Sequence motifs within the nonstructural protein NS3 of members of the 
Flaviviridae family suggest that this protein possesses nucleoside 
triphosphatase (NTPase) and RNA helicase activity. The RNA-stimulated NTPase 
activity of this protein from prototypic members of the Pestivirus and 
Flavivirus genera has recently been established and enzymologically 
characterized. Here, we experimentally demonstrate that the NS3 protein from a 
member of the third genus of Flaviviridae, human hepatitis C virus (HCV), also 
possesses a polynucleotide-stimulated NTPase activity. Characterization of the 
purified HCV NTPase activity showed that it exhibited reaction condition optima 
with respect to pH, MgCl2, and salt identical to those of the representative 
pestivirus and flavivirus enzymes. However, each NTPase also possessed several 
unique properties when compared with one another. Notably, the profile of 
polynucleotide stimulation of the NTPase activity was distinct for the three 
enzymes. The HCV NTPase was the only one whose activity was significantly 
enhanced by a deoxyribopolynucleotide. Additional distinguishing features among 
the three enzymes relating to the kinetic properties of their NTPase activities 
are discussed. These studies provide a foundation for investigation of the 
putative RNA helicase activity of these proteins and for further study of the 
role of the NS3 proteins of members of the Flaviviridae in the replication cycle 
of these viruses.