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 (NS3 FL )-associated NTPase, RTPase, and RNA helicase are presented. The NS3 FL showed an enhanced RNA helicase activity compared with the NS3-pro-minus NS3, which was further enhanced by the presence of the NS2BH (NS2BH-NS3 FL ). 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, 184 RKRK, of NS3 played a crucial role in binding to RNA substrate and modulating the NTPase/RNA helicase and RTPase activities of NS3.The mosquito-borne Flavivirus genus, in the Flaviviridae family, includes human pathogens of global distribution and prevalence (for reviews, see Refs. 1-3), and Dengue viruses (DEN) 1 types 1-4 cause the most common infection encountered in humans (4). The diseases caused by DEN infections include from dengue fever, usually a self-limiting disease, to more severe forms, dengue hemorrhagic fever and dengue shock syndrome. These diseases pose a significant threat to humans living in DEN-infected Aedes aegypti mosquitoes endemic in areas that inhabit two-thirds of world population (5) DEN genome is a single-stranded RNA (10,723 nt in length for DEN2 New Guinea C strain used in this study (6)) of positive polarity. The viral RNA contains a long open reading frame coding for a polyprotein precursor. The polyprotein is processed into mature structural proteins, capsid (C), precursor membrane (prM), and envelope (E) and at least seven nonstructural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5, by cellular signal peptidase and viral serine protease in the endoplasmic reticulum (for review, see Ref. 7). The 5Ј-end of the viral RNA is modified by a type I cap structure (m 7 GpppN; 2Ј-OH moiety of N is methylated). DEN2 NS3 is a multifunctional protein of about 69 kDa. It includes a serine catalytic triad within the N-terminal 185 amino acid residues. The protease domain is activated by the hydrophobic protein NS2B which serves as a cofactor for the protease and forms a complex in the infected cells (8 -12). NS2B has three hydrophobic regions flanking a conserved hydrophilic domain. The hydrophilic domain of NS2B (NS2BH) alone is sufficient for protease act...