Residue-Level Functional Couplings and their Effect on RNA Remodelling by the Zika Virus NS3 Helicase

Abstract

Nucleic acid unwinding carried out by helicases is achieved by efficient coupling of the ATPase hydrolysis to binding, translocation and local melting of the duplexed strands. Understanding this functional coupling between the active sites of the helicase can yield insight into mechanisms that determine the efficiency of RNA remodeling by the helicases from Flaviviridae NS3 family. We employed weighted energy contact networks (wECN) to compare the topology of the NS3 helicases (NS3h) from Zika virus with Hepatitis C virus and Dengue virus. This complemented with fluorescence based binding and unwinding assays allowed us to characterize the dynamics of RNA remodeling events by the ZIKV NS3h and distinguish it from the NS3 helicase family. ZIKV NS3h uniquely unwinds partial duplex RNA 3’ overhangs albeit with low processivity, and its ATPase activity is not stimulated by RNA binding. Using the wECN network model for ZIKA NS3h, we identified residues distal to the binding sites that strongly exert an influence on the RNA unwinding activity and processivity. Analysis of designed mutants with enhanced RNA unwinding activity suggests a contrasting mechanism that dictates helicase efficiency in ZIKV virus NS3h as compared to HCV and DENV, where enhancement is achieved by improving ATPase efficiency. Our assays also allowed us to characterize RNA annealing, a second mode of RNA remodeling mediated by the ZIKV NS3 helicase. The effect of mutations on RNA annealing further provided insights into factors that allow the NS3 protein to sequester RNA at low concentrations. Our mechanistic insights into RNA remodeling events by NS3h will allow rational design of tuned RNA helicases and identify inhibitors for replication by the Zika virus.