Abstract
Dengue virus nonstructural protein 3 (NS3) unwinds double stranded RNA driven by the free energy derived from the hydrolysis of nucleoside triphosphates. This paper presents the first systematic and quantitative characterization of the steady-state NTPase activity of DENV NS3 and their interaction with ssRNA. Substrate curves for ATP, GTP, CTP and UTP were obtained, and the specificity order for these nucleotides - evaluated as the ratio (kcat/KM)- was GTPATPCTP UTP, which showed that NS3 have poor ability to discriminate between different NTPs. Competition experiments between the four substrates indicated that all of them are hydrolyzed in one and the same catalytic site of the enzyme. The effect of ssRNA on the ATPase activity of NS3 was studied using poly(A) and poly(C). Both RNA molecules produced a 10 fold increase in the turnover rate constant (kcat) and a 100 fold decrease in the apparent affinity (KM) for ATP. When the ratio [RNA bases]/[NS3] was between 0 and 20 the ATPase activity was inhibited by increasing both poly(A) and poly(C). Using the theory of binding of large ligands (NS3) to a one-dimensional homogeneous lattice of infinite length (RNA) we tested the hypothesis that inhibition is the result of crowding of NS3 molecules along the RNA lattices. Finally, we discuss why this hypothesis is consistent with the idea that the ATPase catalytic cycle is tightly coupled to the movement of NS3 helicase along the RNA.
Highlights
The dengue virus (DENV) is a member of the family Flaviviridae, which includes other major public health concerns such as yellow fever virus, hepatitis C virus and West Nile virus [1]
In this paper we report quantitative studies on the steady-state kinetics of nucleoside triphosphates (NTPs) hydrolysis catalyzed by DENV nonstructural protein 3 (NS3)
We began the characterization of the kinetics properties of the NS3 helicase studying the hydrolysis of nucleotides ATP, CTP, GTP and UTP catalyzed by NS3 under steady-state conditions
Summary
The dengue virus (DENV) is a member of the family Flaviviridae, which includes other major public health concerns such as yellow fever virus, hepatitis C virus and West Nile virus [1]. Dengue virus exists in four distinct serotypes; all of them are mosquito-borne and cause dengue fever and dengue hemorrhagic fever/dengue shock syndrome. Found in tropical and subtropical regions of the world, DENV causes an estimated of 50 –100 million infections annually and places over 3 billion people at risk for infection [2,3]. The genome of Flaviviruses is a positive-sense single stranded RNA molecule of about 11,000 nucleotides bases constituted by a single open reading frame flanked by conserved and highly structured 59 and 39 untranslated regions [4,5]. At least 10 viral proteins are produced: three structural proteins (capsid, premembrane, and envelope proteins) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 proteins) [4,6,7]
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