College of Veterinary Medicine, Jilin University, Changchun 130062, China.
Department of Biological Sciences and Centre for Bioimaging Sciences, National University of Singapore, 117543, Singapore.
Biosci Rep. 2020 Jun 26;40(6). doi: 10.1042/BSR20201109.
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.
最近发现的致病性猪圆环病毒 3 型（PCV3）可能会在全球范围内大幅减少猪群数量。在我们之前的研究中，成功应用了一种 PCV3 特异性单克隆抗体（mAb-1H11）进行免疫组织化学染色和 ELISA，该抗体特异性识别 PCV3 阳性猪组织中的 PCV3 衣壳蛋白。在本研究中，我们表达和纯化了 PCV3 的可溶性衣壳蛋白。纯化的衣壳蛋白能够自组装成病毒样颗粒（VLPs），这通过透射电子显微镜和动态光散射分析得到了验证。此外，mAb-1H11 的表位被鉴定为 VLPs 表面的 CD 环区域（a.a. 72-79），这通过 PCV2-PCV3 表位交换实验得到了证实。我们首次确定了 PCV3-VLP 的冷冻电镜结构，分辨率为 8.5Å，揭示了 PCV3-VLP 的详细结构信息。在我们的冷冻电镜结构中，PCV3-VLP 由 60 个衣壳蛋白亚基组成，采用 T=1 二十面体对称组装。与我们的生物斑点印迹 Western blot 实验一致，PCV3 和 PCV2 的结构比较显示在表面暴露的环中存在显著的结构差异，包括 CD 环（a.a. 72-79）和 EF 环（a.a. 109-131）。我们的工作为未来 PCV3 疫苗和诊断试剂盒的开发提供了结构框架。
