Reovirus-Induced Apoptosis Requires Mitochondrial Release of Smac/DIABLO and Involves Reduction of Cellular Inhibitor of Apoptosis Protein Levels

Many viruses belonging to diverse viral families with differing structure and replication strategies induce apoptosis both in cultured cells in vitro and in tissues in vivo. Despite this fact, little is known about the specific cellular apoptotic pathways induced during viral infection. We have previously shown that reovirus-induced apoptosis of HEK cells is initiated by death receptor activation but requires augmentation by mitochondrial apoptotic pathways for its maximal expression. We now show that reovirus infection of HEK cells is associated with selective cytosolic release of the mitochondrial proapoptotic factors cytochrome c and Smac/DIABLO, but not the release of apoptosis-inducing factor. Release of these factors is not associated with loss of mitochondrial transmembrane potential and is blocked by overexpression of Bcl-2. Stable expression of caspase-9b, a dominant-negative form of caspase-9, blocks reovirus-induced caspase-9 activation but fails to significantly reduce activation of the key effector caspase, caspase-3. Smac/DIABLO enhances apoptosis through its action on cellular inhibitor of apoptosis proteins (IAPs). Reovirus infection is associated with selective down-regulation of cellular IAPs, including c-IAP1, XIAP, and survivin, effects that are blocked by Bcl-2 expression, establishing the dependence of IAP down-regulation on mitochondrial events. Taken together, these results are consistent with a model in which Smac/DIABLO-mediated inhibition of IAPs, rather than cytochrome c-mediated activation of caspase-9, is the key event responsible for mitochondrial augmentation of reovirus-induced apoptosis. These studies provide the first evidence for the association of Smac/DIABLO with virus-induced apoptosis.