Table 1.
Data collection and refinement statistics.
Fig 1.
Crystal structure of PCV2-His-ΔN45 VLPs.
(A) Ribbon representation of crystal structure of PCV2-His-ΔN45 VLP at 2.8Å assembled from 60 PCV2 capsid protein subunits. (B) The association of subunits in dimer, trimer and pentamer form, presented from VLPs. (C) Structural comparison of crystal structure of PCV2-His-ΔN45 subunit and cryo-EM structure of full-length PCV2 subunit. The crystal structure of PCV2-His-ΔN45 subunit is colored in light yellow, whereas the cryo-EM structure of full-length PCV2 subunit is colored in light blue.
Fig 2.
Cryo-electron microscopy structure of full-length PCV2 VLPs.
(A) Typical raw cryo EM image of PCV2 VLPs imaged at the magnification of 67,000× under Titan Krios electron microscopy. (B) 2D classification of PCV2 VLP particles from the EM images collected under 300 kV. (C) The Fourier Shell Correlation curve of resolution for our current reconstruction is shown. The line corresponds to the gold standard criterion for resolution estimation (FSC 0.143). (D) The 3D reconstructed cryo-EM structure of PCV2 VLP reveals a typical T = 1 icosahedral particle fold. (E) The closest half of the density map removed to reveal internal structural feature of the NLS fragment. (F) Cryo-EM density map surrounding NLS region. The density maps for NLS-B, α-helices etc are indicated. (G) Cryo-EM density map surrounding NLS region. The cation-π interaction between residues 28Trp and 37Arg is labeled.
Table 2.
Data collection and refinement of cryo-EM structure of full-length PCV2 VLP.
Fig 3.
Characterization of PCV2 VLPs derived from in vitro expressed and assembled full-length and truncated PCV2 capsid proteins.
(A) Schematic drawing of full-length and truncated PCV2 capsid proteins. The NLS region was indicated as brace and detailed sequence are shown in S6 Fig. (B) SDS-PAGE gel of purified PCV2 truncated capsid proteins (PCV2-ΔN3, PCV2-ΔN6, PCV2-ΔN9, PCV2-ΔN12, PCV2-ΔN15, PCV2-ΔN18, PCV2-ΔN21, PCV2-ΔN24, and PCV2-ΔN27) with the PCV2 monomers indicated with arrows. (C) Transmission electron microscopy of VLPs assembled from purified truncated PCV2 capsid proteins (PCV2-ΔN3, PCV2-ΔN6, PCV2-ΔN9, PCV2-ΔN12, PCV2-ΔN15, PCV2-ΔN18, PCV2-ΔN21, PCV2-ΔN24, and PCV2-ΔN27). (D) The mouse poly-antibody titers against PCV2 VLPs assembled from truncated PCV2 capsid proteins (PCV2-ΔN3, PCV2-ΔN6, PCV2-ΔN9, PCV2-ΔN12, PCV2-ΔN15, PCV2-ΔN18, PCV2-ΔN21, PCV2-ΔN24, and PCV2-ΔN27).
Table 3.
DLS, SEC and EM of PCV2 truncated proteins.
Fig 4.
PCV2 type-specific neutralizing epitope mapping.
(A) Ribbon diagram of the PCV2 pentamer structure viewed from the side and top orientations, respectively. The seven flexible surface loops of PCV2 pentamer are indicated. (B) Bio-dot western blot detection of affinities between PCV2 type-specific mAb and chimeric PCV2 capsid proteins with swapped loops from PCV1. Swapping of PCV2 capsid protein EF-loop with the corresponding PCV1 capsid protein loop disrupts the binding between PCV2 capsid protein and 3H11 mAb. (C) Cryo-EM density map of full-length PCV2 VLP. The PCV2 type-specific EF-loops are colored in red, whereas the rest of the PCV2 VLP densities are colored in grey.
Fig 5.
The cryo-EM structure of PCV2 VLP in complex with 3H11 Fab.
(A) The 3D reconstructed cryo-EM structure of PCV2 VLP in complex with 3H11-Fab reveals a typical T = 1 icosahedral particle fold. (B) The Fourier Shell Correlation curve of resolution for cryo-EM structure is shown. The line corresponds to the gold standard criterion for resolution estimation (FSC 0.143). (C) Segmentation and of structural model of Fab fragments binding with PCV2 VLP. The crystal structure of PCV2 VLP (PCV2-His-ΔN45) and the homology model structure of mAb-3H11-Fab were fitted into the densities of the 3D-reconstructed complex. The density maps of outer and inner shell are shown as 60% and 10% transparent surfaces, respectively, and the fitted atomic models are represented by ribbons. The densities corresponding to PCV2 VLP and 3H11 Fab fragments are colored in blue and grey, respectively. (D) Structural models of PCV2 VLP and mAb-3H11. (E) The atomic model of PCV2 VLP is colored by subunit and labeled accordingly. The heavy chain and light chain of mAb-3H11 Fab fragment are colored in red and blue, respectively. This structure clearly shows that 3H11 Fab binds to EF-loop located on the PCV2 VLP surface.