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The presence of ATP is known to stimulate helicase activity of the Dengue Virus Non-structural protein 3 helicase (NS3h), and the presence of RNA stimulates NS3h ATPase activity, however this coupling is still mechanistically unclear. Here we use atomistic models and molecular dynamics simulations to evaluate the single-stranded RNA (ssRNA)-length dependence of the NS3h-ssRNA binding affinity and its modulation by bound ATP. Considering complexes with 7, 11, 16 and 26 nucleotides (nts), we observe that both the binding affinity and its modulation by bound ATP are augmented with increased ssRNA lengths. In models with at least 11 nts bound, the binding of ATP results in a shift from a tightly bound to a weakly bound state. We find that the weakly bound state persists during both the ADP-Pi- and ADP-bound stages of the catalytic cycle. We obtain the equilibrium association constants for NS3h binding to an ssRNA 10-mer in vitro, both in the absence and presence of ADP, which further support the alternation between tightly and weakly bound states during the catalytic cycle. The length of bound ssRNA is critical for understanding the NS3h-RNA interaction as well as how it is modulated during the catalytic cycle., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Dengue virus nonstructural protein 3 (NS3) fulfills multiple essential functions during the viral replication and constitutes a prominent drug target. NS3 is composed by a superfamily-2 RNA helicase domain joined to a serine protease domain. Quantitative fluorescence titrations employing a fluorescein-tagged RNA oligonucleotide were used to investigate the effect of salts on the interaction between NS3 and single stranded RNA (ssRNA). We found a strong dependence of the observed equilibrium binding constant, K obs , with the salt concentration, decreasing at least 7-fold for a 1-fold increase on cation concentration. As a result of the effective neutralization of ~10 phosphate groups, binding of helicase domain of NS3 to ssRNA is accompanied by the release of 5 or 7 monovalent cations from an oligonucleotide or a polynucleotide, respectively and of 3 divalent cations from the same oligonucleotide. Such estimates are not affected by the type of cation, either monovalent (KCl, NaCl and RbCl) or divalent (MgCl 2 and CaCl 2 ), nor by the presence of the protease domain or the fluorescein label. Combined effect of mono and divalent cations was well described by a simple equilibrium binding model which allows to predict the values of K obs at any concentration of cations.