Paul D. Friesen

Expression of the Baculovirus p35 Gene Inhibits Mammalian Neural Cell Death

Expression of the apoptosis suppressor genep35, derived from the baculovirus Autographa californica nuclear polyhedrosis virus, markedly inhibited the cell death of stably transfected mammalian neural cells whether the cell death was induced by glucose withdrawal, calcium ionophore, or serum withdrawal. The p35 protein, which is required to block virus‐induced apoptosis of cultured insect cells, is only the second gene product shown to block mammalian neural cell death, with Bcl‐2 being the first. Because there is no apparent homology between p35 and Bcl‐2, the existence of a cellular death program that may be modulated at multiple points is suggested/Furthermore, these findings demonstrate that the putative cellular death program is conserved across species and cell types.

Baculovirus p35 prevents developmentally programmed cell death and rescues a ced-9 mutant in the nematode Caenorhabditis elegans

Programmed cell death, or apoptosis, occurs throughout the course of normal development in most animals and can also be elicited by a number of stimuli such as growth factor deprivation and viral infection. Certain morphological and biochemical characteristics of programmed cell death are similar among different tissues and species. During development of the nematode Caenorhabditis elegans, a single genetic pathway promotes the death of selected cells in a lineally fixed pattern. This pathway appears to be conserved among animal species. The baculovirus p35‐encoding gene (p35) is an inhibitor of virus‐induced apoptosis in insect cells. Here we demonstrate that expression of p35 in C. elegans prevents death of cells normally programmed to die. This suppression of developmentally programmed cell death results in appearance of extra surviving cells. Expression of p35 can rescue the embryonic lethality of a mutation in ced‐9, an endogenous gene homologous to the mammalian apoptotic suppressor bcl‐2, whose absence leads to ectopic cell deaths. These results support the hypothesis that viral infection can activate the same cell death pathway as is used during normal development and suggest that baculovirus p35 may act downstream or independently of ced‐9 in this pathway.

Regulation of Baculovirus Early Gene Expression

Baculovirus multiplication culminates in high-level production of two temporally and morphologically distinct forms of infectious progeny (budded virus and occluded virus) in a process unique among animal viruses. Regulation of the 100 or more open reading frames required to accomplish productive infection is highly complex and involves sequential and coordinated expression of early, late, and very late genes. In the cascade of viral regulatory events, successive stages of virus replication are dependent on proper expression of genes within the preceding stage. Thus, critical to baculovirus replicative success is the appropriate expression and regulation of early genes. The products of early viral genes function to both accelerate replicative events and to prepare the host cell for virus multiplication, which represents an enormous tax on cellular biosynthetic capacity. Specific early genes are also essential for virus-mediated regulation of the host, including the control of larval molting and evasion of host antiviral responses such as apoptosis (see Chapters 10 and 11, this volume). Thus, early baculovirus genes collectively contribute to host range determination.

Crystal structure of baculovirus P35: role of a novel reactive site loop in apoptotic caspase inhibition

The aspartate‐specific caspases are critical protease effectors of programmed cell death and consequently represent important targets for apoptotic intervention. Baculovirus P35 is a potent substrate inhibitor of metazoan caspases, a property that accounts for its unique effectiveness in preventing apoptosis in phylogenetically diverse organisms. Here we report the 2.2 Å resolution crystal structure of P35, the first structure of a protein inhibitor of the death caspases. The P35 monomer possesses a solvent‐exposed loop that projects from the proteins main β‐sheet core and positions the requisite aspartate cleavage site at the loops apex. Distortion or destabilization of this reactive site loop by site‐directed mutagenesis converted P35 to an efficient substrate which, unlike wild‐type P35, failed to interact stably with the target caspase or block protease activity. Thus, cleavage alone is insufficient for caspase inhibition. These data are consistent with a new model wherein the P35 reactive site loop participates in a unique multi‐step mechanism in which the spatial orientation of the loop with respect to the P35 core determines post‐cleavage association and stoichiometric inhibition of target caspases.

Caspase Inhibitor P35 and Inhibitor of Apoptosis Op-IAP Block in Vivo Proteolytic Activation of an Effector Caspase at Different Steps

Signal-induced activation of caspases, the critical protease effectors of apoptosis, requires proteolytic processing of their inactive proenzymes. Consequently, regulation of procaspase processing is critical to apoptotic execution. We report here that baculovirus pancaspase inhibitor P35 and inhibitor of apoptosis Op-IAP prevent caspase activation in vivo, but at different steps. By monitoring proteolytic processing of endogenousSf-caspase-1, an insect group II effector caspase, we show that Op-IAP blocked the first activation cleavage at TETD↓G between the large and small caspase subunits. In contrast, P35 failed to affect this cleavage, but functioned downstream to block maturation cleavages (DXXD↓(G/A)) of the large subunit. Substitution of P35s reactive site residues with TETDG failed to increase its effectiveness for blocking TETD↓G processing of pro-Sf-caspase-1, despite wild-type function for suppressing apoptosis. These data are consistent with the involvement of a novel initiator caspase that is resistant to P35, but directly or indirectly inhibitable by Op-IAP. The conservation of TETD↓G processing sites among insect effector caspases, including Drosophila drICE and DCP-1, suggests that in vivo activation of these group II caspases involves a P35-insensitive caspase and supports a model wherein apical and effector caspases function through a proteolytic cascade to execute apoptosis in insects.

Baculovirus apoptotic suppressor P49 is a substrate inhibitor of initiator caspases resistant to P35 in vivo.

Caspases play a critical role in the execution of metazoan apoptosis and are thus attractive therapeutic targets for apoptosis‐associated diseases. Here we report that baculovirus P49, a homolog of pancaspase inhibitor P35, prevents apoptosis in invertebrates by inhibiting an initiator caspase that is P35 insensitive. Consequently P49 blocked proteolytic activation of effector caspases at a unique step upstream from that affected by P35 but downstream from inhibitor of apoptosis Op‐IAP. Like P35, P49 was cleaved by and stably associated with its caspase target. Ectopically expressed P49 blocked apoptosis in cultured cells from a phylogenetically distinct organism, Drosophila melanogaster. Furthermore, P49 inhibited human caspase‐9, demonstrating its capacity to affect a vertebrate initiator caspase. Thus, P49 is a substrate inhibitor with a novel in vivo specificity for a P35‐insensitive initiator caspase that functions at an evolutionarily conserved step in the caspase cascade. These data indicate that activated initiator caspases provide another effective target for apoptotic intervention by substrate inhibitors.

Baculovirus DNA Replication-Specific Expression Factors Trigger Apoptosis and Shutoff of Host Protein Synthesis during Infection

ABSTRACT 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 (AcMNPV). Transfection with double-stranded RNA (dsRNA) complementary to the AcMNPV late expression factors (lefs) that are designated as replicative lefs (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 lefs (lef-8, lef-9, p47, and pp31) blocked late gene expression without affecting virus DNA replication. Thus, both classes of lefs functioned during infection as defined. Silencing the replicative lefs prevented AcMNPV-induced apoptosis of Spodoptera cells, whereas silencing the nonreplicative lefs did not. Thus, the activity of replicative lefs or virus DNA replication is sufficient to trigger apoptosis. Confirming this conclusion, AcMNPV-induced apoptosis was suppressed by silencing the replicative lefs in cells from a divergent species, Drosophila melanogaster. Silencing replicative but not nonreplicative lefs also abrogated AcMNPV-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.

The BIR motifs mediate dominant interference and oligomerization of inhibitor of apoptosis Op-IAP.

ABSTRACT The defining structural motif of the inhibitor of apoptosis (iap) protein family is the BIR (baculovirusiap repeat), a highly conserved zinc coordination domain of ∼70 residues. Although the BIR is required for inhibitor-of-apoptosis (IAP) function, including caspase inhibition, its molecular role in antiapoptotic activity in vivo is unknown. To define the function of the BIRs, we investigated the activity of these structural motifs within Op-IAP, an efficient, virus-derived IAP. We report here that Op-IAP1–216, a loss-of-function truncation which contains two BIRs but lacks the C-terminal RING motif, potently interfered with Op-IAPs capacity to block apoptosis induced by diverse stimuli. In contrast, Op-IAP1–216 had no effect on apoptotic suppression by caspase inhibitor P35. Consistent with a mechanism of dominant inhibition that involves direct interaction between Op-IAP1–216 and full-length Op-IAP, both proteins formed an immunoprecipitable complex in vivo. Op-IAP also self-associated. In contrast, the RING motif-containing truncation Op-IAP183–268 failed to interact with or interfere with Op-IAP function. Substitution of conserved residues within BIR 2 caused loss of dominant inhibition by Op-IAP1–216 and coincided with loss of interaction with Op-IAP. Thus, residues encompassing the BIRs mediate dominant inhibition and oligomerization of Op-IAP. Consistent with dominant interference by interaction with an endogenous cellular IAP, Op-IAP1–216 also lowered the survival threshold of cultured insect cells. Taken together, these data suggest a new model wherein the antiapoptotic function of IAP requires homo-oligomerization, which in turn mediates specific interactions with cellular apoptotic effectors.

Bidirectional transcription from a solo long terminal repeat of the retrotransposon TED: symmetrical RNA start sites.

A single copy of the retrotransposon TED was found integrated within the DNA genome of the insect baculovirus, Autographa californica nuclear polyhedrosis virus. After excision of the element from the viral genome, a single long terminal repeat (LTR) remained behind. We have examined the effect of this solo TED LTR on the local pattern of viral transcription. Most prominent was the transcription of two sets of abundant RNAs; both originated within the LTR but extended in opposite directions into flanking viral genes. By promoting symmetric transcription of adjacent genes, the solo LTR has the capacity to activate or repress gene expression in two directions. Primer extension analysis demonstrated that the divergent LTR transcripts were initiated near the same point within a 22-base-pair sequence having hyphenated twofold symmetry. Analogous symmetries at the initiation sites of other retrotransposon LTRs, including copia and Ty, suggested that these sequences serve to establish the precise start for transcription.

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 TETD230-G by a DIAP1-inhibitable, P49/P35-resistant protease and then at the prodomain junction DHTD28-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.