NTPase activity of Zika virus NS3 helicase: study of the catalytic site of hydrolysis for NTPs and modulation by ssRNA

MIKKELSEN E; INCICCO JJ; KAUFMAN SB; CABABIE LA; GAMARNIK AV; GEBHARD LG; ROSSI RC

San Luis

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Biofísica; 2019

Sociedad Argentina de Biofísica

Zika virus (ZIKV) is a member of the virus family Flaviviridae that is responsible for human disease [1]. It?s RNA genome encodes structural and non-structural proteins. Among the latter, non-structural protein 3 (NS3) possesses helicase activity [2] and is essential for viral replication [3]. Helicases are molecular motors that couple the mechanical work of unwinding doubled-stranded nucleic acids to the hydrolysis of nucleoside triphosphates (NTPs). In previous work we established the ability of Zika virus NS3 to catalyze the hydrolysis of four NTPs: ATP, CTP, GTP and UTP and determined the specificity order for these nucleotides. In this work we show that these four nucleotides are hydrolyzed by the enzyme in a single and same site. To address this issue we made use of the fact that if two different substrates yield the same product (as in the present case, where orthophosphate is produced from the four nucleotides) it is possible to distinguish between a kinetic model of competition for the same site from others of multiple catalytic sites by measuring the steady-state velocity of product formation in mixtures of both substrates. Additionally we studied the effect of single-stranded RNA (ssRNA) on ATPase activity. To this end we performed measurements of ATPase activity in the presence of homopolyribonucleotide polyA. We obtained substrate curves for ATP in the presence of different concentrations of polyA, these curves were well described by equilateral hyperbolas characterized by parameters kcat and KM. The results show an increase in both the turnover rate constant kcat and in KM for ATP in the presence of polyA.