cis-acting elements in the 3'UTR of dengue virus differentially modulate translation in mosquito and mammalian cells

DIEGO E. ALVAREZ; ANA L. DE LELLA EZCURRA; ANDREA V. GAMARNIK

Davis, California

Congreso; American Society for Virology 22nd Annual Meeting; 2003

American Society for Virology

Dengue virus (DV) is a positive stranded RNA virus responsible of serious illness in humans. The viral genome is used as a template for protein synthesis, RNA amplification, and encapsidation. The regulation of these processes is presumably mediated by signals present at the 5´ and 3´ UTRs. The viral 5´UTR of DV-2 is 97 nts long and has a cap structure while the 3´UTR is 456 nts long and has four defined domains: A1, A2, A3 and A4, lacking a poly(A) tail. To investigate the influence of DV 5´ and 3´ UTRs on translation, we constructed a plasmid encoding luciferase flaked by the complete DV-2 UTR sequences. Based on this construct, we generated a series of RNA molecules with deletions encompassing each domain of the 3´UTR. Translation efficiencies were evaluated after RNA transfections into BHK and mosquito cells or microinjections into Xenopus laevis oocytes. Translation initiation in all systems was absolutely dependent on the cap structure. Interestingly, deletion of A1 causes a profound increase of translation efficiency in mosquito cells while translation in mammalian cells was not significantly modified. Using different assays to assess RNA-protein interaction we observed the formation of specific RNP complexes formed by A1 RNA in the presence of BHK but not with mosquito cell extracts, suggesting a possible modulation of translation by the binding of specific host factors. Deletion of domains A2 and A2-A3 decreased 5 and 10 fold the translation efficiency respectively in both mosquito and mammalian cells. The stability of radiolabeled RNA molecules with or without A2-A3 domains was very similar, suggesting that these RNA structures directly stimulate the translation process. Translation stimulation mediated by A2-A3 domains was dependent on DV2 5´UTR sequences because a capped RNA carrying the beta globine 5´UTR or an IRES sequence was not modified by the presence of DV 3´UTR domains. To understand the role of DV 3´UTR during replication in mammalian and mosquito cells we are currently analyzing the phenotype of viruses with deletions in A1, A2, and A3.