Hepatitis C virus NS3NTPase/helicase: Different stereoselectivity in nucleoside triphosphate utilisation suggests that NTPase and helicase activitiesare coupled by a nucleotide-dependent rate limiting step

Hepatitis C virus (HCV) NS3 protein is a multifunctional enzyme, possessing protease, NTPase and helicase activities within a single polypeptide of 62 5 an-Lino acid residues. These activities are essential for the virus life cycle and are considered attractive targets for anti-HCV chemotherapy. Besi de ATP, the NS3 protein has the ability to utilise deoxynucleoside triphosp hates (dNTPs) as the energy source for nucleic acid unwinding. We have perf ormed an extensive analysis of the substrate specificities of both NS3 NTPa se and helicase activities with respect to all four dNTPs as well as with d ideoxynucleoside triphoshate (ddNTP) analogs, including both D-(beta) and L -(beta)-deoxy and dideoxy-nucleoside triphosphates. Our results show that a lmost all dNTPs and ddNTPs tested were able to inhibit hydrolysis of ATP by the NTPase activity, albeit with different efficiencies. Moreover, this ac tivity showed almost no stereoselectivity, being able to recognise both D-( beta), L-(beta)-deoxy and ddNTPs. On the contrary, the helicase activity ha d more strict substrate selectivity, since, among D-(beta)-nucleotides, onl y ddTTP and its analog 2',3'-didehydro-thymidine triphosphate could be used as substrates with an efficiency comparable to ATP, whereas among L-(beta) -nucleotides, only L-(beta)-dATP was utilised. Comparison of the steady-sta te kinetic parameters for both reactions, suggested that dATP, L-(beta)-dCT P and L-(beta)-dTTP, specifically reduced a rate limiting step present in t he helicase, but not in the NTPase, reaction pathway. These results suggest that NS3-associated NTPase and helicase activities have different sensitiv ities towards different classes of deoxy and dideoxy-nucleoside analogs, de pending on a specific step in the reaction, as well as show different enant ioselectivity for the D-(beta) and L-(beta)-conformations of the sugar ring . These observations provide an essential mechanistic background for the de velopment of specific nucleotide analogs targeting either activity as poten tial anti-HCV agents. (C) 2001 Academic Press.