Characterization And Functional Analysis Of Non-coding 3' Sub-genomic Rnas Of Dengue Viruses

Dengue virus was an important member of flavivirudae which cause dengue fever and hemorhagic fever. Until now dengue vaccines and antiviral drugs were unavailable.Viral genome is a single strand positive RNA molecule, which could serve as viral messenger RNA to initiate translation. All of ten viral proteins were translated together from a single open-reading frame contained in viral genomic RNA(gRNA), then spliced apart by host signalase and viral proteases. In replication, viral gRNA also direct synthesis of progeny gRNAs as template. According to genomic structure and strategy of gene expression, it is believed that none of sub-genomic RNAs were involved in dengue viral replication, except gRNA positive strand and its complementary negative RNA.Translation and replication of dengue viral gRNA were under the control of series genomic cis-acting elements and trans-activators. Most of cis-acting elements were located in genomic 5'and 3'terminal non-coding region, which could formed several highly conserved RNA structures. These structured RNA elements exserted regulation and control function to viral replication by specific binding to trans-activators. According to viral gRNA's organization, it was believed that 5'NCR and 3'NCR played their roles in translation and replication, respectively. several studies had indicated that 3'NCR could also recruite eukaryotic translation initiation factors. Previously, we found translation enhancement elements were located in 3'NCR. Current replication models of dengue virus could not explaine this discrimination. We proposed that other regulation stradegy were empolied by dengue virues.In the perspective of RNA viruse evolution, organization of viral gRNA partly determined the replication strategy which virus used. We were inspired by some plant positive single-strand RNA viruses, which had similar genomic organization as dengue viruses did, but also could generate viral sgRNA which was specifically associated with gRNA 3'terminus. Intrestingly, this viral RNA species played distinct roles in viral life cycle. For dengue viral and other animal RNA viruses, it was still a question. If such 3'sgRNA did exist, it might help us to have a better understanding on viral replication. Furthermore, its potential functions would offer novel ideas for study on viral replication mechanism.To indentify the existence of dengue viral 3'sgRNA, total RNA derived from virus infected cells and tissue were analyized by northern blotting. According to alignment results of 3'terminal sequence derived from four types viruses, an extremely conserved region was chosen to be targets of sepecific probes. RNA probes were labelled by digoxin during in vitro transcription. Northern blotting analysis showed that dengue viruses did generated viral 3'sgRNA during replication. Further experiments confirmed that all of four types of dengue viruses could produce similar 3'sgRNAs both in infected BHK-21 cells and suckling mouse brain tissue, which suggested that generation of 3'sgRNA was a ubiquitous event during viral replication. Unexpectedly, we also witnessed a unique strain of DV2 produces three species of sgRNAs simutaneously in comparison with other isolates. Furthermore, we noticed that 3'sgRNA accumulated in BHK-21 cells along with viral gRNA synthesis, which had strongly indicated that 3'sgRNA properly play roles in viral replication. On the other hand, the quantity of 3'sgRNA produced in distinct host cells were distinct. It had implied that host cell factors also contribute to the rise of 3'sgRNA.To clarify the property of dengue viral 3'sgRNA, its sequence information of 3'sgRNA were obtained by 5'RACE, then sequnences alignment result had showed that the initiation site of 3'sgRNA was all located in 3'non-coding region. It showed that 3'sgRNA was a free 3'NCR fragment per se. Alignment work also revealed an conserved region appeared at the 5'initiation site of 3'sgRNA, which indicated that this sequence could be a promoter-like element. Secondary structure predication further revealed that a RNA stem-loop structures emergered at the iniation site, which suggested that RNA structure might have impact on 3'sgRNA generation. Then ribonuclease degradation experiment was carried out to further tesitify the biochemical propertity of SL structure. It was showed that SL was invuneralbe to degradation of a enkaryotic 5'exo-ribonuclease XRN-1. Both viral full length RNA clone and mini artificial gRNA contained a reporeter gene could generate 3'sgRNA analogs in vitro by XRN-1.On the other hand, XRN-1 was detected in BHK-21 cells, although its expression was at a low level. Taken together, our study proposed a potential mechasim of 3'sgRNA biogenesis, in which host ribonucleases and a stem-loop structure took part in 3'sgRNA generation.Our previous study had showed that 3'sgRNA contained all of cis RNA elements in 3'NCR. Accumulation of 3'sgRNA was also observed. Taken them together, we assumed that 3'sgRNA stand a great possibility to exert its function to viral replication. By 3'sgRNA transfecting, we elevated the quantitiy of 3'sgRNA in host cells. Firstly, we constructed a viral mini gRNA, which contained viral intact NCRs and a reporter gene in order to mimic viral gRNA translation. Then we found 3'sgRNA could suppress reporter gene expression. To investigate 3'sgRNA function in viral gRNA synthesis,3μg 3'sgRNA was transfected into host cells, then copy numbers of viral gRNAs were found increased about 10 times in 105 transfected cells in comparison to untransfected cells. Further observation revealed this replication augument was dependent on transfection dose. Consitently, plague formed by progeny virus particles were also increased. It suggested that 3'sgRNA not only regulate viral gRNA replication and translation, also facilitate viral propergation. To confirm the mechanism of 3'sgRNA maniplutaing viral replication, a pair of cyclization sequences was mutant to depelete RNA-RNA interaction between viral genome 5'and 3'terminus. Mutation partly reduced replication enhancement mediated by 3'sgRNA. We proposed that new discovered 3'sgRNA was a trans-acting ribo-regluatior in viral replication.In our study, a novel 3'sub-genomic RNAs of dengue virues was indentified and characterized for the first time. Sequencing revealed that 3'sgRNA were small non-coding RNA derived from 3'NCR. Structural analysis indicated its interaction with host cell exoribonuclase, by which could give the rise of 3'sgRNA and this mechanism appeared to be a novel pathway in RNA viruses for generating sgRNA. Finally, it showed that 3'sgRNA had influence on both viral gRNA synthesis and translation. We proposed that 3'sgRNA was a trans-acting ribo-regluatior in viral replication. Our study updated our understanding of dengue viral replication, and also shed light on viral replication and host cell interaction.