Dengue virus strain 2 capsid protein switches the annealing pathway and reduces the intrinsic dynamics of the conserved 5' untranslated region

Abstract

The capsid protein of Dengue Virus strain 2 (DENV2C) is a structural protein with RNA chaperone activity that promotes multiple nucleic acid structural rearrangements, critical for transcription of the single-stranded positive-sense DENV2 genomic RNA. Annealing of the conserved 5 untranslated region (5UTR) to either its complementary sequence or to the 3 untranslated region (3UTR) occurs during (+)/(-) ds-RNA formation and (+) RNA circularization, respectively, both essential steps during DENV RNA replication. We investigated the effect of DENV2C on the annealing mechanism of two hairpin structures from the 5UTR region (21-nt upstream AUG region (5UAR) and 23-nt capsid-coding hairpin (5cHP)) to their complementary sequences during (+)/(-) ds-RNA formation and (+) RNA circularization. Using fluorescence spectroscopy, DENV2C was found to switch annealing reactions nucleated mainly through kissing-loop intermediates to stem-stem interactions during (+)/(-) ds-RNA formation while it promotes annealing mainly through kissing-loop interactions during the (+) RNA circularization. Using FRET-FCS and trFRET, we determined that DENV2C exerts RNA chaperone activities by modulating intrinsic dynamics and by reducing the kinetically trapped unfavorable conformations of the 5UTR sequence. Thus, DENV2C is likely to facilitate genome folding into functional conformations required for replication, playing a role in modulating (+)/(-) ds-RNA formation and (+) RNA circularization.