Abstract
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, Kobs, 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 (MgCl2 and CaCl2), 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 Kobs at any concentration of cations.
Highlights
Dengue virus (DENV) is an important human pathogen that belongs to the Flaviviridae family together with other emergent and re-emergent viruses such as Zika, yellow fever, West Nile and hepatitis C viruses[1]
We extend the analysis to characterize the effect of ions on the equilibrium constant for the binding of nonstructural protein 3 (NS3) to single stranded RNA (ssRNA)
In the present study we implemented this analysis as a tool to expand the thermodynamic characterization of the interaction between DENV NS3 helicase domain alone (NS3h) and ssRNA
Summary
Dengue virus (DENV) is an important human pathogen that belongs to the Flaviviridae family together with other emergent and re-emergent viruses such as Zika, yellow fever, West Nile and hepatitis C viruses[1]. At the N-terminal region, amino acid residues 1 to 170, together with part of the NS2B protein, form an active serine protease and at the C-terminal region, amino acid residues 171 to 618 folds into a tri-lobed domain with RNA-helicase, RNA 5’-triphosphatase (RTPase), nucleoside 5’-triphosphatase (NTPase), and RNA annealing activities[8,9,10,11]. In vitro, these two functional domains appear to work independently of each other. ATP), which supplies the driving force for directional movement along RNA strands and for unwinding of double stranded RNA tracks
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