受miRNA-205调控的重组柯萨奇病毒B3的构建及其复制

本研究在柯萨奇病毒B3(coxsackievirus B3，CVB3)基因组P1编码区与P2编码区之间插入一段has-miRNA-205-3p和has-miRNA-205-5p(简称miR-205)的靶序列，得到重组病毒v205T，并比较分析了它在人宫颈癌细胞系HeLa细胞(miR-205低水平表达)和非小细胞肺癌细胞系A549细胞(miR-205高水平表达)中的复制情况。结果表明，插入的miR-205靶序列不影响病毒在HeLa细胞中的复制水平，但抑制了病毒在A549细胞中的复制，病毒滴度为对照的1%以下。为探讨v205T在2株细胞中复制差异的原因，进一步加入miR-205的类似物和抑制物。miR-205类似物可抑制v205T在HeLa细胞中复制和杀伤细胞的水平，而miR-205抑制物可提高v205T在A549细胞中的复制和杀伤细胞的水平。结果表明，v205T的复制确实受miR-205的调控。本研究为开发基于CVB3载体的溶瘤病毒和针对CVB3的减毒活疫苗提供了依据。

Construction of miRNA-205-regulated recombinant Coxsackievirus B3 and its replication

A recombinant Coxsackievirus B3 (CVB3), v205T, was constructed by inserting repeats of a target sequence for miR-205 between the coding sequence of P1 and P2. Its replication was analyzed in Hela and A549 cells, in which the level of miRNA-205 was known to be low and high, respectively. The results showed that the insertion of miRNA-205 targets in the selected site did not affect the infectivity of the recombinant virus in Hela cells, but greatly reduced its replication in A549 cells, resulting in more than 100-fold reduction in virus titer. miR-205 mimics and inhibitors were used to study the mechanism of differential replication of v205T. miR-205 mimics inhibited the replication and cytotoxicity of v205T in Hela cells, whereas miRNA-205 inhibitors increased the replication and cytotoxicity of v205T in A549. These results showed that the replication of v205T was regulated by cellular miR-205. The work provided basis for using CVB3 vectors for cancer viral therapy and vaccine development.