Institute of Preventive Veterinary Medicine, Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, Zhejiang University, Hangzhou, China.
Virol J. 2011 Jul 6;8:341. doi: 10.1186/1743-422X-8-341.
Porcine circovirus type 2 (PCV2) is believed to be the primary causative agent of postweaning multisystemic wasting syndrome (PMWS). It is supposed that capsid protein of PCV may contribute to replication control via interaction between Cap and Rep in the nucleoplasm. In this study, we described the construction and in vitro characterization of NLS-exchanged PCV DNA clones based on a PMWS-associated PCV2b isolate from China to determine the role of ORF2 NLS in PCV replication.
The PCV1, PCV2, PCV2-NLS1 and PCV1-NLS2 DNA clone were generated by ligating a copy of respective genome in tandem with a partial duplication. The PCV2-NLS1 and PCV1-NLS2 DNA clone contained a chimeric genome in which the ORF2 NLS was exchanged. The four DNA clones were all confirmed to be infectious in vitro when transfected into PK-15 cells, as PCV capsid protein were expressed in approximately 10-20% of the transfected cells. The in vitro growth characteristics of the DNA clones were then determined and compared. All the recovered progeny viruses gave rise to increasing infectious titers during passages and were genetically stable by genomic sequencing. The chimeric PCV1-NLS2 and PCV2-NLS1 viruses had the final titers of about 104.2 and 103.8 TCID50/ml, which were significantly lower than that of PCV1 and PCV2 (105.6 and 105.0 TCID50/ml, respectively). When the ORF2 NLS exchanged, the mutant PCV2 (PCV2-NLS1) still replicated less efficiently and showed lower infectious titer than did PCV1 mutant (PCV1-NLS2), which was consistent with the distinction between wild type PCV1 and PCV2.
Recovery of the chimeiric PCV1-NLS2 and PCV2-NLS1 progeny viruses indicate that the nuclear localization signal sequence of capsid protein are functionally exchangeable between PCV1 and PCV2 with respect to the role of nuclear importing and propagation. The findings also reveal that ORF2 NLS play an accessory role in the replication of PCV. However, we found that ORF2 NLS was not responsible for the distinction of in vitro growth characteristic between PCV1 and PCV2. Further studies are required to determine the in vivo viral replication and pathogenicity of the NLS chimeric DNA clones.
猪圆环病毒 2 型（PCV2）被认为是断奶后多系统衰弱综合征（PMWS）的主要病原体。推测 PCV 的衣壳蛋白可能通过核质中 Cap 和 Rep 之间的相互作用来促进复制控制。本研究基于从中国分离到的与 PMWS 相关的 PCV2b 分离株，描述了基于 NLS 交换的 PCV DNA 克隆的构建和体外特性，以确定 ORF2 NLS 在 PCV 复制中的作用。
通过串联连接各自基因组的拷贝并进行部分重复，生成了 PCV1、PCV2、PCV2-NLS1 和 PCV1-NLS2 DNA 克隆。PCV2-NLS1 和 PCV1-NLS2 DNA 克隆包含一个嵌合基因组，其中 ORF2 NLS 被交换。当转染 PK-15 细胞时，这四个 DNA 克隆均被证实具有传染性，大约 10-20%的转染细胞中表达了 PCV 衣壳蛋白。然后确定并比较了 DNA 克隆的体外生长特性。在传代过程中，所有回收的后代病毒的感染滴度均逐渐增加，并且通过基因组测序证明其遗传稳定。嵌合 PCV1-NLS2 和 PCV2-NLS1 病毒的最终滴度约为 104.2 和 103.8 TCID50/ml，明显低于 PCV1 和 PCV2（分别为 105.6 和 105.0 TCID50/ml）。当 ORF2 NLS 交换时，突变型 PCV2（PCV2-NLS1）的复制效率仍然较低，感染滴度也低于 PCV1 突变体（PCV1-NLS2），这与野生型 PCV1 和 PCV2 之间的区别一致。
嵌合 PCV1-NLS2 和 PCV2-NLS1 后代病毒的恢复表明，衣壳蛋白的核定位信号序列在核输入和传播方面在 PCV1 和 PCV2 之间具有功能可互换性。研究结果还表明，ORF2 NLS 在 PCV 的复制中起辅助作用。然而，我们发现 ORF2 NLS 并不是导致 PCV1 和 PCV2 体外生长特性差异的原因。需要进一步研究确定 NLS 嵌合 DNA 克隆的体内病毒复制和致病性。
