Role of dengue virus 3’-untranslated region on translation and RNA stability.

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

Villa Carlos Paz

Congreso; XXXVII Reunión Nacional de la SAIB; 2002

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

Dengue virus (DV) is a positive stranded RNA virus responsible of serious illness in humans. During the viral life cycle the genome is used as a template for protein synthesis, RNA amplification, and encapsidation. The regulation of these processes is mainly mediated by signals present at the 5’ and 3’ untranslated regions (UTRs) of the viral RNA by mechanisms not well understood. To study the role of the UTRs on translation we build a simple construct encoding luciferase flaked by the 5’ and 3’UTRs of DV. The 5’UTR is 100 nucleotides long and has a cap structure while the 3’UTR has four defined domains: A1, A2, A3 and A4, lacking a poly(A) tail. By in vitro transcription we synthesized the RNAs with deletions of each domain of the 3’UTR and studied the translation efficiencies after microinjections into Xenopus laevis oocytes or transfections into BHK and mosquito cells. The deletion of A2 and A3 domains decrease 10 fold the efficiency of translation, suggesting an important role of the 3’UTR on translation initiation or RNA stability. To investigate this last possibility we developed an assay to study the half-life of radiolabeled RNAs into oocytes. The stability of the wild type RNA was very similar to the RNA carrying the A2A3 deletions. These results suggest that the 3’UTR could be directly involved in the translation process. To understand the molecular mechanism of translation stimulation we are using mobility shift and UV crosslinking assays to study RNA-RNA and RNA-protein interactions that involve both the 3’ and the 5’ UTRs.