4.6 Article

Motif V regulates energy transduction between the flavivirus NS3 ATPase and RNA-binding cleft

Journal

JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 295, Issue 6, Pages 1551-1564

Publisher

ELSEVIER
DOI: 10.1074/jbc.RA119.011922

Keywords

flavivirus; RNA helicase; molecular dynamics; computational biology; allosteric regulation; ATPase activity; dengue NS3 helicase; pathway analysis; SF2 helicase; West Nile NS3 helicase; helicase activity; all-atom molecular dynamics; energy transduction; Motif V

Funding

  1. NVIDIA Corporation

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The unwinding of dsRNA intermediates is critical for the replication of flavivirus RNA genomes. This activity is provided by the C-terminal helicase domain of viral nonstructural protein 3 (NS3). As a member of the superfamily 2 (SF2) helicases, NS3 requires the binding and hydrolysis of ATP/NTP to translocate along and unwind double-stranded nucleic acids. However, the mechanism of energy transduction between the ATP- and RNA-binding pockets is not well-understood. Previous molecular dynamics simulations conducted by our group have identified Motif V as a potential ?communication hub? for this energy transduction pathway. To investigate the role of Motif V in this process, here we combined molecular dynamics, biochemistry, and virology approaches. We tested Motif V mutations in both the replicon and recombinant protein systems to investigate viral genome replication, RNA-binding affinity, ATP hydrolysis activity, and helicase-mediated unwinding activity. We found that the T407A and S411A substitutions in NS3 reduce viral replication and increase the helicase-unwinding turnover rates by 1.7- and 3.5-fold, respectively, suggesting that flaviviruses may use suboptimal NS3 helicase activity for optimal genome replication. Additionally, we used simulations of each mutant to probe structural changes within NS3 caused by each mutation. These simulations indicate that Motif V controls communication between the ATP-binding pocket and the helical gate. These results help define the linkage between ATP hydrolysis and helicase activities within NS3 and provide insight into the biophysical mechanisms for ATPase-driven NS3 helicase function.

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