Novel insights into the function of an N-terminal region of DENV2 NS4B for the optimal helicase activity of NS3

• Published in: Virus research 2021-04, Vol.295, p.198318-198318, Article 198318
• Main Authors: Lu, Hongyun, Zhan, Yumeng, Li, Xiaorong, Bai, Xuehui, Yuan, Feifei, Ma, Lulu, Wang, Xue, Xie, Mengjia, Wu, Wei, Chen, Zhongzhou
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; DEAH-box helicase; Dengue virus; Dengue Virus - enzymology; Dengue Virus - genetics; DNA and RNA unwinding; Interaction; Membrane Proteins - metabolism; NS3 helicase; NS4B; Nucleic Acids - metabolism; Protein Binding; RNA Helicases - genetics; RNA Helicases - metabolism; Serine Endopeptidases - metabolism; Unwinding activity; Viral Nonstructural Proteins - metabolism
• ISSN: 0168-1702; 1872-7492
• DOI: 10.1016/j.virusres.2021.198318

•DENV2 NS3 helicase activity was stimulated by the N-terminal residues 51 to 83 of NS4B.•Motif I and motif III of NS3 helicase were required for NS3-NS4B interaction.•NS4B coordinated the ATP binding and single-strand DNA dissociation in the unwinding process. Dengue virus NS3 is a prototypical DEx(H/D) helicase that binds and hydrolyzes NTP to translocate along and unwind double-stranded nucleic acids. NS3 and NS4B are essential components of the flavivirus replication complex. Evidences showed that NS4B interacted with NS3 and modulated the helicase activity of NS3. Despite important insights into structural, mechanistic, and cellular aspects of the NS3 function, there is still a gap in understanding how it coordinates the helicase activities within the replicase complex for efficient replication. Here, using the DENV2 as a model, we redefined the critical region of NS4B required for NS3 function by pull-down and MST assays. The FRET-based unwinding assay showed that NS3 would accelerate unwinding duplex nucleic acids in the presence of NS4B (51-83). The simulated NS3-NS4B complex models based on the rigid-body docking delineated the potential interaction sites located in the conserved motif within the core domain of NS3. Mutations in motif I (I190A) and motif III (P319L) of NS3 interfered with the unwinding activity stimulated by NS4B. Upon binding to the NS3 helicase, NS4B assisted NS3 to dissociate from single-stranded nucleic acid and enabled NS3 helicase to keep high activity at high ATP concentrations. These results suggest that NS4B probably serves as an essential cofactor for NS3 to coordinate the ATP cycles and nucleic acid binding during viral genome replication.