Tang Li, Zhu Wu-Yang, Fu Shi-Hong, He Ying, Wang Zhi-Yu, Liang Guo-Dong
School of Public Health of Shandong University, Jinan 250012, China.
Bing Du Xue Bao. 2010 Mar;26(2):121-7.
To investigate the effects of site-directed mutagenesis at nsP2-726Pro on the characteristics of replicon vector derived from XJ-160 virus, a Sindbis virus (SINV) isolated in China. The mutant vector pBRep-726L, pBRep-726S, pBRep-726V or pBRep-726A was constructed by introducing nsP2-726Pro --> Leu, nsP2-726Pro --> Ser, nsP2-726Pro --> Val or nsP2-726Pro --> Ala into XJ-160 viral replicon vector pBRepXJ respectively. To quantitatively and qualitatively determine the site-directed mutagenesis on the replicon, the recombinant plasmids expressing Neomycinr (Neo(r)), enhanced green fluorescent protein (EGFP) or Renilla luciferase (R. luc) were constructed by cloning report genes into pBRepXJ or mutant XJ-160 vector respectively. And in vitro-synthesized RNA from expression vectors were electroporated into BHK-21 cells. Compared with the wild-type replicon, the mutation nsP2-726Pro --> Val or nsP2-726Pro --> Ala accelerated the processing of CPE on BHK-21 cells and simultaneously enhanced its self-replicating capacity. The mutant vector pBRep-726L with Leu substitution exhibited similar packaging capacity to that of pBRepXJ. In contrast, pBRep-726S exhibited a medium phenotype, including the process of CPE and the activity of R. luc expression in BHK-21 cells. The site-directed mutagenesis at nsP2-726Pro not only regulates directly XJ-160 virus vector-host cell interactions, but also plays an important role in its packaging capacity. All of these results lay a basis for researching the relation between the structure and function of alphavirus genome and developing alphavirus vector system with Chinese intellectual property.
为研究针对nsP2-726Pro进行定点诱变对源自中国分离的辛德毕斯病毒（SINV）XJ-160病毒的复制子载体特性的影响。通过分别将nsP2-726Pro→Leu、nsP2-726Pro→Ser、nsP2-726Pro→Val或nsP2-726Pro→Ala引入XJ-160病毒复制子载体pBRepXJ中，构建了突变载体pBRep-726L、pBRep-726S、pBRep-726V或pBRep-726A。为了定量和定性地确定对复制子的定点诱变，通过将报告基因分别克隆到pBRepXJ或突变的XJ-160载体中，构建了表达新霉素抗性（Neo(r)）、增强型绿色荧光蛋白（EGFP）或海肾荧光素酶（R. luc）的重组质粒。然后将表达载体体外合成的RNA电穿孔导入BHK-21细胞。与野生型复制子相比，nsP2-726Pro→Val或nsP2-726Pro→Ala突变加速了BHK-21细胞上细胞病变效应（CPE）的进程，同时增强了其自我复制能力。具有Leu替代的突变载体pBRep-726L表现出与pBRepXJ相似的包装能力。相比之下，pBRep-726S表现出中等表型，包括BHK-21细胞中CPE的进程和R. luc表达活性。nsP2-726Pro处的定点诱变不仅直接调节XJ-160病毒载体与宿主细胞的相互作用，而且在其包装能力中也起着重要作用。所有这些结果为研究甲病毒基因组的结构与功能之间的关系以及开发具有中国知识产权的甲病毒载体系统奠定了基础。
