Regulation of dengue virus translation and RNA synthesis by the viral 3'-untranslated region

Dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) are caused by the four serotypes of dengue virus (DEN), which are primarily transmitted by Aedes aegypti and Ae. albopictus mosquitoes in tropical and sub-tropical regions of the world. Over 40% of the world's population is at risk for DEN infection. Unfortunately, there is no vaccine or effective treatment to prevent DEN infection at this time. Knowledge of the molecular biology of DEN, specifically, of what is essential for DEN to replicate within a cell, will allow the rational design of attenuated vaccine strains and antiviral therapeutics. The research presented here describes efforts to understand which regions within the DEN genome are required for viral translation and RNA synthesis. Two approaches were taken: mutational analysis of the DEN 3' untranslated region (3' UTR) and screening of antisense molecules complementary to conserved regions within the DEN 3'UTR. Deletion analysis employing reporter mRNAs that contained DEN 5' - and 3'-UTRs flanking a firefly luciferase gene demonstrated that the DEN 3'UTR enhances translation. The DEN 3'UTR appears to stimulate translation independent of the DEN 5'UTR. Approximately half of the translational efficiency is due to the conserved, terminal 3' stem-loop (3'SL) domain, which up-regulates initiation of viral translation. A complementary approach to further explore the role of conserved domains within the DEN 3'UTR in viral translation and RNA synthesis utilized antisense technology. Peptide-conjugated phosphorodiamidate morpholino oligomers (P-PMOs) were synthesized complementary to several conserved regions within the DEN 3'UTR. Three DEN P-PMOs inhibit viral replication in different cell lines without causing significant cytotoxicity. P-PMOs complementary to the top of the 3'SL inhibited viral translation and RNA synthesis. In summary, the DEN genome contains critical regulatory domains within the 3'UTR that control viral translation and RNA synthesis. Further research with antisense compounds complementary to the DEN 3'UTR can potentially lead to effective antiviral therapeutics for DEN infections.