Study On The Unwinding Function Of Zika Virus NS3 Protein And The Mechanism Involved In Virus Replication

Zika virus(ZIKV)is positive single-strand RNA virus,belonging to flavivirus family.ZIKV didn't draw public attention until it spread all over the world in 2015.ZIKV genome is translated into three structural proteins and seven nonstructural proteins,including NS1,NS2A,NS2B,NS3,NS4A,NS4 and NS5.In the process of ZIKV RNA replication,the positive single-RNA is used as the template by NS5 RNA polymerase to synthesize dsRNA.Then the dsRNA is unwound by NS3 to reproduce the single-strand template,which guarantee the continuous RNA amplification.As a result,NS3 helicase activity is of vital importance for ZIKV RNA replication.However,the research about ZIKV NS3 is rare compared with other flaviviruses,and these studies only involved the structure of NS3 helicase domain.In our research,we aim to investigate ZIKV NS3 enzyme activity in respect of biological mechanism.We constructed ZIKV NS3 and NS5 expression vectors and transformed them into E.coli.Then the expression vectors were induced to produce ZIKV proteins which were purified in vitro.ATPase detection kit was used to measure the ATPase activity of ZIKV NS3.We found ZIKV NS3 protein hydrolyze ATP independent of nucleotide.Besides,ZIKV full-length NS3 and its helicase domain have the same ability in ATP hydrolysis.The unwinding system was established in vitro according to FRET(fluorescence resonance energy transfer)principle.Both ZIKV full-length NS3 and helicase domain can unwind dsRNA in vitro with the much weaker helicase activity of helicase domain.ZIKV NS3 tend to unwind dsRNA with 3'overhangs,indicating the 3'to 5'unwinding direction.After the alignment of flavivirus NS3 amino acid,some conserved sites were validated to play key roles in NS3 enzyme activity.G199,K200 and R456/R457 were the vital amino acids to maintain the NS3 ATPase activity.Mutation to these sites impaired ATPase activity of ZIKV NS3,which lead to the weaker helicase activity.Whereas D410 and K431 effect ZIKV NS3 helicase activity by controlling the binding between NS3 and RNA.Mutation of the amino acids mentioned above impaired ZIKV RNA replication and ZIKV production.The result demonstrates that ZIKV NS3 helicase activity is extremely important for ZIKV replication.The tight cooperation between ZIKV NS3 and NS5 indicated they may influence each other's activity.Neither full-length NS3 nor helicase domain's ATPase activity was affected by ZIKV NS5.However,NS5 promote the helicase activity of NS3 to unwind dsDNA and dsRNA with 3' overhangs.The promotion of NS3 helicase activity by NS5 is only applicable to 16bp dsRNA but not 20bp and 23bp dsRNA,which means NS5 improve NS3 unwinding velocity not continuity.The NS5 stimulation is different for ZIKV NS3 and helicase domain.Besides,different NS5 domain show different impact on NS3.The interaction between ZIKV NS3 and NS5 was validated by the co-immunoprecipitation assay.We found N569 and E573 in NS3 protein play a key role for the interaction.ZIKV NS3 with N569A/E573A mutation barely interact with NS5.This NS3 mutant appears normal ATPase and helicase activity compared with wild-type NS3.But NS5 can't improve the helicase activity of N569A/E573A NS3,which indicated the stimulation of NS3 helicase activity by NS5 depends on NS3-NS5 interaction.ZIKV replicon and infectious RNA with N569A/E573A mutation suppress viral RNA replication and ZIKV production.The result demonstrates that the NS3-NS5 interaction is critical for ZIKV survival and replication.To sum up,this work systematically clarified the helicase activity of ZIKV NS3,providing new insights for studying and suppressing ZIKV replication.