Lin Wei-Li, Chien Maw-Sheng, Du Yu-Wen, Wu Pei-Ching, Huang Chienjin
Graduate Institute of Veterinary Microbiology, College of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan, ROC.
Biochem Biophys Res Commun. 2009 Feb 20;379(4):1066-71. doi: 10.1016/j.bbrc.2009.01.001. Epub 2009 Jan 14.
Porcine circovirus type 2 possesses a circular, single-stranded DNA genome that requires the replication protein (Rep) for virus replication. To characterize the DNA binding potential and the significant region that confers the nuclear localization of the Rep protein, the defined coding regions of rep gene were cloned and expressed. All of the recombinant proteins except for the N-terminal 110 residues deletion mutant could bind to the double-stranded minimal binding site of replication origin (ori). In addition, the N-terminal deletion mutant lacking 110 residues exhibited mainly cytoplasmic staining in the transfected cells in contrast to the others, which localized dominantly in the nucleus, suggesting that this N-terminal domain is essential for nuclear localization. Furthermore, a series of green fluorescence proteins (GFP) containing potential nuclear localization signal (NLS) sequences were tested for their cellular distribution. The ability of the utmost 20 residues of the N-terminal region to target the GFP to the nucleus confirmed its role as a functional NLS.
猪圆环病毒2型拥有一个环状单链DNA基因组，该基因组需要复制蛋白（Rep）来进行病毒复制。为了表征Rep蛋白的DNA结合潜力以及赋予其核定位的重要区域，对rep基因的特定编码区域进行了克隆和表达。除了N端缺失110个残基的突变体之外，所有重组蛋白都能与复制起点（ori）的双链最小结合位点结合。此外，与其他主要定位于细胞核的重组蛋白相比，缺少110个残基的N端缺失突变体在转染细胞中主要表现为细胞质染色，这表明该N端结构域对于核定位至关重要。此外，还测试了一系列含有潜在核定位信号（NLS）序列的绿色荧光蛋白（GFP）的细胞分布情况。N端区域最多20个残基将GFP靶向细胞核的能力证实了其作为功能性NLS的作用。
