登革4型病毒5′非编码区SLB元件对病毒复制和翻译的调控作用

Role of SLB elements presented at 5 UTR of dengue virus on translation and RNA synthesis

目的探讨登革4型病毒5′非编码区SLB元件对病毒复制和翻译的调控作用。方法首先通过mfold3·2软件对登革4型病毒5′端RNA二级结构进行预测,确定了3个突变位点:SLB1,缺失位于短茎环82-87位5′UAR环化序列,并破坏其保守茎环结构;SLB2,在次环引入突变破坏其环状结构(M77G/C);SLB3,突变仅涉及核苷酸,不破坏该次环二级结构(M77-78AG/GA)。然后在含有海肾荧光素酶(Renilla Luciferase,R·luc)报告基因的复制子(DEN-R·luc2A-RP)基础上,利用重叠PCR构建上述突变体。将突变体(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报告基因技术和实时定量RT-PCR对突变体的复制和翻译水平进行检测。结果SLB1突变体的翻译水平并未受到显著影响,但其复制水平出现显著下调。SLB2突变体的翻译水平亦未受到显著影响,但其复制水平受到完全抑制;SLB3突变体的复制和翻译水平均受到了完全抑制。结论登革4型病毒5′非编码区SLB元件对基因组RNA复制和翻译具有重要的调控作用。

Objective To investigate the effects of SLB elements presented at 5＇UTR of dengue virus genome on viral translation and RNA replication. Methods The 5 end of RNA secondary structures of dengue virus genome were predicted using mfold 3.2, and three mutants with different modifications in SLB elements were designed： SLB1, part deletion of the 5＇ UAR sequence （82--87nt） ; SLB2, mutation of the sequence on stem （Mut 77G/C）; SLB3, mutation of the sequence on stem （M77-78AG/GA）, respectively. The mutants described above were constructed by OL-PCR based on DEN-R luc2A-RP, respectively. Replicon RNAs corresponding to DEN-R luc2A- RP, DEN-R. Iuc2A△GDD-RP and the 3 mutants mentioned above were in vitro transcribed and equal amounts of RNA were transfected into BHK cells with Lipofectamine 2000. After RNA transfection, the replicons were detected and characterized by RT-PCR, IFA, Renilla Luciferase assay system and real time RT-PCR, respectively. Results It was shown that SLB1 mutant did not significantly affect the translation of the input RNA, but seriously compromised RNA synthesis; SLB2 mutant did not significantly affect the translation of the input RNA either, but its RNA replication was abolished; both the translation and replication of SLB3 mutant were abolished. Conclusion Both the nucleotide sequence and the RNA secondary structure of the second loop and short stem of SLB element are highly conserved. SLB element may play an essential role on viral translation and RNA replication. The findings of present study may set a foundation for elucidating the role of SLB during viral translation and replication.