mAb decorated 80S particle is depicted in the context of the 2-fold related VP2 subunits (yellow) and a model of a Fab (pink). The tube-like density for the N-terminus of VP1 (arrow) is seen above one of the two 2-fold related VP2 helices.

Fig 2. Anti-VP1 antibody trapped genome containing particle (m135) is similar to the canonical 135S particle. (A) Cage density depiction (σ = 0.25) of the mAb-decorated m135 structure in the vicinity of the 2-fold axes showing the rod shaped density for the lower half of the Fab portion of the mAb and the better ordered density for the capsid proteins of the virus (B) The canonical poliovirus protomer consisting of VP1 (blue), VP2 (yellow) and VP3 (red) is shown juxtaposed with 5-fold related copies of VP1(cyan) and VP2 (gold). The cyan, gold, and red copies together form the “5-3-3 triangle” having one 5-fold and two 3-fold symmetry axes at its corners. The quasi 3-fold lies at the center of this triangle. Panels (B) and (C) are viewed from outside the virus. (C) The N-terminus of VP1 (dark blue) is seen exiting the virus capsid through the quasi 3-fold hole. To accommodate this, the GH loop of VP3 (labeled) becomes rearranged and partially disordered. Panels (D-F) are viewed from inside the virus. (D) The exiting N-terminus (dark blue) contacts the tip of the VP2 bottom loop (labeled) (E) The bottom loop of VP2, in m135 (yellow), is rearranged relative to native VP2 (magenta) (F) In…

Fig 3. Receptor-catalyzed, and heat-triggered 135S particles (r/h135). (A) Radially colored isosurface rendering of the “early” 135S particle at a high contour (σ = 5) (left panel) reveals an opening at the 2-fold axis. The panel on the right is shown at a lower contour level (σ = 2) with a clipping plane inserted to highlight the re-organised density inside the capsid. These two panels are based on the r135 map, because the two structures are virtually identical. Bar indicates the radial range that each color corresponds to. (B) Close up of the quasi-3-fold region. Density for the GH loop of VP3, overlaid on the model, is very similar in the r135 (blue) and h135 (purple) maps. In this conformation, the GH loop of VP3 plugs the quasi-3-fold hole. (C) Stereo views compare the GH loops of VP3 from the r135 (red), m135 (salmon), and native (orange) structures. The r135 conformation is more similar to the native conformation, as opposed to the m135 model where the GH loop becomes more extended and forms new beta-sheet interactions with a radially-oriented segment of the VP3 D strand.

Fig 4. Receptor catalyzed 135S particle incubated with the anti-VP1 antibody at 25°C (r135+m25). (A) Radially colored isosurface rendering of the “late” 135S particle shown at high contour (σ = 5) (left) to highlight the hole at the 2-fold axis and at a lower contour (σ = 0.5) (right) to highlight binding of the Fab (pink) relative to the capsid. Bar indicates the radial range that each color corresponds to. (B) The interaction between the Fab density and the capsid is better resolved following a focused classification centered around the 2-fold symmetry axis of the capsid. (C) A focus map from (B) contoured at a σ = 3 compared with the m135 model, helps to visualize the N-terminus of VP1 (blue) exiting the capsid from the quasi-3-fold hole. The density in this region is poorly resolved in the icosahedral averaged reconstruction (D) A focused class obtained from the screening data contoured at σ = 3, using a cylindrical mask centered on the 2-fold axis. This map clearly shows a column of density that accommodates the first 20 amino acids of the N-terminus of VP1 (blue). The helix bound to the top surface of VP2 (yellow) is stabilized in a unique orientation by contacts with the Fab…

Fig 5. Receptor catalyzed 135S particle incubated with the anti-VP1 antibody at 37°C (r135+m37). (A) Radially colored isosurface rendering of the “late” 135S particle that is produced after receptor-catalyzed expanded virus is incubated with anti-VP1 antibody at 37°C for 1h. Bar indicates the radial range that each color corresponds to (A). The panel on the left is contoured at a higher level (σ = 5) to show surface details such as holes at the 2-fold and density in the quasi-3-fold. At a lower contour (σ = 0.1) (right), smeared density due to Fab binding (beige) is apparent. (A and B) The Fab density in r135+m37 appears to be a super-position of the rod-shaped Fab density that extends over the quasi-3-fold (as seen in m135) and the 2-fold-centered density (as seen in the r135+m25 reconstruction). (C) a close-up view of the showing the VP1 N-terminus emerging at quasi-3-fold (compare Figs 2C and 4C).

Fig 6. Location of VP4 before and after capsid expansion. Profile (top panel) and face-on (lower panel) view of VP4 (green) in native (σ = 2), r135 (σ = 1.2), m135 (σ = 0.7) and empty states (σ = 2). The native orientation and location of VP4 has been preserved to highlight the changes in the density, as the capsid expands. Prior to RNA release, VP4 pentamers, somewhat rearranged, appear to remain at their original radius, on the outer surface of the RNA ball, as the rest of the capsid expands outward.

Fig 7. Schematic of VP1 N-terminal stabilization in the r135+m25 state. Four distinct areas in the N-terminal extension of VP1 (dashed blue curve) can be stabilized by binding to the Fab (pink) and the viral capsid (grey). This overall binding pattern is suggested by (i) the observation of residues 70–62 in the m135 complex; (ii) the expectation that amino acids from the 55–42 range (the epitope for the anti-VP1 Ab) will bind to the underside of the anti-peptide mAb; (iii) the anchoring of the N-terminal helix (around residue 21) to the top surface of VP2; and (iv) the nonspecific stabilization of the amphipathic N-terminal helix along the side of the Fab in a focus map. Although the pathway of the N-terminal extension is unlikely to be unique, the diagram does help us to understand why the increased prevalence of Fab moieties at the 2-fold hole (in r135+m25) is evidence for the increased anchoring of the amphipathic helix (residues 1–21) on the top surface of VP2, presumably stabilized by lower temperature and longer incubation before the antibody is introduced.