Abstract
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.
Highlights
The unwinding of dsRNA intermediates is critical for the replication of flavivirus RNA genomes
We found that the T407A and S411A substitutions in nonstructural protein 3 (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
As discussed previously by Davidson et al [24], Motif V may play a role in the communication between the ATP-binding pocket and the RNA-binding cleft/helical gate
Summary
As discussed previously by Davidson et al [24], Motif V may play a role in the communication between the ATP-binding pocket and the RNA-binding cleft/helical gate. The T407A mutant nearly ablates genome replication, whereas the S411A mutation only reduces genome replication by ϳ60% Both mutations should disrupt the hydrogen bond, but T407A and S411A have significantly different replication levels, suggesting that these two mutations have other interactions that potentially affect one or more of the NS3h functions during viral replication. These NS3h functions include the ability of the helicase to bind RNA, the ability to hydrolyze ATP, and the ability to unwind the dsRNA intermediate. B, results from the viral genome replication were projected onto the structure of Motif V with the hydrogen bond highlighted between residues Thr407 and Ser411. C, kcat, Km, and kcat/Km were determined for the helicase-unwinding activity assay for each NS3h variant through fitting the Michaelis–Menten equation to the Kobs versus ATP concentration
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