Insight Into The Non-structural Protein NS5 And Non-coding Region On Tembusu Virus Replication And Production

The 5′and 3′untranslated regions（5′UTR and 3′UTR）and nonstructural protein 5（NS5）of duck Tembusu virus（TMUV）play a critical role in viral life cycle.NS5 is the largest and most conserved viral protein of duck TMUV,which has RNA dependent RNA polymerase（Rd Rp）and methyltransferase（MTase）activities and makes it a potential target of antiviral drugs.The 5′UTR and 3′UTR are characteristic of the conserved RNA structure,which are very important for the formation of virus replication complex（VRC）and regulation of viral genome replication.Herein we aimed to investigate the role of NS5,5′UTR and 3′UTR on viral replication based on duck TMUV reverse genetics system using mutagenesis method.There are three main aspects in the dissertation:1.Determinants of duck TMUV NS5 biological characteristics and regulation on viral replication;2.Secondary structure of 5′UTR and 3′UTR and the impact of its cyclization on viral fitness;3.Trans-complement effect of duck TMUV NS5 on defective virus genome and its co-regulation with UTR.Briefly described as follows:1.Determinants of duck TMUV NS5 biological characteristics and regulation on viral replicationDuck TMUV NS5 protein is mainly located in the endoplasmic reticulum.Based on the dual-luciferase reporter assay system,the overexpression of duck TMUV NS5 activates duck interferon（IFNβ）promoter activity through RIG-I rather than MDA5 pathway.Furthermore,the mutant NS5 Rd Rp active center（GDD）and primer loop（PL）decreased the activation of IFNβpromoter.The immune activation of NS5 depends on the Rd Rp enzyme.Point mutation in PL region resulted in the loss or decrease of viral RNA replication ability,and it was found that there was a positive correlation between replication ability and Rd Rp enzyme activity of NS5.Specially,the S791D mutant resulted in the significant decrease of replication ability,and the higher viral proliferation level and viral load in tissues than those of wild-type recombinant virus（r TMUV-WT）.The interaction characteristics of MTase-Rd Rp in NS5 were studied by Nano Bi T-PPI.The GTR linker region and the conservative interaction interface were found to dominate the MTase-Rd Rp interaction.Only the chimeric Sub-JEV_GTR maintained replication capability among the mutants of NS5 GTR linker deletion(ΔGTR_268-275)and substitution with Japanese encephalitis virus(JEV;Sub-JEV_GTR),West Nile virus(WNV;Sub-WNV_GTR)and Zika virus(ZIKV;Sub-ZIKV_GTR),respectively.Generally,the site-mutagenesis targeting the interface affected the virus replication,proliferation and cell infection ability.Especially for the mutant F467A,the viral replication,proliferation and the mortality of duck embryo were sharply declined without decreasing the MTase-Rd Rp interaction.In conclusion,TMUV NS5regulates TMUV replication through the conserved interaction interface of MTase-Rd Rp and flexible GTR linker.Introduction the arginine into the N390 and Q392 residues of duck TMUV NS5（N390R,Q392R）,facilitated the NS5 to enter the nucleus and decreased the viral replication and proliferation.Subsequently,it was demonstrated that NS5 decreased the interaction with NS3 owing to mutant NS5 transferring into nucleus under overexpressing NS5and virus infection conditions in immunoprecipitation assay（Co-IP）,Nano Luc binary technology for live cell protein-protein interaction system（Nanobi T-PPI）and indirect immunofluorescence assay（IFA）.Notably,the mutant recombinant virus(r TMUV-NS5_NLSmut)induced strong IFNβeven though the transient NS5_NLSmut was proved not to stimulate excessive IFNβthrough RIG-I pathway.Hence r TMUV-NS5_NLSmut induced strong IFNβresponse independent of NS5 synthesizing RNA and then stimulate RIG-I.2.Secondary structure of 5′UTR and 3′UTR and the impact of its cyclization on virus fitnessBased on the putative RNA secondary structure of TMUV 5′UTR and 3′UTR,a series of mutagenesis targeting the specific nucleotides and core secondary structure of 5′UTR and 3′UTR were conducted to study the replication properties using the reverse genetics system.The5′UTR mutants Mut TL_CT/TC,Mut TL_AG/GA,Mut U7^thC,Mut SSD andΔpoly Us decreased the replication ability.Deletion or restoration of 3′TL structure（ΔTL-3′UTR and rt TL-3′UTR）,the substitution of homologous S2-S3 stem in 3′SL(Lg TL_JEV),and the deletion ofΔ3′UTR_299-436nt exhibited detrimental to viral RNA replication.While the Mut A79U、ΔDB1-3nt、ΔDB2-2nt,Mut AG45^thGA and sm TL_JEV（substitute the S3 of 3′SL）decreased RNA replication levels.Additionallly,the impact of RNA cyclization interaction on virus replication was explored by base mismatch and base pairing.We observed that the 5′UTR-3′UTR interaction was not destroyed by any cyclization motif via base mismatch.Except Mut 5′UAR-2nt and Mut 5′DARII-5nt mutants,the remaining mutants abort replication due to base mismatch in the cyclization motif.By restoring base pairing,cy DARI-4nt and cy CS-5nt mutants partially recovered their replication ability to some ectent.Notably,the proliferation of recombinant viruses with base mismatch was decreased,while other mutants with base-pairing proliferate better.In conclusion,TMUV 5′UTR and 3′UTR can regulate the virus replication by both the specific nucleotide and secondary structure.3.Trans-complement effect of duck TMUV NS5 on defective virus genome and its co-regulation with UTRInitially the flavivirus trans-complement system was constructed in U2OS and BHK-21cells.It was found that duck TMUV wild-type replicase（Replicase-WT）and Rd Rp enzyme deficient replicase（Replicase-AAA）could stimulate the activity of template.Furthermore,the replicase can cross-utilize the flavivirus template,indicating that NS5 could trans-participated in the translation or replication process under the control of 5′UTR-3′UTR.Intriguingly,the template was activated independent of the replicase Rd Rp activity.Subsequently,the replication and prolierfation of chimeric 5′UTR,3′UTR and NS5 were further studied.NS5 specifically recognized 5′UTR and coordinately regulated virus proliferation and replication.The interaction between 5′UTR-3′UTR and NS5 protein was verified by transcribed RNA in vitro.Furthermore,the binding site on NS5 was central to replication.Taken together,5′UTR and 3′UTR determine the viral characteristics through both specific nucleotide and secondary RNA structure.From the perspective of NS5 biological properties,we explored the IFNβresponse stimulated by NS5 Rd Rp enzyme activity,the MTase-Rd Rp interaction and the mechanism underlying the NS5 nuclear on decreasing the virus replication.Meanwhile,the core RNA secondary structures and determinants of replication were analyzed.Based on the trans-complement system of flavivirus and mutants related to the interaction between terminal RNA and NS5,it is clear that NS5 and non-coding region cooperate to regulate virus replication.Our results reveal the basis for TMUV replication.