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 (Ca 2+ )-activated calpains are capable of mediating caspaseindependent 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 Ca 2+ -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 Ca 2+ dynamics in the infected cells and found elevated levels of mitochondrial Ca 2+ . Treatment with ruthenium red, a mitochondrial Ca 2+ influx inhibitor, significantly blocked calpain activations and AIF cleavage. Our conclusion was that calpain activation via Ca 2+ 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 Ca 2+ homeostasis on EV71 infection.

Pyrrolidine dithiocarbamate inhibits enterovirus 71 replication by down-regulating ubiquitin–proteasome system

“…The induction of apoptosis in the host cells has been considered one of the cellular defense mechanisms against viral replication and spread (Arzberger et al, 2010;Herold et al, 2012;White, 2006;. In consistent with most reported studies, (Chen et al, 2006Liang et al, 2004;Lu et al, 2013;Shi et al, 2012;Shih et al, 2008), this study demonstrated that EV71 induced apoptosis as early as 8 h of p.i. In contrast, study by Morais et al (2010) and Zheng et al (2014) showed that, when combined with cisplatin, PDTC significantly induced apoptosis in human cancer cells.…”

“…As reported previously, EV71 infection induced apoptosis in both caspase-dependent and caspase-independent manners (Chang et al, 2004;Chen et al, 2007;Liang et al, 2004;Lu et al, 2013;Shi et al, 2013). To determine whether PDTC could influence the apoptosis-inducing property of EV71, cells were infected with EV71 (1.5 × 10 6 TCID50) and cultured in the medium containing various concentrations of PDTC for 7 h. Apoptotic cells were determined by flow cytometry through measuring the exposed phosphotidylserine.…”

Enterovirus 71 infection of motor neuron-like NSC-34 cells undergoes a non-lytic exit pathway

“…EV71 infection has previously been reported to trigger apoptosis in many different cell types including lymphocytes60, endothelial cells61, muscle cells and neural cells3862. The apoptosis pathway that is induced appears to be cell-type specific whereby EV71-induced apoptosis can be either caspase-dependent intrinsic apoptosis or calpain-induced caspase-independent apoptosis63. Proteases 2 A and 3 C were demonstrated to cleave PARP in RD cells64.…”

Enterovirus 71 3C Promotes Apoptosis through Cleavage of PinX1, a Telomere Binding Protein

“…For example, EV71 infection regulates the expression of miR-146a or miR-370, coordinating apoptosis through targeting SOS1 and GADD45␤ (16). EV71 also activates calpain via Ca 2ϩ flux, playing an essential role in the caspase-independent apoptotic pathway (21). Furthermore, it was found that the cleavage of eukaryotic initiation factor 4G (eIF4G) by EV71 2A, which shuts off host translation, also induces apoptosis (22,23).…”