Structural insight into the type-specific epitope of porcine circovirus type 3

Abstract

The recently identified pathogenic Porcine circovirus type 3 (PCV3) may threaten to reduce the pig population dramatically worldwide. In our previous study, a PCV3-specific monoclonal antibody (mAb-1H11) was successfully applied in immune-histochemistry staining and ELISA, which specifically recognize PCV3 capsid protein in PCV3-positive pig tissues. In the present study, we expressed and purified the soluble sole capsid protein of PCV3. The purified capsid protein was capable of self-assembly into virus-like-particles (VLPs), which is validated by transmission electron microscopy and dynamic light scattering assays. Moreover, the epitope of mAb-1H11 was identified in the CD-loop region (a.a. 72-79) on the VLP surface, which is confirmed by PCV2-PCV3 epitope swapping assay. For the first time, we determined the cryo-EM structure of PCV3-VLP at 8.5 Å resolution that reveals the detailed structural information of PCV3-VLP. In our cryo-EM structure, PCV3-VLP is composed of 60 capsid protein subunits assembled with T = 1 icosahedral symmetry. Consistent to our bio-dot Western blot assay, the structural comparison between PCV3 and PCV2 revealed significant structural differences in the surface-exposed loops, including the CD-loop (a.a. 72-79) and the EF-loop (a.a. 109-131). Our work provides a structural framework for engineering future PCV3 vaccine and diagnosis kits development.

Keywords: Porcine circovirus type 3 (PCV3); capsid protein; cryo-EM structure; prophylactic vaccine; type-specific epitope; virus-like-particle (VLP).

Figure 1. Full-length PCV3 capsid proteins assemble into VLPs

(A) Dynamic light scattering measurement of PCV3 VLPs. The enlarged area showed that the average hydrodynamic radius of PCV3 VLP is ∼8.80 nm and the VLP assembly rate is ∼95%. (B) Transmission electron microscopy of PCV3 VLPs. The scale bar is 100 nm long and EM result indicated that the PCV3 capsid proteins are assembled into VLPs.

Figure 2. Cryo-electron microscopy study of PCV3-VLP

(A) Raw cryo-EM micrograph of PCV3-VLP (inverted). (B) 2D classification of PCV3-VLP particles. Most classes showed the structural characteristics of icosahedral particles. (C) Cryo-EM structure of PCV3-VLP at 8.5 Å resolution, the density maps are colored in red. (D) Refine PCV3-VLP model fitting into the cryo-EM map (EMD-6935) of PCV3-VLP, in which PCV3 model structure is shown in blue ribbon mode. (E) Side view of pentamer in the cryo-EM map (EMD-6935) of PCV3-VLP, in which PCV3 pentamer model structure is shown in blue ribbon mode. (F) Refine PCV3-VLP monomer fitting into the cryo-EM map (EMD-6935) of PCV3-VLP, in which PCV3 model structure is shown in blue ribbon mode.

Figure 3. PCV3 type-specific epitope mapping and Structural comparison between PCV2 and PCV3

(A) Overall structural comparisons of PCV3-VLP model and crystal structure of PCV2 VLP (5ZJU), in which the PCV2 VLP and PCV3-VLP are shown in pink and blue ribbon mode, respectively. (B) Structural comparison of capsomers of PCV2 and PCV3 capsid proteins, viewed from the side and top orientations, respectively. Most of the seven flexible surface loops of PCV capsomer are surface exposed. (C) Structural fitting of PCV2 and PCV3 VLPs. PCV3 density maps are colored in red, whereas PCV2 density maps are colored in gray. (D) Structural comparison of PCV2 VLP and PCV3 VLP at loop regions. Detailed structural comparison of CD-loops and EF-loops between PCV2 and PCV3 VLPs. PCV3 VLP density maps are colored in red, whereas PCV2 VLP density maps are colored in gray. (E) Structural details of the N-terminal fragments including the NLS regions of PCV2 cryo-EM structure (EMD-6746) and PCV3 cryo-EM structure (EMD-6935). PCV3 density maps are colored in red, whereas PCV2 density maps are colored in gray. (F) Bio-dot Western blot detection of affinities between PCV3 type-specific mAb and chimeric PCV2 capsid proteins with swapped loops from PCV2. Swapping of PCV2 capsid protein CD-loop with the corresponding PCV1 capsid protein loop disrupts the binding between PCV2 capsid protein and mAb-1H1.