Division of Gastroenterology and Hepatology, Department of Internal Medicine, Saint Louis University School of Medicine, St. Louis, MO 63104, USA.
J Virol Methods. 2013 Aug;191(2):105-12. doi: 10.1016/j.jviromet.2013.04.010. Epub 2013 Apr 17.
The instability of recombinant clones accommodating large or full-length viral genomes is frequently a technical challenge in RNA virus research. In an attempt to establish a rapid plasmid-based reverse genetics system that utilizes long RT-PCR technique (LRP), similar difficulty was encountered in the cloning of 9022-bp LRP amplicon. All HCV genotype 1a strains used for LRP cloning showed a remarkable difference in terms of cloning stability. Subsequent analysis revealed the predictive value of phylogenetic positions in determining the cloning stability. Putative Escherichia coli promoters on the HCV genome might be responsible for such cloning difference. An exhaustive exploration, testing nearly one hundred cloning protocols, did not reveal a general approach that can achieve stable cloning for all HCV 1a strains. The selection of appropriate strains, guided by phylogenetic analysis, appears to be necessary prior to the construction of infectious HCV 1a clones. These observations are not only valuable for potentially establishing an HCV 1a cell culture model but also have general implications for other RNA viruses due to concern about cloning instability.
在 RNA 病毒研究中，容纳大型或全长病毒基因组的重组克隆的不稳定性常常是一个技术挑战。在试图建立一种利用长 RT-PCR 技术 (LRP) 的快速质粒反向遗传学系统时，在克隆 9022bp LRP 扩增子时遇到了类似的克隆稳定性困难。用于 LRP 克隆的所有 HCV 基因型 1a 株在克隆稳定性方面表现出显著差异。随后的分析表明，系统发育位置在确定克隆稳定性方面具有预测价值。HCV 基因组上的推定大肠杆菌启动子可能是导致这种克隆差异的原因。通过将近 100 种克隆方案进行彻底探索和测试，并未发现一种可以为所有 HCV 1a 株实现稳定克隆的通用方法。在构建感染性 HCV 1a 克隆之前，根据系统发育分析选择合适的株系似乎是必要的。这些观察结果不仅对建立 HCV 1a 细胞培养模型具有重要价值，而且由于对克隆不稳定性的关注，对其他 RNA 病毒也具有普遍意义。
