BACULOVIRUS INHIBITOR OF APOPTOSIS FUNCTIONS AT OR UPSTREAM OF THE APOPTOTIC SUPPRESSOR P35 TO PREVENT PROGRAMMED CELL-DEATH

Members of the inhibitor of apoptosis (iap) gene family prevent progra mmed cell death induced by multiple signals in diverse organisms, sugg esting that they act at a conserved step in the apoptotic pathway. To investigate the molecular mechanism of iap function, we expressed epit ope-tagged Op-iap, the prototype viral lap from Olgyia pseudotsugata n uclear polyhedrosis virus, by using novel baculovirus recombinants and stably transfected insect cell lines. Epitope-tagged Op-iap blocked b oth virus- and UV radiation-induced apoptosis. With or without apoptot ic stimuli, Op-IAP protein (31 kDa) cofractionated with cellular membr anes and the cytosol, suggesting a cytoplasmic site of action. To iden tify the step(s) at which Op-iap blocks apoptosis, we monitored the ef fect of Op-iap expression on in vivo activation of the insect CED-31/I CE death proteases (caspases). Op-iap prevented in vivo caspase-mediat ed cleavage of the baculovirus substrate inhibitor P35 and blocked cas pase activity upon viral infection or UV irradiation. However, unlike the stoichiometric inhibitor P35, Op-IAP failed to affect activated ca spase as determined by in vitro protease assays. These findings provid e the first biochemical evidence that Op-iap blocks activation of the host caspase or inhibits its activity by a mechanism distinct from P35 . Moreover, as suggested by the capacity of Op-iap to block apoptosis induced by diverse signals, including virus infection and UV radiation , lap functions at a central point at or upstream from steps involving the death proteases.