1 University of Florida, College of Medicine, Department of Molecular Genetics and Microbiology, PO Box 100266, University of Florida, Gainesville, FL 32610-0266, USA
2 Section of Digestive Disease and Nutrition, University of Illinois at Chicago, Chicago, IL 60612, USA
3 Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA
Correspondence
Richard W. Moyer
rmoyer{at}ufl.edu
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ABSTRACT |
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INTRODUCTION |
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Most serpins function as proteinase inhibitors, although some have functions (including hormone transport and regulation of blood pressure) that do not involve proteinase inhibition (Silverman et al., 2001). Inhibitory serpins form a stable 1 : 1 complex with target enzymes. The interaction is mediated by the reactive centre loop (RCL) of the serpin (Silverman et al., 2001
). The specificity of a serpin is determined primarily by the P1 residue within the RCL. CrmA, with a P1 of aspartate (P1-Asp), is a cross-class inhibitor that targets the serine proteinase, granzyme-B (Quan et al., 1995
) and caspases, which are thiol-proteases (Komiyama et al., 1994
). CrmA is a potent inhibitor of caspase-1 (Komiyama et al., 1994
; Garcia-Calvo et al., 1998
), which processes pro-interleukin (IL)-1
, proIL-1
and proIL-18 to mature, proinflammatory cytokines (Thornberry et al., 1992
; Ghayur et al., 1997
; Gu et al., 1997
). The importance of IL-1
in controlling poxvirus infections is supported by the orthopoxvirus-encoded IL-1
receptor (Spriggs et al., 1992
; Alcami & Smith, 1992
), which controls fever during vaccinia virus (VV) infection (Alcami & Smith, 1996
; Kettle et al., 1997
). IL-18 is a factor that induces synthesis of IFN-
. The significance of IL-18 and IFN-
in poxvirus infections can be inferred because poxviruses encode binding proteins for IL-18 (Born et al., 2000
; Smith et al., 2000
; Calderara et al., 2001
; Xiang & Moss, 2001a
, b
; Symons et al., 2002a
; Reading & Smith, 2003
) and IFN-
(Upton et al., 1992
; Alcami & Smith, 1995
; Mossman et al., 1995
; Verardi et al., 2001
; Smith & Alcami, 2002
; Symons et al., 2002b
).
Infection of embryonated chicken egg chorioallantoic membranes (CAMs) has been used to follow innate immune responses to CPV infection. CAMs at this stage of development lack mature lymphocytes (Chen et al., 1994; Masteller et al., 1997
). Wild-type (wt) CPV produces red, haemorrhagic, non-inflammatory lesions (pocks) on CAMs, whereas CPV deleted for CrmA produces white, inflammatory pocks (Palumbo et al., 1989
). White pocks produce less virus, contain heterophils and reduce nitro blue tetrazolium (NBT), indicating the presence of activated heterophils. Control of inflammation by CrmA may be due to the inhibition of caspase-1-mediated production of IL-1
(Palumbo et al., 1994
).
In addition, CrmA blocks apoptosis in CPV-infected swine cells (Ray & Pickup, 1996). Consistent with these results, CrmA inhibits caspase-8 and -10, which initiate apoptosis (Zhou et al., 1997
; Garcia-Calvo et al., 1998
). In vitro studies have shown that CrmA also inhibits apoptosis induced by allogeneic cytotoxic T lymphocytes (CTLs) (Tewari et al., 1995
); the level of protection differs depending on target-cell and effector-CTL populations (Mullbacher et al., 1999
). The role of CrmA during virus infections in vivo is less well understood.
Deletion of CrmA from CPV caused attenuation in intranasally infected (Thompson et al., 1993) and intracranially infected (Palumbo et al., 1994
) Balb/c mice. In contrast, deletion of SPI-2 from VV had no effect on virulence in intranasally infected Balb/c mice (Kettle et al., 1995
), although intradermal infection of mouse ear pinnae produced larger lesions than wt VV (Tscharke et al., 2002
). An early report (Thompson et al., 1993
) that rabbitpox virus RPV
SPI-2 was attenuated in mice infected intranasally was flawed; the virus contained a second mutation in the K1L gene (R. W. Moyer, unpublished data).
We used CPV-infected CAMs to address the following questions: (i) Does CrmA prevent white pock formation by proteinase inhibition? (ii) Does CrmA function solely as a caspase inhibitor? The first question was addressed by mutating P1-Asp in the CrmA RCL to alanine (D303A), which should abolish serpin function and induce white pocks. The second question was addressed by replacing CrmA in CPV with other caspase inhibitors such as P35 of the baculovirus Autographa californica nuclear polyhedrosis virus or SERP2 from Myxoma virus (MYX).
The baculovirus P35 protein is not a serpin, but it prevents apoptosis in several systems including insect cells, Caenorhabditis elegans, Drosophila melanogaster and mammalian cells (Hay et al., 1994; Xue & Horvitz, 1995
; Izquierdo et al., 1999
). P35 directly inhibits human caspase-1, -3, -6, -7, -8 and -10 (Zhou et al., 1998
) and Spodoptera frugiperda caspase-1 (Ahmad et al., 1997
) but not granzyme B (Zhou et al., 1998
). If the role of CrmA during CPV infection is solely to inhibit caspases, then the virus formed by replacing CrmA in CPV with P35 should resemble wt CPV.
The MYX protein SERP2 is a serpin and contains P1-Asp within the RCL (Petit et al., 1996), but only shares 35 % amino acid identity with CrmA. MYX derivatives lacking SERP2 are attenuated during myxoma infection of European rabbits (Messud-Petit et al., 1998
). SERP2, like CrmA, inhibits caspase-1 and granzyme-B in vitro (Turner et al., 1999
). We set out to ask whether SERP2 could substitute for CrmA in CPV infection of the chicken CAM and whether CrmA could substitute for SERP2 in myxoma infection of the European rabbit.
We found that despite the similarities between CrmA, P35 and SERP2 in vitro, these proteins were not interchangeable in vivo, indicating important differences in biological activity.
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METHODS |
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CrmA site-directed mutagenesis.
The P1-Asp residue at position 303 of CrmA was mutated to alanine using the Altered Sites system (Promega). The CrmA coding region was cloned into pAlterEx1. Primer FS275 (5'-pTGTGCGCTGGTGGCAGCATGCGCATCAACAGTTACA-3') was used to create the mutations (underlined). The resulting plasmid, pAlterEx1CrmA-D303A, was verified by sequencing.
Construction of recombinant cowpox viruses.
The following plasmids were constructed to create CPV recombinant viruses. Underlining in primers indicates the restriction sites XbaI, EcoRI, HindIII, SmaI and NcoI.
pBS-CLF.
The upstream flanking sequence of the CrmA coding region (322 nt) was PCR amplified with Vent polymerase (New England Biolabs) from CPV genomic DNA using primers GM17 (5'-GATCTCTAGAGCGGCCGCGGTTCGGTGGCAAACTTACATGGAA-3') and GM19 (5'-TCGATCGAATTCCATGGCAATCGATTTTGTTGT-3') and then cloned into pBluescriptII KS(+) (Stratagene) using the XbaI and EcoRI sites.
pBS-CCF.
The downstream flanking sequence of the CrmA coding region (347 nt) was PCR amplified from CPV genomic DNA using primers GM21 (5'-ATCGTACGAATTCCCGGGCATATGATCACATTCTTAATATTAGAATATTAG-3') and GM22 (5'-TGCTACAAGCTTGATGAACACTGATTCCGCATC-3'). The right flank PCR product was cloned into the EcoRI and HindIII sites of pBS-CLF.
pBS-CgS/A.
The P7·5-gpt cassette was derived from pBS-gptA (Turner & Moyer, 1992) and cloned into the SalI and ApaI sites of pBS-CCF.
pCglacZ.
The P11-lacZ cassette was PCR amplified from pSC11 (Chakrabarti et al., 1985) with Vent polymerase using primers FS129 (5'-GTCAGATCCATGGTTGAATTCCGAGCTTGGCTG-3') and FS130 (5'-AGTCAACGCCCGGGTACGCTCACAGAATTCCCG-3') and then cloned into pBS-CgS/A using the NcoI and SmaI sites.
pBS-D303ACgS/A.
The CrmA-D303A coding region was PCR amplified from pAlterEx1CrmA-D303A using FS307 (5'-TACGTCCATGGATATCTTCAGGGAAA-3') and FS308 (5'-CAGCTACCCGGGTTATAATTAGTTGTTGGAGAGC-3') and cloned into the NcoI and SmaI sites of pBS-CgS/A.
pC35.
A clone of P35 was provided by Lois Miller (University of Georgia). pC35 was constructed by inserting P35 between the NcoI and SmaI sites of pBS-CgS/A by recombinant PCR (Turner & Moyer, 1992); the start codon of P35 replaced the natural ATG at the NcoI site. The primer pair used to generate the left flank was FS1 (5'-GATCTCTAGAGCGGCCGCGGTTCGGTGGCAAACTTACATGGAA-3') and FS91 (5'-GGCAATCGATTTTGTTG-3'). The primer pair used to generate P35 was FS89 (5'-CAACAAAATCGATTGCCATGTGTGTAATTTTTCCGG-3') with the underlined portion complementary to FS91 and FS74 (5'-TACGTCACCCGGGTTATTTAATTGTGTTTAATATTAC-3'). Finally the left flank was linked to P35 using primers FS1 and FS74. The full-length PCR product was cloned into the XbaI and SmaI sites of pBS-CgS/A.
pCSERP2.
The SERP2 gene was subcloned from pTM1-SERP2 into the NcoI and SmaI sites of pBS-CgS/A.
The CrmA open reading frame (ORF) was replaced with lacZ, CrmA-D303A, P35 or SERP2. CrmA regulatory elements were retained. First, CPVCrmA : : lacZ was generated using pCglacZ, which contained a gpt cassette conferring resistance to mycophenolic acid (MPA), enabling transient dominant selection (TDS) for virus recombinants (Falkner & Moss, 1990
). Wild-type CPV-infected CV-1 cells were transfected with pCglacZ, MPAR plaques selected and MPAS segregants screened for blue staining with X-Gal (5-bromo-4-chloro-3-indolyl
-D-galactopyranoside) to isolate CPV
CrmA : : lacZ. Subsequently, recombinant viruses were obtained from CPV
CrmA : : lacZ by transfection with plasmids containing the appropriate ORF, TDS and loss of X-Gal staining. All viral constructs were verified by sequencing.
Immunoblotting.
CV-1 or LLC-PK1 cells (2x106) were infected at an m.o.i. of 10. Eighteen hours post-infection (p.i.), infected cells were resuspended in buffer (100 mM Tris/HCl, pH 8·0, 100 mM NaCl, 0·5 % Triton X-100) and underwent three freezethaw cycles. Debris was pelleted and 100 µg supernatant protein was electrophoresed on 10 % acrylamide/SDS gels and transferred to nitrocellulose membranes (Micron Separations) in 25 mM Tris/HCl, pH 8·3, 192 mM glycine, 20 % methanol using a semi-dry transfer apparatus (Fisher Scientific). Membranes were blocked in 5 % non-fat milk in PBS, pH 7·4, 0·1 % Tween 20 for 1 h at room temperature and probed with primary antibody for 1 h. Polyclonal rabbit antiserum to P35 was diluted 1 : 10 000 (provided by Paul Friesen, University of Wisconsin). Polyclonal rabbit antiserum to SERP2 diluted 1 : 500 (Turner et al., 1999) and mouse monoclonal antibody to CrmA diluted 1 : 2000 (Macen et al., 1998
) have been described. Membranes were washed three times (5 min each) in wash buffer (PBS, pH 7·4, 0·1 % Tween 20) and incubated with secondary antibodies (diluted 1 : 2500) of either goat anti-mouse IgG conjugated to horseradish peroxidase or goat anti-rabbit IgG conjugated to horseradish peroxidase (Southern Biotechnology Associates) for 1 h at room temperature. After five washes (10 min each) in wash buffer, immunoreactive proteins were visualized by enhanced chemiluminescence (Amersham ECL kit).
CAM infections.
Embryonated chicken eggs from SPAFAS, Inc. were incubated at 38·5 °C with 50 % humidity for 11 days. The dropped CAMs were inoculated with 10 p.f.u. and the eggs incubated for 72 h. Infected CAMs were harvested, washed twice in PBS (pH 7·2) and scanned on a Microtek ScanmakerIII scanner at 500 d.p.i. Individual pocks were excised and stored at 80 °C. Alternatively, membranes were inoculated with 500 p.f.u. and CAMs were harvested 48 h later yielding confluently infected membranes.
Staining reactive oxygen intermediates.
CAMs from eggs were harvested at 72 h p.i. and washed twice with PBS. The membranes were incubated in 0·1 % NBT (Sigma) in PBS at 37 °C for 1 h, the NBT was removed and the membranes were washed once with PBS and scanned.
MTT reduction assay.
Individual pocks were harvested at 72 h p.i. and pooled (each pool weighed 1530 mg), then submerged in 200 µl PBS containing 5 mg 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) ml1 for 30 min at 37 °C. The MTT solution was removed and the reduced formazan extracted by grinding the membranes with a microfuge pestle in 200 µl DMSO (Sigma). The dissolved formazan was clarified by centrifugation at 12 000 g for 2 min. The absorbance of the solution was read at 550 nm. Absorbance values were expressed as A550 (mg tissue)1. The standard error of the mean was calculated from four experiments.
Terminal caspase activity assay.
Individual pocks isolated from CAMs at 72 h p.i. were ground in 100 µl 10 mM HEPES, pH 7·5, 2 mM EDTA, 0·1 % CHAPS and 10 mM DTT, freeze-thawed three times and clarified by centrifugation at 12 000 g for 5 min. Terminal caspase activity in 25 µg of pock extract was measured as increase in fluorescence s1 of aminomethylcoumarin (AMC) from the substrate Ac-DEVD-AMC (Bachem) used at 10 µM in 200 µl of buffer (100 mM HEPES, pH 7·5, 10 % sucrose, 0·1 % CHAPS, 10 mM DTT). Substrate cleavage was monitored using a Tecan SpectraFluor microplate reader with excitation at 380 nm and emission at 460 nm.
Construction of recombinant myxoma viruses.
MYXSERP2 : : lacZ was constructed via a plasmid containing lacZ under the CPV-ATI promoter flanked by SERP2 flanks. Nt 1310 of SERP2 were PCR amplified with primers NP1 (5'-GCGGGTACCATGGAGCTTTTCAAGCATTTCCT-3') and NP2 (5'-GCGAAGCTTTGGCCGCCACCCATCGGTTGA-3') and cloned using KpnI (underlined) and a natural EcoRV site near NP2. Nt 7451002 of SERP2 were amplified with primers NP3 (5'-GCGTCTAGAATCTTCGCACATCCTAACTTCGAGGA-3') and NP4 (5'-GCGGAGCTCTTAGTAATTGGGAGAAGTGACT-3'). The SERP2 flanks were cloned to either side of an MscIXbaI fragment containing PATI-lacZ. The resulting plasmid was transfected into wt MYX-infected RK-13 cells. Blue plaques were obtained and verified by Western blot analysis and sequencing to confirm that lacZ had been inserted into the SERP2 locus.
MYXSERP2 : : CrmA contained the CrmA ORF inserted in place of the SERP2 ORF under the control of the SERP2 promoter. The region upstream from SERP2 including the SERP2 promoter was PCR amplified with primer F1 (5'-GCGGGTACCTCTTGTTTAACAACGCGATACA-3') and M1 (5'-TCCCTGAAGATATCCATAATCGCACTTATACATTATA-3'). The CrmA ORF was amplified with M2 (5'-ATGGATATCTTCAGGGA-3') and M3 (5'-GCGTCTAGATTAATTAGTTGTTGGAGA-3'), then joined to the F1/M1-amplified product by recombinant PCR (Turner & Moyer, 1992
) to create PSERP2-CrmA. The region downstream of SERP2 was PCR amplified with NP3 and NP4 and joined to the PSERP2-CrmA by an XbaI site. The P7·5-gpt cassette was inserted downstream from CrmA. The final construct was transfected into MYX
SERP2 : : lacZ-infected RK-13 cells. MPA-resistant white plaques were selected. The resulting MYX
SERP2 : : CrmA virus containing gpt was confirmed by sequencing.
Infection of rabbits with recombinant myxoma viruses.
All animal procedures were approved by UF-IACUC. Female, specific-pathogen-free New Zealand White (NZW) rabbits (Oryctolagus cuniculus) weighing 2 kg were purchased from Myrtle's Rabbitry (Thompson Station, TN, USA). Areas of 3 cm2 on each thigh were surgically prepared and 500 p.f.u. virus in 0·1 ml PBS was injected intradermally. Rabbits were killed if signs of respiratory distress were observed or by 10 days p.i. Prior to killing, rabbits were anaesthetized by intramuscular injection of 50 mg Ketaject kg1 and 10 mg Xyla-ject kg1 (100 mg ketamine.HCl ml1 and 20 mg xylazine ml1; Phoenix Pharmaceuticals). Blood was collected by cardiac puncture and stored in EDTA. Rabbits were killed by intracardiac injection of 3 ml Beuthanasia-D-Special (Schering-Plough Animal Health).
Tissue sample processing.
Tissue samples from necropsies were stored in 10 % buffered formalin (Protocol; Fisher Scientific) for 24 h. The thickness of the integument at the centre of the virus inoculation site was measured with calipers three times and the mean value was recorded. Tissues were trimmed, placed in cassettes and stored in 10 % buffered formalin before paraffin embedding, sectioning and staining with haematoxylin and eosin (Richard-Allen).
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RESULTS |
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Wild-type CPV infection of CAMs produced red, haemorrhagic pocks, while CPVCrmA : : lacZ produced white pocks as expected (Palumbo et al., 1989
) (Fig. 2
). The white pocks produced by CPV
CrmA : : lacZ stained blue with NBT indicating the presence of activated heterophils. CPV-CrmA-D303A also produced white pocks that stained blue with NBT (Fig. 2
), suggesting that P1-Asp within the RCL is required for CrmA to inhibit inflammation. Surprisingly, both P35- and SERP2-recombinant viruses produced white pocks with positive NBT staining (Fig. 2
). Both P35 and SERP2 failed to inhibit inflammation during CPV infections of CAMs.
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CrmA, P35 and SERP2 block terminal caspase induction within infected pocks
We evaluated apoptosis within infected pocks by measuring the activity of terminal caspases. Extracts were evaluated for cleavage of Ac-DEVD-AMC, a fluorogenic substrate for caspases-3, -6 and -7 (Fig. 6). Little caspase induction was noted in mock-infected or wt CPV-infected CAMs, but infection with CPV
CrmA : : lacZ resulted in cleavage of Ac-DEVD-AMC. CPV
CrmA : : lacZ and CPV-CrmA-D303A induced terminal caspase activity to similar levels, reaffirming that P1-Asp of the CrmA RCL is essential for CrmA function. Expression of either P35 or SERP2 was able to block induction of terminal caspase activity within the context of CPV
CrmA-infected pocks.
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Infection of rabbits with recombinant myxoma viruses lacking SERP2 or expressing CrmA
Poxvirus infection of mammals with fully developed immune systems provides a more complete model for virulence than chicken CAMs. In view of conflicting data about whether deletion of CrmA from CPV (Thompson et al., 1993; Palumbo et al., 1994
) or SPI-2 from VV (Kettle et al., 1995
; Tscharke et al., 2002
) causes virus attenuation in mice, we did not examine CPV
CrmA : : SERP2 for virulence in mice. Instead, we asked whether CrmA could replace the pathogenic function of SERP2 by infecting rabbits with MYX
SERP2 : : CrmA, a recombinant that expresses CrmA in place of SERP2.
Infection of NZW rabbits with wt MYX resulted in the formation of primary dermal lesions that were prominent at day 3 and continued to enlarge until day 8. Lesions were raised, erythematous and ulcerated (Fig. 7A). In contrast, MYX
SERP2 : : lacZ and MYX
SERP2 : : CrmA caused lesions with a central area of necrosis (0·51·5 cm in diameter) surrounded by concentric circles of erythema (1·01·5 cm wide) and blanched skin (0·30·5 cm wide) (Fig. 7A
). Nine days p.i., the mean lesion diameters caused by the mutated viruses were not significantly different from wt MYX lesions.
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Wild-type MYX caused secondary lesions on mucocutaneous junctions and ears by 6 days p.i. Like the primary lesions, the secondary lesions were raised and erythematous (Fig. 7C). Infection with MYX
SERP2 : : lacZ or MYX
SERP2 : : CrmA caused flat secondary lesions that tended to be more numerous in animals that developed more severe signs of respiratory disease (Fig. 7C
). Progression of infection with MYX
SERP2 : : lacZ and MYX
SERP2 : : CrmA followed the same time course observed with wt MYX infection. No lesions were observed on the control rabbit.
Due to production of myxoid extracellular matrix by fibroblasts and severe oedema, the primary lesions caused by wt MYX were markedly thicker than those of MYXSERP2 : : lacZ or MYX
SERP2 : : CrmA. On average, wt MYX lesions were 33 mm in diameter and 9·7 mm thick at necropsy (Fig. 8
). However, the lesions of MYX
SERP2 : : lacZ and MYX
SERP2 : : CrmA were flat, averaging 4·0 mm thick with MYX
SERP2 : : lacZ and 4·9 mm thick with MYX
SERP2 : : CrmA (Fig. 8
).
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At necropsy, all infected rabbits had mild diffuse splenomegaly, patchy consolidation of lung lobes, pulmonary haemorrhage and slightly enlarged sublumbar lymph nodes. Microscopic examination of the lungs revealed non-suppurative interstitial pneumonia with patchy oedema, haemorrhage and congestion. Rabbits with more severe clinical disease had more severe lymphocytic inflammation. Lung tissue showed mild to moderate growth of Bordetella bronchiseptica, a normal inhabitant of the respiratory tract of rabbits that is considered non-pathogenic (Deeb et al., 1990). Sublumbar lymph nodes were examined for apoptosis by TdT-mediated dUTP nick end-labelling (TUNEL) staining, but no appreciable differences between virus infections were observed (data not shown).
The mortality rate of NZW rabbits infected with wt MYX was 100 %; all 11 rabbits were killed between 8 and 10 days p.i. These rabbits developed harsh lung sounds with crackles and wheezes indicative of interstitial lung disease and airway narrowing. The mortality rate of rabbits infected with MYXSERP2 : : lacZ was less than 15 %. Only one of eight rabbits infected with MYX
SERP2 : : lacZ had harsh lung sounds that may have progressed beyond day 10. In contrast, the mortality rate of MYX
SERP2 : : CrmA-infected rabbits was 70 %; seven of ten animals were euthanized between 8 and 10 days p.i. due to respiratory distress.
These results demonstrated that CrmA can partially substitute for SERP2 to restore MYX virulence but cannot replace SERP2 to produce myxomatous lesions.
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DISCUSSION |
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We were surprised by the fact that P35 and SERP2 were unable to block inflammation in the chicken CAM (Figs 24) but did control apoptosis in this system (Fig. 5
). Biochemical data indicate that although P35 is an active inhibitor of a broad range of human caspases, the association rate (ka) of P35 for human caspase-1 is 8·2±1·1x103 M1 s1 (Zhou et al., 1998
) compared with 1·7x107 M1 s1 for CrmA and caspase-1 (Komiyama et al., 1994
). P35 may therefore be less effective as an anti-inflammatory agent than CrmA. In contrast, P35 and CrmA have relatively similar inhibition constants against human caspase-8 with values of 6·4±0·1x104 and 9·1x104 M1 s1, respectively, and against caspase-10 of 1·2±0·4x103 and 1·9±0·4x103 M1 s1, respectively (Zhou et al., 1998
). SERP2 is a weaker inhibitor of human caspase-1 (Ki=420 nM) than CrmA (Ki=4 pM) (Turner et al., 1999
), a difference that may account for the lack of SERP2 activity against inflammation in the CAM. At present, chicken caspase-1 has not been expressed and there is no information regarding inhibition by CrmA, P35 or SERP2.
The mechanism of inflammation in the CAM is not well understood. Mammalian pro-IL-1 is activated by cleavage at Asp-27 followed by Asp-116, with both sites conserved (Swaan et al., 2001
). However, both critical Asp residues are absent from chicken IL-1
(Weining et al., 1998
). We expressed chicken pro-IL-1
using the T7 Quick Coupled Transcription/Translation System (Promega Corporation), but were unable to detect processing by purified caspase-1 or extracts from CAMs infected with wt CPV or CPV
CrmA : : lacZ (data not shown). Chicken pro-IL-18 does have a potential conserved cleavage site when compared with mammalian pro-IL-18. Chicken pro-IL-18, expressed in vitro by transcription/translation, was appropriately processed by 1 U human caspase-1 (data not shown). Extracts from CAMs infected with CPV
CrmA : : lacZ but not wt CPV cleaved chicken pro-IL-18. However experiments with peptide inhibitors showed that the activity was blocked by Ac-DEVD-CHO, an inhibitor of terminal caspases, but not by the caspase-1-specific inhibitor Ac-WEHD-CHO (data not shown). The results indicated the observed cleavage was a by-product of high levels of terminal caspases.
CrmA has been suggested to inhibit generation of pro-inflammatory molecules other than IL-1 or IL-18 (Palumbo et al., 1989). Endothelial injury, which leads to haemorrhage and oedema in CPV and MYX infections, is known to activate factor XII, which activates four cascades that involve pro-inflammatory proteases: the coagulation, fibrinolysis, complement and kinin systems. Vascular injury is also associated with increased arachidonic acid metabolism causing production of pro-inflammatory eicosanoids. Any of these mediators are candidate targets for inhibition by CrmA.
Despite the fact that neither P35 nor SERP2 had anti-inflammatory activity in the CAM, both proteins were functional as apoptosis inhibitors within the context of a CPV infection. Restoration of virus yields under these conditions indicated that control of apoptosis rather than inflammation is required for full virus replication.
CrmA did not substitute fully for SERP2 in MYX-infected rabbits. Although the degree of inflammation in dermal lesions of rabbits infected with wt MYX, MYXSERP2 : : lacZ and MYX
SERP2 : : CrmA was similar, fibroblast reactivity was markedly increased with wt MYX compared with the recombinant viruses (Fig. 7
). This result indicates that CrmA could not function as SERP2 to produce myxomatous lesions. However, MYX
SERP2 : : CrmA caused severe morbidity and mortality, whereas MYX
SERP2 : : lacZ was severely attenuated. This suggested that CrmA can function in place of SERP2 to restore virus virulence. The dichotomy of these results parallels that seen in the CPV-infected CAM and supports the notion that SERP2 has functions both in promoting primary lesion development and in lethality.
We conclude that CrmA can restore partial virulence in MYX deleted for SERP2 but cannot restore lesion morphology. Likewise, replacement of CrmA with known caspase inhibitors prevents apoptosis and allows virus replication during CPV infection of CAMs but does not prevent inflammation. In the CPV/CAM system, the targets for CrmA in blocking apoptosis and inflammation are probably different caspases. At present, we can only speculate that the roles of SERP2 in lesion morphology and virulence may reflect different target proteases or that the same protease is present at different levels in different cells or tissues.
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ACKNOWLEDGEMENTS |
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Received 15 December 2003;
accepted 4 February 2004.