Enterovirus 71 2B Induces Cell Apoptosis by Directly Inducing the Conformational Activation of the Proapoptotic Protein Bax

Abstract

To survive and replicate within a host, many viruses have evolved strategies that target crucial components within the apoptotic cascade, leading to either inhibition or induction of cell apoptosis. Enterovirus 71 (EV71) infections have been demonstrated to impact the mitochondrial apoptotic pathway and induce apoptosis in many cell lines. However, the detailed mechanism of EV71-induced apoptosis remains to be elucidated. In this study, we report that EV71 2B protein (2B) localized to the mitochondria and induced cell apoptosis by interacting directly with and activating the proapoptotic protein Bax. 2B recruited Bax to the mitochondria and induced Bax conformational activation. In addition, mitochondria isolated from 2B-expressing cells that were treated with a recombinant Bax showed increased Bax interaction and cytochrome c (Cyt c) release. Importantly, apoptosis in cells with either EV71 infection or 2B expression was dramatically reduced in Bax knockdown cells but not in Bak knockdown cells, suggesting that Bax played a pivotal role in EV71-or 2B-induced apoptosis. Further studies indicate that a hydrophobic region of 18 amino acids (aa) in the C-terminal region of 2B (aa 63 to 80) was responsible for the location of 2B in the mitochondria. A hydrophilic region of 14 aa in the N-terminal region of 2B was functional in Bax interaction and its subsequent activation. Moreover, overexpression of the antiapoptotic protein Bcl-X L abrogates 2B-induced release of Cyt c and caspase activation. Therefore, this study provides direct evidence that EV71 2B induces cell apoptosis and impacts the mitochondrial apoptotic pathway by directly modulating the redistribution and activation of proapoptotic protein Bax. IMPORTANCEEV71 infections are usually accompanied by severe neurological complications. It has also been postulated that the induction of cell apoptosis resulting from tissue damage is a possible process of EV71-related pathogenesis. In this study, we report that EV71 2B protein (2B) localized to the mitochondria and induced cell apoptosis by interacting directly with and activating the proapoptotic protein Bax. This study provides evidence that EV71 induces cell apoptosis by modulating Bax activation and reveals important clues regarding the mechanism of Cyt c release and mitochondrial permeabilization during EV71 infection. E nterovirus 71 (EV71) is an RNA icosahedral virus that belongs to the human enterovirus species A of the genus Enterovirus within the Picornaviridae family (1, 2). EV71 is thought to be one of the main pathogens that cause foot, hand, and mouth disease (HFMD) in young children (3, 4). In recent years, outbreaks of EV71-related HFMD have been reported in Southeast and East Asia, including Taiwan, Malaysia, Singapore, Japan, and China (5, 6). Particularly, over one million EV71-related HFMD cases were reported each year in China since 2008, including hundreds of fatal cases per year (7). EV71 infections are usually accompanied by severe neurological complications such as aseptic menin...

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