Abstract
ABSTRACT The capsid protein of dengue virus strain 2 (DENV2C) promotes nucleic acid structural rearrangements using chaperone activity. However, the role of DENV2C during the interaction of RNA elements in the conserved 5’ untranslated region (5’UTR) to the 3’ untranslated region (3’UTR) is still unclear. Thus, we investigated the effect of DENV2C on the annealing mechanism of two RNA hairpin elements from the 5’UTR to their complementary sequences during (+)/(-) ds-RNAformation and (+) RNA circularization. DENV2C was found to switch the annealing pathway for RNA elements involved in (+)/(-) ds-RNA formation, but not for RNA elements related to (+) RNA circularization. In addition, we also determined that DENV2C modulates intrinsic dynamics and reduces kinetically trapped unfavourable conformations of the 5’UTR sequence. Thus, our results provide mechanistic insights by which DENV2C chaperones the interactions between RNA elements at the 5’ and 3’ ends during genome recombination, a prerequisite for DENV replication.
Highlights
N translation factors are limiting [4]
We characterized the role of DENV2C as an RNA chaperone by investigating the annealing kinetics of 5’UAR and 5’cHP to their complementary sequences during (+)/(-) double-stranded (+)/(-) RNA (ds-RNA) formation and the (+) RNA circularization
No decrease in annealing reaction rates with the loop mutants indicate that the role of hairpin loops in DENV2C promoted 5’UAR/c5’UAR and DENV2C promoted 5’cHP/c5’cHP annealing is limited and both kinetic reactions are prominently nucleated through the stems. These results showed that DENV2C switches the annealing mechanism of complementary sequences during (+)/(-) ds-RNA formation from predominantly through kissing-loop intermediates to propagate through stem-stem interactions
Summary
N translation factors are limiting [4]. the DENV 3’UTR lacks a poly(A) tail, the DENV genome is known to circularize, noted as (+) RNA circularization, to enhance its translation efficiency, similar to cellular mRNAs (Fig. 1A) [5, 6]. Two complementary sequences at the 5′ and 3′ ends have been identified for such long range 5’-3’ RNA end interactions and are necessary for the (+) RNA circularization [8] One of these sequences, a hairpin structure, known as the 5′ upstream AUG region (5′UAR) element in the 5′UTR, anneals with its complementary 3′UAR counterpart, which is located at the bottom part of 3′ stem loop (3’SL) in the 3’UTR (Fig. 1B) [7, 9, 12]. As the (-) RNA genome is synthesized during transcription, secondary structures of the conserved 5’UTR RNA elements, like 5’UAR and 5’cHP, which is involved in the (-) RNA synthesis dissociates and form double-stranded (+)/(-) RNA (ds-RNA) to facilitate genome recombination [13] During this event, the 5’UAR and the 5’cHP dissociate and anneal to their complementary (-) RNA sequences, c5’UAR and c5’cHP, respectively (Fig. 1B). Our results suggest that DENV2C probably modulates (+)/(-) ds-RNA formation and the (+) RNA circularization events by modulating their annealing pathways
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