Apoptosis is an important antivirus defense. To define the poorly understood pathways by which invertebrates respond to viruses by inducing apoptosis, we have identified replication events that trigger apoptosis in baculovirus-infected cells. We used RNA silencing to ablate factors required for multiplication of Autographa californica multicapsid nucleopolyhedrovirus (Ac M NPV). Transfection with double-stranded RNA (dsRNA) complementary to the Ac M NPV late expression factors ( lef s) that are designated as replicative lef s ( lef-1 , lef-2 , lef-3 , lef-11 , p143 , dnapol , and ie-1 / ie-0 ) blocked virus DNA synthesis and late gene expression in permissive Spodoptera frugiperda cells. dsRNAs specific to designated nonreplicative lef s ( lef-8 , lef-9 , p47 , and pp31 ) blocked late gene expression without affecting virus DNA replication. Thus, both classes of lef s functioned during infection as defined. Silencing the replicative lef s prevented Ac M NPV-induced apoptosis of Spodoptera cells, whereas silencing the nonreplicative lef s did not. Thus, the activity of replicative lef s or virus DNA replication is sufficient to trigger apoptosis. Confirming this conclusion, Ac M NPV-induced apoptosis was suppressed by silencing the replicative lef s in cells from a divergent species, Drosophila melanogaster . Silencing replicative but not nonreplicative lef s also abrogated Ac M NPV-induced shutdown of host protein synthesis, suggesting that virus DNA replication triggers inhibition of host biosynthetic processes and that apoptosis and translational arrest are linked. Our findings suggest that baculovirus DNA replication triggers a host cell response similar to the DNA damage response in vertebrates, which causes translational arrest and apoptosis. Pathways for detecting virus invasion and triggering apoptosis may therefore be conserved between insects and mammals.

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.

Immediate early viral protein IE1 is a potent transcriptional activator encoded by baculoviruses. Although the requirement of IE1 for multiplication of Autographa californica multicapsid nucleopolyhedrovirus (Ac M NPV) is well established, the functional roles of IE1 during infection are unclear. Here, we used RNA interference to ablate IE1, plus its splice variant IE0, and thereby define in vivo activities of these early proteins, including gene-specific regulation and induction of host cell apoptosis. Confirming an essential replicative role, simultaneous ablation of IE1 and IE0 by gene-specific double-stranded RNAs inhibited Ac M NPV late gene expression, reduced yields of budded virus by more than 1,000-fold, and blocked production of occluded virus particles. Depletion of IE1 and IE0 had no effect on early expression of the envelope fusion protein gene gp64 but abolished early expression of the caspase inhibitor gene p35 , which is required for prevention of virus-induced apoptosis. Thus, IE1 is a positive, gene-specific transactivator. Whereas an Ac M NPV p35 deletion mutant caused widespread apoptosis in permissive Spodoptera frugiperda cells, ablation of IE1 and IE0 prevented this apoptosis. Silencing of ie-1 also prevented Ac M NPV-induced apoptosis in nonpermissive Drosophila melanogaster cells. Thus, de novo synthesis of IE1 is required for virus-induced apoptosis. We concluded that IE1 causes apoptosis directly or contributes indirectly by promoting virus replication events that subsequently trigger cell death. This study reveals that IE1 is a gene-selective transcriptional activator which is required not only for expedition of virus multiplication but also for blocking of its own proapoptotic activity by upregulation of baculovirus apoptotic suppressors.

Baculovirus-encoded inhibitor of apoptosis (IAP) proteins likely evolved from their host cell IAP homologs, which function as critical regulators of cell death. Despite their striking relatedness to cellular IAPs, including the conservation of two baculovirus IAP repeat (BIR) domains and a C-terminal RING, viral IAPs use an unresolved mechanism to suppress apoptosis in insects. To define this mechanism, we investigated Op-IAP3, the prototypical IAP from baculovirus OpMNPV. We found that Op-IAP3 forms a stable complex with SfIAP, the native, short-lived IAP of host insect Spodoptera frugiperda . Long-lived Op-IAP3 prevented virus-induced SfIAP degradation, which normally causes caspase activation and apoptosis. In uninfected cells, Op-IAP3 also increased SfIAP steady-state levels and extended SfIAP's half-life. Conversely, SfIAP stabilization was lost or reversed in the presence of mutated Op-IAP3 that was engineered for reduced stability. Thus, Op-IAP3 stabilizes SfIAP and preserves its antiapoptotic function. In contrast to SfIAP, Op-IAP3 failed to bind or inhibit native Spodoptera caspases. Furthermore, BIR mutations that abrogate binding of well-conserved IAP antagonists did not affect Op-IAP3's capacity to prevent virus-induced apoptosis. Remarkably, Op-IAP3 also failed to prevent apoptosis when endogenous SfIAP was ablated by RNA silencing. Thus, Op-IAP3 requires SfIAP as a cofactor. Our findings suggest a new model wherein Op-IAP3 interacts directly with SfIAP to maintain its intracellular level, thereby suppressing virus-induced apoptosis indirectly. Consistent with this model, Op-IAP3 has evolved an intrinsic stability that may serve to repress signal-induced turnover and autoubiquitination when bound to its targeted cellular IAP. IMPORTANCE The IAPs were first discovered in baculoviruses because of their potency for preventing apoptosis. However, the antiapoptotic mechanism of viral IAPs in host insects has been elusive. We show here that the prototypical viral IAP, Op-IAP3, blocks apoptosis indirectly by associating with unstable, autoubiquitinating host IAP in such a way that cellular IAP levels and antiapoptotic activities are maintained. This mechanism explains Op-IAP3's requirement for native cellular IAP as a cofactor and the dispensability of caspase inhibition. Viral IAP-mediated preservation of the host IAP homolog capitalizes on normal IAP-IAP interactions and is likely the result of viral IAP evolution in which degron-mediated destabilization and ubiquitination potential have been reduced. This mechanism illustrates another novel means by which DNA viruses incorporate host death regulators that are modified for resistance to host regulatory controls for the purpose of suppressing host cell apoptosis and acquiring replication advantages.

The molecular mechanisms by which RNA viruses induce apoptosis and apoptosis-associated pathology are not fully understood. Here we show that flock house virus (FHV), one of the simplest RNA viruses (family, Nodaviridae ), induces robust apoptosis of permissive Drosophila Line-1 (DL-1) cells. To define the pathway by which FHV triggers apoptosis in this model invertebrate system, we investigated the potential role of Drosophila apoptotic effectors during infection. Suggesting the involvement of host caspases, the pancaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluromethylketone ( z -VAD-fmk) prevented FHV-induced cytopathology and prolonged cell survival. RNA interference-mediated ablation of the principal Drosophila effector caspase DrICE or its upstream initiator caspase DRONC prevented FHV-induced apoptosis and demonstrated direct participation of this intrinsic caspase pathway. Prior to the FHV-induced activation of DrICE, the intracellular level of inhibitor-of-apoptosis (IAP) protein DIAP1, the principal caspase regulator in Drosophila melanogaster , was dramatically reduced. DIAP1 was depleted despite z -VAD-fmk-mediated caspase inhibition during infection, suggesting that the loss of DIAP1 was caused by an upstream FHV-induced signal. The RNA interference-mediated knockdown of DIAP1 caused rapid and uniform apoptosis of DL-1 cells and thus indicated that DIAP1 depletion is sufficient to trigger apoptosis. Confirming this conclusion, the elevation of intracellular DIAP1 levels in stable diap1 -transfected cells blocked caspase activation and prevented FHV-induced apoptosis. Collectively, our findings suggest that DIAP1 is a critical sensor of virus infection, which upon virus-signaled depletion relieves caspase inhibition, which subsequently executes apoptotic death. Thus, our study supports the hypothesis that altering the level or the activity of cellular IAP proteins is a general mechanism by which RNA viruses trigger apoptosis.

Immediate-early protein IE1 is a principal regulator of viral transcription and a contributor to origin-specific DNA replication of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (Ac M NPV). Since these viral functions involve interaction of dimeric IE1 with palindromic homologous region ( hr ) enhancer-origin elements of the Ac M NPV genome within the nucleus, it is presumed that proper nuclear transport of IE1 is essential for productive infection. To investigate the mechanisms of IE1 nuclear import, we analyzed the effect of site-directed mutations on IE1 subcellular distribution. As demonstrated by fluorescence microscopy and biochemical fractionation of plasmid-transfected cells, wild-type IE1 localized predominantly to the nucleus. Substitution or deletion of amino acid residues within a positively charged domain (residues 534 to 538) adjacent to IE1's oligomerization motif impaired nuclear import and caused loss of transactivation. Moreover, upon coexpression, these import-defective mutations prevented nuclear entry of wild-type IE1. In contrast, double-mutated IE1 defective for both nuclear import and dimerization failed to block nuclear entry or transactivation by wild-type IE1. Thus, import-defective IE1 dominantly interfered with wild-type IE1 by direct interaction and cytosolic trapping. Collectively, our data indicate that the small basic domain encompassing residues R ⁵³⁷ and R ⁵³⁸ constitutes a novel nuclear localization element that functions only upon IE1 dimerization. These findings support a model wherein IE1 oligomerizes within the cytosol as a prerequisite for nuclear entry and subsequent high-affinity interaction with the symmetrical binding sites comprising Ac M NPV hr enhancer-origin elements.