Study On Interaction Of Japanese Encephalitis Virus NS1’ Protein And Host Cyclin-dependent Kinase 1(CDK1) And MiR-22

Japanese encephalitis virus（JEV）is a transmosquito-borne zoonotic pathogen,belongs to the genus flavivirus of the Flaviviridae,which is susceptible to a variety of animals and has obvious encephalitis symptoms after human infection.Pig infection mainly leads to reproductive disorder diseases.The JEV NS1’protein,present only in JE serogroup of the flavivirus genus category,is a nonstructural protein produced by ribosomal frameshifting and can significantly enhance the neuroaggressiveness of the virus.Our previous study found that JEV NS1’protein was able to inhibit MAVS-mediated type I interferon（IFN-I）production by targeting miR-22,thereby enhancing viremia and increasing the viral load of JEV invading brain tissue.But very little is known about how the JEV NS1’protein regulates miR-22 expression.Based on this,the molecular mechanism of miR-22 regulation by JEV NS1’protein was mainly investigated in this paper.1.Preparation of monoclonal antibodies recognizing only the JEV NS1’protein4-week-old Balb/C mice were immunized with the JEV NS1’_52aaprotein（ribosome frameshift producing 52 amino acids unique to the NS1’protein）and,using fusion and subcloning techniques,two hybridoma cell lines were finally obtained:3D11 and 4H1,both of which stably secreted antibodies bound to the JEV NS1’_52aaprotein.Both m Abs showed good specificity as verified by Western blot and indirect immunofluorescence experiments.2.The JEV NS1’protein enhances the binding of transcription factors to the miR-22 promoter regionOur previous study found that the JEV NS1’protein was able to inhibit MAVS-mediated IFN-I production by targeting miR-22,but does the miR-22-regulated MAVS pathway play a dominant role in this process?In vivo experiments in MAVS^-/-mice or knockdown of MAVS（or miR-22）on Hela cells revealed that after MAVS（or miR-22）was knockdown,the ability of JEV NS1’protein to inhibit IFN-βwas completely abolished,indicating that JEV NS1’protein reduces IFN-βproduction mainly by targeting the miR-22-MAVS pathway.To further explore how the JEV NS1’protein regulates miR-22expression,we constructed a dual-fluorescein reporter system for the miR-22 promoter and used this system to confirm that the transcription factors CREB and c-Rel are able to bind directly to the promoter region of miR-22 to regulate miR-22 transcription.In addition,the results of chromatin coimmunoprecipitation（Ch IP）experiments indicate that JEV NS1’protein cannot directly bind to the promoter region of miR-22,but can indirectly enhance the binding of CREB and c-Rel to the promoter region of miR-22,and subsequently upregulate miR-22 expression.3.Screening and validation of host interaction proteins regulating miR-22transcription by the NS1’proteinTo further explore how NS1’protein enhances binding of CREB and c-Rel to the promoter region of miR-22,we used affinity chromatography combined with mass spectrometry analysis to screen host proteins that only interact with the JEV NS1’protein in Hela cells infected with JEV by identifying only JEV NS1’protein monoclonal antibodies.Validation of the screened proteins by co-immunoprecipitation revealed that the host protein cyclin-dependent kinase 1（CDK1）interacted with the JEV NS1’protein,but not with the JEV NS1 protein.To verify whether the screened CDK1 protein is involved in the transcription of miR-22,CDK1 positively regulates miR-22 transcription,and expression levels were found after overexpressing CDK1 or inhibiting CKD1 function in Hela cells.4.Interaction of JEV NS1’protein and CDK1 protein enhances miR-22transcriptionTo further confirm that the JEV NS1’protein enhances miR-22 transcription by interacting with the CDK1 protein,upon inhibition of CDK1 function in Hela cells,it was found that the ability of JEV NS1’protein to upregulate miR-22 expression was completely abolished.Meanwhile,JEV infection showed that the differences of NS1’deletion virus,the induction of IFN-β,and the virus proliferation were removed.It indicates that CDK1 is a key interacting protein that the JEV NS1’protein upregulates miR-22 expression.Next,CDK1 was verified to promote the nucleation of CREB and c-Rel to enhance miR-22transcription,which in turn inhibited MAVS-mediated IFN-βproduction.To validate the promoting effect of CDK1 protein on JEV replication in a mouse model,CDK1 function was inhibited in mice by mouse tail vein injection of the CDK1 inhibitor RO3306.The results of the in vivo tests showed that after CDK1 inhibition,the JEV proliferation in mice was significantly reduced,and the mouse onset and death caused by JEV infection in mice were significantly reduced.However,in MAVS^-/-mice,CDK1 inhibition did not affect JEV replication in mice.Our study is the first to fully reveal the important role of the CDK1 protein in the JEV NS1’regulation of the miR-22-MAVS-IFN-βpathway.The molecular mechanism underlying the interaction of JEV NS1’and CDK1 to induce miR-22 expression to regulate the type I interferon was initially elucidated.The key role of CDK1 in JEV replication is revealed.It helps to further reveal the mechanism of JEV immune escape.It also provides new ideas and potential drug targets for the treatment of JEV.