CrmA/SPI-2 inhibition of an endogenous ICE-related protease responsible for lamin A cleavage and apoptotic nuclear fragmentation.

CrmA, a poxvirus gene product with a serpin-like structure, blocks a variety of apoptotic death events in cultured cells. Based on the ability of CrmA to inhibit the interleukin-1β converting enzyme in vitro, it has been speculated that interleukin-1β converting enzyme-related proteases (caspases) essential for apoptosis are the cellular targets of CrmA. Here we found that rabbitpox virus CrmA/SPI-2 inhibits the cleavage of lamin A mediated by a caspase in our cell-free system of apoptosis. In the presence of CrmA/SPI-2, nuclear apoptosis in vitro was blocked at an intermediate stage after collapse of the chromatin against the nuclear periphery and before nuclear shrinkage and disintegration into apoptotic body-like fragments. Using N-(acetyltyrosinylvalinyl-Nε-biotinyllysyl) aspartic acid [(2,6-dimethylbenzoyl)oxy] methyl ketone, which derivatizes the active forms of caspases, we could show that one of five caspases active in the extracts is inhibited both by CrmA/SPI-2 and by a peptide spanning the lamin A apoptotic cleavage site. These results reveal that CrmA/SPI-2 can inhibit a caspase responsible both for lamin A cleavage and for the nuclear disintegration characteristic of apoptosis.

shrinkage, and fragmentation of the cell into apoptotic bodies (1), is the most widely studied form of programmed cell death. Genetic analysis of programmed cell death in the nematode Caenorhabditis elegans identified ced-3 as a master regulatory gene (2) that encodes a cysteine protease homologous to the interleukin-1␤ converting enzyme (ICE) 1 (3,4). In mammalian cells, in contrast, multiple cDNAs have been identified that encode ICE-related proteases (now termed caspases, for cysteine aspartases) (6). In addition to caspase-1 (ICE), nine additional human caspases have been described (see Ref. 5 for review). Of these, the enzymes most widely studied with respect with their role in apoptosis include caspase-3 (CPP32/ YAMA/Apopain (7)(8)(9)) and caspase-6 (Mch-2 (10)).
CrmA/SPI-2 (11) is a poxvirus gene product with homology to members of the serpin superfamily that appears to assist these viruses in evading the host inflammatory responses (12). Cowpox virus-derived (13)(14)(15)(16) and vaccinia virus-derived (17) crmA cDNAs transfected into cells can inhibit apoptosis induced by nerve growth factor depletion (13), serum withdrawal (14), Fas ligation (15)(16)(17), and activation of the tumor necrosis factor receptor (15). Based on the ability of purified CrmA protein to inhibit the cleavage of pro-interleukin-1␤ by ICE (12), it has been assumed that CrmA inhibits apoptosis by blocking the activity of caspases within the transfected cells (16,17). However, cotransfection of crmA cDNA poorly inhibits cell death induced by overexpression of some caspases such as caspase-2 (ICH-1 (18)) and CED-3 (4,19). Also, CrmA inhibits the cleavage of poly(ADP-ribose) polymerase (PARP) by purified caspase-3 (CPP32) only poorly (15,20). Moreover, the recent report that CrmA binds granzyme B (21), a serine protease not structurally related to ICE, reveals that the action of CrmA may not be restricted to caspases. Therefore, it remains to be determined whether the inhibition of apoptosis by CrmA results solely from the blockade of caspase(s).
We have established a cell-free system in which isolated nuclei undergo morphological and biochemical changes characteristic of apoptosis when exposed to cytoplasmic extracts (S/M extracts) from chicken DU249 cells committed to apoptotic cell death (22). Using this in vitro system, PARP was identified as the first confirmed substrate for any caspase in apoptotic cells (23). We subsequently found that lamin cleavage during nuclear apoptosis in vitro requires an enzyme distinct from the PARP-cleaving caspase (24) and showed that Mch2 can act as a lamin protease (20). Labeling of active caspases in S/M extracts using YV(bio)KD-aomk, a biotinylated peptide synthesized based on the ICE cleavage site in pro-interleukin-1␤ (25), revealed at least five distinct labeled polypeptides, which we termed prICE 1 -prICE 5 (20). Here we show that rabbitpox virus (RPV) CrmA/SPI-2 inhibits the caspase(s) catalyzing the cleavage of lamin A and the disintegration of nuclei into apoptotic body-like fragments in the cell-free system.

EXPERIMENTAL PROCEDURES
Reagents-Peptides RLVEIDNGKQR (D peptide) and RLVEIA-NGKQR (A peptide) were synthesized based on the apoptosis-specific * This work was supported by a grant from the Wellcome Trust (to W. C. E.). Other support included National Institutes of Health Grants CA69008 (to W. C. E.) and AI 15722 (to R. W. M.), as well as grants from the Canadian Medical Research Council and the National Cancer Institute of Canada (to G. G. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported by a "Programa Gulbenkian de Doutoramento em Biologia e Medicina" studentship.
Recombinant viruses were generated by transfecting pTM1-His-Spi2 and pTM1-His-Spi7 plasmids into sub-confluent CV-1 cells (ATCC) using LipofectACE Reagent (Life Technologies, Inc.). These cells were co-infected with vaccinia virus (VV) strain WR at a multiplicity of infection of 0.05. The resulting progeny virus was plaqued on Rat2 thymidine kinase Ϫ cells (ATCC) under 0.5% agarose and selected with 100 g/ml 5-bromo-2Ј-deoxyuridine. Three days post-infection, well isolated plaques were picked and subjected to two subsequent rounds of plaque purification. The resultant thymidine kinase Ϫ recombinant viruses (VV-His-Spi2 and VV-His-Spi7) were further selected with 5-bromo-2Ј-deoxyuridine, expanded, and titered.
Confluent RK-13 cells (ATCC) in a 150-mm dish were infected at an multiplicity of infection of 5 of either VV-His-Spi2 or VV-His-Spi7, both in the presence of vTF7-3 (ATCC VR-2153) that expresses the bacteriophage T7 RNA polymerase. At 16 -19 h post-infection, the cells were harvested and resuspended in 1 ml of 20 mM Tris, 10 mM NaCl, pH 8.0, at 4°C. All subsequent steps were performed at 4°C. The cell suspension was supplemented with 250 l of 0.5% Triton X-100, 1.5 M NaCl, 1 mM phenylmethylsulfonyl fluoride, 2 g/ml aprotinin, 50% glycerol, placed on ice for 10 min, and centrifuged at 4800 ϫ g for 5 min. The supernatant was cleared by centrifugation at 40,000 ϫ g for 30 min.
Cell-free Apoptosis Reaction-Cytoplasmic extracts from preapoptotic chicken DU249 cells (S/M extracts) were prepared as described previously (22). For CrmA/SPI-2 inhibition, S/M extracts (10 -25 mg/ml) were preincubated at 37°C for 25 min with 10% serpin solvent, with CrmA/SPI-2 (1-30 g/ml), with SPI-7 (1-20 g/ml), or with 100 M YVAD-cmk (Bachem). Incubations were performed in the presence of an ATP regeneration system consisting of 2 mM ATP, 10 mM creatine phosphate (Calbiochem), and 50 g/ml creatine kinase. Nuclei (1.5 ϫ 10 6 ) isolated from HeLa S3 cells as described (22) were added to 10 l of the pretreated S/M extracts, and the reaction mixtures were incubated at 37°C for 90 min. Nuclei in 1 l of the mixtures were stained with 1 g/ml 4Ј,6-diamidino-2-phenylindole, a fluorescent probe for DNA, to assess morphological changes. Proteins were recovered from 3 l of the reaction mixtures to analyze cleavage of PARP and lamin A by immunoblotting as described (24). Nuclear DNA was recovered from 6 l of the mixtures by a standard method (27) to analyze DNA fragmentation by agarose gel electrophoresis.
Labeling of Active Caspases in S/M Extracts with YV(bio)KD-aomk-S/M extracts were preincubated with 100 M YVAD-cmk, 100 M TLCK, 0.5% methanol (solvent for TLCK), 30 g/ml CrmA/SPI-2, 20 g/ml SPI-7, or 10% serpin solvent for 15 min at 37°C. In other experiments, S/M extracts were pretreated for 1 min on ice with 5 mg/ml of the D peptide, with 5 mg/ml of the A peptide, or with phosphate-buffered saline containing 7% Me 2 SO to control for solvent effects. YV(bio)KDaomk (20,25) was added to the pretreated S/M extracts and incubated at 37°C for 5 min. Labeled proteins were electrophoresed in 16% SDS-polyacrylamide gels, transferred to nitrocellulose membranes (Schleicher & Schuell), and visualized by horseradish peroxidase-conjugated streptavidin and ECL (Amersham Corp.).

RESULTS AND DISCUSSION
We first examined whether RPV CrmA/SPI-2 affects the apoptotic morphological changes of nuclei in our cell-free system. In the presence of CrmA/SPI-2, the initial condensation of chromatin proceeded uninterrupted. However, overall shrinkage of nuclei and the disintegration of the nuclei into apoptotic body-like fragments were blocked in a dose-dependent manner (Fig. 1, D-G). At 30 g/ml of CrmA/SPI-2, the nuclear apoptotic changes were halted at an intermediate stage, with the chromatin condensed against the periphery of the nuclei (Fig. 1G). In contrast, SPI-7, another poxvirus serpin predicted to have Asp at its P1 site 2 did not affect the progression of apoptotic changes in vitro (Fig. 1H). Despite this blockade of the morphological changes at an intermediate stage, CrmA/SPI-2 did not affect the internucleosomal fragmentation of nuclear DNA at any concentration tested (Fig. 2, lanes 4 -8). SPI-7 also had 2 R. W. Moyer, unpublished observations. no significant effect on DNA fragmentation (Fig. 2, lane 9). YVAD-cmk, a broad spectrum caspase inhibitor at the concentration used here, completely blocked both the morphological changes in nuclei (Fig. 1I) and DNA ladder formation (Fig. 2, lane 10), as described previously (23).
To begin to examine the biochemical mechanism of the inhibition of nuclear apoptotic changes by CrmA/SPI-2, we examined the effect of purified CrmA/SPI-2 on the proteolysis of substrates catalyzed by the endogenous caspases in S/M extracts. Cleavage of PARP was not significantly inhibited at any concentration of CrmA/SPI-2 (Fig. 3, upper lanes 4 -7), consistent with a recent report that showed a very poor inhibition of PARP cleavage in apoptotic osteosarcoma cell extracts by cowpox virus-derived CrmA/SPI-2 (9). However, the cleavage of nuclear lamin A, another hallmark of apoptosis (28,29), was inhibited by the CrmA/SPI-2 protein in a dose-dependent manner (Fig. 3, lower lanes 4 -7). In contrast, SPI-7 did not inhibit either PARP or lamin A cleavage (data not shown). In control experiments, proteolysis of both PARP and lamin A was blocked by YVAD-cmk, consistent with the cleavage of both substrates by caspases (Fig. 3, lane 8).
Recent studies revealed that the cleavage of both PARP (15) and the 70-kDa protein component of the U1 small ribonucleoprotein particle (30) as well as the morphological changes of apoptosis induced by tumor necrosis factor and Fas (15) are inhibited by transfection of the crmA cDNA into cells. However, those experiments do not necessarily mean that the CrmAsensitive protease(s) are directly responsible for the observed effects; the results could be explained equally well if CrmA inhibits an upstream protease whose action leads to the activation of downstream proteases that cleave PARP and the U1 70-kDa protein.
Further experiments suggested that the inhibition of lamin A cleavage by CrmA/SPI-2 in our cell-free system is likely to be mediated by direct inhibition of a caspase. This conclusion is based on the use of YV(bio)KD-aomk (25), which covalently derivatizes residues within the active site of active caspases, enabling them to be revealed directly on immunoblots using horseradish peroxidase-conjugated streptavidin and ECL (20). Incubation of S/M extracts with YV(bio)KD-aomk revealed five labeled polypeptides ranging from 19.5 to 17.5 kDa (Fig. 4A,  lane 1). We previously termed these species prICE 1 -prICE 5 (20).
Labeling with YV(bio)KD-aomk was used to assess the effects of a series of inhibitors on the activities of prICE 1 -prICE 5 . These results are most consistent with the conclusion that prICE 5 is the endogenous lamin protease in S/M extracts. Competition experiments using a peptide spanning the cleavage site within PARP had previously suggested that prICE 1 is a PARP-cleaving caspase (20). This peptide had no effect on the binding of YV(bio)KD-aomk to prICE 4 and prICE 5 (20). TLCK inhibits lamin cleavage by an endogenous caspase in S/M extracts (24). Pretreatment of S/M extracts with TLCK preferentially inhibited labeling of prICE 2 , prICE 3 , and prICE 5 (Fig.  4A, lane 3). CrmA/SPI-2 inhibited the binding of YV(bio)KDaomk to prICE 5 and, to a lesser extent, prICE 4 (Fig. 4B, lane 5), whereas in a control experiment, SPI-7 had no effect (Fig. 4B,  lane 6).
We previously demonstrated that the cleavage of recombinant human lamin A by S/M extracts is inhibited by a peptide spanning the cleavage site within human lamin A (RLVEIDNGKQR ϭ D peptide) but not by a mutant peptide in which the P1 residue (Asp) was mutated to Ala (RLVE-IANGKQR ϭ A peptide) (20). As shown in Fig. 4B, the D peptide preferentially inhibited the binding of YV(bio)KDaomk to prICE 5 (lane 2), whereas the A peptide had no significant effect (lane 3). In control experiments, the labeling of prICE 1 -prICE 5 was inhibited by 100 M YVAD-cmk (Fig. 4A,  lane 2), a broad spectrum inhibitor of caspases at this concentration.
The data of Fig. 4 show competitions between noncovalent inhibitors (CrmA/SPI-2 and the cleavage site peptides) and the covalent inhibitor YV(bio)KD-aomk. We have been unable to observe effective competition at the high concentrations of YV(bio)KD-aomk required to label prICE 2 and prICE 3 , and therefore the effect of lamin cleavage site peptides, CrmA/ SPI-2, or SPI-7 on the activity of these two caspases is not known (data not shown).
Because the cleavage of lamin A in S/M extracts is inhibited by TLCK (24), CrmA/SPI-2 (Fig. 3, lanes 4 -7) and the peptide corresponding to the cleavage site in lamin A (20), the caspase that is inactivated by these three inhibitors, prICE 5 , is most likely a lamin-cleaving enzyme. The fact that TLCK and CrmA/ SPI-2 inhibit both the cleavage of lamin A and the completion of morphological apoptosis in S/M extracts suggests that the activity of the lamin protease (probably prICE 5 ) is required for completion of morphological apoptosis. This makes excellent sense given the structural role of the lamin intermediate filament network in providing a structural support for the nuclear envelope. Lamin cleavage may thus serve not only to release lamin-chromatin interactions as previously suggested (24) but also to render the nuclear envelope malleable so that it can be packaged into condensed apoptotic bodies.
Accumulating evidence suggests that apoptosis is mediated by combinations of caspases acting in concert (20) that may be cell type-specific and stimulus-dependent. Further studies of the effects of inhibitors such as CrmA/SPI-2 on caspases active in apoptosis may ultimately provide us a means of controlling apoptotic cell death in order to suppress unwanted apoptosis in a tissue-specific manner (31) or to induce apoptosis in selected cell populations (32).