The 2.3-angstrom structure of porcine circovirus 2

Abstract

Porcine circovirus 2 (PCV2) is a T=1 nonenveloped icosahedral virus that has had severe impact on the swine industry. Here we report the crystal structure of an N-terminally truncated PCV2 virus-like particle at 2.3-Å resolution, and the cryo-electron microscopy (cryo-EM) image reconstruction of a full-length PCV2 virus-like particle at 9.6-Å resolution. This is the first atomic structure of a circovirus. The crystal structure revealed that the capsid protein fold is a canonical viral jelly roll. The loops connecting the strands of the jelly roll define the limited features of the surface. Sulfate ions interacting with the surface and electrostatic potential calculations strongly suggest a heparan sulfate binding site that allows PCV2 to gain entry into the cell. The crystal structure also allowed previously determined epitopes of the capsid to be visualized. The cryo-EM image reconstruction showed that the location of the N terminus, absent in the crystal structure, is inside the capsid. As the N terminus was previously shown to be antigenic, it may externalize through viral "breathing."

Fig. 1.

PCV2^CS VLP assembly. (A) Micrograph of a crushed PCV2^CS crystal stained with 2% (wt/vol) uranyl acetate. (B) The condition was optimized to produce VLPs without crystal growth.

Fig. 2.

Crystal structure of PCV2^CS VLP. (A) Ribbon diagram of the PCV2 capsid protein with the secondary structures labeled according to the convention first described for tomato bushy stunt virus (TBSV). The N and C termini are labeled using blue and red spheres, respectively. (B) Stereoscopic representation of a ribbon diagram of the PCV2^CS VLP. The loops connecting the different strands have been color coded according to the bar diagram at the top right. N-term, N termini.

Fig. 3.

Surface mapping of the PCV2^CS VLP. Sulfate ions are shown as sphere models. (A) Sequence conservation among the deposited PCV2 capsid protein sequences. The sequences are shown colored from a gradient of red (no conservation) to blue (absolute conservation). The PCV2 residues responsible for discriminating the reported MAbs (20) are color coded as follows: epitope A is blue, epitope B is red, epitope C is magenta, epitope D is yellow, and epitope E is cyan. Epitope F is not shown. (B) Electrostatic potential map of the PCV2 crystal structure. Sulfates surrounding the icosahedral 3-fold axes sit within a canyon of positively charged residues that may bind heparin sulfate. This figure was made using University of California, San Francisco Chimera (28).

Fig. 4.

PCV2 coat protein sequence conservation and epitope mapping. The aligned coat protein sequences of the PCV2^N12, PCV2^CS, and the PCV1 and PCV2 strains used by Mahe et al (21). The secondary structure for the PCV2^CS crystal structure is shown at the top (β-strands are shown as cyan arrows, α-helices as yellow ovals, and loops as gray bars). A BLAST search of the PCV2^N12 coat protein sequence identified 199 coat protein sequences belonging to various strains of PCV2. Sequence alignment using the T-Coffee server produced the conservation bars shown above. The blue, red, magenta, yellow, and cyan boxes highlight the epitopes identified by Mahe et al. (21), and the correspondingly colored circles identify the surface residues responsible for discriminating MAb binding to PCV1 and PCV2. This figure was produced using Jalview (38).

Fig. 5.

Subnanometer resolution cryo-EM image reconstruction of the PCV2^N12 VLPs. (A) Surface cutaway view showing the capsid (transparent gray) with the fitted subunits (ribbon diagram) and packaged cellular nucleic acid (red). (B) The crystal structure of the PCV2^CS is overlaid onto the difference map (see text). The N and C termini are identified by blue and red spheres, respectively. Sulfate ions in the vicinity of the difference map are shown as sphere models and believed to represent the phosphate backbones of the packaged cellular nucleic acid in the PCV2^N12 VLPs used for the cryo-EM image reconstruction. The N-terminal segment, not present in the crystal structure, is believed to be located proximal to the icosahedral 5-fold axes.