title: Calcium flux and calpain-mediated activation of the apoptosis-inducing factor contribute to enterovirus 71-induced apoptosis.
abstract: Enterovirus 71 (EV71) is a causative agent of an array of childhood diseases with severe neurological manifestations implicated. EV71 infection is known to induce caspase-dependent apoptosis in cell cultures and animal models. However, whether an alternative apoptotic pathway independent of caspase activation can be triggered by EV71 infection has not been explored. In this study, we showed that calcium (Ca²⁺)-activated calpains are capable of mediating caspase-independent pathway activation during EV71-induced apoptosis in HeLa cells. Results from subcellular fractionation analysis and confocal imaging indicated that during EV71 infection, apoptosis-inducing factor (AIF), a primary mediator of the caspase-independent pathway, became truncated and translocated from the mitochondrion to nucleus. This was accompanied by the release of cytochrome c, and sharply decreased mitochondrial membrane potential. AIF knockdown data indicated significant protection against apoptotic cell death, with greater protection provided by the addition of a pan-caspase inhibitor. The Ca²⁺-dependent, calpain isoforms 1 and 2, but not cathepsins, were proven crucial for the altered AIF behaviour as studied by the pharmacological inhibitor and the knockdown approaches. We then analysed Ca²⁺ dynamics in the infected cells and found elevated levels of mitochondrial Ca²⁺. Treatment with ruthenium red, a mitochondrial Ca²⁺ influx inhibitor, significantly blocked calpain activations and AIF cleavage. Our conclusion was that calpain activation via Ca²⁺ flux plays an essential role in eliciting an AIF-mediated, caspase-independent apoptotic pathway in EV71-infected cells. These findings should be useful for understanding the virus-induced cytopathology and the impact of Ca²⁺ homeostasis on EV71 infection.
authors.affiliation: Department of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei, Taiwan, Republic of China.
authors.forename: Jia-Rong
authors.initials: JR
authors.lastname: Lu
authors.affiliation: Department of Clinical Pathology, Cheng Hsin Rehabilitation Medical Center, Taipei, Taiwan, Republic of China.
authors.forename: Wen-Wen
authors.initials: WW
authors.lastname: Lu
authors.affiliation: Department of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei, Taiwan, Republic of China.
authors.forename: Jian-Zhong
authors.initials: JZ
authors.lastname: Lai
authors.affiliation: Department of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei, Taiwan, Republic of China.
authors.forename: Fu-Lian
authors.initials: FL
authors.lastname: Tsai
authors.affiliation: Department of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei, Taiwan, Republic of China.
authors.forename: Szu-Hsien
authors.initials: SH
authors.lastname: Wu
authors.affiliation: Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan, Republic of China.
authors.forename: Cheng-Wen
authors.initials: CW
authors.lastname: Lin
authors.affiliation: Department of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei, Taiwan, Republic of China.
authors.forename: Szu-Hao
authors.initials: SH
authors.lastname: Kung
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D051033:Apoptosis Inducing Factor / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D004770:Enterovirus / Q000472:pathogenicity*; D004769:Enterovirus Infections / Q000821:virology; D006367:HeLa Cells; D006801:Humans; D008928:Mitochondria / Q000378:metabolism; D014709:Vero Cells
chemical_list: D051033:Apoptosis Inducing Factor; D002154:Calpain; D002118:Calcium