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

Du Pont, Kelly E; Davidson, Russell B; McCullagh, Martin; Geiss, Brian J

Du Pont, Kelly E; Davidson, Russell B; McCullagh, Martin; Geiss, Brian J.

Afiliación

Du Pont KE; Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523; Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado 80523.
Davidson RB; Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523.
McCullagh M; Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523. Electronic address: Martin.McCullagh@colostate.edu.
Geiss BJ; Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado 80523; School of Biomedical Engineering, Colorado State University, Fort Collins, Colorado 80523. Electronic address: Brian.Geiss@colostate.edu.

J Biol Chem ; 295(6): 1551-1564, 2020 02 07.

Article en En | MEDLINE | ID: mdl-31914411

RESUMEN

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.

Asunto(s)

Adenosina Trifosfatasas/metabolismo; Virus del Dengue/metabolismo; ARN Helicasas/metabolismo; Proteínas no Estructurales Virales/metabolismo; Adenosina Trifosfatasas/química; Adenosina Trifosfato/metabolismo; Secuencia de Aminoácidos; Animales; Línea Celular; Cricetinae; Dengue/virología; Virus del Dengue/química; Virus del Dengue/fisiología; Hidrólisis; Modelos Moleculares; Dominios y Motivos de Interacción de Proteínas; ARN Helicasas/química; ARN Viral/metabolismo; Serina Endopeptidasas/química; Serina Endopeptidasas/metabolismo; Proteínas no Estructurales Virales/química; Replicación Viral

Palabras clave

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

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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Proteínas no Estructurales Virales / Adenosina Trifosfatasas / ARN Helicasas / Virus del Dengue Límite: Animals Idioma: En Revista: J Biol Chem Año: 2020 Tipo del documento: Article Pais de publicación: Estados Unidos

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