Role of SLB elements presented at 5 UTR of dengue virus on translation and RNA synthesis / 解放军医学杂志

ABSTRACT

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 designedSLB1,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.luc2AGDD-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.