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

Craig Meyers,Junhua Deng,Adam Yuan,Bo Yin,Xiangdong Li,Xiaobing Mo,Kegong Tian

doi:10.1371/journal.ppat.1007562

Abstract

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.

Highlights

In early 1990s, a postweaning multisystemic wasting syndrome (PMWS) outbreak in Western Canada [1] was reported due to the spreading of a small, non-enveloped virus containing a single-stranded, circular DNA genome, which was later diagnosed as the porcine circovirus (PCV) [2]
Prophylactic efficacy of VLP-based porcine circovirus type 2 (PCV2) vaccine largely relies on the correct VLP assembly from the individual PCV2 capsid protein
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

Summary

Introduction

In early 1990s, a postweaning multisystemic wasting syndrome (PMWS) outbreak in Western Canada [1] was reported due to the spreading of a small, non-enveloped virus containing a single-stranded, circular DNA genome, which was later diagnosed as the porcine circovirus (PCV) [2]. PCV2 is the dominant pathogen causing PMWS and a number of associated diseases in pigs [5,6,7], severely affecting swine production worldwide [8,9,10,11,12,13,14,15]. Only PCV1 and PCV2 (PCV2a, PCV2b, PCV2c, PCV2d, PCV2e and PCV2f) were identified, a distantly related porcine circovirus (PCV3) were recently diagnosed both in US and China [16,17]. PCV1 is characterized as a non-pathogenic virus, PCV2 is identified as a causative agent for severe economic losses in the swine industry, whereas PCV3 is found to be associated with porcine dermatitis nephropathy syndrome (PDNS), reproductive failure, etc [18,19]. PCV2 capsid protein (233/234 amino acids) encoded by the open reading frame 2 (ORF2) of PCV2 is capable of self-assembly into VLP and resembling the icosahedral morphology of the native

Methods

Results

Discussion

Conclusion