Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, China.
Key Laboratory of Urban Agriculture (North) of Ministry of Agriculture China, Beijing University of Agriculture, Beijing, China.
Virus Res. 2018 Feb 15;246:12-22. doi: 10.1016/j.virusres.2017.12.012. Epub 2017 Dec 30.
The open reading frame 2 (ORF2) of Porcine circovirus type 2 (PCV2) encodes the major Capsid (Cap) protein, which self-assembles into virus-like particle (VLP) of similar morphology to the PCV2 virion and accumulates in the nucleus through the N-terminal arginine-rich nuclear localization signal (NLS). In this study, PCV2 Cap protein and its derivates were expressed via the baculovirus expression system, and the cellular localization of the recombinant proteins were investigated using anti-Cap mAb by imaging flow cytometry. Analysis of subcellular localization of Cap protein and its variants demonstrated that NLS mediated Cap protein nuclear export as well as nuclear import, and a phosphorylation site (S17) was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the NLS domain to regulate Cap protein nuclear export. Phosphorylation of NLS regulating the PCV2 Cap protein nuclear export was also demonstrated in PK15 cells by fluorescence microscopy. Moreover, the influence of Rep and Rep' protein on Cap protein subcellular localization was investigated in PK15 cells. Phosphorylation of NLS regulating Cap protein nuclear export provides more detailed knowledge of the PCV2 viral life cycle.
猪圆环病毒 2 型（PCV2）的开放阅读框 2（ORF2）编码主要衣壳（Cap）蛋白，该蛋白自我组装成与 PCV2 病毒粒子相似形态的病毒样颗粒（VLP），并通过 N 端富含精氨酸的核定位信号（NLS）积累在核内。在本研究中，通过杆状病毒表达系统表达了 PCV2 Cap 蛋白及其衍生物，并通过成像流式细胞术使用抗 Cap mAb 研究了重组蛋白的细胞定位。Cap 蛋白及其变体的亚细胞定位分析表明，NLS 介导 Cap 蛋白的核输出和核输入，通过液相色谱-串联质谱（LC-MS/MS）在 NLS 结构域中鉴定出一个磷酸化位点（S17）来调节 Cap 蛋白的核输出。荧光显微镜还证明了 NLS 磷酸化调节 PCV2 Cap 蛋白核输出在 PK15 细胞中的作用。此外，还研究了 Rep 和 Rep'蛋白对 Cap 蛋白亚细胞定位的影响。NLS 调节 Cap 蛋白核输出的磷酸化，为 PCV2 病毒生命周期提供了更详细的知识。
