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

• Published in: The Journal of biological chemistry Vol. 295; no. 6; pp. 1551 - 1564
• Main Authors: Du Pont, Kelly E., Davidson, Russell B., McCullagh, Martin, Geiss, Brian J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 07-02-2020; American Society for Biochemistry and Molecular Biology
• Subjects: Adenosine Triphosphatases - chemistry; Adenosine Triphosphatases - metabolism; Adenosine Triphosphate - metabolism; all-atom molecular dynamics; allosteric regulation; Amino Acid Sequence; Animals; ATPase activity; Cell Line; computational biology; Cricetinae; Dengue - virology; dengue NS3 helicase; Dengue Virus - chemistry; Dengue Virus - metabolism; Dengue Virus - physiology; energy transduction; flavivirus; helicase activity; Hydrolysis; Microbiology; Models, Molecular; molecular dynamics; Motif V; pathway analysis; Protein Interaction Domains and Motifs; RNA helicase; RNA Helicases - chemistry; RNA Helicases - metabolism; RNA, Viral - metabolism; Serine Endopeptidases - chemistry; Serine Endopeptidases - metabolism; SF2 helicase; Viral Nonstructural Proteins - chemistry; Viral Nonstructural Proteins - metabolism; Virus Replication; West Nile NS3 helicase; computational biology; energy transduction; helicase activity; West Nile NS3 helicase; allosteric regulation; dengue NS3 helicase; molecular dynamics; pathway analysis; SF2 helicase; ATPase activity; all-atom molecular dynamics; Motif V; flavivirus; RNA helicase
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA119.011922

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.