Abstract
The dengue virus (DENV) poses a significant threat to human health, accounting for approximately 400 million infections each year. Its genome features a circular structure that facilitates replication through long-range RNA-RNA interactions, utilizing cyclization sequences located in the untranslated regions (UTRs). To gain new insights into the organization of the DENV genome, we purified the 5' and 3' UTRs of DENV in vitro and examined their structural and binding properties using various biophysical techniques combined with computational methods. Through our biophysical characterization, we determined the 5' and 3' UTR regions to bind with an affinity of 40 nM in a 1:1 stoichiometry. By using small-angle x-ray scattering, we provide the first structural characterization of the 3' and 5' UTR regions, revealing several plausible conformations that the viral UTRs may adopt during replication. This comprehensive investigation revealed key features that provide mechanistic insights into the different structural states during DENV replication, as tracked through the accessibility of various RNA conformations. Overall, our research enhances the understanding of DENV cyclization, emphasizing the structural adaptability, dynamic folding, and flexibility of these RNA molecules in solution. By uncovering details at the atomic level, we aim to contribute to the development of targeted drugs that can disrupt crucial stages of viral replication.