Analysis of the interaction between dengue virus NS3 helicase and ssRNA under equilibrium conditions

J. JEREMIAS INCICCO; LEOPOLDO G. GEBHARD; RODOLFO M. GONZÁLEZ LEBRERO; ANDREA V. GAMARNIK; SERGIO B. KAUFMAN

Córdoba

Encuentro; XLII Reunión Anual de la Sociedad Argentina de Biofísica; 2013

Sociedad Argentina de Biofísica

Dengue virus nonstructural protein 3 (NS3) is a molecular motor protein that unwinds RNA duplexes driven by the free energy derived from the hydrolysis of nucleoside triphosphates. This RNA-helicase was shown to bind preferentially to single stranded (ss) RNA along which it would move with 3´ → 5´ directionality. Here we report results from studies of the interaction of dengue virus NS3 helicase with ssRNA using a quantitative fluorescence titration technique. Binding experiments were performed with a series of fluorescein-labeled ssRNA oligomers of different lengths. Model-independent analysis of the titration curves provided estimates for the stoichiometry of binding. Experimental results were well described by a statistical thermodynamic model that takes into account the overlap of potential binding sites and cooperative interactions between adjacent NS3 molecules. Global fitting of this model to all titration curves gave a binding site size of 9.1 ± 0.1 bases per NS3h; an intrinsic association constant of 0.0048 ± 0.0001 nM-1 and a cooperativity factor ω of 3.4 ± 0.7.