Mechanism Of BAK Protein Affecting The Proliferation Of Japanese Encephalitis Virus In PK-15 Cells

Japanese encephalitis virus(JEV)belongs to the Flaviviridae family and is a single-stranded,enveloped RNA virus.As a zoonotic disease,JEV has brought great harm to the pig industry and public health in my country.In-depth exploration of the mechanism of JEV-induced inflammation in different tissues can screen out targeted proteins for effective treatment of the disease,which is very important for the prevention and control of the disease.In the previous study,through the analysis and preliminary verification of the transcriptome of JEV-infected cells,we identified the BCL2 family cell apoptosis-promoting protein BAK,which has a significant impact on the proliferation of JEV.In order to clarify the role and mechanism of BAK affecting the proliferation of JEV on PK-15 cells.Therefore,this article aims to explore the effect and mechanism of BAK on JEV proliferation on PK-15 cells.This research paper is divided into three parts:(1)Effects of BAK knockdown expression on JEV replication.First,a BAK.KD monoclonal cell line was constructed using CRISPR/Cas9 technology and a q PCR detection method for the virus E gene was established.Then the growth curve and viral adsorption and internalization of JEV on BAK.KD cells were measured.Finally,the BAK eukaryotic expression plasmid was constructed,and the effects of BAK complementation and overexpression on JEV replication were determined.The results showed that the replication of JEV and the internalization of virus adsorption were significantly reduced after the expression of BAK.KD.After replenishment and overexpression of BAK,the proliferation of JEV was enhanced.Therefore,the expression of BAK had a significant promoting effect on the proliferation of JEV.(2)Effects of BAK knockdown expression on cytokine-related pathways after JEV infection.JEV was inoculated into BAK.KD cells,and the transcription levels of cytokines,and cell death gene transcription levels and types were detected after JEV infection.The results showed that knockdown expression of BAK inhibited the transcriptional expression of various inflammatory factors,cell deathrelated genes and JEV gene in cells.Therefore,BAK could promote the inflammatory response induced by JEV,lead to the increase of ROS in the cytoplasm,and activate the subsequent necrotic death pathway.(3)Effects of BAK-mediated pyroptosis on JEV.After infecting BAK.KD cells with JEV,the gene transcription level and protein level in the pyroptosis pathway were measured,and the co-localization of GSDMD-N protein and the inhibitory effect of pore formation were studied.The results showed that knockdown of BAK expression significantly reduced gene transcription and protein levels in the pyroptosis pathway;there was co-localization between GSDMD-N protein and JEV,and after GSDMD-N protein was inhibited,the release of virions from JEV was reduced.The results of the study showed that BAK-mediated activation of pyroptosis could induce the activation of GSDMD protein to form pores,promote the release of inflammatory factors and JEV,and then promote the proliferation of JEV.In summary,JEV infection of PK-15 cells promoted the expression of BAK,and the expression of BAK activated the transcription and expression of inflammatory factors and cell death pathways.When BAK activates the pyroptosis pathway of NLRP3 inflammasome,it promotes the activation of GSDMD protein to form pores,and enhances the release of inflammatory factors and JEV virions,thereby affecting the viral proliferation of JEV.The results of this study showed that the pyroptosis pathway mediated by BAK could promote the proliferation of JEV on PK-15 cells,and the results of this study could provide a theoretical basis for the antiviral treatment of JEV.