Baculovirus Caspase Inhibitors P49 and P35 Block Virus-Induced Apoptosis Downstream of Effector Caspase DrICE Activation in Drosophila melanogaster Cells

Abstract

Baculoviruses induce widespread apoptosis in invertebrates. To better understand the pathways by which these DNA viruses trigger apoptosis, we have used a combination of RNA silencing and overexpression of viral and host apoptotic regulators to identify cell death components in the model system of Drosophila melanogaster. Here we report that the principal effector caspase DrICE is required for baculovirus-induced apoptosis of Drosophila DL-1 cells as demonstrated by RNA silencing. proDrICE was proteolytically cleaved and activated during infection. Activation was blocked by overexpression of the cellular inhibitor-of-apoptosis proteins DIAP1 and SfIAP but not by the baculovirus caspase inhibitor P49 or P35. Rather, the substrate inhibitors P49 and P35 prevented virus-induced apoptosis by arresting active DrICE through formation of stable inhibitory complexes. Consistent with a two-step activation mechanism, proDrICE was cleaved at the large/small subunit junction TETD 230 -G by a DIAP1-inhibitable, P49/P35-resistant protease and then at the prodomain junction DHTD 28 -A by a P49/P35-sensitive protease. Confirming that P49 targeted DrICE and not the initiator caspase DRONC, depletion of DrICE by RNA silencing suppressed virus-induced cleavage of P49. Collectively, our findings indicate that whereas DIAP1 functions upstream to block DrICE activation, P49 and P35 act downstream by inhibiting active DrICE. Given that P49 has the potential to inhibit both upstream initiator caspases and downstream effector caspases, we conclude that P49 is a broad-spectrum caspase inhibitor that likely provides a selective advantage to baculoviruses in different cellular backgrounds.

p53-Independent Apoptosis Limits DNA Damage-Induced Aneuploidy

“…Similarly, in discs expressing the anti-apoptotic baculovirus protein p35, which inhibits the effector caspases Drice and DCP-1 (Xue and Horvitz 1995; Kondo et al 2006;Xu et al 2006;Baum et al 2007;Lannan et al 2007), no acridine orange staining was observed at any time point ( Figure S2E). Because p35 inhibits effector caspase activity after proteolytic cleavage, cleaved caspase-3 staining can still be observed once these caspases are activated ( Yuet al 2002).…”

Nodavirus-Induced Membrane Rearrangement in Replication Complex Assembly Requires Replicase Protein A, RNA Templates, and Polymerase Activity

“…The generation of dsRNA and transfection methods were performed as previously described (30). Cells were transfected with ago2-specific dsRNA in suspension and maintained in monolayer for 1 day at 27°C prior to use.…”

Spatial, temporal and molecular hierarchies in the link between death, delamination and dorsal closure

“…The effects of these perturbations fell into two classes. All anti-apoptotic genotypes showed a complete [bantam overexpression, known to downregulate Drosophila hid (Brennecke et al, 2003) and expression of p35, a baculoviral inhibitor of effector caspases (Lannan et al, 2007)] or nearly complete (DIAP1 overexpression) suppression of delamination and delayed closure, evident from the absence of rosettes. Pro-apoptotic perturbations (overexpression of hid or reaper) significantly increased the number of delaminations and hastened closure ( Fig.…”