Motif-VI loop acts as a nucleotide valve in the West Nile Virus NS3 Helicase.

The Orthoflavivirus NS3 helicase (NS3h) is crucial in virus replication, representing a potential drug target for pathogenesis. NS3h utilizes nucleotide triphosphate (ATP) for hydrolysis energy to translocate on single-stranded nucleic acids, which is an important step in the unwinding of double-stranded nucleic acids. Intermediate states along the ATP hydrolysis cycle and conformational changes between these states, represent important yet difficult-to-identify targets for potential inhibitors. Extensive molecular dynamics simulations of West Nile virus NS3h+ssRNA in the apo, ATP, ADP+Pi and ADP bound states were used to model the conformational ensembles along this cycle. Energetic and structural clustering analyses depict a clear trend of differential enthalpic affinity of NS3h with ADP, demonstrating a probable mechanism of hydrolysis turnover regulated by the motif-VI loop (MVIL). Based on these results, MVIL mutants (D471L, D471N and D471E) were found to have a substantial reduction in ATPase activity and RNA replication compared to the wild-type. Simulations of the mutants in the apo state indicate a shift in MVIL populations favoring either a closed or open 'valve' conformation, affecting ATP entry or stabilization, respectively. Combining our molecular modeling with experimental evidence highlights a conformation-dependent role for MVIL as a 'valve' for the ATP-pocket, presenting a promising target for antiviral development.

Motif-VI Loop Acts as a Nucleotide Valve in the West Nile Virus NS3 Helicase.

The flavivirus NS3 helicase (NS3h), a highly conserved protein, plays a pivotal role in virus replication and thus represents a potential drug target for flavivirus pathogenesis. NS3h utilizes nucleotide triphosphate, such as ATP, for hydrolysis energy (ATPase) to translocate on single-stranded nucleic acids, which is an important step in the unwinding of double-stranded nucleic acids. The intermediate states along the ATP binding and hydrolysis cycle, as well as the conformational changes between these states, represent important yet difficult-to-identify targets for potential inhibitors. We use extensive molecular dynamics simulations of apo, ATP, ADP+Pi, and ADP bound to WNV NS3h+ssRNA to model the conformational ensembles along this cycle. Energetic and structural clustering analyses on these trajectories depict a clear trend of differential enthalpic affinity of NS3h with ADP, demonstrating a probable mechanism of hydrolysis turnover regulated by the motif-VI loop (MVIL). These findings were experimentally corroborated using viral replicons encoding three mutations at the D471 position. Replication assays using these mutants demonstrated a substantial reduction in viral replication compared to the wild-type. Molecular simulations of the D471 mutants in the apo state indicate a shift in MVIL populations favoring either a closed or open 'valve' conformation, affecting ATP entry or stabilization, respectively. Combining our molecular modeling with experimental evidence highlights a conformation-dependent role for MVIL as a 'valve' for the ATP-pocket, presenting a promising target for antiviral development.