Japanese encephalitis virus infection activates caspase-8 and -9 in a FADD-independent and mitochondrion-dependent manner Chang-Huei Tsao, 1 Hong-Lin Su, 2 Yi-Ling Lin, 3 Han-Pang Yu, 3 Shu-Ming Kuo, 2 Ching-I Shen, 4 Ching-Wen Chen 2 and Ching-Len Liao 5 Correspondence Ching-Len Liao chinglen@ms1.hinet.net 1 Graduate Institute of Life Sciences, National Defense Medical Center, Taiwan, ROC 2 The Department of Life Sciences, National Chung-Hsing University, Taiwan, ROC 3 Institute of Biomedical Sciences, Academia Sinica, Taiwan, ROC 4 The Department of Veterinary Medicine, National Chung-Hsing University, Taiwan, ROC 5 Department of Microbiology and Immunology, National Defense Medical Center, Taiwan, ROC Received 9 January 2008 Accepted 10 April 2008 Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, replicates primarily at the endoplasmic reticulum and thereby triggers apoptosis of infected cells. This study investigated the hierarchical activation of the caspase network induced by JEV infection. It was found that JEV activated the initiators caspase-8 and -9, as well as effector caspase-3, in infected baby hamster kidney and mouse neuroblastoma (N18) cells. In neuronal N18 cells, JEV infection triggered cytochrome c release from mitochondria, which in turn activated caspase-9 and -3. Treatment of JEV-infected N18 cells with cyclosporin A or ruthenium red, which attenuate mitochondrial injuries, blocked activation of caspase-9 or -3, typifying that, in neuronal cells, this apoptosis involves the mitochondrial pathway. Alternatively, in caspase-3-deficient MCF-7 cells, JEV persisted and readily triggered a typical apoptotic response, including cytochrome c release and full activation of caspase-9 and -8 along with caspase-6, indicating that JEV did not require caspase-3 to manifest caspase-8 activation and apoptosis. Interestingly, a Fas-associated death- domain-containing protein (FADD) dominant-negative mutant, which interfered with transmission of the extracellular death signals into cells through the Fas/tumour necrosis factor (TNF) receptor, failed to block JEV-induced apoptosis and caspase-8 activation, implying that receptor oligomerization of the Fas/TNF pathway might not participate in JEV-induced apoptosis. Taken together, these results illustrate that JEV infection triggers caspase cascades involving the initiators caspase-8 and -9, probably through FADD-independent but mitochondrion-dependent pathways. INTRODUCTION Caspases are the central executioners for most apoptotic responses when activated in proteolytic cascades (Cohen, 1997; Villa et al., 1997). Apoptosis is implemented by effector caspases, including caspase-3, -6 and -7, which cleave a variety of essential cellular targets, and their activations are controlled hierarchically by initiator cas- pases such as caspase-8 and -9. Caspase-8 activation is triggered by an extracellular death-receptor signalling pathway involving the Fas/tumour necrosis factor receptor (TNFR), whilst caspase-9 is activated by intracellular apoptotic stimuli through a mitochondrial pathway. The mitochondrial apoptosis pathway is initiated by holocyto- chrome c (Cyto-c) released from mitochondria, which causes apoptosome formation and in turn triggers caspase- 9 autoactivation (Adams & Cory, 2002; Green & Kroemer, 2004). The death-receptor pathway, via CD95/Fas/Apo1 or TNFR, is activated through oligomerization of membran- ous receptors that recruit procaspase-8 and Fas-associated death-domain-containing protein (FADD) to form a death-inducing signalling complex (DISC). This induced proximity to DISC formation subsequently stimulates caspase-8 autoactivation (Chen & Wang, 2002; Muzio et al., 1998). These two pathways function separately in response to diverse death signals, but may converge to the same downstream effector caspases, caspase-3, -6 and -7 (Cohen, 1997). However, the death-receptor pathway has been reported to potentiate the mitochondrial pathway by triggering Cyto-c release through caspase-8-cleaved Bid protein, a pro-apoptotic member of the Bcl-2 family that targets mitochondria (Li et al., 1998). Caspase-3 and -7 may also participate directly in causing Cyto-c release from Journal of General Virology (2008), 89, 1930–1941 DOI 10.1099/vir.0.2008/000182-0 1930 2008/000182 G 2008 SGM Printed in Great Britain