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We have previously reported that EV71 induces reactive oxygen species (ROS). However, the underlying mechanism remains elusive. Co-immunoprecipitation-proteomic analysis revealed that enteroviral 2B protein interacted with mitochondrial voltage-dependent anion channel 3 (VDAC3). Knockdown (KD) of VDAC3 expression specifically inhibited enteroviral replication. Single-round viral replication was also inhibited in KD cells, suggesting that VDAC3 plays an essential role in replication. Consistent with this, VDAC3 gene KD significantly reduced the EV71-induced mitochondrial ROS generation. Exogenous 2B expression could induce the mitochondrial ROS generation that was significantly reduced in VDAC3-KD cells or in the Mito-TEMPO-treated cells. Moreover, VDAC3 appears to be necessary for regulation of antioxidant metabolism. VDAC3 gene KD led to the enhancement of such pathways as hypotaurine\/taurine synthesis in the infected cells. Taken together, these findings suggest that 2B and VDAC3 interact to enhance mitochondrial ROS generation, which promotes viral replication.<\/jats:p>","DOI":"10.3390\/v14081717","type":"journal-article","created":{"date-parts":[[2022,8,5]],"date-time":"2022-08-05T02:12:39Z","timestamp":1659665559000},"page":"1717","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":26,"title":["Enteroviral 2B Interacts with VDAC3 to Regulate Reactive Oxygen Species Generation That Is Essential to Viral Replication"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2006-133X","authenticated-orcid":false,"given":"Mei-Ling","family":"Cheng","sequence":"first","affiliation":[{"name":"Department of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan City 33302, Taiwan"},{"name":"Healthy Aging Research Center, Chang Gung University, Taoyuan City 33302, Taiwan"},{"name":"Metabolomics Core 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Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chien-Hsueh","family":"Lai","sequence":"additional","affiliation":[{"name":"Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan City 33302, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4595-5754","authenticated-orcid":false,"given":"Guan-Jie","family":"Li","sequence":"additional","affiliation":[{"name":"Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan City 33302, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuan-Yu","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan City 33302, 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