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

• We develop a new mAb to ORF3 recognizing natural ORF3 protein in PCV2 infected cell.
• We analyze the linear epitope of ORF3 protein using mAbs to ORF3 protein.
• We analyze the distributions of ORF3 protein in PCV2 infected cells and in ORF3 transfected cells.
• Finally, we identify a nuclear export signal (NES) in ORF3.

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 recognized by a panel of monoclonal antibodies (mAbs) and to characterize putative nuclear localization (NLS) and nuclear export (NES) sequences in ORF3. The linear epitopes targeted by two previously published mAbs 3B1 and 1H3 and a novel mouse mAb 3C3 were defined using overlapping pools of peptides. Here, 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. These findings provide a novel insight that deepens our understanding of the biological function of PCV2 ORF3.