登革4型病毒基因组cHP发卡结构对病毒复制和翻译的调控作用

Role of cHP element present at the C gene of dengne virus type-4 in viral translation and RNA replication

目的:探讨登革4型病毒C基因中保守的cHP发卡结构对病毒复制和翻译的调控作用。方法:首先在获得含有海肾荧光素酶(Renilla luciferase,R.luc)报告基因的复制子(DEN-R.luc2A-RP)基础上,利用重叠PCR构建cHP发卡结构系列突变体(cHP1～cHP6)。将构建的突变体(包括相关的阳性和阴性对照复制子)分别线性化并体外转录成RNA后,取等量各转录体RNA,以脂质体法分别转染BHK-21细胞。通过间接免疫荧光(IFA)、RT-PCR、R.luc报告基因检测技术和实时定量RT-PCR对突变体的复制和翻译进行检测。结果:cHP发卡结构系列突变体均可在BHK-21细胞中复制并稳定表达病毒特异蛋白,在该发卡结构中引入缺失或突变对病毒早期翻译并无显著影响,但病毒RNA复制均显著下调。结论:cHP发卡结构在基因组RNA复制过程中可能具有重要的调控作用。

Objective：To investigate the role of cliP element present at the C gene of dengue virus genome in translation and RNA replication, Methods： Six replicons with different modification in cliP element were constructed by OL-PCR based on DEN-R. luc2A-RP, respectively, cliP1, complete deletion of cliP; cliP2 and cliP3, mutation of the top loop; cliP4, cliP5 and cliP6, deletion, mutation and reverse mutation of the lower stem, respectively. Replicon RNAs corresponding to DEN-R. luc2A-RP, DEN-R. luc2△GDD-RP and the 6 mutants described above were in vitro transcribed and equal amounts of RNA were transfected into BHK cells. After RNA transfection, the replicons were further detected and characterized by RT-PCR, IFA, Renilla luciferase assay system and real time RT-PCR, respectively. Results：Our results showed that all the 6 cliP mutants constructed here did not significantly affect translation of the input RNA but seriously compromised RNA replication, Conclusion.cliP element may play an essential role in viral translation and RNA replication.