The Carboxyl Terminus of the Porcine Circovirus Type 2 Capsid Protein Is Critical to Virus-Like Particle Assembly, Cell Entry, and Propagation

Abstract

The capsid protein (Cap) is the sole structural protein and the main antigen of porcine circovirus type 2 (PCV2). Structural loops of the Cap play crucial roles in viral genome packaging, capsid assembly, and virus-host interactions. Although the molecular mechanisms are yet unknown, the carboxyl terminus (CT) of the PCV2 Cap is known to play critical roles in the evolution, pathogenesis, and proliferation of this virus. In this study, we investigated functions of CT. Removal of this loop leads to abrogation of the in vitro Cap self-assembly into virus-like particles (VLPs). Likewise, the mutated virus resists rescue from PK15 cell culture. A conserved PXXP motif in the CT is dispensable for VLP assembly and subsequent cell entry. However, its removal leads to the subsequent failure of virus rescued from PK15 cells. Furthermore, substituting either the PCV1 counterpart or an AXXA for the PXXP motif still supports virus rescue from cell culture but results in a dramatic decrease in viral titers compared with wild type. In particular, a strictly conserved residue (227K) in the CT is essential for VLP entry into PK15 cells, and its mutation to alanine greatly attenuates cell entry of the VLPs, supporting a mechanism for the failure to rescue a mutated PCV2 infectious DNA clone (K227A) from PK15 cell culture. These results suggest the CT of the PCV2 Cap plays critical roles in virus assembly, viral-host cell interaction(s), and virus propagation in vitro. IMPORTANCE The carboxyl terminus (CT) of porcine circovirus type 2 (PCV2) capsid protein (Cap) was previously reported to be associated with immunorecognition, alterations of viral titer in swine sera, and pathogenicity. However, the molecular mechanisms underlying these effects remain unknown. In this study, roles of the critical residues and motifs of the CT are investigated with respect to virus-like particle (VLP) assembly, cell entry, and viral proliferation. The results revealed that the positively charged 227K of the CT is essential for both cell entry of PCV2 VLPs and virus proliferation. Our findings, therefore, suggest that the CT should be considered one of the key epitopes, recognized by neutralizing antibodies, for vaccine design and a target for drug development to prevent PCV2-associated diseases (PCVADs). Furthermore, it is important to respect the function of 227K for its role in cell entry if using either PCV2 VLPs for nanoscale DNA/drug cell delivery or using PCV2 VLPs to display a variety of foreign epitopes for immunization.

Structural Perspectives of Beak and Feather Disease Virus and Porcine Circovirus Proteins

“…A recent study discovered a critical PXXP motif in the C-terminal loop of the PCV2 Cap plays a critical role in self-assembly of VLPs in vitro. In this study, using site-directed mutagenesis approaches, they demonstrated that a strictly conserved residue (K227A) in the CT loop is essential for VLPs entry into PK15 cells and mutation of this residue results in abrogated infectivity compared with wild-type viruses (99).…”

Mitochondrial Localization Signal of Porcine Circovirus Type 2 Capsid Protein Plays a Critical Role in Cap-Induced Apoptosis

“…In a previous study, PCV2 was rescued from PK-15 cells transfected with an infectious DNA clone containing two copies of stem loops, but not rescued from cells transfected with a DNA clone carrying only one copy of the stem loop [24]. To determine the function of the MLS, an infectious DNA clone mutant, with deletion of the MLS, was prepared and transfected into PK-15 cells.…”

“…The Cap plasmid was obtained by deleting EGFP from Cap-EGFP, and the Cap-DMLS (deletion of 16-41) plasmid was obtained by deletion of the MLS of Cap. PCV2 infectious clone DNA was pre-constructed in our laboratory [24], and the infectious clone DNA mutant of PCV2 was derived from it, with the MLS sequence deleted.…”

Advances in Crosstalk between Porcine Circoviruses and Host

“…The structural loops of the PCV Cap play critical roles in viral genome packaging, virus particle assembly, and virus–host interactions [ 31 , 32 , 33 , 77 , 78 ]. The CT domain of viral Cap, especially conserved positively charged residue 227K, is essential for the cell entry of VLP or virion [ 78 ].…”

“…The structural loops of the PCV Cap play critical roles in viral genome packaging, virus particle assembly, and virus–host interactions [ 31 , 32 , 33 , 77 , 78 ]. The CT domain of viral Cap, especially conserved positively charged residue 227K, is essential for the cell entry of VLP or virion [ 78 ]. The conserved PXXP motif in the CT is not necessary for VLP assembly and subsequent cell entry, but it may hijack tyrosine kinases Abl and Fyn to enhance virus replication [ 77 , 78 ].…”