Steady-state NTPase activity of Zika virus NS3 helicase and its coupling with mechanical work (unwinding activity)

LEILA A. CABABIE; ANDREA V. GAMARNIK; LEOPOLDO G. GEBHARD; ROLANDO C. ROSSI; EVELYN B. MIKKELSEN; J. JEREMIAS INCICCO; SERGIO B. KAUFMAN

La Plata

Congreso; XLVII Reunión Anual de la Sociedad Argentina de Biofísica; 2018

Zika virus (ZIKV) nonstructural protein 3 (NS3) unwinds double-stranded RNA driven by the free energy derived from the hydrolysis of nucleoside triphosphates (NTPs). In this work, we present an initial characterization of the properties of the steady-state NTPase activity of ZIKV NS3 helicase. Initial rates of NTP hydrolysis were measured and the corresponding substrate curves for ATP, GTP, CTP, and UTP were obtained. In all cases, the resulting substrate curves were well described by hyperbolic functions whose parameter values (kcat and KM) were obtained by nonlinear regression analysis. The order of specificity of NS3 for these nucleotides was evaluated according to the value of the ratio (kcat/KM). Additionally, different nucleotides were tested as substrates for the RNA unwinding activity of NS3. Time courses of RNA unwinding reactions were carried-out and samples were resolved by polyacrylamide gel electrophoresis. Results indicate that NS3 acts as an energy transducer using any of the four nucleotides tested as substrates. That is, in all four cases NS3 couples its catalytic properties (NTPase activity) with the ability to perform the mechanical work of translocation along single-stranded RNA and unwinding of double stranded RNA.