Kinetic analysis of the interaction between dengue virus NS3 helicase and ssRNA

J. JEREMÍAS INCICCO; LEILA A. CABABIE; LEOPOLDO G. GEBHARD; ANDREA V. GAMARNIK; RODOLFO M. GONZÁLEZ-LEBRERO; SERGIO B. KAUFMAN

Congreso; Latin American Conference on Mathematical Modeling of Biological Systems; 2015

p { margin-bottom: 2.47mm; direction: ltr; color: rgb(0, 0, 0); line-height: 120%; text-align: left; }p.western { font-family: "Calibri",serif; font-size: 11pt; }p.cjk { font-family: "Liberation Serif"; font-size: 11pt; }p.ctl { font-family: "Liberation Serif"; font-size: 12pt; }Denguevirus nonstructural protein 3 (NS3) is a molecular motor protein thatunwinds duplex RNA driven by the free energy derived from thehydrolysis of nucleoside triphosphates. Inprevious studies we examined the interaction of NS3h with ssRNA underequilibrium conditions and we found that the interaction ischaracterized by minimum and occluded site sizes both of 10nucleotides. In this presentation we show results from the study ofthe kinetic mechanism of this interaction obtained from stopped-flowexperiments. A series of 5?-fluorescein-labeled oligonucleotides ofdifferent lengths (11 to 16 nucleotides) with a repetitive sequenceof five nucleotides (AGUUG) were employed and the binding timecourses to the NS3h was monitored by the fluorescence increaseobserved upon excitation at 495 nm. Reaction were carried out at 25ºCin buffer B (MOPS 25 mM, pH 6.4, KCl 100 mM (total K+),EDTA 0.50 mM, MgCl2 2.0 mM).Thebinding time courses observed under pseudofirst order conditionsrequired a minimum of three exponential terms for an adequate fit. Akinetic model of three sequential steps was able to describe theexperimental time courses and provided best-fitting estimates of therate constants for each oligonucleotide tested.