Characterization of specific antigenic epitopes and the nuclear export signal of the porcine circovirus 2 ORF3 protein

Porcine circovirus 2 (PCV2) is the etiological agent of postweaning multisystemic wasting syndrome. PCV2 ORF3 protein is a nonstructural protein known to induce apoptosis, but little is known about the biological function of ORF3 protein. Therefore, we undertook this study to map ORF3 protein epitopes, characterize putative nuclear localization (NLS) and nuclear export (NES) sequences in ORF3, compare Virus kinetics and animal pathogenicity between wild type PCV2 and ORF3-deficient PCV2 (mPCV2).

In the present study, recombinant GST-ORF3 and his-ORF3 proteins were expressed in E. coli and mAbs were produced by these two fused proteins, peptide-ELISA and peptide-dot-blot, ELISA additivity test, western blot and Indirect immunofluorescence assay (IFA) analysis were carried out to analyze the epitopes targeted by these mAbs. Recombinant plasmids containing putative NLS, NES, mutated NES were transfected into PK15 cells and confocal microscopy were used to analyze the function of NLS and NES in ORF3. Localization of ORF3 in PK15 cells is analyzed by both infection and transfection forms. Virus kinetics and animal pathogenicity test of PCV2 and mPCV2 were tested in PK15 cells and in Balb/c mice, respectively.

mAbs 3B1, 1H3 were generated against GST-ORF3 and mAb 3C3 was generated against his-ORF3. We find that ORF3 in PCV2 infected cells contains a conformational epitope targeted by the antibody 3C3, which is distinct from linear epitopes recognized by the antibodies 3B1 and 1H3 in recombinant ORF3 protein. These results suggest that the linear epitope recognized by 3B1 and 1H3 is masked in PCV2 infected cells, and that the conformational epitope is unique to PCV2 infection. Furthermore, we find that ORF3 protein expressed in cytoplasm in early stages of PCV2 infection and then accumulated in nucleus over time. Moreover, we localize a NES at the N-terminus (residues 1-35aa) of ORF3 which plays critical role in nuclear export activity. PCV2 and mPCV2 were rescued by transfection of infectious clones, the growth curves of PCV2 and mPCV2 show that there was no significant difference between these two viruses (P> 0.05), indicating that the ORF3 protein is not required for replication in cell culture. There was no significant differences between PCV2 and mPCV2 groups in viremia and induced antibody levels in animal experiment, but the wild type PCV2 can cause more obvious pathological changes of spleen than ORF3-deficient PCV2, indicating that ORF3 may related to pathogenicity of PCV2.

These findings provide a novel insight that deepens our understanding of the biological function of PCV2 ORF3.

Disclosure of Interest: None Declared.