Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome in pigs. In this study, transcription and translation of a novel viral gene (termed ORF3 here) was detected during productive infection of PCV2 in PK15 cells. The results of infection with ORF3-deficient PCV2 by site-directed mutagenesis indicated that the protein is not essential for viral replication. To investigate the underlying mechanism of cell death caused by replication of PCV2, apoptosis characterized by chromosomal condensation and fragmentation, formation of apoptotic bodies, and significant increase in hypodiploids were detected in infected cells. We further demonstrated that PCV2-induced apoptosis required the activation of caspase-8 but not caspase-9. The activation of caspase-8 results in the activation of caspase-3 as shown by an increase in the cleavage of the caspase substrate in the infected cells. To determine whether ORF3 protein could trigger apoptosis, ORF3 as well as ORF1 and ORF2 genes were transiently expressed in PK15 and Cos-7 cells for apoptotic activity assay. Transfection of cells with the ORF3 alone induced apoptosis using a pathway similar to that described in the context of viral infection. This is further confirmed by a significant decrease in apoptotic activity of infected cells in the absence of the ORF3 expression, suggesting that the protein plays a major role in the induction of virus-induced apoptosis. Altogether, these results indicate that ORF3 is a novel PCV2 protein that is not essential for viral replication in cultured cells but is involved in PCV2-induced apoptosis by activating caspase-8 and caspase-3 pathways.

Porcine circovirus type 2 (PCV2) is the primary causative agent of an emerging swine disease, postweaning multisystemic wasting syndrome. We previously showed that a novel identified protein, ORF3, was not essential for PCV2 replication in cultured PK15 cells and played a major role in virus-induced apoptosis. To evaluate the role of the ORF3 protein in viral pathogenesis in vivo, we inoculated 8-week-old BALB/c mice that have been developed for PCV2 replication with ORF3-deficient mutant PCV2 (mPCV2). By 42 days postinoculation, all of the mice inoculated with the mPCV2, as well as with wild-type PCV2 (wPCV2), had seroconverted to PCV2 capsid antibody, and the mutant induced levels of PCV2 antibodies that were higher than those of the wPCV2. The PCV2 genomic copy numbers in serum were significantly higher (P < 0.05) in the wPCV2-inoculated mice than in mice inoculated with the mPCV2. Also, the wPCV2 caused microscopic lesions characterized by lymphocyte depletion with histiocytic infiltration of lymphoid organs, but the mutant virus failed to induce any obvious pathological lesions. In situ hybridization and immunohistochemical analyses also showed that larger amounts of viral DNA and antigens were detected in the lymph nodes of the wPCV2-inoculated than mPCV2-inoculated mice. Furthermore, animals of the wPCV2-inoculated group showed significant downshifts of CD8 ؉ T-cell subsets of peripheral blood lymphocytes compared to the control mice (P < 0.05) at various time points postinoculation. Also, the proportions of the CD4 ؉ and CD4 ؉ CD8 ؉ cells were significantly reduced in wPCV2-inoculated mice at some time points postinoculation. In contrast, there are some reductions in the proportions of these subsets in the mutant virus-inoculated mice, but the proportions do not decrease significantly. Taken together, these results demonstrate that the ORF3 protein is also dispensable for viral replication in vivo and that it plays an important role in viral pathogenesis.

Porcine circovirus type 2 (PCV2) is the primary causative agent of an emerging swine disease, postweaning multisystemic wasting syndrome. We previously showed that a newly identified protein, ORF3, plays a major role in virus-induced apoptosis and is involved in viral pathogenesis in vitro and in vivo. To characterize the role of the ORF3 protein in modulation of cellular function, a yeast two-hybrid system was used to screen a porcine cDNA library to find its interacting partner. We have isolated and characterized pPirh2 (for "porcine p53-induced RING-H2"), an E3 ubiquitin ligase, which specifically interacts with the ORF3 protein of PCV2. This interaction was further confirmed when the ORF3 protein coimmunoprecipitated with and colocalized to pPirh2 in PK15 cells. The ORF3 protein has been found to interact with the p53 binding domain of pPirh2 in yeast cells. Expression of the protein results in less pPirh2 expression in PCV2-infected cells. Furthermore, increases in p53 expression were observed in PCV2-infected and ORF3 (alone)-transfected cells. Phosphorylation of p53 at Ser-46, which is related to p53-induced apoptosis, was also time-dependently activated in PCV-infected and ORF3-transfected cells. Taken together, our results show that the PCV2 ORF3 protein specifically interacts with pPirh2 and inhibits its stabilization; this may lead to increasing p53 expression, resulting in apoptosis.