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.

由猪圆环病毒2型（PCV2）引起的仔猪断奶后多系统浪费病（PMWS）是全世界大多数养猪场的主要威胁之一。在所有PCV类型中，PCV2是导致PMWS和相关疾病的主要基因型。为了建立工程化PCV2疫苗开发的坚实基础，已经进行了相当大的努力来研究病毒样颗粒（VLP）装配体和与PCV2相关的特定表位。尽管包括核定位信号（NLS）序列的N端片段对于重组PCV2衣壳蛋白表达和VLP组装似乎很重要，但有关此重要片段的详细结构和功能信息仍然未知。在这项研究中，我们报告了由N末端NLS截短的PCV2衣壳蛋白2组装而成的PCV2 VLP的晶体结构。由全长PCV2衣壳蛋白以4.1Å分辨率组装而成的PCV2 VLP的8Å分辨率和cryo-EM结构。我们的体外PCV2 VLP组装结果显示，NLS截短的PCV2衣壳蛋白仅形成不稳定的VLP，很容易在溶液中分解，而全长PCV2衣壳蛋白由于^15个PRSHLGQILRRRP ²⁷ （α-螺旋）和³³ RHRYRWRRKN之间的相互作用而形成稳定的VLP。⁴² （NLS-B）反复地 此外，我们的结果还表明，PCV2衣壳蛋白的多达27个残基的N端截短仍在溶液中形成大小和免疫原性相似的PCV2颗粒，而PCV2衣壳蛋白的超过30个残基的N端截短不能在溶液中形成稳定的PCV2颗粒，证明了PCV2 VLP形成过程中N末端的α-螺旋和NLS-B之间相互作用的重要性。此外，我们还报告了PCV2 VLP与3H11-Fab（一种PCV2型特异性中和抗体）复合物的冷冻EM结构，分辨率为15Å。MAb-3H11特异性识别位于VLP表面EF环上的一个暴露表位（残基128-143），这可通过PCV1-PCV2表位交换测定进一步证实。因此，