Identification of cis-acting elements in the 3'-untranslated region of the dengue virus type 2 RNA that modulate translation and replication

Abstract

Using the Massively Parallel Genetic Algorithm for RNA folding, we show that the core region of the 3'-untranslated region of the dengue virus (DENV) RNA can form two dumbbell structures (5' and 3'DBs) of unequal frequencies of occurrence. These structures have the propensity to form two potential pseudoknots between identical five-nucleotide terminal loops 1 and 2 (TL1 and TL2) and their complementary motifs, PK2 and PK1. Mutagenesis of a DENV2 replicon RNA encoding the Renilla luciferase reporter indicated that all four motifs and the Conserved Sequence 2 element (CS2) within the 3' DB are important for replication. However, for translation, mutation of TL1 alone does not have any effect; TL2 mutation has only a modest effect, but translation is reduced by ~60% in the TL1/TL2 double mutant, indicating that TL1 exhibits a cooperative synergy with TL2 in translation. Despite the variable contributions of individual TL and PK motifs in translation, WT levels are achieved when the complementarity between TL1/PK2 and TL2/PK1 is maintained even under conditions of inhibition of the translation initiation factor 4E function mediated by LY294002 via a non-canonical pathway. Taken together, our results indicate that the cis-acting RNA elements in the core region of DENV2 RNA that include two DB structures are required not only for RNA replication but also for optimal translation.