Murine Coronavirus-Induced Apoptosis in 17Cl-1 Cells Involves a Mitochondria-Mediated Pathway and Its Downstream Caspase-8 Activation and Bid Cleavage Chun-Jen Chen and Shinji Makino1 Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019; and Department of Microbiology and Institute of Molecular and Cellular Biology, The University of Texas at Austin, Austin, Texas 78712-1095 Received January 29, 2002; returned to author for revision June 7, 2002; accepted June 16, 2002 Mouse hepatitis virus (MHV) infection in murine 17Cl-1 cells results in apoptotic cell death. Inhibition of MHV-induced apoptosis by the pancaspase inhibitor Z-VAD-FMK promoted virus production late in infection, indicating that apoptosis could be a host response to limit the production of viral progeny. Activation of the mitochondria-mediated apoptotic pathway was indicated by the activation of caspase-9 and delay of apoptosis by Bcl-2 overexpression. Analyses of the subcellular distribution of cytochrome c, procaspase-9, and Apaf-1 suggested an aberrant apoptosome formation in the vicinity of the mitochondria, which could be a cell type-specific event. An increase in the amount of Fas (APO-1/CD95), caspase-8 activation, caspase-8-mediated Bid cleavage, and subsequent translocation of truncated Bid to mitochondria, all of which relate to the Fas-mediated pathway, also occurred in MHV-infected 17Cl-1 cells, whereas the formation of the death-inducing signaling complex, a direct indication of the activation of Fas-mediated pathway, was undetectable. Caspase-8 and Bid activation appeared to be downstream of mitochondria, because Bcl-2 overexpression suppressed both events, suggesting that infected 17Cl-1 cells might have activated a receptor-mediated “type II” signaling pathway, in which primary and low levels of receptor-mediated pathway activation lead to the activation of the mitochondria-mediated pathway. All our data indicate that a mitochondria-mediated pathway played a major regulatory role in apoptosis in MHV-infected 17Cl-1 cells. 2002 Elsevier Science (USA) INTRODUCTION Viral infection may trigger a variety of host cellular responses, and one outcome of virushost interaction is the activation of an innate cell death machinery, called programmed cell death, or apoptosis; many viruses and viral proteins are capable of inducing apoptosis (re- viewed by OBrien, 1998; Teodoro and Branton, 1997). The induction of apoptosis upon viral infection has been hypothesized to be a host-defense response, since early death of virus-infected cells would prevent viral replica- tion. Many viruses have developed a countermeasure(s) that effectively blocks or delays cell death by expressing antiapoptotic proteins to maximize the production of viral progeny (reviewed by Hardwick, 1998; OBrien, 1998; Te- odoro and Branton, 1997). On the other hand, apoptosis induction at the end of the viral replication cycle might assist in viral dissemination, while attenuating an inflam- matory response. Apparently viruses have evolved to regulate apoptosis, which may directly or indirectly con- tribute to viral pathogenesis. Apoptosis usually involves the activation of a family of cysteine proteases named caspases, which cleave a variety of cellular substrates, and the cleavage of certain important protein targets leads to detrimental biochem- ical and morphological changes and eventual cell de- struction (reviewed by Earnshaw et al., 1999; Nicholson, 1999). Caspases are synthesized as proenzymes, which require cleavage for their activation. Initiator caspases, such as caspase-8 and -9, undergo self-cleavage and activation upon receiving apoptotic signals. These up- stream caspases then activate effector caspases, such as caspase-3, -6, and -7, which execute the cleavage of other cellular substrates. Caspase cascades can be ac- tivated by two major pathways. In one pathway, different stimuli converge death signals to mitochondria, resulting in mitochondrial damage and the release of cytochrome c to the cytoplasm, where it forms apoptosome com- plexes with Apaf-1 and procaspase-9 in the presence of dATP, leading to the activation of caspase-9 (Li et al., 1997; Saleh et al., 1999; Zou et al., 1999). The other pathway initiates from cell surface death receptors, such as Fas (CD95/APO-1) and TNF-R1. Engagement of these receptors by their cognate ligands results in the aggre- gation of intracellular death domains and the recruitment of FADD and procaspase-8, forming the death-inducing signaling complex (DISC), where caspase-8 is activated (Kischkel et al., 1995; Medema et al., 1997). Bcl-2 family proteins play an important role in regulat- ing apoptotic signaling at the mitochondrial level (re- 1To whom reprint requests should be addressed at Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, MRB 4.146, 301 University Blvd., Galveston, Texas 77555- 1019. Fax: