Liu Jue, Zhu Yu, Chen Isabelle, Lau Jennifer, He Fang, Lau Adeline, Wang Zhilong, Karuppannan Anbu K, Kwang Jimmy
Animal Health Biotechnology Group, Temasek Life Sciences Laboratory, National University of Singapore, 1 Research Link, Singapore.
J Virol. 2007 Sep;81(17):9560-7. doi: 10.1128/JVI.00681-07. Epub 2007 Jun 20.
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.
猪圆环病毒2型（PCV2）是一种新出现的猪病——断奶后多系统消耗综合征的主要病原体。我们之前表明，一种新鉴定的蛋白质ORF3在病毒诱导的细胞凋亡中起主要作用，并在体外和体内参与病毒发病机制。为了阐明ORF3蛋白在调节细胞功能中的作用，我们利用酵母双杂交系统筛选猪cDNA文库以寻找其相互作用伴侣。我们分离并鉴定了一种E3泛素连接酶pPirh2（“猪p53诱导的RING-H2”），它与PCV2的ORF3蛋白特异性相互作用。当ORF3蛋白在PK15细胞中与pPirh2共免疫沉淀并共定位时，这种相互作用得到进一步证实。在酵母细胞中发现ORF3蛋白与pPirh2的p53结合结构域相互作用。该蛋白的表达导致PCV2感染细胞中pPirh2表达减少。此外，在PCV2感染和ORF3（单独）转染的细胞中观察到p53表达增加。在PCV感染和ORF3转染的细胞中，与p53诱导的细胞凋亡相关的Ser-46位点的p53磷酸化也呈时间依赖性激活。综上所述，我们的结果表明，PCV2 ORF3蛋白与pPirh2特异性相互作用并抑制其稳定性；这可能导致p53表达增加从而引发细胞凋亡。
