| Dengue virus (DEN), a member of genus Flavivirus, including four types, can cause human dengue fever, dengue haemorrhagia fever, dengue shock syndrome. DEN contains a single plus-sense RNA genome of approximately 11 kb in length, which consists of a 5′untranslated region (UTR), a single long open reading frame (ORF), and a 3′UTR. The ORF encodes 3 structural proteins and 7 nonstructural proteins. The 5′UTR is about 450 nucleotides in length, and the 3′UTR, which is 100 nucleotides in length, contains 5 RNA elements: variable region (VR), repeated conserved sequence (RCS2), CS2, CS1 and stem-loop (SL) structure. It has been suggested that the 3′UTR play key role during virus translation and RNA replication. However none of these mechanisms is as yet well defined. Genome replication and virus translation are two important stages for virus lifecycle. Therefore, the exploration of 3′UTR mechanism on RNA replication and virus translation should be warranted. In order to elucidate the mechanism of DEN translation, the 3′UTR and RNA elements within it of DEN genome are subjected to investigation through virus induced reporter gene (VIRG) and deoxyribozyme (DRz) assays. Following are the main results:1. Construction and characterization of VIRGs containing DEN UTRsTo develop a reporting system to study the effect of the 3′UTR on translation, a VIRG composed of firefly luciferase gene flanked by the 5′and 3′UTRs of DEN was firstly constructed. Based on the different chemical structure and substrate between firefly luciferase and Renilla luciferase and the same reaction situation, a control VIRG carrying Renilla luciferase was constructed simultaneously. The constructed VIRGs were then transfected into BHK-21 cells by electroporation or Lipofection 2000. The expression of these VIRGs were further examined by luciferase and western blot assays. The results showed that both the reporter genes were sustainably and efficiently expressed in BHK-21 cells, which provided a basic platform to explore the 3′UTR effects on translation.2. Control of translation by the 3′UTR of DEN genomeUsing the system described above, we found that the translational efficiency of reporter gene was enhanced by RCS2-CS2 of 3′UTR and diminished by RCS2-CS2 of 3′UTR. The translation of the reporter in the construct lacking full-length 3′UTR was hardly detected. The translational efficiency of VIRG with RNA element VR was comparable to that of VIRG with the naive 3′UTR, demonstrating the importance of VR on translation. In addition, VIRGs with or without poly(A) tail were constructed in parallel and no difference was observe between them, indicating that VR might increase RNA stability just like poly(A) tail. In addition, the translational efficiency of CS1-SL that base-paired with 5′UTR was lower than that of RCS2-CS2. These results suggested cyclization through 5′UTR and CS1-SL was dispensable for translation. And cyclization might block the interaction between the 5′UTR and ribosome and resulted in lower translational efficiency.Furthermore, to observe the effect of CS1-SL on DEN translation, we designed and selected 5 deoxyribozymes (DRz) targeted with the 3′UTR of DEN, and verified their specific RNA-cleavage activity in vitro. The results showed the amount of 3′UTR decreased with cleavaged segments increasing Then VIRG with the naive 3′UTR was cleavaged by DRz and the translational efficiency of that was further observed. We then observed the translational efficiency of VIRG with 3′UTR different length caused by cleavaged of DRz. At last, we evaluated the effect of 3′UTR different length on dengue type 2 virus translation by DRz. These results suggested the 3′UTR participated in translation and CS1-SL diminished translation.3. Translational Regulation by the RNA elements within the 3′UTR of DENOn the basis of above, we explored the contribution of 3′UTR single element on translation. We constructed a series of VIRGs with a single element or with a single element deleted. The data showed RCS and CS2 element might up-regulate translation and CS1 and SL element might down-regulate translation. To rule out the possible interference by mRNA stability, we analyzed the half-life of mRNA by RT-PCR and real-time RT-PCR. The result showed the difference of translation was irrelative of mRNA stability. As to SL element, we constructed VIRGs with mutation and retro-mutation through base-paired broken, and the results indicated the SL bases that paired with the 5′UTR were responsible for the down-regulation of translation, not the secondary SL structure.In this study, we systematically explored the effect of VIRG with multiple and single element of the 3′UTR of DEN genome on translation for the first time. Our results suggested that the 3′UTR of DEN participated in translation and VR element increased mRNA stability, and RCS2/CS2 might up-regulate and CS1/SL down-regulate translation, and the SL bases that paired with the 5′UTR were responsible for the down-regulation of translation. These results laid a solid foundation for exploring the translational mechanism of dengue virus.
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