Structural roles of PCV2 capsid protein N-terminus in PCV2 particle assembly and identification of PCV2 type-specific neutralizing epitope

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 angstrom resolution and cryo-EM structure of PCV2 VLP assembled from full-length PCV2 capsid protein at 4.1 angstrom 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 (15)PRSHLGQILRRRP(27) (-helix) and (RHRYRWRRKN42)-R-33 (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 -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 angstrom 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. Author summary Porcine circovirus type 2 (PCV2) is considered as one of the most wide-spread pathogens threatening swine production by causing postweaning multisystemic wasting disease (PMWS) in piglets worldwide. Several VLP-based PCV2 vaccines are commercially available which significantly reduce the viral burden and virally induced lesions. However, prophylactic efficacy of VLP-based PCV2 vaccine largely relies on the correct VLP assembly from the individual PCV2 capsid protein. Notably, limited structural information of PCV2 N-terminal fragment containing arginine-rich patches significantly delays our understanding of PCV2 assembly at the molecular level, and the lack of solid evidence in identification of PCV2 type-specific epitope delays the development of PCV2 type-specific diagnosis kits. In this study, through the combination of structural and immunological approaches, we are able, for the first time, to disclose the structural details of the N-terminal Nuclear Localization Signal (NLS) region of PCV2 capsid protein. We show that the interaction between the -helix from one capsid protein and the NLS-B from an adjacent capsid protein within the pentamer stabilizes the assembled PCV2 VLP in solution. Moreover, by the combination of structural determination and biochemical mapping, we have identified that a short linear sequence ((134)KATALT(139)) located within PCV2 EF-loop is a unique PCV2 type-specific neutralizing epitope. Therefore, our work has revealed the detailed structural information of PCV2 particle assembly and a PCV2 type-specific neutralizing epitope, which should provide insightful information for virus-host interaction studies and next-generation PCV2 vaccine and type-specific diagnostic kits development.