Role Of SLB And CHP Elements Present At 5' End Of Dengue Virus Genome On Viral Translation And RNA Replication

Dengue virus (DEN), a member of Flavivirus, is the pathogen of human dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Transmitted mainly by Ae. aegypti and Ae. albopictus, dengue virus infections was one of the most important human arboviral diseases in tropical and subtropical areas. The frequency and intensity of the outbreaks increased dramatically during last 3 decades. Despite the wide morbidity and mortality associated with dengue virus infections, the pathogenesis of this virus is not well understood, and at present, neither specific antiviral therapy nor licensed vaccine exists. Thus, defining the molecular determinants that regulatory role of the viral RNA in the infected cells is of central importance for understanding dengue virus pathogenesis.RNA elements present at the 5′and 3′end of dengue virus genome play essential regulatory roles during viral translation and RNA replication by participate in genome cyclization and interaction with associated viral and host proteins. Among the 3 elements(SLA, SLB and cHP)within the 5′end, the function of SLA has been extensively studied, while the function of SLB and cHP remain elusive. SLB and cHP are highly conserved among flaviviruses, here we performed a systematic mutation analysis to elucidate their effects on viral translation and replication. The research included three parts.1. Construction and identification of the sub-genomic replicons of dengue virus type 4 To construct the sub-genomic replicon system of dengue virus type 4, a dengue replicon (DENRP) with deletion in prM-E gene was constructed by site-directed mutagenesis PCR technique firstly, then the Renilla Luciferase and EGFP reporter gene was respectively inserted to the corresponding region of the DENRP by CHYSEL (cis-acting hydrolase element) technique to obtain the desired replicon DEN-R.luc2A-RP and DEN-GFP2A-RP. In addition, negative control replicons named DEN-△GDD-RP, DEN-GFP2A△GDD-RP and DEN-R.luc2A△GDD-RP were constructed with GDD motif deleted, respectively. The replicons were linearizd and transcripted in vitro, then equal amounts of the transcription RNA were transfected into BHK-21 cells by electroporation or with Lipofectamine 2000. The replicons were detected and characterized by RT-PCR, IFA and Renilla Luciferase assay system, respectively. Our results show that all 3 replicons constructed in this study could self-replicate and express specific proteins of dengue virus type 4 in BHK-21 cells. And the translation and RNA replication of DEN-R.luc2A-RP can be monitored through the expression of Luc as a function of time in replicon transfected BHK cells. Our date show that the level of Luc 3 h post transfection reflected the translation of the input RNA, while the Luc signal 72 h post transfection can be used to assess RNA replication. GFP signals can be readily detected in BHK-21cells transfected with DEN-GFP2A-RP 30 h later, and the positive cells were less than 5%. R.luc and EGFP were successfully expressed by DENRP, it could be a stable viral vector system for protein production after further modification. Lipofectamine 2000 should be the first choice for RNA transfection, compare to electroporation, Lipofectamine 2000 method could be handled easily with stable efficiency. To conclude, the replicons constructed here will be a useful tool to study the regulation mechanisms of replication and translation of dengue virus.2. Analysis of the role of SLB element present at the 5′UTR on viral translation and replication.To investigate the role of SLB present at the 5′UTR of dengue virus on translation and RNA synthesis, we performed deletions and mutations of SLB using the DEN-R.luc2A-RP system mentioned above. Three replicons with different modification were constructed respectively: 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). Replicon RNAs corresponding to DEN-R.luc2A-RP, DEN-R.luc2A△GDD-RP and the 3 mutants described above were in vitro transcribed and equal amounts of RNA were transfected into BHK cells. Luc activities were then monitored at different time. The normalized Luc signal at 3 h post transfection was representative of the translation activity. The 72h signal representative RNA replication. Our results show that SLB1 mutant did not significantly affect translation of the input RNA but seriously compromised RNA synthesis; SLB2 mutant did not significantly affect translation of the input RNA either, but its RNA replication was abolished; both the translation and replication of SLB3 mutant were abolished. Those date indicate the sequence and second structure of SLB was highly conserved. Our results lay a solid foundation for elucidating the role of SLB during viral translation and replication. 3. Assay of the role of cHP element present at the C gene on viral translation and RNA replication.To investigate the role of cHP element present at the C gene of dengue virus genome on translation and RNA replication, Six replicons with different modification in cHP element were constructed based on DEN-R.luc2A-RP ,respectively: H1, complete deletion of cHP; H2 and H3, mutation of the top loop; H4, H5 and H6 , deletion and mutation of lower stem, respectively. Replicon RNAs corresponding to DEN-R.luc2A-RP, DEN-R.luc2A△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, we monitored Luc activities at different time. The normalized Luc signal a t 3 h was representative of the translation activity. The 72h signal representative RNA amplification. Our results showed that all the 6 cHP mutants constructed here did not significantly affect translation of the input RNA but seriously compromised RNA replication.To conclude, we developed a dengue virus replicon system that could be used to dissect RNA elements of the viral genome involved in translation and/or RNA replication. This replicon has a Renilla Luciferase gene fused in frame to the viral polyprotein in place of the viral structural proteins. Using this system, we analyzed the functional role of SLB and cHP RNA elements present at the 5′end of dengue virus. Our results show that the SLB elements present at 5′UTR is highly conserved. SLB1 mutant did not significantly affect translation of the input RNA but seriously compromised RNA synthesis; SLB2 mutant did not significantly affect translation of the input RNA either, but it's RNA replication was abolished.; both the translation and replication of SLB3 mutant were abolished; in addition, deletion and mutation of cHP did not significantly affect translation of the input RNA but seriously compromised RNA synthesis. These lay a solid foundation for elucidate the role of SLB and cHP during viral translation and replication finally.