The ORF3 protein of porcine circovirus type 2 interacts with porcine ubiquitin E3 ligase Pirh2 and facilitates p53 expression in viral infection.
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.
Liu
Jue
J
Animal Health Biotechnology Group, Temasek Life Sciences Laboratory, National University of Singapore, 1 Research Link, Singapore.
Zhu
Yu
Y
Chen
Isabelle
I
Lau
Jennifer
J
He
Fang
F
Lau
Adeline
A
Wang
Zhilong
Z
Karuppannan
Anbu K
AK
Kwang
Jimmy
J
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D017925:Circovirus / Q000502:physiology*; D018076:DNA, Complementary; D015723:Gene Library; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017422:Sequence Analysis, DNA; D013552:Swine; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis* / Q000235:genetics; D020798:Two-Hybrid System Techniques; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D016159:Tumor Suppressor Protein p53; D014764:Viral Proteins; D044767:Ubiquitin-Protein Ligases