An externalized polypeptide partitions between two distinct sites on genome-released poliovirus particles

Abstract

During cell entry, native poliovirus (160S) converts to a cell-entry intermediate (135S) particle, resulting in the externalization of capsid proteins VP4 and the amino terminus of VP1 (residues 1 to 53). Externalization of these entities is followed by release of the RNA genome (uncoating), leaving an empty (80S) particle. The antigen-binding fragment (Fab) of a monospecific peptide 1 (P1) antibody, which was raised against a peptide corresponding to amino-terminal residues 24 to 40 of VP1, was utilized to track the location of the amino terminus of VP1 in the 135S and 80S states of poliovirus particles via cryogenic electron microscopy (cryo-EM) and three-dimensional image reconstruction. On 135S, P1 Fabs bind to a prominent feature on the external surface known as the "propeller tip." In contrast, our initial 80S-P1 reconstruction showed P1 Fabs also binding to a second site, at least 50 Å distant, at the icosahedral 2-fold axes. Further analysis showed that the overall population of 80S-P1 particles consisted of three kinds of capsids: those with P1 Fabs bound only at the propeller tips, P1 Fabs bound only at the 2-fold axes, or P1 Fabs simultaneously bound at both positions. Our results indicate that, in 80S particles, a significant fraction of VP1 can deviate from icosahedral symmetry. Hence, this portion of VP1 does not change conformation synchronously when switching from the 135S state. These conclusions are compatible with previous observations of multiple conformations of the 80S state and suggest that movement of the amino terminus of VP1 has a role in uncoating. Similar deviations from icosahedral symmetry may be biologically significant during other viral transitions.

Fig. 1.

Structural features of 160S and 135S particles. (Top) Prominent structural features on the exterior of the 160S poliovirus particle (2). Fivefold (pentagon), 3-fold (triangle), and 2-fold (oval) symmetry axes are labeled. A second 3-fold axis is labeled to show an asymmetric unit, which is the triangle formed with the 5-fold and two 3-fold axes as vertices. (The line connecting 3-fold axes passes through the 2-fold axis.) The asymmetric unit is the unique portion of the structure. The rest of the structure (59 other equivalent portions) is made by symmetry operations. (Bottom) Close-up view of four prominent structural features on the exterior of the 135S poliovirus particle (8), with one symmetry-related copy of the “propeller tip” and “bridge” labeled. The predicted helices in the canyon are residues 42 to 52 from the amino terminus of VP1 (black wire diagram). The gray net is a cryo-EM reconstruction. For both 160S and 135S particles, the mesa is formed solely by VP1, and the canyon and propeller are formed by VP1, VP2, and VP3. The propeller tip is formed by the EF loop (loop between E and F β-strands) of VP2 and flanking polypeptide sequences from VP1 and VP3. Each mesa is centered on a 5-fold symmetry axis, and each propeller is centered on a 3-fold symmetry axis.

Fig. 2.

Stereo views of reconstructed 135S and 135S-P1 complex. (A) 135S-P1 complex (resolution, 26 Å) reconstruction at a contour level of 0σ. Fab coordinates (cyan) are shown with the variable domain fitted into the P1 density. The fitting here provides only an estimate of the average Fab position. In the Fab variable domain, the volume occupied by the coordinates is significantly larger than the cryo-EM density, indicating that the epitope is flexible or that polyclonal Fabs overlap. (B and C) Coordinates (wire diagram) of the 135S model (8) (Protein Data Bank accession number 1XYR) fitted in the 135S-P1 (B) and 135S (C) (8) maps (fine mesh). Coordinates for VP1 (blue), VP2 (yellow), VP3 (red), and residues 42 to 52 of the amino terminus of VP1 (green) are shown.

Fig. 3.

Views of reconstructed 80S-P1 complexes. (A to C) Stereo views of 80S-P1 complex reconstructions. P1 Fabs bind 80S capsid (white) at the propeller tips (dark gray) and 2-fold axes (light gray). (A) The unclassified 80S-P1 (resolution, 21 Å) reconstruction at a contour level of 0.2σ. (B and C) Reconstructions of 80S-P1 complexes (resolution, 18 Å) with Fab bound to the propeller tip (B) and with Fab bound at the 2-fold axis (C), both at a contour level of 0.5σ. (D) Fab coordinates (47) (ribbon structures) fitted into the two different binding sites in the 80S-P1 map (mesh). The Fab bound to the propeller tip is shown in the left panel. The Fab bound at the 2-fold axis is shown in the right panel. In both cases, the density in the antigen-binding domain (of the Fab) was not complete. The fitting shown here provides only an estimate of where the Fabs might be positioned. The constant (non-antigen-binding) domain of the Fab bound at the 2-fold axis is larger than the volume occupied by the ribbon model. The extra density likely results from 2-fold positioning of the epitope (but only one Fab can bind at a time), the monospecific nature of the antibody, or the flexibility of the constant domain.

Fig. 4.

Binding of P1 Fab to poliovirus. Central slices from density maps of P1 Fab complexed with 80S particle (A and B) and 135S particle (C) and poliovirus (160S form) complexed with poliovirus receptor (Pvr) (4) (D). The highest densities are white. Arrows indicate P1 Fab-related density: Fabs bound at the propeller tip (unfilled arrowhead) or 2-fold axis (filled arrowhead). The 80S-P1 complex maps were computed from unclassified (A) and classified (B) particles. (A) Left, resolution of 21 Å; right, resolution of 13 Å. (B) Left, Fab bound to the propeller tip; right, Fab bound at the 2-fold axis (both structures at 18-Å resolution). (C) 135S-P1. Left, resolution of 26 Å (from classified particles); right, resolution of 12 Å (from unclassified particles). For relative density estimates, a sphere with a radius of 3 pixels was centered on the designated spots (A, right) and the average density was computed. In the unclassified 80S-P1 map at a resolution of 21 Å (A, left), P1 Fab at the propeller tip (unfilled arrowhead) has 15% of capsid density and P1 Fab at the 2-fold axis (filled arrowhead) has 24%. In the classified 80S-P1 maps at a resolution of 18 Å (B), P1 Fab bound to the propeller tip (unfilled arrowhead) has 25% of capsid density (B, left) and P1 Fab bound at the 2-fold axis (filled arrowhead) has 23% (B, right). In the 135S-P1 map at a resolution of 21 Å (C, left), P1 Fab has 16% of capsid density. (Insets) Smaller, unlabeled versions of each panel, given to allow the faint Fab density to be seen more easily. (D) Control image showing Pvr attached to poliovirus 160S particle (4). Bars, longer bar for larger images, shorter bar for inset images. (Note that a faint bit of density appears on the 2-fold axes in panel B, left. This faint density does not appear in the surface rendering in Fig. 3B and therefore is much lower than the density observed at the propeller tip. We attribute this faint density to errors in classification and to artifacts that often appear along symmetry axes in reconstructions.).

Fig. 5.

One- or eight-particle reconstructions of 80S-P1 complexes. (A to C) Central slices of one-particle reconstructions of 80S-P1 complex with P1 Fab bound at the propeller tip (A), at the 2-fold axis (B), and at both binding sites (propeller tip and 2-fold axis) (C). Arrowheads, Fab bound at the propeller tip; asterisks, Fab bound at the 2-fold axis. (D) Central slices of eight-particle reconstructions of 80S-P1. The same particle images that were used to make the one-particle reconstructions in panels A to C were combined to give the structures seen here. From left to right, eight-particle reconstruction of 80S-P1 with P1 Fab bound at the propeller tip, eight-particle reconstruction of 80S-P1 with P1 Fab bound at the 2-fold axis, mixed eight-particle reconstruction (four particles of 80S-P1 with P1 Fab bound at the propeller tip and four particles of 80S-P1 with P1 Fab bound at the 2-fold axis), and eight-particle reconstruction of 80S-P1 with P1 Fab bound at both binding sites. (E) Control structures. Central slices of one-particle reconstructions from a different poliovirus-antibody (C3) complex (top row) (J. Lin et al., unpublished data) and from the poliovirus-Pvr complex (bottom row) (4). The left three panels in each row are one-particle reconstructions, and the rightmost panel is an eight-particle reconstruction. The one-particle reconstructions show bound Fab (top row) or bound receptor (bottom row). The C3 antibody binds very tightly to poliovirus 160S, 135S, and 80S particles (18), and Fabs were found to bind to the tips of the mesa (Lin et al., unpublished). Pvr binds to one side of the propeller tip and bridges the canyon (4). The monotone gray circular center seen in some slices was added artificially to mask very high positive or negative density values at the center of the 3D reconstruction. Extreme values at the center are common artifacts in one-particle, icosahedral reconstructions.