Department of Biological Sciences and Centre for Bioimaging Sciences, National University of Singapore, Singapore.
National Research Center for Veterinary Medicine, Luoyang, Henan, China.
PLoS Pathog. 2019 Mar 1;15(3):e1007562. doi: 10.1371/journal.ppat.1007562. eCollection 2019 Mar.
Postweaning multisystemic wasting disease (PMWS) in piglets caused by porcine circovirus type 2 (PCV2) is one of the major threats to most pig farms worldwide. Among all the PCV types, PCV2 is the dominant genotype causing PMWS and associated diseases. Considerable efforts were made to study the virus-like-particle (VLP) assembly and the specific PCV2-associated epitope(s) in order to establish the solid foundation for engineered PCV2 vaccine development. Although the N-terminal fragment including Nuclear Localization Signal (NLS) sequence seems important for recombinant PCV2 capsid protein expression and VLP assembly, the detailed structural and functional information regarding this important fragment are largely unknown. In this study, we report crystal structure of PCV2 VLP assembled from N-terminal NLS truncated PCV2 capsid protein at 2.8 Å resolution and cryo-EM structure of PCV2 VLP assembled from full-length PCV2 capsid protein at 4.1Å resolution. Our in vitro PCV2 VLP assembly results show that NLS-truncated PCV2 capsid protein only forms instable VLPs which were easily disassembled in solution, whereas full-length PCV2 capsid protein forms stable VLPs due to interaction between 15PRSHLGQILRRRP27 (α-helix) and 33RHRYRWRRKN42 (NLS-B) in a repeated manner. In addition, our results also showed that N-terminal truncation of PCV2 capsid protein up to 27 residues still forms PCV2 particles in solution with similar size and immunogenicity, while N-terminal truncation of PCV2 capsid protein with more than 30 residues is not able to form stable PCV2 particles in solution, demonstrating the importance of interaction between the α-helix at N-terminal and NLS-B in PCV2 VLP formation. Moreover, we also report the cryo-EM structure of PCV2 VLP in complex with 3H11-Fab, a PCV2 type-specific neutralizing antibody, at 15 Å resolution. MAb-3H11 specifically recognizes one exposed epitope located on the VLP surface EF-loop (residues 128-143), which is further confirmed by PCV1-PCV2 epitope swapping assay. Hence, our results have revealed the structural roles of N-terminal fragment of PCV2 capsid protein in PCV2 particle assembly and pinpointed one PCV2 type-specific neutralizing epitope for the first time, which could provide clear clue for next generation PCV2 vaccine and diagnostic kits development.
断奶后多系统消耗综合征（PMWS）是由猪圆环病毒 2 型（PCV2）引起的仔猪疾病，是世界范围内大多数猪场的主要威胁之一。在所有的 PCV 类型中，PCV2 是导致 PMWS 和相关疾病的主要基因型。为了研究病毒样颗粒（VLP）的组装和特定的 PCV2 相关表位，已经做出了相当大的努力，以便为工程化 PCV2 疫苗的开发奠定坚实的基础。尽管包括核定位信号（NLS）序列在内的 N 端片段似乎对重组 PCV2 衣壳蛋白的表达和 VLP 组装很重要，但关于这个重要片段的详细结构和功能信息在很大程度上仍然未知。在这项研究中，我们报告了在 2.8 Å 分辨率下由 N 端 NLS 截断的 PCV2 衣壳蛋白组装的 PCV2 VLP 的晶体结构，以及在 4.1 Å 分辨率下由全长 PCV2 衣壳蛋白组装的 PCV2 VLP 的 cryo-EM 结构。我们的体外 PCV2 VLP 组装结果表明，NLS 截断的 PCV2 衣壳蛋白仅形成不稳定的 VLPs，容易在溶液中解组装，而全长 PCV2 衣壳蛋白由于在重复的情况下相互作用 15PRSHLGQILRRRP27（α-螺旋）和 33RHRYRWRRKN42（NLS-B），形成稳定的 VLPs。此外，我们的结果还表明，PCV2 衣壳蛋白的 N 端截断至 27 个残基仍然可以在溶液中形成具有相似大小和免疫原性的 PCV2 颗粒，而 N 端截断超过 30 个残基的 PCV2 衣壳蛋白则不能在溶液中形成稳定的 PCV2 颗粒，这表明 N 端的 α-螺旋和 NLS-B 之间相互作用在 PCV2 VLP 形成中的重要性。此外，我们还报告了在 15 Å 分辨率下与 3H11-Fab（一种 PCV2 型特异性中和抗体）复合物的 PCV2 VLP 的 cryo-EM 结构。MAb-3H11 特异性识别位于 VLP 表面 EF 环（残基 128-143）上的一个暴露表位，这进一步通过 PCV1-PCV2 表位交换实验得到证实。因此，我们的结果揭示了 PCV2 衣壳蛋白 N 端片段在 PCV2 颗粒组装中的结构作用，并首次确定了一个 PCV2 型特异性中和表位，这可为下一代 PCV2 疫苗和诊断试剂盒的开发提供明确线索。
