Structure, Antigenic Properties, and Highly Efficient Assembly of PCV4 Capsid Protein

Abstract

Porcine circovirus type 4 (PCV4), a recently reported circovirus, was first identified in pigs with clinical signs similar to porcine dermatitis nephropathy syndrome (PDNS), in Hunan province, China, in 2019. More knowledge regarding the assembly of capsid protein (Cap) into virus-like particles (VLPs), their structure and antigenic properties, are needed to provide new knowledge for diagnosis and further characterization of PCV4. In this study, high-level expression of PCV4 Cap was achieved in Escherichia coli with purified Cap self-assembling into VLPs (~20 nm) in vitro. Furthermore, these VLPs were internalized in vitro by PK15 and 3D4/21 cell lines. Significant structural differences between PCV4 and PCV2 capsids were demonstrated among loops (loop BC, CD, DE, EF, and GH), based on comparisons of 3D structures. In addition, five potential B cell epitopes identified in silico were mostly located in surface-exposed loops of PCV4 capsid. Cross-reaction between PCV4 and PCV2 or PCV3 conferred by humoral immune responses was deemed unlikely on the basis of ELISA and Western blotting for assessment of VLPs and using PCV4 or PCV2 VLPs. In conclusion, these studies provided new knowledge regarding PCV4 capsid surface patterns. It is noteworthy that the PCV4 VLPs prepared in our study have much potential for development of serological diagnostics for PCV4 and to further characterize this virus.

Current knowledge on epidemiology and evolution of novel porcine circovirus 4

“…Two studies demonstrated that the established PCV4 ELISA, based on Rep or Cap, had no serological cross-reactions with positive sera of other PCV [ 5 , 27 ]. Moreover, our previous study also demonstrated an absence of cross-reactions between PCV4 and either PCV2 or PCV3 [ 23 ]. VLP, morphologically and immunogenically similar to their native viruses, are widely used for novel vaccine design and serological diagnosis.…”

“…The Cap is the sole structural protein, capable of eliciting robust immune responses and regarded as the major target antigen for PCV4 serological diagnosis and vaccine design [ 23 ]. Since the three-dimensional (3D) structure of PCV4 Cap has not been resolved, we modeled the 3D structure of the PCV4 Cap via homology modeling based on PCV2 Cap structure (PDB ID: 3R0R) in the SWISS-MODEL, as described [ 23 ]. There was a typical jelly-roll structure in the PCV4 Cap, although surface-exposed loops were distinctly different between PCV2 and PCV4, similar to the PCV2 Cap [ 23 ].…”

“…Since the three-dimensional (3D) structure of PCV4 Cap has not been resolved, we modeled the 3D structure of the PCV4 Cap via homology modeling based on PCV2 Cap structure (PDB ID: 3R0R) in the SWISS-MODEL, as described [ 23 ]. There was a typical jelly-roll structure in the PCV4 Cap, although surface-exposed loops were distinctly different between PCV2 and PCV4, similar to the PCV2 Cap [ 23 ]. As a non-enveloped virus, the Cap has a vital role in entry of the virus into cells.…”

“…Therefore, a better environment is created for receptor binding by hydrogen bonds, increasing its polarity by increasing polar residues on PCV4 capsid surface. Although the receptor and cell entry mechanism of PCV4 are unknown, we reported that PCV4 virus-like particles (VLP) can efficiently enter porcine cell lines (PK15 and 3D4/21) [ 23 ]. Thus, we hypothesized that PCV4 may have evolved with increasing polar residues via nonspecific interactions of the virus with negatively charged HS, CSB or perhaps other cell membrane receptors during PCV4 infection.…”

Porcine Circovirus Type 4 Strains Circulating in China Are Relatively Stable and Have Higher Homology with Mink Circovirus than Other Porcine Circovirus Types

“…The homology of Cap genes of different PCV2 strains is consistent with that of the whole genome, and the difference of the Cap genes may determine the genetic distance between different strains. Furthermore, it was found that the mutation rates of PCV3 and PCV4 viruses were low [ 7 , 17 , 18 ]. We also found in this study that PCV4 strains had a high degree of genetic stability.…”