The RNA helicase, nucleotide 5′-triphosphatase, and RNA 5′-triphosphatase activities of Dengue virus protein NS3 are Mg 2+-dependent and require a functional Walker B motif in the helicase catalytic core

Abstract

The nonstructural protein 3 (NS3) of Dengue virus (DV) is a multifunctional enzyme carrying activities involved in viral RNA replication and capping: helicase, nucleoside 5′-triphosphatase (NTPase), and RNA 5′-triphosphatase (RTPase). Here, a 54-kDa C-terminal domain of NS3 (ΔNS3) bearing all three activities was expressed as a recombinant protein. Structure-based sequence analysis in comparison with Hepatitis C virus (HCV) helicase indicates the presence of a HCV-helicase-like catalytic core domain in the N-terminal part of ΔNS3, whereas the C-terminal part seems to be different. In this report, we show that the RTPase activity of ΔNS3 is Mg 2+-dependent as are both helicase and NTPase activities. Mutational analysis shows that the RTPase activity requires an intact NTPase/helicase Walker B motif in the helicase core, consistent with the fact that such motifs are involved in the coordination of Mg 2+. The R513A substitution in the C-terminal domain of ΔNS3 abrogates helicase activity and strongly diminishes RTPase activity, indicating that both activities are functionally coupled. DV RTPase seems to belong to a new class of Mg 2+-dependent RTPases, which use the active center of the helicase/NTPase catalytic core in conjunction with elements in the C-terminal domain.