Baculovirus and host factors regulating apoptosis: Mechanistic clues on viral Op-IAP anti-apoptotic function

Apoptosis or programmed cell death (PCD) is used by a cell to eliminate itself. Infection of Spodoptera frugiperda (SF21) cells by baculoviruses causes apoptosis through a mechanism that involves activation of host death proteases known as caspases. The molecular mechanism by which baculoviruses activate these caspases is not known. Membrane permeable peptide inhibitors to various classes of caspases revealed the activity of a novel upstream caspase (Sf caspase-X) in vivo, which is crucial for propagating the caspase cascade and thus causing apoptosis. To evade the host apoptosis response, baculoviruses have evolved apoptotic inhibitors that allow successful virus replication. Inhibitor of apoptosis (IAP) and P35 are two distinct classes of baculovirus apoptotic inhibitors. The iap gene family consists of homologs from diverse animal species. To gain insight into the mechanism by which baculovirus Op-IAP functions, its subcellular localization and site of function in the PCD pathway were determined. Op-IAP localized to the cytosol and associated with cellular membranes, consistent with a role in regulation of the apoptotic signal. Upon coexpression, Op-IAP functioned upstream of the caspase substrate-inhibitor P35 within the apoptotic pathway. The step at which Op-IAP functioned is most consistent with it targeting Sf-caspase-X, an apical caspase required for apoptotic signaling. Subsequent structure-function analyses of Op-IAP revealed that the conserved baculovirus IAP repeat (BIR) motifs are necessary for function. Protein interaction assays indicated that BIR2, but not BIR1, is required for interaction with Drosophila Reaper, an apoptosis-inducing protein. BIRs from Drosophila IAP homologs did not functionally substitute for Op-IAP BIRs, suggesting that the cellular and viral IAP homologs function by distinct mechanisms. Consistent with this hypothesis, Op-IAP and DIAP1 failed to block apoptosis in dipteran (Drosophila) and lepidopteran (Spodoptera) cell lines, respectively. Collectively, these results suggested that Op-IAP is a unique member of the IAP family since it functions to block apoptosis by selectively targeting an apical caspase (Sf-caspase-X) as well as cellular pro-apoptotic proteins.