PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Annu Rev Microbiol. Author manuscript; available in PMC Oct 7, 2008.
Published in final edited form as:
PMCID: PMC2562643
NIHMSID: NIHMS54264
Viral Subversion of Apoptotic Enzymes: Escape from Death Row*
Sonja M. Best
Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana 59840; email: sbest/at/niaid.nih.gov
To prolong cell viability and facilitate replication, viruses have evolved multiple mechanisms to inhibit the host apoptotic response. Cellular proteases such as caspases and serine proteases are instrumental in promoting apoptosis. Thus, these enzymes are logical targets for virus-mediated modulation to suppress cell death. Four major classes of viral inhibitors antagonize caspase function: serpins, p35 family members, inhibitor of apoptosis proteins, and viral FLICE-inhibitory proteins. Viruses also subvert activity of the serine proteases, granzyme B and HtrA2/Omi, to avoid cell death. The combined efforts of viruses to suppress apoptosis suggest that this response should be avoided at all costs. However, some viruses utilize caspases during replication to aid virus protein maturation, progeny release, or both. Hence, a multifaceted relationship exists between viruses and the apoptotic response they induce. Examination of these interactions contributes to our understanding of both virus pathogenesis and the regulation of apoptotic enzymes in normal cellular functions.
Keywords: caspase inhibition, serine protease, CrmA, p35, IAP, vFLIP
The successful replication of a virus within an individual cell requires a remarkable cascade of interactions between virus and host, beginning at the first engagement of the cell receptor to the final release of progeny virions. Viruses utilize everything from cellular enzymes and transcription factors to membranes and organelles to facilitate their replication (11). However, hijacking of the cell by a virus is not without a potent antiviral response. One such response is apoptosis, the genetically and biochemically controlled process of cell death that functions in development and homeostasis of multicellular organisms through selective removal of unwanted or damaged cells (120). Apoptosis can also serve as an innate cellular response to infection that limits both the time and cellular machinery available for virus replication (120). Many of the key biochemical events that occur during apoptosis are mediated by proteases (62), the most important of which are caspases (cysteine-dependent aspartate-specific proteases). Not surprisingly, viruses from diverse families have evolved mechanisms to evade or delay cell death by suppressing the activity of caspases and other enzymes with central roles in the implementation of apoptosis. Examination of interactions between viruses and the host apoptotic machinery has contributed extensively to our understanding of virus replication and pathogenesis. Furthermore, the elucidation of how viruses modulate these responses has furthered our knowledge of apoptotic pathways and how they contribute to both normal and disease states. This review enumerates the specific mechanisms underlying virus-induced suppression of enzyme activity during apoptosis.
In its simplest form, apoptosis can be considered a two-step proteolytic pathway (92). The first is an initiating phase resulting in activation of initiator caspases. These caspases are responsible for the second execution phase by cleaving and activating executioner or effector caspases. During this second phase, effector caspases cleave target host proteins, culminating in the step-wise demise of the cell (66). Apoptosis is initiated through two general mechanisms: from outside the cell (extrinsic) or from within (intrinsic) (Figure 1). Extrinsic triggering of apoptosis occurs following ligation of death receptors by the tumor necrosis factor (TNF) superfamily including TNF, Fas ligand (FasL), and TNF-related apoptosis-inducing ligand (TRAIL) (Figure 1). Ligation of the TNF receptor family results in the formation of the death-inducing signaling complex (DISC) (63) required for activation of initiator caspases, particularly caspase-8 and caspase-10.
Apoptosis: refers to morphological changes occurring in a controlled process of cell death, resulting in membrane-bound cell fragments usually eliminated by phagocytosis
Caspase: cysteine-dependent aspartate-specific protease
Death induced signaling complex (DISC): multimeric assembly platform required for caspase activation induced after death receptor ligation
Figure 1
Figure 1
Apoptosis signaling pathways. Apoptosis is initiated extrinsically following ligation of death receptors including the Fas receptor. This results in the recruitment of the adaptor protein FADD and the formation of the death-inducing signaling complex (more ...)
Intrinsic activation of apoptosis is triggered following translocation of a proapoptotic Bcl-2 family member, Bid or Bax, to the mitochondria (133, 135) (Figure 1). In particular, cytosolic Bid is cleaved by caspases or other proapoptotic proteases to form truncated Bid (tBid), which then localizes to the mitochondria. Here, tBid interacts with other proapoptotic Bcl-2 member proteins such as Bax and/or Bak, resulting in pore formation and permeabilization of the outer mitochondrial membrane (45, 133). This initiates the release of proteins from the inner membrane into the cytosol. One such protein, cytochrome c, is central to this apoptosis program (45) because it binds to apoptosis protease-activating factor (Apaf-1) to induce the formation of an oligomeric assembly platform, termed the apoptosome (7, 92). The initiator caspase-9 is subsequently recruited to the apoptosome, resulting in its activation (92). A wide variety of cellular insults result in loss of mitochondrial membrane integrity, including aberrant calcium signaling, growth factor withdrawal, treatment with various cytotoxic agents, and endoplasmic reticulum stress. However, receptor-mediated cell death can also result in caspase-8-mediated cleavage of Bid, mitochondrial membrane permeabilization, and activation of caspase-9, thus amplifying apoptotic signaling pathways (133) (Figure 1). Active caspase-8, -10, and -9 then target effector caspases for cleavage. The individual pathways involved in initiating and regulating apoptosis are expanded upon in the following sections.
Apoptosome: multimeric assembly platform required for caspase-9 activation induced following disruption of mitochondrial membrane potential
As highlighted above, the key effector proteins activated during apoptosis and targeted by viruses for inhibition are caspases (1). There are currently 13 members of the mammalian caspase family, characterized by a near-absolute specificity for substrates containing an Asp in the P1 cleavage position. In addition, these enzymes contain both a Cys and a His in the active site that assist in peptide bond hydrolysis (24, 115). Caspases that function in apoptosis include caspase-2, -8, -9, -10, and -12 (the initiator caspases) as well as caspase-3, -6, -7, and -14 (the effector caspases). Caspase-1, -4, -5, and -11 function in inflammation.
Caspases exist in the cell as inactive procaspases, or zymogens, and based on the length of their prodomains, they can be classified in two main categories (66). The first category is the large-prodomain caspases (caspase-1, -2, -4, -5, -8, -9, -10, -11, and -12), which have a long prodomain characterized by death domain (DD) motifs including death effector domains (DEDs) and caspase activation and recruitment domains (CARDs) (90). These caspases are present in the cell as monomers and, following recruitment into multimeric protein platforms such as the DISC (63) or apoptosome (7, 92), dimerize and undergo a conformational change resulting in activation. The second category is the short-prodomain caspases (the initiator caspase-3, -6, -7, and -14). These caspases exist as preformed dimers and, in contrast to the large-prodomain enzymes, require proteolysis to be activated (99). Active caspases are obligate homodimers with each monomer composed of a large (17- to 20-kDa) subunit and a small (10- to 12-kDa) subunit (42).
Because of their central role in controlling cell fate decisions, caspases are subject to regulation at multiple levels, through prevention of activation as well as inhibition of activity following maturation (28). This property renders caspases a logical target for inhibition by viruses. Inhibition can be achieved through indirect mechanisms such as the virus-induced downregulation of death receptor expression (116) or the expression of secreted viral TNF receptor homologs (9), both of which prevent signaling at the cell surface. An important general mechanism viruses use to suppress caspase activation is inhibition of mitochondrial membrane permeabilization, thus preventing or delaying the release of cytochrome c. These approaches utilized by viruses to prevent apoptosis have been reviewed in detail elsewhere (2, 14, 17, 39, 47, 61, 107, 113).
Viruses can also directly antagonize enzyme function. This is achieved either by interacting with the caspase active site or by acting as competitive inhibitors of signaling molecules required for caspase activation. Four major classes of virus-encoded inhibitors directly regulate caspases: (a) the serine protease inhibitor (serpin) family, (b) the p35 family (for which no cellular homologs are known), (c) viral inhibitor of apoptosis proteins (vIAPs), and (d ) viral FLICE (Fas-associated death domain-like interleukin-1β-converting enzyme) inhibitory proteins, or viral FLICE inhibitory proteins (vFLIPs) (Table 1). Each class of inhibitor is discussed below.
DD: death domain
DED: death effector domain
Serpin: serine protease inhibitor
IAP: inhibitor of apoptosis protein
vFLIP: viral FLICE inhibitory protein
CrmA: cytokine response modifier A
Table 1
Table 1
Virus-encoded caspase inhibitors
CrmA: Building a Better Mousetrap
The first caspase inhibitor discovered was cytokine response modifier A (CrmA) encoded by cowpox virus, an orthopoxvirus. CrmA is a member of the serpin superfamily. It was originally identified as an inhibitor of interleukin-1β (IL-1β)-converting enzyme (ICE), now known as caspase-1, by preventing its ability to process the precursor of IL-1β into its active, secreted form (89). This discovery was made before it was understood that caspases were the central proteases of cell death (16). However, when the critical nature of caspases in apoptosis was determined following the discovery of CED-3 (a caspase that functions as both an initiator and an executioner) from Caenorhabditis elegans (84, 136), the ability of CrmA to inhibit apoptosis was tested. CrmA can inhibit apoptosis triggered by overexpression of caspase-1 (84) as well as by treatment of cells with TNF or ligation of Fas receptors. In addition to direct inhibition of caspase-1 in the low picomolar range, CrmA can also suppress caspase-8 and caspase-10 activity (44, 44, 139, 140), thus explaining its protective properties when apoptosis is initiated through death receptors (Table 1). CrmA can inhibit a wide range of caspases including caspase-3, -6, -7, and -9 (140). However, the efficiency of inhibition is generally lower than that demonstrated for caspase-1 and caspase-8, and caspase-9 is not thought to be an important target in vivo (27).
CrmA is unique among the serpins, as this family normally functions to suppress the activity of serine proteases rather than that of cysteine proteases. Serpins have a conserved core globular domain consisting of three β-sheets and eight to nine α-helices. CrmA maintains the serpin architecture and fold even though it lacks conserved features of the serpin superfamily including the D-helix, half of the A-helix, and part of the E-helix (91, 101). In addition to these differences, CrmA contains a pseudosubstrate of the caspases (Leu-Val-Ala-Asp303) within the serpin flexible reactive site loop (RSL), with Asp303 in the P1 position an unusual residue for a serpin.
RSL: reactive site loop
CrmA inhibits caspase-1 by a mechanism of conformational trapping similar to that determined for serpin inhibition of serine proteases (106, 110). The exposed RSL of CrmA is inserted into the caspase active site where the caspase catalytic cysteine is covalently linked to the serpin P1 residue. The RSL is then cleaved at the P1-P1' bond, leading to a radical conformational change whereby the N terminus of the newly cleaved loop inserts into the center of the major β-sheet of CrmA (sheet A) (Figure 2). This causes the caspase tethered to the P1 residue to be translocated to the opposite end of the serpin. The caspase is inactivated as its active site becomes conformationally distorted and trapped (106). In addition, the small subunit of caspase-1 and caspase-8 can disassociate from the large subunit during the trapping process, providing an additional irreversible inhibition of the protease (32). The structure of the serpin in complex with its target is less energy rich than its native structure (101), thus providing the energetics for this virus-encoded mousetrap.
Figure 2
Figure 2
Model of CrmA-mediated caspase inhibition. (a) Ribbon diagram of a serpin in its native state demonstrating the position of the reactive site loop (RSL). The crystal structure of native CrmA is not known so the structure of the related α1-antitrypsin (more ...)
Other members of the poxviruses encode serpins homologous to CrmA, including serine protease inhibitor (SPI)-1 and SPI-2 [in orthopoxviruses including vaccinia (31), ectromelia (118), and rabbitpox (76) viruses] or serpin-2 (SERP-2) from myxoma virus, a Leporipoxvirus (87). SERP-2 shares 34.9% identity at the amino acid level, and 57.3% similarity (87), with CrmA and can inhibit caspase-1, -8, and -10 in vitro (77) (Table 1). Both CrmA and SERP-2 are required for full virus virulence in their models of infection, chicken chorioallantoic membranes (CAMs) and rabbits, respectively. This property is dependent on the P1 Asp residue present in both proteins (77). Despite these similarities, SERP-2 and CrmA are not functionally interchangeable. For example, replacement of CrmA by SERP-2 enabled the resulting recombinant cowpox virus to inhibit apoptosis and reach wild-type virus yields in infected CAMs. However, SERP-2 did not block inflammation in this context (86), which is the original hallmark of CrmA function (89). Similarly, the reciprocal recombinant myxoma virus containing CrmA in place of SERP-2 was not fully virulent in its European rabbit host and did not cause the fulminant skin lesions characteristic of infection with wild-type virus (86). Both recombinant viruses could inhibit apoptosis of infected cells, suggesting that poxvirus serpins may have host-specific roles in control of inflammation.
DRONC: Drosophila Nedd2-like caspase
p35 Inhibition of Caspases: A Ménage à Trois
p35 is a broad-spectrum caspase inhibitor identified from the baculovirus, Autographa californica multiple Nucleopolyhedrovirus (AcMNPV) (23). This protein inhibits caspases from C. elegans (CED-3), Drosophila melanogaster (the effector caspase DrICE), Spodoptera frugiperda (the effector Sf-caspase-1), and mammals (caspase-1, -3, -6, -8, -7, and -10) (67, 130, 139) (Table 1). p35 is least effective against caspase-2 and caspase-9. The related p49 protein from Spodoptera littoralis Nucleopolyhedrovirus (SlNPV) (34) inhibits a wider range of caspases compared with p35, including caspase-2 and caspase-9 as well as Drosophila Nedd2-like caspase (DRONC), a caspase-9-like initiator caspase (56, 142) (Table 1). However, unlike p35, p49 cannot suppress DrICE activity, suggesting host-specific roles for this class of inhibitors (70). Recently, a homolog of the p35 protein, called p33, was discovered in Amsacta moorei entomopoxvirus (AmEPV), and it is the first homolog found outside of the baculoviruses (78). p33 is only 25% identical to p35 at the amino acid level but has strikingly similar predicted secondary structure and can inhibit similar effector caspases in vitro (78).
Similar to CrmA, p35 is a suicide inhibitor of caspases, acting as bait for the active caspase to attack. In addition, the exposed RSL of p35 contains P4-P1 residues, Asp-Gln-Met-Asp87, with the critical Asp residue in the P1 position. However, the molecular mechanism of inhibition differs between p35 and CrmA, as demonstrated for the p35-mediated inhibition of caspase-8 (74). Following insertion of the RSL into the caspase active site and cleavage of p35, the cleavage residue Asp87 forms a thioester intermediate with the caspase active site Cys (Cyscasp8). This enables the portion of the RSL distal to Asp87 to dissociate from the caspase, leading to dramatic conformational changes in p35 (Figure 3). This conformational change liberates the N terminus of p35 from where it is buried in the core of the molecule and enables it to insert into the caspase active site (74). During normal substrate cleavage by caspase-8, a water molecule is activated by the active site His to quickly hydrolyze the thioester intermediate. However, the p35 N-terminal cysteine residue (Cys2) attacks the thioester bond of Asp87 and excludes solvent, thereby trapping Asp87 at the thioester intermediate step. In addition, once attacked by the thiol of Cys2, the carbonyl carbon of Asp87 is no longer accessible to the free amino group of Cys2 much in the same way that water is excluded from attacking the Asp87-Cyscasp8 bond (74). In the absence of this second step, chemical ligation of the two ends is not completed and p35 does not become circularized. Instead, an equilibrium of reversible trans-thioesterification between Asp87-Cyscasp8 and Asp87-Cys2 is established (74), trapping the caspase active site in a futile ménage à trois (Figure 3). It is currently unknown if this mechanism of inhibition is limited to p35/caspase-8 interactions or if this is a general mechanism that contributes to the promiscuity of the p35 class of caspase inhibitors (103).
Suicide inhibitor: a protein that inhibits by irreversibly binding to its target
Figure 3
Figure 3
Mechanism of p35-mediated inhibition of caspase-8. (a) Worm diagram of p35 in its native state. The extreme N terminus (red ) is buried in the core of the protein. (b) Diagram of p35 after complex formation with caspase-8. Cleavage of the reactive site (more ...)
Viral Inhibitor of Apoptosis Proteins: The Ol' Bait and Switch
The first IAP was discovered in a genetic screen of baculoviruses searching for genes that could complement the deletion of p35 from AcMNPV (26). In addition to the baculoviruses, homologous IAPs are encoded by entomopoxviruses, iridoviruses, and African swine fever virus (21, 22, 29). Cellular homologs of IAPs also exist in diverse organisms, from yeast to humans, with eight identified in the human genome. However, all IAPs do not function in modulating apoptotic responses, as they have roles in diverse cellular functions including innate immunity to bacterial infection and the regulation of chromosome segregation during mitosis (reviewed in References 95 and 121). The IAP family is characterized by the presence of baculovirus IAP repeat domains (BIRs), a novel zinc binding fold of approximately 70 amino acids (52, 83). Cellular IAPs (cIAPs) contain up to three BIRs while viral IAPs (vIAPs) only contain one or two. In addition, the IAPs contain a really interesting new gene (RING) domain that possesses E3 ubiquitin ligase activity and is required for IAP-mediated suppression of apoptosis (121, 122).
To understand how vIAPs function to suppress caspase activity, it is important to understand the function and regulation of cIAPs during apoptosis. Of the human IAPs, X-linked IAP (XIAP), cellular IAP1 (c-IAP1), and c-IAP2 can directly bind specific caspases, including caspase-3, -7, and -9 in vitro (35, 98, 109). However, only XIAP can directly inhibit caspase activity, and therefore it is thought to be the primary IAP responsible for caspase inhibition in vivo (36). XIAP has two distinct domains that bind either to caspase-9 (via its BIR3) or to caspase-3 and caspase-7 (via the region immediately N-terminal to BIR2) (30), resulting in caspase inhibition (Figure 4a). Similar to XIAP, Drosophila IAP1 (DIAP1) is essential for negative regulation of the initiator caspase DRONC and the effector caspases DrICE and Drosophila caspase-1 (DCP-1). However, in this case, binding of caspases by the BIR1 domain of DIAP1 alone is not sufficient for inhibition. Following binding, DIAP1 is cleaved by Drosophila effector caspases at its N terminus, resulting in the targeting of DIAP1 for ubiquitin-dependent proteasome-mediated degradation. Presumably, the active caspase tethered to DIAP1 is also degraded (108, 109, 131).
Figure 4
Figure 4
Mechanism of vIAP inhibition of caspase activity. (a) Direct inhibition of caspases by cellular XIAP. The BIR3 domain of XIAP binds to caspase-9, and the region proximal to the XIAP BIR2 binds to caspase-3 and caspase-7 and suppresses their activity. (more ...)
Activity of the cellular antiapoptotic IAPs is modulated by IAP-interacting proteins often termed IAP antagonists. In humans and mice, the major IAP antagonist is termed SMAC (second mitochondrial activator of caspases) or DIABLO (direct IAP binding protein with low pI), respectively, and resides in the intermembrane space of the mitochondria (33, 123). The release of SMAC/DIABLO occurs in response to apoptotic stimuli along with cytochrome c, at which time it can bind to XIAP (72, 129) and displace the active caspase(s) to promote apoptosis. Drosophila has multiple IAP antagonists, including Reaper, HID (head-involution defective), Grim, and Sickle, that can bind to DIAP1 and antagonize its ability to bind to DRONC. These IAP antagonists share a common IAP binding motif [IBM (98), also known as the Reaper-HID-Grim (RHG) motif] that enables them to bind to and suppress the function of DIAP1 (128) and XIAP (Figure 4b), suggesting a mechanism of binding conserved between flies and humans. One function of Reaper, HID, and Grim, as well as the Drosophila ubiquitin conjugase-related protein termed Morgue, is to promote degradation of Drosophila IAPs (54, 95, 134). Degradation is dependent on a functional RING domain in the IAP itself (93, 127), suggesting that binding by the antagonist induces IAP autoubiquitination (95).
BIR: baculovirus IAP repeat
RING domain: specialized type of zinc-finger domain involved in protein-protein interactions that possesses E3-ubiquitin ligase activity
Given the complex roles of cIAPs in apoptosis suppression, how do vIAPs with anti-apoptotic activity function? vIAPs such as the baculovirus Op-IAP (from Orgyia pseudotsugata NPV) do not bind to insect or mammalian caspases (127), nor do they undergo caspase-mediated cleavage (108). As these two events are essential for XIAP- and DIAP1-mediated inhibition of caspases, respectively, it is unlikely that vIAPs function as direct caspase inhibitors. Instead, it is thought that vIAPs act as decoys for the IAP antagonists. Op-IAP contains two BIR domains and one RING domain. The BIR2 domain of Op-IAP can bind to Reaper, HID, and Grim (124126). Op-IAP can also efficiently bind SMAC/DIABLO via its BIR2 domain, a property required for its ability to prevent apoptosis when expressed in human cells (127). Furthermore, the RING domain of Op-IAP also contributes to IAP function, facilitating ubiquitination of itself and of HID (22, 49) or SMAC/DIABLO (127) and presumably degradation. Thus, Op-IAP and probably vIAPs in general function in an old-fashioned bait-and-switch tactic. They deceive the IAP antagonists and then disable them, permitting endogenous IAPs such as DIAP1 to inhibit caspases (Figure 4c).
The observations that cIAPs are conserved in diverse genomes, including flies and humans, and function in apoptosis by conserved mechanisms suggest that vIAPs might be encoded by a wide variety of viruses. However, such homologs have only been found in viruses that infect arthropods (22). This restricted presence of viral homologs is consistent with the relative role of IAPs in apoptotic programs of insects versus humans. The prevailing view is that induction of apoptosis in humans is regulated by the activation of inactive zymogens, whereas apoptosis in Drosophila is initiated by the liberation of constitutively active caspases from their complex with IAPs (94). Hence, vIAPs may impart a greater degree of caspase control if the virus's host is an arthropod rather than a higher vertebrate, thus creating the selective pressure required to retain IAPs as principal modulators of apoptosis. This suggestion is supported by the observation that known genomes of two entomopoxviruses do not encode serpins like their mammalian counterparts, although at least one of these, Amsacta moorei (AmEPV), has a functional IAP (71).
FADD: Fas-associated via death domain
Viruses FLIP Out Over the DISC
Signaling through death receptors, including TNF and Fas, generates oligomeric signaling assemblies such as the DISC (reviewed in References 7, 15, and 92) (Figure 5). The intracellular region of death receptors contains a DD, which in the case of Fas recruits the Fas-associated via death domain (FADD) adaptor protein via a homotypic interaction with the DD of FADD. FADD contains an additional DED that interacts with DEDs in the prodomains of caspase-8 and caspase-10. The recruitment of these initiator caspases into the DISC results in their dimerization and maturation. During this activation process, caspase N-terminal DEDs are removed by intermolecular processing, resulting in the release of caspases into the cytosol where they activate effector caspases (24).
Figure 5
Figure 5
Mechanism of vFLIP-mediated inhibition of caspase activation. Death-receptor induced apoptosis occurs following ligation of the receptor, recruitment of the adaptor protein FADD via its DD and higher-order DISC assembly. Procaspase-8 or procaspase-10 (more ...)
DISC assembly and caspase activation are negatively regulated by cellular FLIP (cFLIP) that directly competes with caspase-8 for recruitment to FADD (65) (Figure 5). Two major isoforms of cFLIP exist, the 26-kDa short form (cFLIPS) and the 55-kDa long form (cFLIPL) (112). cFLIPS contains two DEDs and completely inhibits proteolytic processing of caspase-8 (65). In contrast, in addition to two DEDs, cFLIPL contains a longer C terminus than does cFLIPS that closely resembles the overall structure of caspase-8 and caspase-10 (65). cFLIPL can form a heterodimer with caspase-8 through interactions between the DEDs and caspase-like domains. This association causes a conformational change in caspase-8, exposing the caspase active site (19, 81). The result is a partial autoprocessing of caspase-8 that in turn cleaves cFLIPL (19, 82). Cleavage of cFLIPL augments its ability to recruit TNF-receptor-associated factor 2 (TRAF2) and receptor-interacting protein 1 (RIP1) (60). These events facilitate NF-κB activation, which is the quintessential role of TNF signaling. A similar partial activation of caspase-8 may also be important in T-cell activation (reviewed in Reference 15).
vFLIPs (111) are encoded by several γ-herpesviruses and by the molluscum contagiosum virus (MCV) (10, 10, 55, 112). vFLIPs contain two DED domains and, similar to their cellular counterparts, interfere with recruitment, processing, and release of caspase-8 and caspase-10 following ligation of death receptors. However, the precise mechanism utilized by various viruses differs, as illustrated by their interactions with Fas receptor-induced DISC formation. vFLIP encoded by γ-herpesviruses is analogous to cFLIPS outcompeting caspases for recruitment to FADD (132) (Figure 5). In contrast, the MCV genome contains two vFLIPs, termed MC159 and MC160. MC159 interacts with both FADD and Fas (100) but does not compete with caspase-8 for recruitment. Instead, it binds at least two FADD molecules and disrupts FADD oligomerization (132). Hence, caspase inhibition results from the absence of FADD self-association and DISC formation (132) (Figure 5). In addition, MCV FLIPs are unique in that they have C-terminal extensions responsible for binding TRAF2 and TRAF3 and recruiting them to the Fas DISC in a FasL-dependent manner. The binding of TRAF3 in particular contributes to inhibition of Fas receptor internalization (114), a step that occurs after DISC formation and caspase-8 activation and is required for optimal induction of apoptosis. MCV-encoded vFLIPs also interact with proteins that regulate NF-κB activation including TRAFs, RIP1, NF-κB-inducing kinase (NIK), and inhibitor of κB-kinase 2 (IKK2) (20). Thus, MCV vFLIPs may impart multiple blocks following death receptor ligation, resulting in the inhibition of initiator caspase activation as well as the promotion of cellular survival signals such as NF-κB activation.
Although not a vFLIP, the E6 protein of human papillomavirus type 16 (HPV-16) also targets DISC assembly at multiple levels to suppress death receptor-induced apoptosis. The long isoform of E6 can bind to the DD of TNF receptor 1 and interfere with the recruitment of TNF receptor-associated via death domain (TRADD) and therefore DISC formation (41). In addition, E6 can bind to the DEDs of both FADD and procaspase-8. The consequences of this binding are twofold. First, E6 prevents the normal association of caspase-8 with FADD, and second, it accelerates the degradation of both molecules (40, 41) and prevents DISC formation. Furthermore, the large isoform of E6 accelerates degradation of the tumor suppressor p53 (40, 97), thus rendering cells less sensitive to apoptosis initiated by genotoxic stress. E6 is therefore a multifunctional protein (with functions additional to those described here) that blocks numerous initiating signals of apoptosis. Finally, the human cytomegalovirus UL36 gene product termed vICA also associates with procaspase-8 and blocks its activation, although it possesses no sequence homology with vFLIPs (102).
Effective antiviral immune responses involve the generation of virus-specific cytotoxic T lymphocytes (CTLs), whose function is to kill virus-infected cells. The two main weapons utilized by CTLs are perforin-/granzyme- and Fas-mediated apoptosis. Following recognition of virus-infected cells, perforin and granzymes are released by CTLs and are endocytosed by the target cell (5, 73). Perforin then polymerizes and inserts into the membrane of the endocytic vesicle, forming a pore through which granzymes including granzyme B are transported into the cytosol (73). Granzyme B is a unique serine protease owing to its strict requirement for Asp in the substrate P1 position. Apoptosis is initiated following direct cleavage and activation of caspase-3 by granzyme B, or following cleavage and activation of Bid (8, 51, 73). Truncated Bid translocates to the mitochondria and recruits Bax to facilitate alterations in membrane permeabilization, release of cytochrome c, and activation of downstream caspases (8, 51).
In addition to potently inhibiting caspases, CrmA can antagonize the proteolytic activity of granzyme B (88). Granzyme B is structurally and mechanistically distinct from the caspases, demonstrating a remarkable cross-class ability of CrmA to inhibit proteolysis (64). The 100,000-kDa (100 K) assembly protein of human adenovirus type 5 (Ad5−100K) is also a strong inhibitor of granzyme B (3, 5). The Ad5−100K protein has several essential functions in the adenovirus life cycle, including virus assembly, activation of late viral protein synthesis, and inhibition of cellular protein synthesis (53). Inhibition of granzyme B is absolutely dependent on Asp48 within a granzyme B consensus recognition sequence in the Ad5−100K protein (3). Although granzyme B from multiple species (human, rat, and mouse) can cleave Ad5−100K, these proteins cleave at different sites within the viral protein and only activity of human granzyme B is inhibited (4). The precise molecular mechanisms by which Ad5−100K prevents granzyme B activity are not known, although at least two intermolecular interactions are required. These are between the viral protein RSL and the protease active site, resulting in the cleavage of the former, as well as an additional interaction involving the extreme C-terminal residues of Ad5−100K (residues 688−781) that may stabilize interactions between protease and inhibitor (4).
The fencing match between host and virus does not end following the initial thrust with granzyme B and the parry of the adenovirus inhibitor. The human host has a riposte in the form of granzyme H that is also delivered to cells by activated CTLs. Granzyme H cleaves and inactivates Ad5−100K, as well as the adenoviral DNA binding protein (6). Cleavage of these proteins not only significantly restricts virus replication, but also relieves virus-mediated inhibition of granzyme B and restores its ability to process caspase-3 (6). Granzyme H does not have orthologs in species other than humans, and the inhibitory activity of Ad5−100K is specifically directed toward human granzyme B. Thus, despite generally well-conserved processes of apoptosis, these findings suggest a remarkably complex evolutionary relationship between the induction and suppression of host cell apoptosis and virus replication.
Granzyme: serine esterases present within granules of cytotoxic T cells and natural killer cells
Viruses target proapoptotic serine proteases other than granzymes to suppress the cell death response. The high-temperature requirement protein A2 (HtrA2/Omi) is a serine pro-tease released from the mitochondrial inner membrane space along with cytochrome c and SMAC/DIABLO following membrane permeabilization (50). Once released, HtrA2/Omi can degrade XIAP and thereby relieve its inhibition of caspases (105). When used in combination as clinical therapy for some tumors, interferon (IFN) and retinoic acid promote apoptosis in part through the upregulation of genes associated with retinoid-interferon-induced mortality (GRIM) protein expression (59). In particular, GRIM-19 associates with HtrA2/Omi and augments XIAP degradation (75). The Kaposi's sarcoma–associated herpesvirus oncoprotein termed viral IFN-regulatory factor 1 (vIRF1) binds to GRIM-19 and prevents its interaction with HtrA2/Omi (75). This preserves cellular XIAP levels in response to IFN and retinoic acid and ablates caspase-9 activation.
The original function identified for vIRF1 was a suppressor of IFN signaling (141), a potent antiviral response that viruses devote large amounts of their genomes to evade. It is well known that IFN stimulation can eventually lead to apoptosis through the increased expression of proapoptotic genes. However, the targeting of GRIM-19 and HtrA2/Omi by a viral homolog of the IFN signal transduction pathway suggests that there may be direct regulation, or cross-talk, between the two cellular pathways. The recent identification of HtrA2/Omi as a mitochondria-resident protein with roles in apoptosis in Drosophila (18) suggests a level of conservation that facilitates virus coevolution. Therefore, it is of interest to determine if additional oncogenic viruses, which may persist in the presence of chronic expression of proapoptotic cytokines such as IFN, also target HtrA2/Omi to suppress apoptosis.
The combined efforts of viruses to suppress apoptosis in general and caspase activity in particular suggest that this cellular response is to be avoided at all costs. However, caspases also have roles in cell proliferation, differentiation, and NF-κB activation (69). These roles for caspase activation raise the question of whether viruses can positively modulate or utilize caspase activity to facilitate replication. The role of the HPV-16 E6 long isoform in caspase-8 degradation has already been highlighted. The E6 short isoform, which is only produced by so-called high-risk HPV types, can also bind to caspase-8. However, this molecular interaction results in caspase-8 stabilization (40). The implications from these findings are that, like inhibition of caspase activity, stabilization of limited caspase-8 activity may be important for cell survival or maintaining a cell at a particular stage of differentiation. Thus, caspases may be targeted by certain viruses to prolong cell viability or aid in cellular transformation.
Examples of viruses that directly utilize caspase activity to facilitate replication also exist. Permissive replication of Aleutian mink disease parvovirus (ADV) in cell culture is associated with an apoptotic response that can be blocked by treatment with caspase inhibitors. There are numerous examples of virus infection in which blockage of apoptosis increases the virus titer recovered from treated cells, consistent with the antiviral role of apoptosis (23, 38, 137). However, in the case of ADV, inhibition of specific caspases results in decreased virus yield, suggesting that caspase activity facilitates virus replication (13). The major nonstructural protein of ADV, NS1, is required for many replication functions including control of viral and cellular gene transcription, viral DNA replication, and capsid assembly. Caspases mediate cleavage of NS1 early in virus replication, particularly by effector caspase-3, caspase-7, or both (12; S.M. Best, unpublished data). In the absence of cleavage, translocation of NS1 to the site of virus replication in the nucleus is impaired. It appears that the role of caspases is to generate a C-terminal cleavage product that contains the NS1 nuclear localization sequence. This product forms oligomers with full-length NS1 and facilitates transport of the latter to the nucleus (M.E. Bloom & S.M. Best, unpublished data).
Although a clear requirement exists for caspases in ADV replication, it is currently unknown how this contributes to virus pathogenesis. In infected mink kits, permissive replication of ADV occurs in pulmonary type II pneumocytes, resulting in high levels of cytopathology and mortality. In contrast, infection of adult mink results in restricted replication in lymph node macrophages and persistent infection. It is possible that cell-type-specific regulation of caspase activity modulates the degree of NS1 nuclear translocation and hence its function in DNA replication and viral gene expression. In adult mink, for example, tight regulation of caspase activity in macrophages may restrict nuclear translocation of NS1 and virus replication, contributing to persistent infection. However, apoptosis may be triggered during virus infection of pneumocytes, resulting in elevated caspase activation and permissive replication in mink kits.
Adenoviral proteins are also cleaved during virus replication. The early transcription units E1A (which encodes two proteins, 12S and 13S) and E1B encode proteins involved in transactivation of both cellular and viral transcription as well as cellular transformation. Following transient expression of the E1A proteins from Ad2 or Ad12, caspases cleave both 12S and 13S at multiple sites, resulting in progressive truncation from the N termini (48). This cleavage disrupts interactions of E1A with cellular transcription-regulating proteins that bind to the N terminus but not to the C terminus (48). Thus, caspases have a potential regulatory role in E1A-mediated gene expression. E1A is also proteolytically cleaved by caspases during Ad5 replication. However, cleavage of E1A proteins, as well as cellular proteins normally cleaved during apoptosis, is limited and dependent on cell type (48). The degree of cleavage is likely influenced by temporal expression of adenovirus-encoded inhibitors of apoptosis such as E3, the protein responsible for the downregulation of cell surface death receptors (116). Thus, an intricate interplay between caspase activation and inhibition in a particular cell type may be required for optimal virus replication.
Additional examples of viruses that utilize caspase activity include human astroviruses that cause viral gasteroenteritis. In this case, caspase-mediated cleavage of the capsid precursor protein facilitates the release of viral particles from the cell (80). Other examples of virus-encoded proteins cleaved by caspases include NS5A of hepatitis C virus (46, 58, 96), the nucleocapsid protein of influenza A virus (138), and ICP22 of herpes simplex virus (85), although the role of protein cleavage in replication of these viruses is unclear (11). Cleavage may facilitate protein degradation and negatively impact virus replication as described for the granzyme H-mediated cleavage of the Ad5−100K protein. However, because mutation of the crucial Asp residue would eliminate the enzyme recognition site, conservation of these sequences suggests a selection for caspase-mediated cleavage in virus replication (11). Hence, in addition to facilitating virus release, caspases may have roles in regulation of virus protein maturation and function as suggested by studies using ADV and adenoviruses.
To prolong cell viability after infection, the function of enzymes essential for the promotion of host cell death, such as caspases and serine proteases, are obvious targets for suppression by viruses. Although not discussed here, additional proteases including cathepsins, calpains (104, 117, 119), phosphatidylinositol 3-kinase (PI3K) (25), mitogen-activated protein kinases (MAPK) (57), and protein kinase R (PKR) (43) have important roles in regulating apoptosis. PI3K, MAPK and PKR are all targeted by various viruses to modulate cell death. It is possible that other proteases like the cathepsins or calpains are also targeted by viruses for this purpose.
The discovery of virus-encoded inhibitors of apoptotic proteases has been invaluable, both in our understanding of virus requirements for replication and for tools used to study apoptosis. In this arena, CrmA, p35, vIAPs, and vFLIPs have yielded enormous insight into apoptotic signaling pathways stimulated by a plethora of insults. These proteins also have potential use as therapeutics to treat conditions in which apoptosis is a major contributor to disease. Owing to the observation that many viruses encode several inhibitors of apoptosis and that individual proteins have multiple functions, the full spectrum of viral protein activity in modulating cell death is probably not fully realized. Furthermore, caspases have roles in nonapoptotic cellular processes (68, 69) as well as in the facilitation of virus replication (11). Thus, it is likely that further knowledge of both cell death and cell survival programs will be gained from understanding specific mechanisms by which virus proteins modulate these enzymes.
Taken together, the studies highlighted in this review suggest that complex relationships exist between enzyme activity, virus replication, and cell survival and transformation. Furthermore, although general mechanisms of apoptosis are conserved from Drosophila to humans, many of the viral proteins that modulate enzyme activity during apoptosis do so in a host-specific context, including CrmA, p35 and p49, vIAPs, and Ad5−100K. Combined with the fact that apoptosis is a strong antiviral response, these studies suggest that virus-encoded proteins are important contributors to virus cell tropism and host range specificity. Thus, challenges remain in understanding how these multifaceted virus-host interactions within the individual cell contribute to the outcome of virus infection at the level of the organism.
SUMMARY POINTS
1. Viruses suppress the function of proteases with central roles in promoting apoptosis to prolong cell viability and facilitate replication. The functions of caspases and serine proteases are most commonly modulated by viruses to suppress apoptosis.
2. Four classes of viral proteins directly suppress caspase function. Two of these, serpins and p35 family members, have in common a RSL that is inserted into the caspase active site. However, serpins function by conformational distortion of the caspase active site, whereas p35 engages the active site in a process of chemical ligation.
3. Two other classes of virus inhibitors suppress caspase activity by competing with signaling molecules involved in caspase activation. vIAPs act as decoys for cellular IAP antagonists, thus enabling cellular IAPs to inhibit caspase activity. vFLIPs prevent DISC assembly in response to ligation of death receptors, resulting in suppression of caspase-8 and caspase-10 activation.
4. Viral gene products also antagonize the proapoptotic function of the serine proteases, granzyme B and Htr2A/Omi.
5. Despite the antiviral role of apoptosis, some viruses appear to exploit this response by utilizing caspase activity to cleave viral proteins and facilitate replication. Thus, complex relationships exist between caspase activity, virus replication, and cell survival.
FUTURE ISSUES
1. Elucidation of protein structures in complex with their targets has provided enormous insight into distinct mechanisms utilized by p35 to suppress apoptosis. Additional structural studies, for example, of Ad5−100K in complex with granzyme B, are required to fully elucidate the mechanisms underlying virus protein function in apoptosis suppression.
2. The diverse functions of cIAPs in the regulation of apoptosis are beginning to be revealed. Hence, functions of vIAPs in addition to their ability to subvert cellular IAP antagonists may exist and remain to be identified.
3. In examples in which viruses utilize caspases for replication, little is known regarding how caspase activity is positively regulated while limited to complete replication. Thus, the temporal relationships between viral pro- and antiapoptotic signals are poorly understood. Furthermore, it is unknown how caspase-dependent replication contributes to virus pathogenesis.
4. A major challenge continues to be converting knowledge of viral protease inhibitors into viable therapeutics to treat conditions involving aberrant apoptosis or inflammation.
ACKNOWLEDGMENTS
Thank you to Marshall Bloom, Dana Mitzel, Shelly Robertson, Travis Taylor, and Dan Voth for critique of the manuscript and to Anita Mora for graphical expertise. This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.
Footnotes
*The U.S. Government has the right to retain a nonexclusive, royalty-free license in and to any copyright covering this paper.
DISCLOSURE STATEMENT
The author is not aware of any biases that might be perceived as affecting the objectivity of this review.
1. Alnemri ES, Livingston DJ, Nicholson DW, Salvesen G, Thornberry NA, et al. Human ICE/CED-3 protease nomenclature. Cell. 1996;87:171. [PubMed]
2. Andoniou CE, Degli-Esposti MA. Insights into the mechanisms of CMV-mediated interference with cellular apoptosis. Immunol. Cell Biol. 2006;84:99–106. [PubMed]
3. Andrade F, Bull HG, Thornberry NA, Ketner GW, Casciola-Rosen LA, Rosen A. Adenovirus L4−100K assembly protein is a granzyme B substrate that potently inhibits granzyme B-mediated cell death. Immunity. 2001;14:751–61. [PubMed]Together with Reference 6 reports the interplay between Ad5-100K and granzymes B and H.
4. Andrade F, Casciola-Rosen LA, Rosen A. A novel domain in adenovirus L4−100K is required for stable binding and efficient inhibition of human granzyme B: possible interaction with a species-specific exosite. Mol. Cell Biol. 2003;23:6315–26. [PMC free article] [PubMed]
5. Andrade F, Casciola-Rosen LA, Rosen A. Granzyme B-induced cell death. Acta Haematol. 2004;111:28–41. [PubMed]
6. Andrade F, Fellows E, Jenne DE, Rosen A, Young CS. Granzyme H destroys the function of critical adenoviral proteins required for viral DNA replication and granzyme B inhibition. EMBO J. 2007;26:2148–57. [PubMed]
7. Bao Q, Shi Y. Apoptosome: a platform for the activation of initiator caspases. Cell Death Differ. 2007;14:56–65. [PubMed]
8. Barry M, Heibein JA, Pinkoski MJ, Lee SF, Moyer RW, et al. Granzyme B short-circuits the need for caspase 8 activity during granule-mediated cytotoxic T-lymphocyte killing by directly cleaving Bid. Mol. Cell Biol. 2000;20:3781–94. [PMC free article] [PubMed]
9. Benedict CA, Norris PS, Ware CF. To kill or be killed: viral evasion of apoptosis. Nat. Immunol. 2002;3:1013–18. [PubMed]
10. Bertin J, Armstrong RC, Ottilie S, Martin DA, Wang Y, et al. Death effector domain-containing herpesvirus and poxvirus proteins inhibit both Fas- and TNFR1-induced apoptosis. Proc. Natl. Acad. Sci. USA. 1997;94:1172–76. [PubMed]Together with Reference 111 provided the first description of vFLIPs before the discovery of cFLIP.
11. Best SM, Bloom ME. Caspase activation during virus infection: more than just the kiss of death? Virology. 2004;320:191–94. [PubMed]
12. Best SM, Shelton JF, Pompey JM, Wolfinbarger JB, Bloom ME. Caspase cleavage of the nonstructural protein NS1 mediates replication of Aleutian mink disease parvovirus. J. Virol. 2003;77:5305–12. [PMC free article] [PubMed]
13. Best SM, Wolfinbarger JB, Bloom ME. Caspase activation is required for permissive replication of Aleutian mink disease parvovirus in vitro. Virology. 2002;292:224–34. [PubMed]
14. Boya P, Pauleau AL, Poncet D, Gonzalez-Polo RA, Zamzami N, Kroemer G. Viral proteins targeting mitochondria: controlling cell death. Biochim. Biophys. Acta. 2004;1659:178–89. [PubMed]
15. Budd RC, Yeh WC, Tschopp J. cFLIP regulation of lymphocyte activation and development. Nat. Rev. Immunol. 2006;6:196–204. [PubMed]
16. Callus BA, Vaux DL. Caspase inhibitors: viral, cellular and chemical. Cell Death Differ. 2007;14:73–78. [PubMed]
17. Cassens U, Lewinski G, Samraj AK, von BH, Baust H, et al. Viral modulation of cell death by inhibition of caspases. Arch. Immunol. Ther. Exp. 2003;51:19–27. [PubMed]
18. Challa M, Malladi S, Pellock BJ, Dresnek D, Varadarajan S, et al. Drosophila Omi, a mitochondrial-localized IAP antagonist and proapoptotic serine protease. EMBO J. 2007;26:3144–56. [PubMed]
19. Chang DW, Xing Z, Pan Y, Geciras-Schimnich A, Barnhart BC, et al. c-FLIP(L) is a dual function regulator for caspase-8 activation and CD95-mediated apoptosis. EMBO J. 2002;21:3704–14. [PubMed]
20. Chaudhary PM, Jasmin A, Eby MT, Hood L. Modulation of the NF-kappa B pathway by virally encoded death effector domains-containing proteins. Oncogene. 1999;18:5738–46. [PubMed]
21. Clem RJ. The role of apoptosis in defense against baculovirus infection in insects. Curr. Top. Microbiol. Immunol. 2005;289:113–29. [PubMed]
22. Clem RJ. Baculoviruses and apoptosis: a diversity of genes and responses. Curr. Drug Targets. 2007;8:1069–74. [PubMed]
23. Clem RJ, Fechheimer M, Miller LK. Prevention of apoptosis by a baculovirus gene during infection of insect cells. Science. 1991;254:1388–90. [PubMed]The first description of p35.
24. Cohen GM. Caspases: the executioners of apoptosis. Biochem. J. 1997;326(Pt 1):1–16. [PubMed]
25. Cooray S. The pivotal role of phosphatidylinositol 3-kinase-Akt signal transduction in virus survival. J. Gen. Virol. 2004;85:1065–76. [PubMed]
26. Crook NE, Clem RJ, Miller LK. An apoptosis-inhibiting baculovirus gene with a zinc finger-like motif. J. Virol. 1993;67:2168–74. [PMC free article] [PubMed]The first description of an IAP as a viral product before the discovery of cIAPs.
27. Datta R, Kojima H, Banach D, Bump NJ, Talanian RV, et al. Activation of a CrmA-insensitive, p35-sensitive pathway in ionizing radiation-induced apoptosis. J. Biol. Chem. 1997;272:1965–69. [PubMed]
28. Degterev A, Boyce M, Yuan J. A decade of caspases. Oncogene. 2003;22:8543–67. [PubMed]
29. Delhon G, Tulman ER, Afonso CL, Lu Z, Becnel JJ, et al. Genome of invertebrate iridescent virus type 3 (mosquito iridescent virus). J. Virol. 2006;80:8439–49. [PMC free article] [PubMed]
30. Deveraux QL, Leo E, Stennicke HR, Welsh K, Salvesen GS, Reed JC. Cleavage of human inhibitor of apoptosis protein XIAP results in fragments with distinct specificities for caspases. EMBO J. 1999;18:5242–51. [PubMed]
31. Dobbelstein M, Shenk T. Protection against apoptosis by the vaccinia virus SPI-2 (B13R) gene product. J. Virol. 1996;70:6479–85. [PMC free article] [PubMed]
32. Dobo J, Swanson R, Salvesen GS, Olson ST, Gettins PG. Cytokine response modifier a inhibition of initiator caspases results in covalent complex formation and dissociation of the caspase tetramer. J. Biol. Chem. 2006;281:38781–90. [PubMed]
33. Du C, Fang M, Li Y, Li L, Wang X. Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell. 2000;102:33–42. [PubMed]
34. Du Q, Lehavi D, Faktor O, Qi Y, Chejanovsky N. Isolation of an apoptosis suppressor gene of the Spodoptera littoralis nucleopolyhedrovirus. J. Virol. 1999;73:1278–85. [PMC free article] [PubMed]
35. Eckelman BP, Salvesen GS. The human antiapoptotic proteins cIAP1 and cIAP2 bind but do not inhibit caspases. J. Biol. Chem. 2006;281:3254–60. [PubMed]
36. Eckelman BP, Salvesen GS, Scott FL. Human inhibitor of apoptosis proteins: why XIAP is the black sheep of the family. EMBO Rep. 2006;7:988–94. [PubMed]
37. Elliott PR, Lomas DA, Carrell RW, Abrahams JP. Inhibitory conformation of the reactive loop of alpha 1-antitrypsin. Nat. Struct. Biol. 1996;3:676–81. [PubMed]
38. Everett H, McFadden G. Apoptosis: an innate immune response to virus infection. Trends Microbiol. 1999;7:160–65. [PubMed]
39. Everett H, McFadden G. Viruses and apoptosis: meddling with mitochondria. Virology. 2001;288:1–7. [PubMed]
40. Filippova M, Johnson MM, Bautista M, Filippov V, Fodor N, et al. The large and small isoforms of human papillomavirus type 16 E6 bind to and differentially affect procaspase 8 stability and activity. J. Virol. 2007;81:4116–29. [PMC free article] [PubMed]Provides insight into the role of HPV E6 in modulation of cell survival and transformation.
41. Filippova M, Parkhurst L, Duerksen-Hughes PJ. The human papillomavirus 16 E6 protein binds to Fas-associated death domain and protects cells from Fas-triggered apoptosis. J. Biol. Chem. 2004;279:25729–44. [PubMed]
42. Fuentes-Prior P, Salvesen GS. The protein structures that shape caspase activity, specificity, activation and inhibition. Biochem. J. 2004;384:201–32. [PubMed]
43. Garcia MA, Meurs EF, Esteban M. The dsRNA protein kinase PKR: virus and cell control. Biochimie. 2007;89:799–811. [PubMed]
44. Garcia-Calvo M, Peterson EP, Leiting B, Ruel R, Nicholson DW, Thornberry NA. Inhibition of human caspases by peptide-based and macromolecular inhibitors. J. Biol. Chem. 1998;273:32608–13. [PubMed]
45. Garrido C, Galluzzi L, Brunet M, Puig PE, Didelot C, Kroemer G. Mechanisms of cytochrome c release from mitochondria. Cell Death Differ. 2006;13:1423–33. [PubMed]
46. Goh PY, Tan YJ, Lim SP, Lim SG, Tan YH, Hong WJ. The hepatitis C virus core protein interacts with NS5A and activates its caspase-mediated proteolytic cleavage. Virology. 2001;290:224–36. [PubMed]
47. Goldmacher VS. Cell death suppression by cytomegaloviruses. Apoptosis. 2005;10:251–65. [PubMed]
48. Grand RJ, Schmeiser K, Gordon EM, Zhang X, Gallimore PH, Turnell AS. Caspase-mediated cleavage of adenovirus early region 1A proteins. Virology. 2002;301:255–71. [PubMed]
49. Green MC, Monser KP, Clem RJ. Ubiquitin protein ligase activity of the antiapoptotic baculovirus protein Op-IAP3. Virus Res. 2004;105:89–96. [PubMed]
50. Hegde R, Srinivasula SM, Zhang Z, Wassell R, Mukattash R, et al. Identification of Omi/HtrA2 as a mitochondrial apoptotic serine protease that disrupts inhibitor of apoptosis protein-caspase interaction. J. Biol. Chem. 2002;277:432–38. [PubMed]
51. Heibein JA, Goping IS, Barry M, Pinkoski MJ, Shore GC, et al. Granzyme B-mediated cytochrome c release is regulated by the Bcl-2 family members Bid and Bax. J. Exp. Med. 2000;192:1391–402. [PMC free article] [PubMed]
52. Hinds MG, Norton RS, Vaux DL, Day CL. Solution structure of a baculoviral inhibitor of apoptosis (IAP) repeat. Nat. Struct. Biol. 1999;6:648–51. [PubMed]
53. Hodges BL, Evans HK, Everett RS, Ding EY, Serra D, Amalfitano A. Adenovirus vectors with the 100K gene deleted and their potential for multiple gene therapy applications. J. Virol. 2001;75:5913–20. [PMC free article] [PubMed]
54. Holley CL, Olson MR, Colon-Ramos DA, Kornbluth S. Reaper eliminates IAP proteins through stimulated IAP degradation and generalized translational inhibition. Nat. Cell Biol. 2002;4:439–44. [PMC free article] [PubMed]
55. Hu S, Vincenz C, Buller M, Dixit VM. A novel family of viral death effector domain-containing molecules that inhibit both CD-95- and tumor necrosis factor receptor-1-induced apoptosis. J. Biol. Chem. 1997;272:9621–24. [PubMed]
56. Jabbour AM, Ekert PG, Coulson EJ, Knight MJ, Ashley DM, Hawkins CJ. The p35 relative, p49, inhibits mammalian and Drosophila caspases including DRONC and protects against apoptosis. Cell Death Differ. 2002;9:1311–20. [PubMed]
57. Junttila MR, Li SP, Westermarck J. Phosphatase-mediated crosstalk between MAPK signaling pathways in the regulation of cell survival. FASEB J. 2008;22:1–12. [PubMed]
58. Kalamvoki M, Georgopoulou U, Mavromara P. The NS5A protein of the hepatitis C virus genotype 1a is cleaved by caspases to produce C-terminal-truncated forms of the protein that reside mainly in the cytosol. J. Biol. Chem. 2006;281:13449–62. [PubMed]
59. Kalvakolanu DV. The GRIMs: a new interface between cell death regulation and interferon/retinoid induced growth suppression. Cytokine Growth Factor Rev. 2004;15:169–94. [PubMed]
60. Kataoka T, Tschopp J. N-terminal fragment of c-FLIP(L) processed by caspase 8 specifically interacts with TRAF2 and induces activation of the NF-kappaB signaling pathway. Mol. Cell Biol. 2004;24:2627–36. [PMC free article] [PubMed]
61. Keckler MS. Dodging the CTL response: viral evasion of Fas and granzyme induced apoptosis. Front. Biosci. 2007;12:725–32. [PubMed]
62. Kidd VJ, Lahti JM, Teitz T. Proteolytic regulation of apoptosis. Semin. Cell Dev. Biol. 2000;11:191–201. [PubMed]
63. Kischkel FC, Hellbardt S, Behrmann I, Germer M, Pawlita M, et al. Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor. EMBO J. 1995;14:5579–88. [PubMed]
64. Komiyama T, Ray CA, Pickup DJ, Howard AD, Thornberry NA, et al. Inhibition of interleukin-1 beta converting enzyme by the cowpox virus serpin CrmA. An example of cross-class inhibition. J. Biol. Chem. 1994;269:19331–37. [PubMed]
65. Krueger A, Schmitz I, Baumann S, Krammer PH, Kirchhoff S. Cellular FLICE-inhibitory protein splice variants inhibit different steps of caspase-8 activation at the CD95 death-inducing signaling complex. J. Biol. Chem. 2001;276:20633–40. [PubMed]
66. Kumar S. Caspase function in programmed cell death. Cell Death Differ. 2007;14:32–43. [PubMed]
67. LaCount DJ, Hanson SF, Schneider CL, Friesen PD. Caspase inhibitor P35 and inhibitor of apoptosis Op-IAP block in vivo proteolytic activation of an effector caspase at different steps. J. Biol. Chem. 2000;275:15657–64. [PubMed]
68. Lamkanfi M, Declercq W, Vanden BT, Vandenabeele P. Caspases leave the beaten track: caspase-mediated activation of NF-kappaB. J. Cell Biol. 2006;173:165–71. [PMC free article] [PubMed]
69. Lamkanfi M, Festjens N, Declercq W, Vanden BT, Vandenabeele P. Caspases in cell survival, proliferation and differentiation. Cell Death Differ. 2007;14:44–55. [PubMed]
70. Lannan E, Vandergaast R, Friesen PD. Baculovirus caspase inhibitors P49 and P35 block virus-induced apoptosis downstream of effector caspase DrICE activation in Drosophila melanogaster cells. J. Virol. 2007;81:9319–30. [PMC free article] [PubMed]
71. Li Q, Liston P, Moyer RW. Functional analysis of the inhibitor of apoptosis (iap) gene carried by the entomopoxvirus of Amsacta moorei. J. Virol. 2005;79:2335–45. [PMC free article] [PubMed]
72. Liu Z, Sun C, Olejniczak ET, Meadows RP, Betz SF, et al. Structural basis for binding of Smac/DIABLO to the XIAP BIR3 domain. Nature. 2000;408:1004–8. [PubMed]
73. Lord SJ, Rajotte RV, Korbutt GS, Bleackley RC. Granzyme B: a natural born killer. Immunol. Rev. 2003;193:31–38. [PubMed]
74. Lu M, Min T, Eliezer D, Wu H. Native chemical ligation in covalent caspase inhibition by p35. Chem. Biol. 2006;13:117–22. [PubMed]Reports the structure of p35 complexed with caspase-8 and demonstrates inhibition by chemical ligation.
75. Ma X, Kalakonda S, Srinivasula SM, Reddy SP, Platanias LC, Kalvakolanu DV. GRIM-19 associates with the serine protease HtrA2 for promoting cell death. Oncogene. 2007;26:4842–49. [PubMed]
76. Macen JL, Garner RS, Musy PY, Brooks MA, Turner PC, et al. Differential inhibition of the Fasand granule-mediated cytolysis pathways by the orthopoxvirus cytokine response modifier A/SPI-2 and SPI-1 protein. Proc. Natl. Acad. Sci. USA. 1996;93:9108–13. [PubMed]
77. MacNeill AL, Turner PC, Moyer RW. Mutation of the Myxoma virus SERP2 P1-site to prevent proteinase inhibition causes apoptosis in cultured RK-13 cells and attenuates disease in rabbits, but mutation to alter specificity causes apoptosis without reducing virulence. Virology. 2006;356:12–22. [PubMed]
78. Means JC, Penabaz T, Clem RJ. Identification and functional characterization of AMVp33, a novel homolog of the baculovirus caspase inhibitor p35 found in Amsacta moorei entomopoxvirus. Virology. 2007;358:436–47. [PMC free article] [PubMed]
79. Meier P, Silke J, Leevers SJ, Evan GI. The Drosophila caspase DRONC is regulated by DIAP1. EMBO J. 2000;19:598–611. [PubMed]
80. Mendez E, Salas-Ocampo E, Arias CF. Caspases mediate processing of the capsid precursor and cell release of human astroviruses. J. Virol. 2004;78:8601–8. [PMC free article] [PubMed]
81. Micheau O, Thome M, Schneider P, Holler N, Tschopp J, et al. The long form of FLIP is an activator of caspase-8 at the Fas death-inducing signaling complex. J. Biol. Chem. 2002;277:45162–71. [PubMed]
82. Micheau O, Tschopp J. Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes. Cell. 2003;114:181–90. [PubMed]
83. Miller LK. An exegesis of IAPs: salvation and surprises from BIR motifs. Trends Cell Biol. 1999;9:323–28. [PubMed]
84. Miura M, Zhu H, Rotello R, Hartwieg EA, Yuan J. Induction of apoptosis in fibroblasts by IL-1 beta-converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3. Cell. 1993;75:653–60. [PubMed]The original demonstration that caspase-1 could induce apoptosis and that CrmA could inhibit it.
85. Munger J, Hagglund R, Roizman B. Infected cell protein no. 22 is subject to proteolytic cleavage by caspases activated by a mutant that induces apoptosis. Virology. 2003;305:364–70. [PubMed]
86. Nathaniel R, MacNeill AL, Wang YX, Turner PC, Moyer RW. Cowpox virus CrmA, myxoma virus SERP2 and baculovirus P35 are not functionally interchangeable caspase inhibitors in poxvirus infections. J. Gen. Virol. 2004;85:1267–78. [PubMed]
87. Petit F, Bertagnoli S, Gelfi J, Fassy F, Boucraut-Baralon C, Milon A. Characterization of a myxoma virus-encoded serpin-like protein with activity against interleukin-1 beta-converting enzyme. J. Virol. 1996;70:5860–66. [PMC free article] [PubMed]
88. Quan LT, Caputo A, Bleackley RC, Pickup DJ, Salvesen GS. Granzyme B is inhibited by the cowpox virus serpin cytokine response modifier A. J. Biol. Chem. 1995;270:10377–79. [PubMed]
89. Ray CA, Black RA, Kronheim SR, Greenstreet TA, Sleath PR, et al. Viral inhibition of inflammation: cowpox virus encodes an inhibitor of the interleukin-1 beta converting enzyme. Cell. 1992;69:597–604. [PubMed]
90. Reed JC, Doctor KS, Godzik A. The domains of apoptosis: a genomics perspective. Sci. STKE. 2004;2004:re9. [PubMed]The original description of CrmA.
91. Renatus M, Zhou Q, Stennicke HR, Snipas SJ, Turk D, et al. Crystal structure of the apoptotic suppressor CrmA in its cleaved form. Structure. 2000;8:789–97. [PubMed]Together with Reference 101 reports the crystal structure of cleaved CrmA.
92. Riedl SJ, Salvesen GS. The apoptosome: signalling platform of cell death. Nat. Rev. Mol. Cell Biol. 2007;8:405–13. [PubMed]
93. Ryoo HD, Bergmann A, Gonen H, Ciechanover A, Steller H. Regulation of Drosophila IAP1 degradation and apoptosis by reaper and ubcD1. Nat. Cell Biol. 2002;4:432–38. [PubMed]
94. Salvesen GS, Abrams JM. Caspase activation: stepping on the gas or releasing the brakes? Lessons from humans and flies. Oncogene. 2004;23:2774–84. [PubMed]
95. Salvesen GS, Duckett CS. IAP proteins: blocking the road to death's door. Nat. Rev. Mol. Cell Biol. 2002;3:401–10. [PubMed]
96. Satoh S, Hirota M, Noguchi T, Hijikata M, Handa H, Shimotohno K. Cleavage of hepatitis C virus nonstructural protein 5A by a caspase-like protease(s) in mammalian cells. Virology. 2000;270:476–87. [PubMed]
97. Scheffner M, Werness BA, Huibregtse JM, Levine AJ, Howley PM. The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell. 1990;63:1129–36. [PubMed]
98. Scott FL, Denault JB, Riedl SJ, Shin H, Renatus M, Salvesen GS. XIAP inhibits caspase-3 and -7 using two binding sites: evolutionarily conserved mechanism of IAPs. EMBO J. 2005;24:645–55. [PubMed]
99. Shi Y. Mechanisms of caspase activation and inhibition during apoptosis. Mol. Cell. 2002;9:459–70. [PubMed]
100. Shisler JL, Moss B. Molluscum contagiosum virus inhibitors of apoptosis: The MC159 v-FLIP protein blocks Fas-induced activation of procaspases and degradation of the related MC160 protein. Virology. 2001;282:14–25. [PubMed]
101. Simonovic M, Gettins PGW, Volz K. Crystal structure of viral serpin crmA provides insights into its mechanism of cysteine proteinase inhibition. Protein Sci. 2000;9:1423–27. [PubMed]
102. Skaletskaya A, Bartle LM, Chittenden T, McCormick AL, Mocarski ES, Goldmacher VS. A cytomegalovirus-encoded inhibitor of apoptosis that suppresses caspase-8 activation. Proc. Natl. Acad. Sci. USA. 2001;98:7829–34. [PubMed]
103. Stennicke HR, Salvesen GS. Chemical ligation—an unusual paradigm in protease inhibition. Mol. Cell. 2006;21:727–28. [PubMed]
104. Stoka V, Turk V, Turk B. Lysosomal cysteine cathepsins: signaling pathways in apoptosis. Biol. Chem. 2007;388:555–60. [PubMed]
105. Suzuki Y, Imai Y, Nakayama H, Takahashi K, Takio K, Takahashi R. A serine protease, HtrA2, is released from the mitochondria and interacts with XIAP, inducing cell death. Mol. Cell. 2001;8:613–21. [PubMed]
106. Swanson R, Raghavendra MP, Zhang W, Froelich C, Gettins PG, Olson ST. Serine and cysteine proteases are translocated to similar extents upon formation of covalent complexes with serpins. Fluorescence perturbation and fluorescence resonance energy transfer mapping of the protease binding site in CrmA complexes with granzyme B and caspase-1. J. Biol. Chem. 2007;282:2305–13. [PubMed]
107. Taylor JM, Barry M. Near death experiences: poxvirus regulation of apoptotic death. Virology. 2006;344:139–50. [PubMed]
108. Tenev T, Ditzel M, Zachariou A, Meier P. The antiapoptotic activity of insect IAPs requires activation by an evolutionarily conserved mechanism. Cell Death Differ. 2007;14:1191–201. [PubMed]
109. Tenev T, Zachariou A, Wilson R, Ditzel M, Meier P. IAPs are functionally nonequivalent and regulate effector caspases through distinct mechanisms. Nat. Cell Biol. 2005;7:70–77. [PubMed]
110. Tesch LD, Raghavendra MP, Bedsted-Faarvang T, Gettins PG, Olson ST. Specificity and reactive loop length requirements for crmA inhibition of serine proteases. Protein Sci. 2005;14:533–42. [PubMed]
111. Thome M, Schneider P, Hofmann K, Fickenscher H, Meinl E, et al. Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors. Nature. 1997;386:517–21. [PubMed]
112. Thome M, Tschopp J. Regulation of lymphocyte proliferation and death by FLIP. Nat. Rev. Immunol. 2001;1:50–58. [PubMed]
113. Thomson BJ. Viruses and apoptosis. Int. J. Exp. Pathol. 2001;82:65–76. [PubMed]
114. Thurau M, Everett H, Tapernoux M, Tschopp J, Thome M. The TRAF3-binding site of human molluscipox virus FLIP molecule MC159 is critical for its capacity to inhibit Fas-induced apoptosis. Cell Death Differ. 2006;13:1577–85. [PubMed]
115. Timmer JC, Salvesen GS. Caspase substrates. Cell Death Differ. 2007;14:66–72. [PubMed]
116. Tollefson AE, Hermiston TW, Lichtenstein DL, Colle CF, Tripp RA, et al. Forced degradation of Fas inhibits apoptosis in adenovirus-infected cells. Nature. 1998;392:726–30. [PubMed]
117. Turk B, Stoka V. Protease signalling in cell death: caspases versus cysteine cathepsins. FEBS Lett. 2007;581:2761–67. [PubMed]
118. Turner SJ, Silke J, Kenshole B, Ruby J. Characterization of the ectromelia virus serpin, SPI-2. J. Gen. Virol. 2000;81:2425–30. [PubMed]
119. Vandenabeele P, Orrenius S, Zhivotovsky B. Serine proteases and calpains fulfill important supporting roles in the apoptotic tragedy of the cellular opera. Cell Death Differ. 2005;12:1219–24. [PubMed]
120. Vaux DL, Haecker G, Strasser A. An evolutionary perspective on apoptosis. Cell. 1994;76:777–79. [PubMed]
121. Vaux DL, Silke J. IAPs, RINGs and ubiquitylation. Nat. Rev. Mol. Cell Biol. 2005;6:287–97. [PubMed]
122. Vaux DL, Silke J. IAPs—the ubiquitin connection. Cell Death Differ. 2005;12:1205–7. [PubMed]
123. Verhagen AM, Ekert PG, Pakusch M, Silke J, Connolly LM, et al. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell. 2000;102:43–53. [PubMed]The first study to suggest that vIAPs interact with IAP antagonists.
124. Vucic D, Kaiser WJ, Harvey AJ, Miller LK. Inhibition of reaper-induced apoptosis by interaction with inhibitor of apoptosis proteins (IAPs). Proc. Natl. Acad. Sci. USA. 1997;94:10183–88. [PubMed]
125. Vucic D, Kaiser WJ, Miller LK. A mutational analysis of the baculovirus inhibitor of apoptosis Op-IAP. J. Biol. Chem. 1998;273:33915–21. [PubMed]
126. Vucic D, Kaiser WJ, Miller LK. Inhibitor of apoptosis proteins physically interact with and block apoptosis induced by Drosophila proteins HID and GRIM. Mol. Cell Biol. 1998;18:3300–9. [PMC free article] [PubMed]
127. Wilkinson JC, Wilkinson AS, Scott FL, Csomos RA, Salvesen GS, Duckett CS. Neutralization of Smac/Diablo by inhibitors of apoptosis (IAPs). A caspase-independent mechanism for apoptotic inhibition. J. Biol. Chem. 2004;279:51082–90. [PubMed]
128. Wing JP, Schwartz LM, Nambu JR. The RHG motifs of Drosophila Reaper and Grim are important for their distinct cell death-inducing abilities. Mech. Dev. 2001;102:193–203. [PubMed]
129. Wu G, Chai J, Suber TL, Wu JW, Du C, et al. Structural basis of IAP recognition by Smac/DIABLO. Nature. 2000;408:1008–12. [PubMed]
130. Xu G, Rich RL, Steegborn C, Min T, Huang Y, et al. Mutational analyses of the p35-caspase interaction. A bowstring kinetic model of caspase inhibition by p35. J. Biol. Chem. 2003;278:5455–61. [PubMed]
131. Yan N, Wu JW, Chai J, Li W, Shi Y. Molecular mechanisms of DrICE inhibition by DIAP1 and removal of inhibition by Reaper, Hid and Grim. Nat. Struct. Mol. Biol. 2004;11:420–28. [PubMed]
132. Yang JK, Wang L, Zheng L, Wan F, Ahmed M, et al. Crystal structure of MC159 reveals molecular mechanism of DISC assembly and FLIP inhibition. Mol. Cell. 2005;20:939–49. [PMC free article] [PubMed]
133. Yin XM. Bid, a BH3-only multi-functional molecule, is at the cross road of life and death. Gene. 2006;369:7–19. [PubMed]
134. Yoo SJ, Huh JR, Muro I, Yu H, Wang L, et al. Hid, Rpr and Grim negatively regulate DIAP1 levels through distinct mechanisms. Nat. Cell Biol. 2002;4:416–24. [PubMed]
135. Youle RJ, Strasser A. The BCL-2 protein family: opposing activities that mediate cell death. Nat. Rev. Mol. Cell Biol. 2008;9:47–59. [PubMed]
136. Yuan J, Shaham S, Ledoux S, Ellis HM, Horvitz HR. The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1 beta-converting enzyme. Cell. 1993;75:641–52. [PubMed]
137. Zaragoza C, Saura M, Padalko EY, Lopez-Rivera E, Lizarbe TR, et al. Viral protease cleavage of inhibitor of kappaBalpha triggers host cell apoptosis. Proc. Natl. Acad. Sci. USA. 2006;103:19051–56. [PubMed]
138. Zhirnov OP, Konakova TE, Garten W, Klenk H. Caspase-dependent N-terminal cleavage of influenza virus nucleocapsid protein in infected cells. J. Virol. 1999;73:10158–63. [PMC free article] [PubMed]
139. Zhou Q, Krebs JF, Snipas SJ, Price A, Alnemri ES, et al. Interaction of the baculovirus antiapoptotic protein p35 with caspases. Specificity, kinetics, and characterization of the caspase/p35 complex. Biochemistry. 1998;37:10757–65. [PubMed]
140. Zhou Q, Snipas S, Orth K, Muzio M, Dixit VM, Salvesen GS. Target protease specificity of the viral serpin CrmA. Analysis of five caspases. J. Biol. Chem. 1997;272:7797–800. [PubMed]
141. Zimring JC, Goodbourn S, Offermann MK. Human herpesvirus 8 encodes an interferon regulatory factor (IRF) homolog that represses IRF-1-mediated transcription. J. Virol. 1998;72:701–7. [PMC free article] [PubMed]
142. Zoog SJ, Schiller JJ, Wetter JA, Chejanovsky N, Friesen PD. Baculovirus apoptotic suppressor P49 is a substrate inhibitor of initiator caspases resistant to P35 in vivo. EMBO J. 2002;21:5130–40. [PubMed]