Nuclear localization of the ORF2 protein encoded by porcine circovirus type 2

Infectious porcine circovirus type 2 (PCV2) was generated following transfection of a porcine retina cell line (VIDO R1) with cloned circovirus DNA. Expression of open reading frame 2 (ORF2) was detected at 24 h postinfection and onwards increasingly throughout the infection by Western blot analysis using ORF2 specific polyclonal antibody. Moreover, the ORF2 protein was also detected in purified PCV2 virus, indicating that ORF2 is a structural component of PCV2 viral capsid. Nuclear localization of PCV2 ORF2 was demonstrated by immunofluorescence assay in PCV2-infected cells. An analysis of the subcellular localization of a series of truncation mutants of ORF2 fused with the green fluorescent protein indicated that the nuclear localization signal of ORF2 was conferred by the N-terminal 41 amino acids. This domain was further analyzed through site-directed mutagenesis, suggesting that the presence of basic amino acid residues at positions 12 to 18 and 34 to 41 are important for the strict nuclear targeting of PCV2 ORF2.