Study On Interaction Of Japanese Enceplalitis Virus E Protein With Host KARS Protein

Japanese encephalitis virus(JEV),a mosquito-borne virus,is a member of the Flavivirus genus,Flaviviridae family.JEV is closely associated with other pathogenic flaviviruses,including West N ile virus(WN V),tick borne encephalitis virus(TBEV),dengue virus(DENV)and yellow fever virus(YFV),et al.JEV is the causative agent of Japanese encephalitis(JE),an epidemic characterized by acute infection of the central nervous system in humans and stillbirths in swine.Today,Approximate 3 billion people live in countries where the JE is endemic,and JE has become a serious public health issue.There is no specific treatment available for JE,and vaccination is the only effective way to prevent JEV infection in humans and domestic animals.Currently,little is known about the life cycle and pathogenic mechanism of Japanese encephalitis virus.There are extensive virus-host interactions during the life cycle of Japanese encephalitis virus.JEV envelope(E)glycoprotein,a 53 kDa protein,is the main component on the surface of the virus particles and plays major role in multiple steps of viral life cycle,including virus attachment,receptor binding,penetration,membrane fusion and virusbudding.A comprehensive understanding of the molecular mechanisms underlying protein–protein interactions(PPIs)between host and virus E protien would enable the utilization of these mechanisms for the discovery of antiviral drugs and the identification of virally altered host proteins.In our study,the E gene of JEV BJB and SA14-14-2were fusedthe Strep tag at its C-terminus and cloned into the eukaryotic expression vector p RE-3×Flag-M.Plsmids were transfected into HEK293 cells,and cells collected 36 h posttranfection were subjected to Pull Down assay.Then the protein samples were sbjected to liquid chromatography-tandem mass spectrometry(LC-MS/MS)analysis to identify.81 host proteins were indentified as the binding partners of JEV E protein,including the Lysine--tRNA ligase(KARS)and Aminoacyl tRNA synthase complex-interacting multifunctional protein 2(AIMP2).Then,we find BJB E protein colocalized with KARS in the cytoplasm,and we also identified BJB E protein intetaction with KARS by Co-Immunoprecipitation(Co-IP)and Bimolecular fluorescence complementation(BiFC).Fourthermore,we indentified the 1-193 aa of BJB E protein interaction with C-terminus of Aminoacylation of KARS by Co-IP.Furthermore,the virus titers were significantly increased after transfection of siRNA-KARS,compared to negative control andblankgroup.Then,another protein AIMP2 also was indentified could interacte with BJB E and KARS.K nockout KARS and AIMP2 by CRISPR-Cas9,we also find the virus titers was significantly increased in the late life cycle.In summary,we identified the host cellar proteins that interact with the JEV E protein,helping to draw an interactome profile of E with host cellular proteins,providing important clues and evidence for exploring the interactions between E and cellular proteins.O ur study also confirmed the interactions between KARS,AIMP2 and BJB E,and then couldsuppressthe productionof virus.In the meantime,this research could makea foundation for further exploration of biological functions and molecular mechanisms of E which is involved in the life cycle and pathogenecity of JEV.