The Concerted Conformational Changes during Human Rhinovirus 2 Uncoating  Elizabeth A Hewat, Emmanuelle Neumann, Dieter Blaas  Molecular Cell  Volume 10, Issue 2, Pages 317-326 (August 2002) DOI: 10.1016/S1097-2765(02)00603-2

Figure 1 Schematic Diagram of the Pseudo T = 3 HRV2 Capsid and Electron Micrographs of Frozen Hydrated Native HRV2 (A) Schematic diagram of the pseudo T = 3 HRV2 capsid. The VPs 1, 2, and 3 are color coded blue, green, and red, respectively. The icosahedral 5-, 3-, and 2-fold symmetry axes are indicated for one asymmetric unit. The pseudo 3-fold axes are marked with yellow triangles. The canyon around each 5-fold axis is indicated in yellow. Electron micrographs of frozen hydrated native HRV2 and empty HRV2 are shown in (B) and (C), respectively. Both these images were taken at an underfocus of 1.8 μm on a FEG electron microscope operating at 200 kV. They each have a good CTF which extends beyond 6 Å, and hence they appear with low contrast because the high frequencies contribute with alternating positive and negative contrast. Molecular Cell 2002 10, 317-326DOI: (10.1016/S1097-2765(02)00603-2)

Figure 2 Surface Representations and Density Maps Derived from the X-Ray Map of Native HRV2 and the Reconstructed Cryo-Electron Microscopy Map of the Empty HRV2 Surface representations of the X-ray map of native HRV2 capsid only (no RNA and no VP4) limited to 12 Å resolution with a temperature factor of 500 Å2. (A) is a stereo view, and (B) has the front half cut away. (C) is a stereo view of the surface representation of the reconstructed cryo-electron microscopy map of the empty HRV2, and (D) has the front half cut away. All maps are viewed down the 2-fold axis. (E), (F), and (G) represent central sections of the native HRV2 X-ray map in the 2-, 5-, and 3-fold orientations, respectively. (H), (I), and (J) represent central sections of the empty HRV2 particle in the same orientations. White arrowheads mark the VP3 β-barrel. Density from the N terminus of VP1 is marked with black arrowheads. The position of the missing VP1 N terminus and missing VP2 density is marked with an open white arrowhead, and the N-terminal loop of VP2 is marked with an open black arrowhead. The scale bar represents 100 Å. Molecular Cell 2002 10, 317-326DOI: (10.1016/S1097-2765(02)00603-2)

Figure 3 Surface Representations of Sections and Close Up Views of the X-Ray Map of Native HRV2 and the Reconstructed Cryo-Electron Microscopy Map of the Empty HRV2 Seven angstrom thick central section of the isosurfaces of native (dark blue) and empty (gold) HRV2 (A) viewed down a 2-fold axis. (B) Fifteen angstrom thick central section of the isosurfaces of native (dark blue) and empty (gold) HRV2 viewed down a 5-fold axis. The section is cut near the apex of the 5-fold axis of native HRV2. A clockwise rotation of the dome of the empty capsid is apparent. (C) and (D) are the isosurfaces of native and empty HRV2, respectively, viewed down a 5-fold axis. (E) is a stereo view of the protuberance on the 2-fold axis inside the empty capsid. Molecular Cell 2002 10, 317-326DOI: (10.1016/S1097-2765(02)00603-2)

Figure 4 Ribbon Representations of VP1, 2, and 3 Color Coded by Their Temperature Factor from Blue to White to Red for 0 to 100 Å2 The strands of each VP, which were fitted separately, are represented, in yellow, in their new position. They are 1052 to 1059, 2036 to 2061, and 3001 to 3013. Note that the N terminus of VP1 (1001 to 1059), which has been removed, corresponds to the most disordered region of VP1, as indicated by the high temperature factor; residue 1059 has the highest temperature factor of VP1, 2, and 3. This is in keeping with its pivotal position in the model we propose. The N-terminal loop of VP2 (2036 to 2061) is also relatively mobile. In contrast, the N terminus of VP3 is well defined and must be fixed in position in the β-barrel. However, it is notable that Giranda et al. (1992) reported that the N terminus of VP3 of HRV14 becomes disordered at acidic pH. Molecular Cell 2002 10, 317-326DOI: (10.1016/S1097-2765(02)00603-2)

Figure 5 Fit of the Atomic Structures of VP1, VP2, and VP3 into the Cryo-Electron Microscopy Map of the Empty HRV2 Capsid The Cα backbones of VP1, VP2, and VP3 are shaded light blue, green, and red, respectively. The N termini of VP1 and VP3, which have been fitted separately, are shaded in purple and yellow, respectively. The density maps are represented in gray. For comparison, (A) represents the X-ray map of native HRV2 viewed down a 5-fold axis. For clarity only the VP1 and the 13 N-terminal residues of VP3 are represented. The pocket factor is shown in magenta. (B) shows the same view of the empty capsid with an opening on the 5-fold axis. (C) is a side-on view of the 5-fold axis of the empty capsid. (D) shows the 2-fold axis seen from inside the capsid. The isodensity surface is depicted as an opaque surface for clarity. VP1 and VP2 have only been fitted as single rigid bodies in this figure. The 59 residues of VP1 are seen to lie just outside the density map of the empty HRV2 capsid, as does much of the N-terminal loop of VP2 (2036–2061). These two chains are seen to clash. The N terminus of VP1 was subsequently remodeled to exit across the capsid just above Ser1059 as seen in (C) and Figures 6B and 6C, and the N-terminal loop of VP2 was bent to occupy the lobe of density visible in (D). Molecular Cell 2002 10, 317-326DOI: (10.1016/S1097-2765(02)00603-2)

Figure 6 Stereo Views of the Atomic Structures of VP1, VP2, and VP3 Fitted into the Cryo-Electron Microscopy Map of the Empty HRV2 Capsid The Cα backbones of VP1, VP2, and VP3 are shaded light blue, green, and red, respectively. The N termini of VP1 and VP3, which have been fitted separately, are shaded in purple and yellow, respectively, and the N-terminal loop of VP2, which has bent inward, is shaded in dark green. The density maps are represented in gray. (A) represents VP2. The 2-fold axis is indicated by an arrow; the pocket, which has opened up as the N-terminal loop bends inward, is marked with an asterisk; and Trp2038 is indicated by a dot. (B) and (C) show residues 1052 to 1059 of VP1 (purple) modeled to exit the capsid at the pseudo 3-fold axis, i.e., at the junction of VP1, VP2, and VP3. For comparison with (C), the same view of the native HRV2 capsid is shown in (D), with the pocket factor represented in magenta. An asterisk in (C) and (D) marks the bubble of low/solvent density, which is present in the native structure but almost filled in the empty capsid. In the empty capsid there is a larger distance between the VPs on the pseudo 3-fold axis than in the native capsid. However, this larger space is largely filled with density. We interpret this as being due to the N terminus of VP1 passing across the capsid at this point. Note that the VP1 pocket is visible at the top right of (C). Molecular Cell 2002 10, 317-326DOI: (10.1016/S1097-2765(02)00603-2)

Figure 7 Schematic Diagram of the Reorganization of the HRV2 Capsid during Release of the RNA into the Cell In the native capsid (A), the RNA is bound to Trp2038 of VP2, and the pocket is filled with a pocket factor. In the low pH (<5.6) environment of the late endosome, the capsid undergoes a 4% expansion (B). The pocket factor is probably expelled at this stage, thus allowing VP1 greater flexibility. Each VP1 is cantilevered up and away from the 5-fold axis while it swivels around to open a 10 Å diameter channel. The β-barrels formed by the N termini of VP3 on each 5-fold axis are rearranged to become larger with a similar 10 Å opening to allow exit of the RNA. The N terminus of VP1 exits the capsid at the pseudo 3-fold axis at the junction of VP1, 2, and 3. We propose that it then anchors itself in the membrane to retain the virus close to the membrane, and in association with VP4, which probably exits via the 5-fold axis, it forms a pore or channel in the membrane to allow passage of the RNA. VP4 may remain attached to the external lip of the opening formed by VP1. The N-terminal loop of VP2 bends inward to detach the RNA bound to the conserved Trp2038, and the RNA exits via the passage on the 5-fold axis. Molecular Cell 2002 10, 317-326DOI: (10.1016/S1097-2765(02)00603-2)