Insect Inhibitor-of-Apoptosis (IAP) Proteins Are Negatively Regulated by Signal-Induced N-Terminal Degrons Absent within Viral IAP Proteins

Abstract

Inhibitor-of-apoptosis (IAP) proteins are key regulators of the innate antiviral response by virtue of their capacity to respond to signals affecting cell survival. In insects, wherein the host IAP provides a primary restriction to apoptosis, diverse viruses trigger rapid IAP depletion that initiates caspase-mediated apoptosis, thereby limiting virus multiplication. We report here that the Nterminal leader of two insect IAPs, Spodoptera frugiperda SfIAP and Drosophila melanogaster DIAP1, contain distinct instability motifs that regulate IAP turnover and apoptotic consequences. Functioning as a protein degron, the cellular IAP leader dramatically shortened the life span of a long-lived viral IAP (Op-IAP3) when fused to its N terminus. The SfIAP degron contains mitogen-activated kinase (MAPK)-like regulatory sites, responsible for MAPK inhibitor-sensitive phosphorylation of SfIAP. Hyperphosphorylation correlated with increased SfIAP turnover independent of the E3 ubiquitin-ligase activity of the SfIAP RING, which also regulated IAP stability. Together, our findings suggest that the SfIAP phospho-degron responds rapidly to a signal-activated kinase cascade, which regulates SfIAP levels and thus apoptosis. The N-terminal leader of dipteran DIAP1 also conferred virus-induced IAP depletion by a caspase-independent mechanism. DIAP1 instability mapped to previously unrecognized motifs that are not found in lepidopteran IAPs. Thus, the leaders of cellular IAPs from diverse insects carry unique signalresponsive degrons that control IAP turnover. Rapid response pathways that trigger IAP degradation and initiate apoptosis independent of canonical prodeath gene (Reaper-Grim-Hid) expression may provide important innate immune advantages. Furthermore, the elimination of these response motifs within viral IAPs, including those of baculoviruses, explains their unusual stability and their potent antiapoptotic activity. IMPORTANCEApoptosis is an effective means by which a host controls virus infection. In insects, inhibitor-of-apoptosis (IAP) proteins act as regulatory sentinels by responding to cellular signals that determine the fate of infected cells. We discovered that lepidopteran (moth and butterfly) IAPs, which are degraded upon baculovirus infection, are controlled by a conserved phosphorylation-sensitive degron within the IAP N-terminal leader. The degron likely responds to virus-induced kinase-specific signals for degradation through SKP1/ Cullin/F-box complex-mediated ubiquitination. Such signal-induced destruction of cellular IAPs is distinct from degradation caused by well-known IAP antagonists, which act to expel IAP-bound caspases. The major implication of this study is that insects have multiple signal-responsive mechanisms by which the sentinel IAPs are actively degraded to initiate host apoptosis. Such diversity of pathways likely provides insects with rapid and efficient strategies for pathogen control. Furthermore, the absence of analogous degrons in virus-encoded IAPs explains their relative stability ...

Protection of Insects against Viral Infection by Apoptosis-Dependent Phagocytosis

“…We made a similar observation: DIAP1 decreased in S2 cells after DCV infection. The same research group recently reported another mechanism for the degradation of DIAP1 that is independent of Reaper, Hid, and Grim (66). Therefore, it is important to elucidate the mechanism by which the productive infection of DCV brings about a reduction in the level of this caspase inhibitor for the induction of apoptosis in Drosophila cells.…”

Baculovirus Inhibitor-of-Apoptosis Op-IAP3 Blocks Apoptosis by Interaction with and Stabilization of a Host Insect Cellular IAP

“…Thus, as expected for critical regulators of cell death, SfIAP and DIAP1 are highly responsive to host degradative signals (8,21,(27)(28)(29)(30)(31). Consequently, factors that positively or negatively affect IAP levels modulate apoptosis, including that triggered by virus infection.…”

“…Similarly, SfIAP and Op-IAP3 also exist as homodimeric complexes, which can be dominantly inhibited by various truncations of the same (8,11). Viral Op-IAP3, which lacks a degron-containing leader, is highly stable and resistant to signal-induced turnover (27). It inhibits apoptosis in baculovirus-infected Spodoptera cells, acting at a step upstream of initiator caspase activation (34)(35)(36)(37).…”

“…Consequently, factors that positively or negatively affect IAP levels modulate apoptosis, including that triggered by virus infection. In the case of SfIAP, a phospho-degron embedded within the N-terminal leader contributes to this turnover via proteosome-mediated degradation (27). The C-terminal RING also negatively regulates SfIAP stability, probably by autoubiquitination.…”

“…For infections, extracellular budded virus (BV) was added to monolayers at a multiplicity of infection (MOI) of 10 PFU per cell. AcMNPV reporter assays used recombinant v⌬p35/lacZ wherein lacZ is under the control of the polyhedrin promoter (8,26,27,45). Intracellular ␤-galactosidase was measured 24 h after infection by using a Galacto-Light Plus ␤-galactosidase chemiluminescent assay (Tropix) and is reported as the average activities Ϯ standard deviations obtained from triplicate assays.…”