Dengue virus type 2 (DEN2), a member of the Flaviviridae family, is a 
re-emerging human pathogen of global significance. DEN2 nonstructural protein 3 
(NS3) has a serine protease domain (NS3-pro) and requires the hydrophilic domain 
of NS2B (NS2BH) for activation. NS3 is also an RNA-stimulated nucleoside 
triphosphatase (NTPase)/RNA helicase and a 5'-RNA triphosphatase (RTPase). In 
this study the first biochemical and kinetic properties of full-length NS3 
(NS3FL)-associated NTPase, RTPase, and RNA helicase are presented. The NS3FL 
showed an enhanced RNA helicase activity compared with the NS3-pro-minus NS3, 
which was further enhanced by the presence of the NS2BH (NS2BH-NS3FL). An active 
protease catalytic triad is not required for the stimulatory effect, suggesting 
that the overall folding of the N-terminal protease domain contributes to this 
enhancement. In DEN2-infected mammalian cells, NS3 and NS5, the viral 5'-RNA 
methyltransferase/polymerase, exist as a complex. Therefore, the effect of NS5 
on the NS3 NTPase activity was examined. The results show that NS5 stimulated 
the NS3 NTPase and RTPase activities. The NS5 stimulation of NS3 NTPase was 
dose-dependent until an equimolar ratio was reached. Moreover, the conserved 
motif, 184RKRK, of NS3 played a crucial role in binding to RNA substrate and 
modulating the NTPase/RNA helicase and RTPase activities of NS3.