{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T20:20:27Z","timestamp":1791404427217,"version":"4.3.3"},"reference-count":53,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2024,10,28]],"date-time":"2024-10-28T00:00:00Z","timestamp":1730073600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:p>The Japanese encephalitis virus (JEV), a zoonotic flavivirus, is Asia\u2019s primary cause of viral encephalitis. JEV induces apoptosis in a variety of cells; however, the precise mechanisms underlying this apoptosis resulting from JEV infection remain to be elucidated. Our previous studies showed that the proapoptosis gene BAX may have a role in JEV proliferation. In this study, we constructed a PK-15 cell line (BAX.KO) with a knockout of the BAX gene using CRISPR\/Cas9. The knockout of the BAX gene effectively inhibited the proliferation of JEV, resulting in a 39.9% decrease in viral protein levels, while BAX overexpression produced the opposite effect. We confirmed that JEV induces apoptosis of PK-15 using 4\u2032,6-diamidino-2-phenylindole (DAPI) staining and Annexin V-FITC\/PI staining. Furthermore, we found that the phosphorylation of P53 and the expression levels of BAX, NOXA, PUMA, and cleaved-caspase-3\/9 were significantly upregulated after JEV infection. Moreover, we found that JEV infection not only caused mitochondrial damage, the release of mitochondrial cytochrome C (Cyt C), and the downregulation of the apoptosis-inhibiting protein BCL-2 but also reduced the mitochondrial membrane potential (MOMP) and the accumulation of intracellular reactive oxygen species (ROS). These factors collectively encourage the activation of the mitochondrial apoptosis pathway. In contrast, BAX gene knockout significantly reduces the apoptotic changes caused by JEV infection. Treatment with the caspase3 inhibitor attenuated JEV-induced viral proliferation and release, leading to a decrease in viral protein levels of 46% in PK-15 cells and 30% in BAX.KO cells. In conclusion, this study clarified the molecular mechanisms of JEV-induced apoptosis and provided a theoretical basis for revealing the pathogenic mechanisms of JEV infection.<\/jats:p>","DOI":"10.3389\/fmicb.2024.1485667","type":"journal-article","created":{"date-parts":[[2024,10,28]],"date-time":"2024-10-28T04:45:56Z","timestamp":1730090756000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":11,"title":["Japanese encephalitis virus infection induces mitochondrial-mediated apoptosis through the proapoptotic protein BAX"],"prefix":"10.3389","volume":"15","author":[{"given":"Ke","family":"Yang","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinran","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shuqing","family":"Yang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yi","family":"Zheng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sanjie","family":"Cao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qigui","family":"Yan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaobo","family":"Huang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yiping","family":"Wen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qin","family":"Zhao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Senyan","family":"Du","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yifei","family":"Lang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shan","family":"Zhao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rui","family":"Wu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2024,10,28]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1007\/s10495-019-01575-3","article-title":"Influenza a virus-induced apoptosis and virus propagation","volume":"25","author":"Ampomah","year":"2020","journal-title":"Apoptosis"},{"key":"ref2","doi-asserted-by":"publisher","first-page":"337","DOI":"10.1146\/annurev-cellbio-100913-013226","article-title":"Regulated cell death: signaling and mechanisms","volume":"30","author":"Avi","year":"2014","journal-title":"Annu. 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Dis."},{"key":"ref25","doi-asserted-by":"publisher","first-page":"e1000018","DOI":"10.1371\/journal.ppat.1000018","article-title":"Viral control of mitochondrial apoptosis","volume":"4","author":"Lorenzo","year":"2008","journal-title":"PLoS Pathog."},{"key":"ref26","doi-asserted-by":"publisher","first-page":"16652","DOI":"10.1038\/s41598-017-16952-8","article-title":"Ribosomal stress and Tp53-mediated neuronal apoptosis in response to capsid protein of the Zika virus","volume":"7","author":"Lukasz","year":"2017","journal-title":"Sci. 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Microbiol."},{"key":"ref36","doi-asserted-by":"publisher","first-page":"496","DOI":"10.3389\/fmicb.2016.00496","article-title":"Zika virus: the latest newcomer","volume":"7","author":"Saiz","year":"2016","journal-title":"Front. Microbiol."},{"key":"ref37","doi-asserted-by":"publisher","first-page":"985363","DOI":"10.3389\/fonc.2022.985363","article-title":"The role of BCL-2 family proteins in regulating apoptosis and cancer therapy","volume":"12","author":"Shanna","year":"2022","journal-title":"Front. Oncol."},{"key":"ref38","doi-asserted-by":"publisher","first-page":"100994","DOI":"10.1016\/j.mam.2021.100994","article-title":"Pathobiology of Japanese encephalitis virus infection","volume":"81","author":"Sharma","year":"2021","journal-title":"Mol. Asp. Med."},{"key":"ref39","doi-asserted-by":"publisher","first-page":"7","DOI":"10.1038\/nri.2015.7","article-title":"Converging roles of caspases in inflammasome activation, cell death and innate immunity","volume":"16","author":"Si Ming","year":"2015","journal-title":"Nat. Rev. Immunol."},{"key":"ref40","doi-asserted-by":"publisher","first-page":"7951","DOI":"10.1038\/s41598-018-26208-8","article-title":"North American domestic pigs are susceptible to experimental infection with Japanese encephalitis virus","volume":"8","author":"So Lee","year":"2018","journal-title":"Sci. Rep."},{"key":"ref41","doi-asserted-by":"publisher","first-page":"370","DOI":"10.1056\/NEJMra030476","article-title":"Flavivirus encephalitis","volume":"351","author":"Solomon","year":"2004","journal-title":"N. Engl. J. Med."},{"key":"ref42","doi-asserted-by":"publisher","first-page":"405","DOI":"10.1136\/jnnp.68.4.405","article-title":"Japanese encephalitis","volume":"68","author":"Solomon","year":"2000","journal-title":"J. Neurol. Neurosurg. Psychiatry"},{"key":"ref43","doi-asserted-by":"publisher","first-page":"e1007299","DOI":"10.1371\/journal.ppat.1007299","article-title":"Infection with flaviviruses requires BCLXL for cell survival","volume":"14","author":"Suzuki","year":"2018","journal-title":"PLoS Pathog."},{"key":"ref44","doi-asserted-by":"publisher","first-page":"402","DOI":"10.1038\/nrm2395","article-title":"Transcriptional control of human p53-regulated genes","volume":"9","author":"Todd","year":"2008","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref45","doi-asserted-by":"publisher","first-page":"243","DOI":"10.3390\/v9090243","article-title":"Regulation of apoptosis during flavivirus infection","volume":"9","author":"Toru","year":"2017","journal-title":"Viruses"},{"key":"ref46","doi-asserted-by":"publisher","first-page":"3724","DOI":"10.1038\/s41598-017-03912-5","article-title":"An essential role of PI3K in the control of West Nile virus infection","volume":"7","author":"Wang","year":"2017","journal-title":"Sci. Rep."},{"key":"ref47","doi-asserted-by":"publisher","first-page":"974","DOI":"10.3390\/v15040974","article-title":"BAK-mediated pyroptosis promotes Japanese encephalitis virus proliferation in porcine kidney 15 cells","volume":"15","author":"Weimin","year":"2023","journal-title":"Viruses"},{"key":"ref48","doi-asserted-by":"publisher","first-page":"578","DOI":"10.1006\/excr.2000.4859","article-title":"Cleavage of Bax enhances its cell death function","volume":"256","author":"Wood","year":"2000","journal-title":"Exp. Cell Res."},{"key":"ref49","doi-asserted-by":"publisher","first-page":"e01445-20","DOI":"10.1128\/JVI.01445-20","article-title":"Zika virus infection induced apoptosis by modulating the recruitment and activation of pro-apoptotic protein Bax","volume":"95","author":"Xiaodong","year":"2021","journal-title":"J. Virol."},{"key":"ref50","doi-asserted-by":"publisher","first-page":"2605","DOI":"10.1182\/blood-2003-01-0211","article-title":"Cleavage of Bax to p18 Bax accelerates stress-induced apoptosis, and a cathepsin-like protease may rapidly degrade p18 Bax","volume":"102","author":"Xuefang","year":"2003","journal-title":"Blood"},{"key":"ref51","doi-asserted-by":"publisher","first-page":"598203","DOI":"10.3389\/fmicb.2020.598203","article-title":"Zika virus-induced neuronal apoptosis via increased mitochondrial fragmentation","volume":"11","author":"Yang","year":"2020","journal-title":"Front. Microbiol."},{"key":"ref52","doi-asserted-by":"publisher","first-page":"e99347","DOI":"10.15252\/embj.201899347","article-title":"Zika virus elicits inflammation to evade antiviral response by cleaving cGAS via NS1-caspase-1 axis","volume":"37","author":"Zheng","year":"2018","journal-title":"EMBO J."},{"key":"ref53","doi-asserted-by":"publisher","first-page":"313","DOI":"10.1002\/med.21379","article-title":"Direct activation of Bax protein for cancer therapy","volume":"36","author":"Zhiqing","year":"2015","journal-title":"Med. Res. 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