Structure And Kinetics Of Zika Virus NS3 Helicase

Zika virus(ZIKV)is a single-stranded positive RNA virus belonging to a group of mosquito-borne flaviviruses.Although most cases of ZIKV infection produce no symptoms,previous studies showed that ZIKV infection is associated with various severe neural disorders,including a brain defect in newborn called microcephaly and Guillain-Barré syndrome(GBS).Its non-structural protein 3(NS3)contains a protease domain and a helicase domain,both of which play essential roles during the viral life cycle.N-terminal serine protease domain requires the membrane-bound NS2B protein cofactor for its activity,and C-terminal helicase domain belongs to the superfamily 2 group of helicases and uses energy from NTP hydrolysis to unwind nucleic acid duplexes.The NS3 is an essential component of the viral replication,and therefore a potentially important drug target.Divalent metal cations play essential roles in the initiation of NTP hydrolysis,and the generated energy is used to drive RNA/DNA translocation in flavivirus family NS3 helicases.Because of the lack of detailed structural information regarding the pre-activation state of the metal-free NS3 helicase in complex with NTP substrate,little is known about the molecular mechanisms underlying the metalinduced conformational changes of both NTP ligand and key residues at the Walker motifs that are involved in NTP binding.Here,we determined ZIKV NS3hapo,NS3h-GTPγS,NS3h-MgGTPyS,NS3h-MnATP2-(V04)and NS3h-MnADP-crystal structures to reveal the mechanism of divalent-metal-induced activation of NS3 helicase.We obtained an NTP binding pre-activation state structure,in which only a GTPyS was bound in the NTP-binding pocket and GTPγS adopts a compact conformation.Both the NTP-binding site and the NTP ligand undergo substantial conformational changes compared with the activation transition state observed in the complex structures containing MgGTPyS,MnATP2-and MnADP-.In the presence of a divalent cation,GTPyS adopts an extended conformation,and the Walker A motif undergoes substantial conformational changes.Both features contribute to more extensive interactions between the GTPyS and the enzyme and y phosphate is cleaved more easily by a nucleophilic attack initiated by an active water.From these results,we identified the allosteric regulatory roles of divalent metals in promoting NTP binding and local structural rearrangement,which may be applicable to other flaviviral NS3 helicases.ZIKV NS3 binds to the 3’ overhang of double-stranded nucleic acids and unwinds the duplex in a 3’-to-5’direction.Compared to other viral helicases,ZIKV NS3 is a weak enzyme,unable to separate RNA duplexes longer than～20 base pairs(bp).This raises the question of how its helicase activity can be enhanced to a level necessary for efficient viral genome replication.How this activity is stimulated to process the viral genome and whether the two domains of NS3 are functionally coupled remain unclear.Moreover,even though both enzymatic activities of NS3(namely protease and helicase)are essential for viral assembly and replication,it remains unclear whether these two domains with disparate functions influence each other’s activities for ZIKV and for flaviviruses in general.In this work,we used an optical tweezers assay to visualize RNA unwinding by ZIKV NS3 in real time and characterize the RNA unwinding properties of ZIKV NS3—including its processivity,velocity,and step size at the single-molecule level.We used optical tweezers to study the RNA unwinding activity of NS3 under different forces applied to the end of RNA substrate.We show that ZIKV NS3 has ATP-dependent RNA helicase activity and applying piconewton forces to the RNA substrate increases the unwinding velocity,and processivity of NS3,leading to a～3-fold enhancement of catalytic activity.External forces that weaken the substrate stability significantly enhance the NS3 helicase activity.The reason of NS3 compasses two domains with seemingly disconnected activities remains elusive for flavivirus family,both enzymatic domains may be functionally coupled and that the helicase activity may be enhanced by the presence of the protease domain.Our single molecule results show that deletion of protease subdomain has no significant effect on unwinding activity.Furthermore,we show that the cyclic movement of NS3 is coordinated by ATP in discrete steps of estimating 3 base pairs.Our results indicate that the RNA unwinding activity of NS3 can be regulated by force which may be provided by NS5 in the context of viral replication and the replication complex can efficiently unwind the ZIKV genome.Overall,fromstructural results,we identified the allosteric regulatory roles of divalent motals in promoting NTP binding and local structural rearrangement,which may be applicable to other flaviviral NS3 helicases.The compact conformation of inhibitory NTP identified in this study provides precise information for the rational drug design of small molecule inhibitors for the treatment of ZIKV infection.We also systematically characterized the enzymatic activity of the ZIKV NS3at single molecule level and the results suggests that that mechanical regulation may be a means to activate the unwinding activity of ZIKV NS3 in vivo.