The apoptotic pathways leading to cell death can be generally divided into two nonexclusive signaling cascades involving death receptors (extrinsic pathways) or mitochondria (intrinsic pathway) (2, 37). Induction of apoptosis through the death receptor pathway is initiated by the binding of ligand to the receptor, causing oligomerization of the receptor. This induces the formation of the death-induced signaling complex, leading to the activation of the initiator caspase, caspase-8 (2). Caspase-8 can then activate downstream effector caspases. Apoptosis via the mitochondrial pathway involves specific signals that allow the release of proapoptotic molecules from the inner-membrane space, including cytochrome c (28), second mitochondrion-derived activator of caspase (Smac/DIABLO) (11, 55), apoptosis-inducing factor (AIF) (50), and endonuclease G (26). Cytosolic cytochrome c, Apaf-1, and procaspase-9 form a complex termed the apoptosome (63). Formation of the apoptosome leads to the activation of caspase-9 and subsequent effector caspase activation. Relocalization of Smac/DIABLO to the cytoplasm promotes caspase activation through inhibition of the inhibitor of apoptosis (IAP) protein family (11, 55). IAP proteins are negative regulators of apoptosis that inhibit caspase activity. IAP family members are characterized by the presence of one or more baculoviral IAP repeat domains (9). Six human IAP family members have been identified: NAIP, c-IAP1, c-IAP2, XIAP, survivin, and BRUCE (1, 12, 15, 27, 39). Four of these proteins—c-IAP1, c-IAP2, XIAP, and survivin—have been shown to directly interact with caspase-3, caspase-7, and caspase-9 and, at least in the cases of c-IAP1, c-IAP2, and XIAP, inhibit caspase activity (10, 41, 51). Additionally, reduction of c-IAP2 and XIAP protein levels is associated with apoptosis (25). The proapoptotic mitochondrial protein Smac/DIABLO has been shown to directly interact with XIAP eliminating XIAP's ability to bind to and inhibit caspases and thereby promoting apoptosis (14). The Bcl-2 protein family is of central importance in the regulation of the mitochondrial apoptotic pathway. Members of this family may be either antiapoptotic (e.g., Bcl-2 and Bcl-xl) or proapoptotic (e.g., Bid, Bax, and Bak). Bcl-2 appears to play a role in the maintenance of mitochondrial integrity and inhibits mitochondrial release of proapoptotic factors. Conversely, Bid, Bax, and Bak appear to facilitate the release of these factors (22). Activation of the death receptor and mitochondrion-associated death pathways are not mutually exclusive, and these pathways may interact (cross talk) at many levels. One important link between these two pathways appears to involve the caspase-8-dependent cleavage of Bid. Truncated Bid translocates to the mitochondrion, where it facilitates release of mitochondrial proteins, presumably by inducing the homo-oligomerization of Bax or Bak (58). This process may result in alteration of the mitochondrial permeability transition pore and loss of mitochondrial membrane potential (ΔΨm). However, recent evidence has shown that Bid-dependent release of mitochondrial proteins can occur without perturbing mitochondrial structure and function (20, 57). Mammalian reoviruses are nonenveloped double-stranded RNA viruses that replicate exclusively in the cytoplasm. Reoviruses have been shown to induce apoptosis both in cultured cells in vitro (33, 38, 54) and in specific tissues, including the heart and brain, in vivo (6, 34). Apoptosis is an important mechanism of reovirus-induced tissue injury in vivo, and inhibition of apoptosis dramatically reduces the severity of disease (6). Reovirus-induced apoptosis has been shown to involve death receptor 4 (DR4), death receptor 5 (DR5), and their cognate ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Inhibition of the TRAIL/death receptor interaction with anti-TRAIL antibodies or soluble forms of DR4/DR5 inhibits cell death (5). Recently we have shown that reovirus-induced apoptosis also requires activation of the mitochondrial apoptotic pathway (21). In reovirus-infected HEK cells, there is a caspase-8-dependent cleavage of Bid leading to the subsequent release of cytochrome c and activation of caspase-9 and the effector caspase, caspase-3. Caspase-3 activation is inhibited in cells overexpressing a dominant-negative form of the adaptor protein FADD (FADD-DN) and in cells overexpressing Bcl-2, consistent with the importance of both extrinsic and intrinsic pathways in reovirus-induced apoptosis (21). In this study we set out to better characterize the mitochondrion-dependent apoptotic processes induced following reovirus infection. Release of the mitochondrial proteins Smac/DIABLO and AIF were examined. Additionally, the fate of a number of IAP protein family members was determined. We find that Smac/DIABLO is released into the cytosol of infected HEK 293 cells, while AIF remained sequestered in the mitochondria. The release of cytochrome c and Smac/DIABLO occur without disturbing the mitochondrial membrane potential. Additionally, by utilizing a dominant-negative isoform of caspase-9, we find that while the mitochondrial pathway is required for caspase-3 activation, caspase-9 is dispensable for this process. Finally, we find that a specific subset of IAP proteins are down-regulated following reovirus infection. These results suggest that activation of Smac/DIABLO-dependent rather that caspase-9-dependent pathways represents the key mitochondrial event during reovirus-induced apoptosis and provide the first evidence for involvement of Smac/DIABLO in virus-induced apoptosis.