Active Depletion of Host Cell Inhibitor-of-Apoptosis Proteins Triggers Apoptosis upon Baculovirus DNA Replication

Abstract

Apoptosis is an important antivirus defense by virtue of its impact on virus multiplication and pathogenesis. To define molecular mechanisms by which viruses are detected and the apoptotic response is initiated, we examined the antiviral role of host inhibitor-of-apoptosis (IAP) proteins in insect cells. We report here that the principal IAPs, DIAP1 and SfIAP, of the model insects Drosophila melanogaster and Spodoptera frugiperda, respectively, are rapidly depleted and thereby inactivated upon infection with the apoptosis-inducing baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Virus-induced loss of these host IAPs triggered caspase activation and apoptotic death. Elevation of IAP levels by ectopic expression repressed caspase activation. Loss of host IAP in both species was triggered by AcMNPV DNA replication. By using selected inhibitors, we found that virus-induced IAP depletion was mediated in part by the proteasome but not by caspase cleavage. Consistent with this conclusion, mutagenic disruption of the SfIAP RING motif, which acts as an E3 ubiquitin ligase, stabilized SfIAP during infection. Importantly, SfIAP was also stabilized upon the removal of its 99-residue N-terminal leader, which serves as a critical determinant of IAP turnover. These data indicated that a host pathway initiated by virus DNA replication and acting through instability motifs embedded within IAP triggers IAP depletion and thereby causes apoptosis. Taken together, the results of our study suggest that host modulation of cellular IAP levels is a conserved mechanism by which insects mount an apoptotic antiviral response. Thus, host IAPs may function as critical sentinels of virus invasion in insects.

Baculoviruses Modulate a Proapoptotic DNA Damage Response To Promote Virus Multiplication

“…3B, lanes 3 and 4). Confirming the efficacy of zVAD-fmk, virus-induced processing of the Drosophila effector caspase DrICE to its active large and small subunits (27,64) was fully blocked (compare lanes 3 and 4). We concluded that the DDR was caspase independent and thus triggered by vDNA replication-dependent events upstream of apoptosis.…”

“…4C). Active DrICE is a principal mediator of baculovirus-induced apoptosis in DL-1 cells (27,64). Ruling out the possibility that caffeine blocked virus entry or early gene expression, levels of intracellular IE1 were comparable up through 10 mM caffeine (Fig.…”

“…Through unknown events associated with AcMNPV DNA replication, the principal IAP of Spodoptera is rapidly depleted as a result of instability motifs embedded within its N-terminal leader domain (9,64). Likewise, AcMNPV DNA replication causes depletion of the Drosophila melanogaster IAP (DIAP1), which leads to widespread apoptosis in cultured cells derived from this distantly related insect species (54,64). Thus, baculoviruses provide a unique opportunity to define conserved pathways by which vDNA replication engages the host apoptotic program.…”

“…Caspase activation occurs upon virus-signaled destruction of the host's inhibitor-of-apoptosis (IAP) proteins, which normally repress caspase activity (reviewed in reference 17). Through unknown events associated with AcMNPV DNA replication, the principal IAP of Spodoptera is rapidly depleted as a result of instability motifs embedded within its N-terminal leader domain (9,64). Likewise, AcMNPV DNA replication causes depletion of the Drosophila melanogaster IAP (DIAP1), which leads to widespread apoptosis in cultured cells derived from this distantly related insect species (54,64).…”

“…A critical role for the host DDR in baculovirus-induced apoptosis was suggested by the finding that the prototype baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) triggers apoptosis through events associated with the replication of its 134-kb DNA genome ( Fig. 1) (54,64). The replication of mammalian virus DNA (vDNA) is a well-established initiator of the DDR, which is also a trigger for apoptosis in both vertebrates and invertebrates (reviewed in references 61 and 66).…”