Schematic presentation of human CD155α. The ectodomain is divided into the Ig-like D1, D2, and D3 domains (circles), followed by the transmembrane (TM) domain and the cytoplasmic domain (Cyt). The number of amino acids within each structural segment is indicated. The predicted N-glycosylation sites are depicted as open squares.

The structure of domains 1 and 2 of CD155. (A) The β-strands in D1 and D2 are labeled A–F using the normal nomenclature for a “variable” and “constant” Ig-like fold, respectively (21). The 160° elbow angle between domains D1 and D2 is shown. (B) The Cα backbones (red) of the fitted D1 and D2 domains are shown in the CD155-PV1 cryoEM density (blue). The elbow angle between D1 and D2 found in crystal structure has been adjusted to fit in the cryoEM density.

Stereoviews showing the cryoEM reconstructions of PV1 complexed with deglycosylated CD155, viewed down an icosahedral 2-fold axis (Tables S1 and S2). The black triangle defines 1 icosahedral asymmetric unit on the viral surface.

Mutational studies of CD155 domain D1. (A) Ribbon diagram of D1. Residues in the virus–receptor binding interfaces are shown as colored spheres. Residues identified as being in the interface in only 1 serotype are shown in black, residues identified in 2 serotypes are in blue, and residues identified in all 3 serotypes are in red. (B) Amino acid sequence and the secondary structural elements of CD155 in D1. Residues are colored as in A. Residues that have no impact on CD155 binding, as shown by mutagenesis studies, are labeled with a black +. Residues that have impact on CD155 binding, as shown by mutagenesis studies, are labeled with a red +. The strong correlation between colored amino acids with a red + (residues that impact CD155 binding) is visually evident. Similarly, there is an obvious correlation between residues that have been mutated, but are not in the virus–CD155 interface with a black + (residues that do not impact CD155 binding).

Pocket factor density. (A) Diagrammatic view of the pocket factor position within the reference icosahedral asymmetric unit. The arrow shows the direction of viewing the difference maps in B–D. (B) The control showing a 10-Å-thick slab of the difference map at 9-Å resolution between the electron density generated from the known PV-CD155 atomic structure with and without the presence of the pocket factor (see Materials and Methods). The pocket factor density (contoured at a 15σ level) in the difference map is much bigger than the largest noise peak (which has a height of 1.7σ level) and showed the characteristic elongated shape of the pocket factor. The labels “virus exterior” and “RNA core” define the radial edge of the capsid shell. The edges of the capsid structure are indicated by continuous red lines. (C) The actual difference map at 9-Å resolution between the EM-reconstruction of the PV–CD155 complex and the electron density map based on the known PV–CD155 atomic structure without the presence of the pocket factor. The difference map showed the presence of the pocket factor in the PV–CD155 complex, and significant density at the internal and external edges of the capsid protein. (D) A further control map at 9-Å resolution in which the calculated structure omitted 5 aa (residues 1197–1201). This difference map showed positive density at the site of the pocket factor and at the site of the missing residues (colored green), and essentially the same positive and negative noise density in the regions outside the PV protein capsid.

Mutational studies of PV1. (A) The Cα backbone of VP1, VP2, and VP3 in 1 icosahedral asymmetric unit are colored blue, green, and red respectively. Residues that have been mutated and characterized are colored yellow, black, magenta, or gray. Residues that are not in the footprint and do not affect CD155 binding when mutated are colored yellow. The impact on CD155 binding when mutating PV1 residues is color-coded as shown. In general, residues that are not in the CD155 footprint do not affect CD155 binding (yellow spheres). Also, residues that are in the CD155 footprint do affect CD155 binding (magenta spheres). However, there are some residues that are not in the CD155 footprint but do affect CD155 binding (black spheres). These black-labeled residues all are in the vicinity of the pocket factor or in the interfaces between the viral subunits, suggesting that these become available to CD155 subsequent to the first binding event. (B) A diagrammatic representation of CD155 interacting with PV. Ovals, triangles, and pentagons indicate the positions of the 2-, 3-, and 5-fold axes, respectively. During the first recognition stage of PV by CD155, the pocket factor (PF) stays in the hydrophobic pocket in VP1. The interaction between PV and CD155 results in the expulsion of the pocket factor, thereby decreasing the stability of the virus and permitting CD155 to move further into the canyon and causing a separation of the viral subunits. This causes exposure of residues that were previously hidden in the interface between the VPs, consistent with the earlier structural studies of 135S particles (25).