Study Of Interactions Of 5'-untranslated Region Of WNV Chin-01 Strain With Viral Proteins Involved In Virus Replication

West Nile virus(WNV)belongs to genus Flavivirus within the the family Flaviviridae.WNV possesses a single-stranded,positive-sense RNA genome of approximately 11 kb,which contains a long open reading frame coding for a polyprotein precursor.The polyprotein is processed into three mature structural proteins(C,prM,and E)and seven nonstructural proteins(NS1,NS2A,NS2B,NS3, NS4A,NS4B,and NS5)by cellular and viral proteases.Flanking the genome were 5' and 3'untranslated regions(UTR)containing conserved sequences and secondary structures which appear to be essential for viral RNA replication.NS3 and NS5, which are thought to be the main structural components of RNA replication complex,are the key proteins responsible for the viral RNA replication.The function of 3'-UTR has been characterized in great detail,the common strategy used in the study of which is to observe the biological consequences of mutations introduced into the infectious full-length cDNA clone.While the enzymatic functions of NS3 and NS5 have been well characterized,the interactions of NS3 and NS5 with UTR of genome remain unclear.In addition,there still have been no reports on interactions of NS3 and NS5 with WNV 5'-UTR.Therefore,study of functions of interactions of viral and cellular proteins with conserved sequences and secondary structures in 5'-UTR of WNV genome will help to clarify molecular details of the viral replication.In this study,firstly an infectious full-length cDNA clone of WNV Chin-01 strain was constructed.Then full-length NS3,RNA-dependent RNA polymerase (RdRp)domain of NS5(hereafter named NS5pol)and complete NS5(hereafter named NS5F)with an N-terminal histidine tag were expressed in E.coli and purified, respectively,which were enzymatically active.Further a bioinformatic analysis of 5'-UTRs of several WNV strains including strain Chin-01 was performed and several nucleotides were selected as mutations introduced into the 5'-UTR of WNV.Then RdRp activity of NS5pol and NS5 were observed using different mutant 5'-UTRs as templates,which was followed by the observation of the effects of NS3 on the polymerase activity of NS5 using mutant 5'-UTRs and capability of binding of NS5 to mutant templates.Finally,the mutations in elements related to NS3 and NS5 in 5'-UTR involved in viral replication were introduced into the infectious full-length cDNA clone of Chin-01 strain.Then the biological characteristics of the recovered virus were observed.A model for replication complex formed in the stage of initiation of negative-strand synthesis was proposed.The results in this study may shed light on molecular mechanisms of genome replication of WNV and other flaviviruses.1.Construction and identification of two infectious full-length cDNA clones of WNV Chin-01 strainFirstly,a full-length cDNA clone of Chin-01 strain was constructed.Five cDNA fragments(F1～F5)were synthesized from genomic RNA through RT-PCR to cover the complete WNV genome.Then F1～F3 and F4～F5 were cloned together into plasmids to yield 5' and 3'-halves of genome of Chin-01 strain,respectively.In order to obtain RNA possessing authentic 3'-end,two XbaI sites in WNV gemome were abolished and a unique XbaI site was introduced into the 3'-end of the 3'-half of genome of Chin-01 strain by site-directed PCR mutagenesis.The 5' and 3'-halves of genome were assembled into plasmid pBluescriptⅡKS to form the full-length cDNA clone of WNV(pBS-F-N and pBS-F-X).The complete WNV cDNA is positioned under the control of SP6 promoter elements for in vitro transcription.The virus-specific sequence of each intermediate cloning product and junction sequences were validated by sequence analysis and restriction digestion.In order to observe the infectivity of RNA from the full-length cDNA clones, Capped RNA transcript synthesized from the linearized full-length cDNA plasmid by using SP6 RNA polymerase was electroporated into BHK-21 cells and recovered virus BS-F-N and BS-F-X were obtained.The fragments spanning the genetic markers were amplified through RT-PCR from RNA extracted from either parental or recovered viruses and validated by sequence analysis,the results of which clearly showed that virus recovered from the transfected cells was derived from the infectious full-length RNA;The results of IFA used to detect viral protein expression in BHK-21 cells transfected with WNV RNA transcript also indicated that specific viral proteins were expressed by progeny virus.No qualitative differences in CPE were observed between parental and recovered viruses,but the CPE from recovered virus showed after a delay of approximately 12～16 h.Furthermore,there was no difference morphology on Vero cells between the recombinant and parental virus,but the size of plaques of recovered virus,which appeared after a delay of approximately 15～20 h, was marginally smaller in diameter than that of parental virus.One-step growth curves were similar for both recombinant and parental viruses on BHK-21,the peak titers of which were 10~5 PFU/ml at 72 h p.i and 10~6 PFU/ml at 48 h p.i,respectively. These data suggest that the parental and recovered viruses are indistinguishable in replication and spread in mammalian cells.In addition,the levels of viral protein expression and RNA replication of recovered virus BS-F-N whose 3'-end contained several nucleiotides unrelated to virus were lower than that of BS-F-X.This disparity is likely due to the nucleiotides unrelated to virus postioned at 3'-end of BS-F-N.The results above suggested two infectious full-length cDNA clones of WNV Chin-01 strain were obtained successfully(pBS-F-N and pBS-F-X).The pBS-F-X whose infectivity was similar to parental virus was used to construct the mutant in further.2.Expression and purification of enzymatically activive of WNV Chin-01 strain NS3,NS5pol and NS5FIt is crucial for study of interactions between viral proteins and genome of WNV to obtain NS3 and NS5 possessing enzymatic activity.In this regard,firstly the PCR-fragments encoding NS3,NS5pol and NS5F were cloned into prokaryotic expression plasmid pET-28a resulting in the corresponding recombinant expression plasmids. Expressions for all proteins were carried out at 15℃after induction with 200μM IPTG.Recombinant proteins were identified after SDS-PAGE by Western blotting using anti-6His monoclonal antibody and polyclonal rabbit antiserum against the WNV,respectively.The purified recombinant proteins NS3,NS5pol and NS5F with purity above 90%were obtained through affinity chromatography on a Ni-column. The results of RNA helicase assay in vitro indicated that recombinant protein NS3 possessed the intact helicase activity;In vitro RdRp assay and EMSA undertaken further showed that the recombinant NS5pol and NS5 exhibited high RdRp activity, and that the RdRp activity was dependent on the sequence and secondary structure of RNA.The recombinant fusion NS3,NS5pol and NS5F of WNV obtained layed a foundation to identify elements in WNV genome essential for viral RNA synthesis. 3.Interactions between viral proteins related to viral replication and 5'-UTR of WNV Chin-01 strain.In order to screen the key nucleotides in 5'-UTR as targets,the secondary structures of 5'-UTRs of several strains of WNV firstly were predicted firsly,the results of which showed that 5'-UTR consists of subdomain A',B' and C'.Then mutations were introduced into several positions selected as targets in these subdomains by PCR mutagenesis,and ten mutants containing 5'-UTR,secondary structures of subdomains of which are different from each other,were constructed. Given to the effect of 3'-UTR on the interactions of viral proteins with viral genome, the corresponding ten mutants containing intact UTR were constructed further.Then RdRp activity and capability of binding to templates of NS5pol and NS5F were evaluated through RdRp assay in vitro and EMSA respectively using the mutant 5'-UTRs mentioned above.In these experiments,mutations at nucleotides 28,37,38 and 43 simultaneously and deletion of nucleotides 46～60 all resulted in deficiency of RdRp activity of NS5pol and NS5F;but mutations at nucleotides 8 and 9 resulted in deficiency of RdRp activity and capability of binding to templates of two proteins using mutants containing 5'-UTR as templates;However,Only mutations at nucleotides 8,9,69 and 70 simultaneously resulted in deficiency of RdRp activity and capability of binding to templates using mutants containing intact UTRs as templates. These results indicated that the RdRp activity of NS5 might be dependent on the nucleotides 28,37,38 and 43 in subdomain C' and nucleotides 45～60 in subdomain B' in 5'-UTR,while nucleotides 8,9,69 and 70 in subdomain A' might play a key role in RNA replication through interaction with 3'-UTR.In addition,mutatations in 5'-UTR could affect the mechanisms used in initiation of RNA synthesis of NS5.And then the effect of NS3 on the polymerase activity of NS5 using mutant 5'-UTRs and capability of binding of NS5 to mutant templates were evaluated.After addition of NS3,the template activity of mutant with deletion of nucleotide 46～60 was still almost abolished.But RNA synthesis was restored using NS5F and mutants containing intact UTR with mutations at nucleotides 28,37,38 and 43 simultaneously, and that the amount of RNA synthesized was increased with increasing concentrations and prolonged incubation of NS3.In addition,the mutant containing intact UTR with mutations at nucleotides 8,9,69 and 70 simultaneously was active for RNA synthesis using NS5F.Besides,NS3 had a capability of binding to 3'-UTR specifically.The results above indicated that NS3 might be able to promote the RdRp activity and improve the capability of binding to template of NS5 by interactions with 3'-UTR and (or)the N-terminal region of non-RdRp without NLS ofNS5.Altogether,these results suggested that nucleotides 45～60 in subdomain B' of 5'-UTR of WNV might be NS3-independent and closely related to RdRp activity of NS5,While nucleotides 28,37,38 and 43 in subdomain C' and stem-loop kept by nucleotides 8,9,69 and 70 in subdomain A' were likely to contain the sites interact with NS3 and NS5.In addition,NS3 might play its role through interaction with 3'-UTR and the N-terminal region of non-RdRp without NLS ofNS5. 4.Biological characteristics of recovered virus with mutations in 5'-UTRIn order to verify the results obtained in vitro,mutations at nucleotides 8,9,69 and 70 in subdomain A',28,37,38 and 43 in subdomain C' and deletion of nucleotides 45～60 in subdomain B' were introduced into the correspondent region of infectious full-length cDNA clone of pBS-F-X.In addition,deletion of nucleotides 20～45 in subdomain C' was also introduced so that the effects of subdomain C' on RNA replication could be evaluated.RNA transcript synthesized from the linearized full-length cDNA plasmids was electroporated into BHK-21 cells.Then the biological characteristics of mutant recovered viruses obtained were observed by IFA,plaque assay,one-step-growth and RT-PCR of genome.When the region of nucleotides 45～60 in subdomain B' was deleted and the stem-loop in subdomain A' was destroyed, The mutant recovered viruses were almost completely replication defective,but the deletion of nucleotides 20～45 and mutations 28,37,38 and 43 in subdomain C' had no effect on viral replication.These results indicated that stem-loop and side-loop in subdomain A' and B' respectively may play an essential role in viral RNA replication.In this study,the results above suggested that stem-loop and side-loop in subdomain A' and B' respectively were critical for WNV Chin-01 strain replication; NS3 had a capability of binding to 3'-UTR specifically,and might be able to promote the RdRp activity and improve the capability of binding to template of NS5.Based on these results,A possible model for RNA replication complex formed in the stage of initiation of negative-strand synthesis was proposed,in which NS5 firstly recognizes 5'-UTR,and then binds to NS3 directly or through other factors.In virtue of the interaction of NS3 with 3'-UTR and(or)long-range RNA-RNA interactions between 5' and 3'-UTR.the 5' and 3'-end of the genome are brought together to facilitate RNA synthesis.