SPI-1-Dependent Host Range of Rabbitpox Virus and Complex Formation with Cathepsin G Is Associated with Serpin Motifs

Serpins are a superfamily of serine proteinase inhibitors which function to regulate a number of key biological processes including fibrinolysis, inflammation, and cell migration. Poxviruses are the only viruses known to encode functional serpins. While some poxvirus serpins regulate inflammation (myxoma virus SERP1 and cowpox virus [CPV] crmA/SPI-2) or apoptosis (myxoma virus SERP2 and CPV crmA/SPI-2), the function of other poxvirus serpins remains unknown. The rabbitpox virus (RPV) SPI-1 protein is 47% identical to crmA and shares all of the serpin structural motifs. However, no serpin-like activity has been demonstrated for SPI-1 to date. Earlier we showed that RPV with the SPI-1 gene deleted, unlike wild-type virus, fails to grow on A549 or PK15 cells (A. Ali, P. C. Turner, M. A. Brooks, and R. W. Moyer, Virology 202:306-314, 1994). Here we demonstrate that in the absence of a functional SPI-1 protein, infected nonpermissive cells which exhibit the morphological features of apoptosis fail to activate terminal caspases or cleave the death substrates PARP or lamin A. We show that SPI-1 forms a stable complex in vitro with cathepsin G, a member of the chymotrypsin family of serine proteinases, consistent with serpin activity. SPI-1 reactive-site loop (RSL) mutations of the critical P1 and P14 residues abolish this activity. Viruses containing the SPI-1 RSL P1 or P14 mutations also fail to grow on A549 or PK15 cells. These results suggest that the full virus host range depends on the serpin activity of SPI-1 and that in restrictive cells SPI-1 inhibits a proteinase with chymotrypsin-like activity and may function to inhibit a caspase-independent pathway of apoptosis.     

A rabbitpox virus serpin gene controls host range by inhibiting apoptosis in restrictive cells.

Poxviruses are unique among viruses in encoding members of the serine proteinase inhibitor (serpin) superfamily. Orthopoxviruses contain three serpins, designated SPI-1, SPI-2, and SPI-3. SPI-1 encodes a 40-kDa protein that is required for the replication of rabbitpox virus (RPV) in PK-15 or A549 cells in culture (A. N. Ali, P. C. Turner, M. A. Brooks, and R. W. Moyer, Virology 202:305-314, 1994). Examination of nonpermissive human A549 cells infected with an RPV mutant disrupted in the SPI-1 gene (RPV delta SPI-1) suggests there are no gross defects in protein or DNA synthesis. The proteolytic processing of late viral structural proteins, a feature of orthopoxvirus infections associated with the maturation of virus particles, also appears relatively normal. However, very few mature virus particles of any kind are produced compared with the level found in infections with wild-type RPV. Morphological examination of RPV delta SPI-1-infected A549 cells, together with an observed fragmentation of cellular DNA, suggests that the host range defect is associated with the onset of apoptosis. Apoptosis is seen only in RPV delta SPI-1 infection of nonpermissive (A549 or PK-15) cells and is absent in all wild-type RPV infections and RPV delta SPI-2 mutant infections examined to date. Although the SPI-1 gene is expressed early, before DNA replication, the triggering apoptotic event occurs late in the infection, as RPV delta SPI-1-infected A549 cells do not undergo apoptosis when infections are carried out in the presence of cytosine arabinoside. While the SPI-2 (crmA) gene, when transfected into cells, has been shown to inhibit apoptosis, our experiments provide the first indication that a poxvirus serpin protein can inhibit apoptosis during a poxvirus infection.     

Activation of Caspases in Pig Kidney Cells Infected with Wild-Type and CrmA/SPI-2 Mutants of Cowpox and Rabbitpox Viruses

The cowpox virus (CPV) CrmA and the equivalent rabbitpox virus (RPV) SPI-2 proteins have anti-inflammatory and antiapoptosis activity by virtue of their ability to inhibit caspases, including the interleukin-1β-converting enzyme (ICE; caspase-1). Infection of LLC-PK1 pig kidney cells with a CPV CrmA mutant, but not with wild-type (wt) CPV, results in the induction of many of the morphological features of apoptosis (C. A. Ray and D. J. Pickup, Virology 217:384–391, 1996). In our study, LLC-PK1 cells infected with CPVΔcrmA, but not those infected with wt CPV, showed induction of poly(ADP-ribose) polymerase (PARP)- and lamin A-cleaving activities and processing of the CPP32 (caspase-3) precursor to a mature 18-kDa form. Surprisingly, infection of LLC-PK1 cells with either wt RPV (despite the presence of the SPI-2 protein) or RPVΔSPI-2 resulted in cleavage activity against PARP and lamin A and the appearance of the mature subunit of CPP32/caspase-3. The biotinylated specific peptide inhibitor Ac-Tyr-Val-Lys(biotinyl)-Asp-2,6-dimethylbenzoyloxymethylketone [AcYV(bio)KD-aomk] labeled active caspase subunits of 18, 19, and 21 kDa in extracts from LLC-PK1 cells infected with CPVΔcrmA, wt RPV, or RPVΔSPI-2 but not wt CPV. Mixed infection of LLC-PK1 cells with wt RPV and wt CPV gave no PARP-cleaving activity, and all PARP cleavage mediated by SPI-2 and CrmA mutants of RPV and CPV, respectively, could be eliminated by coinfection with wt CPV. These results suggest that the RPV SPI-2 and CPV CrmA proteins are not functionally equivalent and that CrmA, but not SPI-2 protein, can completely prevent apoptosis in LLC-PK1 cells under these conditions.     

The ectromelia virus SPI-2 protein causes lethal mousepox by preventing NK cell responses

Ectromelia virus (ECTV) is a natural pathogen of mice that causes mousepox, and many of its genes have been implicated in the modulation of host immune responses. Serine protease inhibitor 2 (SPI-2) is one of these putative ECTV host response modifier proteins. SPI-2 is conserved across orthopoxviruses, but results defining its mechanism of action and in vivo function are lacking or contradictory. We studied the role of SPI-2 in mousepox by deleting the SPI-2 gene or its serine protease inhibitor reactive site. We found that SPI-2 does not affect viral replication or cell-intrinsic apoptosis pathways, since mutant viruses replicate in vitro as efficiently as wild-type virus. However, in the absence of SPI-2 protein, ECTV is attenuated in mousepox-susceptible mice, resulting in lower viral loads in the liver, decreased spleen pathology, and substantially improved host survival. This attenuation correlates with more effective immune responses in the absence of SPI-2, including an earlier serum gamma interferon (IFN-γ) response, raised serum interleukin 18 (IL-18), increased numbers of granzyme B(+) CD8(+) T cells, and, most notably, increased numbers and activation of NK cells. Both virus attenuation and the improved immune responses associated with SPI-2 deletion from ECTV are lost when mice are depleted of NK cells. Consequently, SPI-2 renders mousepox lethal in susceptible strains by preventing protective NK cell defenses.     

Inhibition of apoptosis and clonogenic survival of cells expressing crmA variants: optimal caspase substrates are not necessarily optimal inhibitors.

To study the role of various caspases during apoptosis, we have designed a series of caspase inhibitors based on the cowpox virus cytokine response modifier A (crmA) protein. Wild-type crmA inhibits caspases 1 and 8 and thereby protects cells from apoptosis triggered by ligation of CD95 or tumour necrosis factor (TNF) receptors, but it does not protect against death mediated by other caspases. By replacing the tetrapeptide pseudosubstrate region of crmA (LVAD) with tetrapeptides that are optimal substrates for the different families of caspases, or with the four residues from the cleavage site of the baculovirus protein p35 (DQMD), we have generated a family of caspase inhibitors that show altered ability to protect against cell death. Although DEVD is the optimal substrate for caspase 3, crmA DEVD was degraded rapidly and was a weaker inhibitor than crmA DQMD, which was not degraded. Unlike wild-type crmA and crmA DEVD, crmA DQMD was able to inhibit apoptosis caused by direct activation of caspase 3 and protected lymphoid cells from death induced by radiation and dexamethasone. Significantly, the protected cells were capable of sustained growth.     

Viral modulation of the host response via crmA/SPI-2 expression.

Viruses have evolved numerous strategies to modulate the host response to infection. Poxviruses cause acute infections and need to replicate quickly to promote efficient transmission. Consequently, it is not surprising to learn that poxviruses encode a large number of proteins designed to target various arms of the host inflammatory response. One of the earliest described and most well-studied viral modulatory proteins is crmA/SPI-2. While the biochemical targets and possible modes of action have been well characterized in vitro, the role that crmA/SPI-2 plays during natural infection is less clear. It may have effects in modulating host responses involving apoptosis and inflammation. It is important to further understand the precise mode of action of viral proteins, such as crmA/SPI-2, because this may lead to better therapeutic strategies to combat a range of inflammatory and autoimmune diseases.     

CrmA expression in T lymphocytes of transgenic mice inhibits CD95 (Fas/APO-1)-transduced apoptosis, but does not cause lymphadenopathy or autoimmune disease.

The cysteine protease interleukin-1beta converting enzyme (ICE) is implicated as an effector of apoptosis in mammalian cells. Proteolytic activity of ICE can be blocked in vitro by the cytokine response modifier A (crmA), a serpin-like protease inhibitor encoded by cowpox virus. Here we show that CD2 enhancer-driven expression of crmA in T lymphocytes of transgenic mice (CD2-crmA mice) reduces CD95 (Fas/APO-1)-transduced apoptosis in vitro to the level seen in CD95-deficient mutant lpr mice, but does not protect against gamma-radiation or corticosteroid-induced cell death. Unlike lpr mice, CD2-crmA transgenic mice developed neither T cell hyperplasia nor serum autoantibodies. These results provide evidence that the phenotype of lpr mice is not simply due to failure of CD95 to trigger T cell apoptosis mediated by ICE.     

Multigenic evasion of inflammation by poxviruses.

Analyses of different cowpox virus (Brighton Red strain [CPV-BR]) mutants indicate that there is a minimum of three genes encoded by CPV-BR that are nonessential for virus replication in tissue culture but are involved in inhibiting the generation of an inflammatory response in the chicken embryo chorioallantoic membrane (CAM) model. The CPV-BR-encoded anti-inflammatory genes include the gene encoding the 38-kDa protein (also called 38K, crmA, SPI-2, or VV-WR-ORF-B13R), a tumor necrosis factor receptor homolog, and an unidentified gene that maps to the right end of the CPV genome. The kinetics of triggering of an inflammatory response at the site of virus infection as well as the magnitude of the response is dependent on the virus-encoded inhibitor that is deleted. Virus yields recovered from pocks decreased in proportion to the magnitude of the inflammatory response. The deletion of these identified inhibitors of inflammation was associated with attenuation of the mutant viruses in mice. These data confirm the existence of multiple poxvirus-encoded host defense modifiers whose function is to block the generation of an inflammatory response at the site of virus infection, which allows enhanced virus replication and potentially facilitates virus transmission.     

An orthopoxvirus serpinlike gene controls the ability of infected cells to fuse.

Most orthopoxviruses encode a functional hemagglutinin (HA), which is nonessential for virus growth in cell culture. However, inactivation of the HA gene leads to the formation of polykaryocytes (syncytia) by fusion of infected cells at neutral pH. Fusion is not observed when a functional HA gene is present. Deletion of open reading frames (ORFs) K2, K3, and K4 within the HindIII K fragment of the HA-positive (HA+) vaccinia virus strain WR also led to fusion of cells upon infection at neutral pH. A novel ORF inactivation procedure utilizing the polymerase chain reaction was used to specifically implicate the K2 ORF in this phenomenon. The K2 ORF (the viral SPI-3 gene) encodes a protein resembling serine protease inhibitors (serpins). Inactivation of the SPI-3 gene in any of the HA+ orthopoxviruses tested caused infected cells to fuse in a manner which appeared identical to that seen for HA- mutants, although fusion was most pronounced with cowpox virus. SPI-3-negative strains fused despite the fact that the HA was expressed and processed normally, i.e., cells infected with SPI-3 mutants remained functionally hemadsorption positive, and analysis of the HA protein by Western immunoblot suggested that posttranslational modifications of the HA protein appeared normal. Fusion triggered by SPI-3 mutants, like that for HA- mutants, was inhibited by the monoclonal antibody C3 directed against the vaccinia virus 14-kDa envelope protein. Therefore SPI-3- and HA-mediated fusion share a requirement for the 14-kDa protein, suggesting linkage of the seemingly disparate SPI-3 and HA genes through a common pathway which normally acts to prevent fusion of cells infected with wild-type virus.     

Suppressors of a host range mutation in the rabbitpox virus serpin SPI-1 map to proteins essential for viral DNA replication

The orthopoxvirus serpin SPI-1 is an intracellular serine protease inhibitor that is active against cathepsin G in vitro. Rabbitpox virus (RPV) mutants with deletions of the SPI-1 gene grow on monkey kidney cells (CV-1) but do not plaque on normally permissive human lung carcinoma cells (A549). This reduced-host-range (hr) phenotype suggests that SPI-1 may interact with cellular and/or other viral proteins. We devised a genetic screen for suppressors of SPI-1 hr mutations by first introducing a mutation into SPI-1 (T309R) at residue P14 of the serpin reactive center loop. The SPI-1 T309R serpin is inactive as a protease inhibitor in vitro. Introduction of the mutation into RPV leads to the same restricted hr phenotype as deletion of the SPI-1 gene. Second-site suppressors were selected by restoration of growth of the RPV SPI-1 T309R hr mutant on A549 cells. Both intragenic and extragenic suppressors of the T309R mutation were identified. One novel intragenic suppressor mutation, T309C, restored protease inhibition by SPI-1 in vitro. Extragenic suppressor mutations were mapped by a new procedure utilizing overlapping PCR products encompassing the entire genome in conjunction with marker rescue. One suppressor mutation, which also rendered the virus temperature sensitive for growth, mapped to the DNA polymerase gene (E9L). Several other suppressors mapped to gene D5R, an NTPase required for DNA replication. These results unexpectedly suggest that the host range function of SPI-1 may be associated with viral DNA replication by an as yet unknown mechanism.     

Granzyme B short-circuits the need for caspase 8 activity during granule-mediated cytotoxic T-lymphocyte killing by directly cleaving Bid

Cytotoxic T lymphocytes (CTL) can trigger an apoptotic signal through the Fas receptor or by the exocytosis of granzyme B and perforin. Caspase activation is an important component of both pathways. Granzyme B, a serine proteinase contained in granules, has been shown to proteolytically process and activate members of the caspase family in vitro. In order to gain an understanding of the contributions of caspases 8 and 3 during granule-induced apoptosis in intact cells, we have used target cells that either stably express the rabbitpox virus-encoded caspase inhibitor SPI-2 or are devoid of caspase 3. The overexpression of SPI-2 in target cells significantly inhibited DNA fragmentation, phosphatidylserine externalization, and mitochondrial disruption during Fas-mediated cell death. In contrast, SPI-2 expression in target cells provided no protection against granzyme-mediated apoptosis, mitochondrial collapse, or cytolysis, leading us to conclude that SPI-2-inhibited caspases are not an essential requirement for the granzyme pathway. Caspase 3-deficient MCF-7 cells were found to be resistant to CTL-mediated DNA fragmentation but not to CTL-mediated cytolysis and loss of the mitochondrial inner membrane potential. Furthermore, we demonstrate that granzyme B directly cleaves the proapoptotic molecule Bid, bypassing the need for caspase 8 activation of Bid. These results provide evidence for a two-pronged strategy for mediating target cell destruction and provide evidence of a direct link between granzyme B activity, Bid cleavage, and caspase 3 activation in whole cells.     

Orthopoxvirus fusion inhibitor glycoprotein SPI-3 (open reading frame K2L) contains motifs characteristic of serine proteinase inhibitors that are not required for control of cell fusion.

The cowpox virus (CPV) SPI-3 gene (open reading frame K2L in vaccinia virus) is one of three orthopoxvirus genes whose products are members of the serpin (serine proteinase inhibitor) superfamily. The CPV SPI-3 gene, when overexpressed by using the vaccinia virus/T7 expression system, synthesized two proteins of 50 and 48 kDa. Treatment with the N glycosylation inhibitor tunicamycin converted the two SPI-3 proteins to a single 40-kDa protein, close to the size of 42 kDa predicted from the DNA sequence, suggesting that the SPI-3 protein, unlike the other two orthopoxvirus serpins, is a glycoprotein. Immunoblotting with an anti-SPI-3 antibody showed that the SPI-3 protein is synthesized early in infection prior to DNA replication. SPI-3 inhibits cell-cell fusion during infections with both CPV and vaccinia virus. A transfection assay was devised to test engineered mutants of SPI-3 for the ability to inhibit fusion. Two mutants with C-terminal deletions of 156 and 70 amino acids were completely inactive in fusion inhibition. Site-directed mutations were constructed near the C terminus of SPI-3, in or near the predicted reactive-site loop which is conserved in inhibitory serpins. Substitutions within the loop at the P1 to P1' positions and P5 to P5' positions, inclusive, did not result in any loss of activity, nor did changes at the P17 to P10 residues in the stalk of the reactive loop. Therefore, SPI-3 does not appear to control cell fusion by acting as a serine proteinase inhibitor.     

Vaccinia Virus Infection Disarms the Mitochondrion-Mediated Pathway of the Apoptotic Cascade by Modulating the Permeability Transition Pore

Many viruses have evolved strategies that target crucial components within the apoptotic cascade. One of the best studied is the caspase 8 inhibitor, crmA/Spi-2, encoded by members of the poxvirus family. Since many proapoptotic stimuli induce apoptosis through a mitochondrion-dependent, caspase 8-independent pathway, we hypothesized that vaccinia virus would encode a mechanism to directly modulate the mitochondrial apoptotic pathway. In support of this, we observed that Jurkat cells, which undergo Fas-mediated apoptosis exclusively through the mitochondrial route, were resistant to Fas-induced death following infection with a crmA/Spi-2-deficient strain of vaccinia virus. In addition, vaccinia virus-infected cells subjected to the proapoptotic stimulus staurosporine exhibited decreased levels of both cytochrome c released from the mitochondria and caspase 3 activation. In all cases we found that the loss of the mitochondrial membrane potential, which occurs as a result of opening the multimeric permeability transition pore complex, was prevented in vaccinia virus-infected cells. Moreover, vaccinia virus infection specifically inhibited opening of the permeability transition pore following treatment with the permeability transition pore ligand atractyloside and t-butylhydroperoxide. These studies indicate that vaccinia virus infection directly impacts the mitochondrial apoptotic cascade by influencing the permeability transition pore.     

Intrahepatic lymphocyte expression of dipeptidyl peptidase I-processed granzyme B and perforin induces hepatocyte expression of serine proteinase inhibitor 6 (Serpinb9/SPI-6)

Human proteinase inhibitor 9 (PI-9/serpinB9) and the murine ortholog, serine proteinase inhibitor 6 (SPI-6/serpinb9) are members of a family of intracellular serine proteinase inhibitors (serpins). PI-9 and SPI-6 expression in immune-privileged cells, APCs, and CTLs protects these cells against the actions of granzyme B, and when expressed in tumor cells or virally infected hepatocytes, confers resistance to killing by CTL and NK cells. The present studies were designed to assess the existence of any correlation between granzyme B activity in intrahepatic lymphocytes and induction of hepatic SPI-6 expression. To this end, SPI-6, PI-9, and serpinB9 homolog expression was examined in response to IFN-alpha treatment and during in vivo adenoviral infection of the liver. SPI-6 mRNA expression increased 10- to 100-fold in the liver after IFN-alpha stimulation and during the course of viral infection, whereas no significant up-regulation of SPI-8 and <5-fold increases in other PI-9/serpinB9 homolog mRNAs was observed. Increased SPI-6 gene expression during viral infection correlated with influxes of NK cells and CTL. Moreover, IFN-alpha-induced up-regulation of hepatocyte SPI-6 mRNA expression was not observed in NK cell-depleted mice. Additional experiments using genetically altered mice either deficient in perforin or unable to process or express granzyme B indicated that SPI-6 is selectively up-regulated in hepatocytes in response to infiltration of the liver by NK cells that express perforin and enzymatically active granzyme B.     

The ps/hr gene (B5R open reading frame homolog) of rabbitpox virus controls pock color, is a component of extracellular enveloped virus, and is secreted into the medium.

Wild-type rabbitpox virus (RPV) produces red hemorrhagic pocks on the chorioallantoic membranes (CAMs) of embryonated chicken eggs. Like the crmA (SPI-2) gene of cowpox virus, disruption of the RPV ps/hr gene results in a mutant which produces white pocks on the CAMs. An examination of the properties of the RPV(ps/hr) mutant in cell culture also reveals a significantly reduced host range, defined as the inability to form plaques, compared with wild-type virus. One of several cell types on which RPV(ps/hr) mutants fail to produce plaques is chicken embryo fibroblasts, cells which have been traditionally used to propagate spontaneously arising white pock mutants isolated from CAMs. The inability of the RPV(ps/hr) mutant to form plaques in chicken embryo fibroblasts correlates with a failure of a low multiplicity of infection to spread to neighboring cells and to form extracellular enveloped virus (EEV), although the formation and yields of infectious intracellular naked virus appear relatively normal. The gene product of the ps/hr gene, initially synthesized as a 45-kDa glycoprotein, is found as a component of EEV, but not intracellular naked virus, and as a smaller, secreted soluble protein of 35 kDa. Production of the secreted 35-kDa protein was found to be independent of any viral morphogenesis, suggesting two distinct pathways for release of the ps/hr gene product from the cell, i.e., as a component of the EEV particle and as a separately secreted glycoprotein.     

Serpins prevent granzyme-induced death in a species-specific manner

Expression of serine protease inhibitors (serpins) is one of the mechanisms used by tumour cells to escape immune surveillance. Previously, we have shown that expression of serpins SPI-6 and SPI-CI, respectively, renders tumour cells resistant to granzyme B (GrB)-mediated death and granzyme M (GrM)-mediated death. To obtain better insight into the interaction between serpins and their target proteases, we investigated the roles of protease inhibitor (PI)-9 and SPI-6 in the resistance to GrB-mediated and CD95-mediated death in further detail. Neither human PI-9 nor its murine orthologue SPI-6 was capable of preventing CD95-induced apoptosis in murine or human cells, indicating that these serpins do not inhibit the activation of apical caspases in this pathway. High expression of PI-9 or SPI-6 did prevent apoptosis induced by human GrB. Strikingly, only SPI-6, and not PI-9, was capable of inhibiting murine GrB, suggesting that a difference in enzymatic specificity exists between the mouse and the human granzymes. In agreement with this suggestion, murine GrB was clearly less effective in inducing apoptosis in human cells. Similar species specificity was also observed for SPI-CI and GrM when either their capacity to associate or the effectiveness of GrM-induced cytotoxicity was analysed. Our findings therefore indicate a species diversity that has a clear effect on mixed in vitro effector target settings.     

Prevention of apoptosis in CNTF-dependent neurons by a mutant ICE and by viral protein CrmA but not by proto-oncogene product Bcl-2

The interleukin-1beta converting enzyme (ICE) gene family, (homologues of C. elegans cell death gene product Ced-3) plays an important role in controlling programmed cell death. Nerve growth factor (NGF) promotes survival of cultured embryonic chicken dorsal root ganglion neurons. Ciliary ganglion neurons depend exclusively on ciliary neurotrophic factor (CNTF) for survival. Complete depletion of NGF or CNTF from culture medium induces apoptosis in both types of neurons. We can prevent apoptosis, due either to NGF or CNTF withdrawal and in either type of neuron, by overexpression of a mutant inactive ICE and an ICE inhibitor, the product of cowpox virus gene crmA. Bcl-2 does not prevent apoptosis in CNTF-dependent ciliary neurons or DRG neurons as it does in NGF-dependent neurons. These results suggest that neuronal cell death is mediated through a common effector mechanism involving the Ice family of genes, whereas different suppression mechanisms are engaged depending upon the specific neurotrophic factors present.     

Effective Blockage of Both the Extrinsic and Intrinsic Pathways of Apoptosis in Mice by TAT-crmA

Evidence accumulates that in clinically relevant cell death, both the intrinsic and extrinsic apoptotic pathway synergistically contribute to organ failure. In search for an inhibitor of apoptosis that provides effective blockage of these pathways, we analyzed viral proteins that evolved to protect the infected host cells. In particular, the cowpox virus protein crmA has been demonstrated to be capable of blocking key caspases of both pro-apoptotic pathways. To deliver crmA into eukaryotic cells, we fused the TAT protein transduction domain of HIV to the N terminus of crmA. In vitro, the TAT-crmA fusion protein was efficiently translocated into target cells and inhibited apoptosis mediated through caspase-8, caspase-9, and caspase-3 after stimulation with α-Fas, etoposide, doxorubicin, or staurosporine. The extrinsic apoptotic pathway was investigated following α-Fas stimulation. In vivo 90% of TAT-crmA-treated animals survived an otherwise lethal dose of α-Fas and showed protection from Fas-induced organ failure. To examine the intrinsic apoptotic pathway, we investigated the survival of mice treated with an otherwise lethal dose of doxorubicin. Whereas all control mice died within 31 days, 40% of mice that concomitantly received intraperitoneal injections of TAT-crmA survived. To test the ability to comprehensively block both the intrinsic and extrinsic apoptotic pathway in a clinically relevant setting, we employed a murine cardiac ischemia-reperfusion model. TAT-crmA reduced infarction size by 40% and preserved left ventricular function. In summary, these results provide a proof of principle for the inhibition of apoptosis with TAT-crmA, which might provide a new treatment option for ischemia-reperfusion injuries.     

Recruitment of apoptotic cysteine proteases (caspases) in influenza virus-induced cell death.

Influenza virus infection induces apoptosis in cultured cells with an augmented expression of Fas (APO-1/CD95). Caspases, a family of cysteine proteases structurally related to interleukin-1-beta-converting enzyme (ICE), play crucial roles in apoptosis induced by various stimuli, including Fas. However, activation of the caspase-cascade seems to be different in various pathways of apoptotic stimuli. We therefore examined the involvement of caspases in influenza virus-induced apoptosis using caspase inhibitors. We found that z-VAD-fmk and z-IETD-fmk effectively inhibited virus-induced apoptosis, whereas Ac-DEVD-CHO and Ac-YVAD-CHO showed partial and little effect on virus-induced cell death, respectively. Consistently, caspase-3-like activity, but not caspase-1-like activity, was increased in the virus-infected cells. The transfection of plasmids encoding viral inhibitors of caspase (v-FLIP or crmA) into HeLa cells inhibited apoptosis by virus infection. The peptide inhibitors of caspases used in this study did not inhibit viral replication. We conclude that influenza virus infection activates some caspases, and that this activation may be downstream of viral replication.     

Crystal structure of viral serpin crmA provides insights into its mechanism of cysteine proteinase inhibition

CrmA is an unusual viral serpin that inhibits both cysteine and serine proteinases involved in the regulation of host inflammatory and apoptosis processes. It differs from other members of the serpin superfamily by having a reactive center loop that is one residue shorter, and by its apparent inability to form SDS-stable covalent complexes with cysteine proteinases. To obtain insight into the inhibitory mechanism of crmA, we determined the crystal structure of reactive center loop-cleaved crmA to 2.9 A resolution. The structure, which is the first of a viral serpin, suggests that crmA can inhibit cysteine proteinases by a mechanism analogous to that used by other serpins against serine proteinases. However, one striking difference from other serpins, which may be significant for in vivo function, is an additional highly charged antiparallel strand for b sheet A, whose sequence and length are unique to crmA.