Department of Microbiology, School of Medical Sciences, University of Otago, PO Box 56, Dunedin, New Zealand1
Author for correspondence: Vernon Ward. Fax +64 3 479 8540. e-mail vernon.ward{at}stonebow.otago.ac.nz
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Abstract |
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Introduction |
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The dysregulation of apoptosis is important in many disease states (Thompson, 1995 ; Barr & Tomei, 1994
). Apoptosis is an important factor in the replication of many viruses, with cellular suicide being an important virus defence mechanism (OBrien, 1998
). Viruses often carry genes that can intercede directly in the apoptosis signalling pathways or act as inhibitors of caspases (Tschopp et al., 1998
). In common with several other large DNA viruses, baculoviruses contain genes that can inhibit apoptosis (Miller, 1997
). The p35 gene of Autographa californica nucleopolyhedrovirus (AcMNPV) (Clem et al., 1991
) encodes a direct inhibitor of caspases (Bump et al., 1995
), while the inhibitor of apoptosis (iap) genes are thought to prevent the activation of caspases (Seshagiri & Miller, 1997
). A p35-like gene called Slp49 has been identified in Spodoptera littoralis nucleopolyhedrovirus (Du et al., 1999
). The baculovirus p35 gene has been identified in very few baculoviruses (Clem et al., 1991
; Kamita et al., 1993
), while the iap genes have been identified in all baculoviruses studied to date, and in insect iridescent viruses (Crook et al., 1993
). The role of the viral iap genes was determined by functional replacement of the p35 gene of AcMNPV with an iap gene from the granulovirus of Cydia pomonella (CpGV) (Crook et al., 1993
). Subsequently, an iap gene from Orgyia pseudotsugata nucleopolyhedrovirus (OpMNPV) has been identified as an active inhibitor of apoptosis (Birnbaum et al., 1994
).
Most NPVs carry multiple genes that have the characteristic iap motifs of baculovirus inhibitor repeats (BIRs) and a RING finger domain, yet functionality has only been shown for the two iaps described above (Crook et al., 1993 ; Birnbaum et al., 1994
). The domain requirements of IAPs are variable. Mammalian cellular IAPs have been shown not to require a RING finger domain for functionality (Roy et al., 1997
), unlike invertebrate IAPs of both cellular and viral origin, which require this domain to be active (Clem & Miller, 1994
; Huang et al., 2000
; Seshagiri et al., 1999
).
There is very little information on what apoptotic pathways IAPs can block. Recent evidence that an IAP from OpMNPV can block apoptosis in mammalian cells (Hawkins et al., 1996 ), combined with the similarity to IAPs from the cells of vertebrate organisms, suggests that IAPs target a universal point(s) in apoptosis. The importance of iap genes has been reinforced by the discovery of cellular homologues of baculovirus iaps in a variety of mammalian (Liston et al., 1996
), dipteran (Hay et al., 1995
) and avian (You & Bose, 1998
) cells.
In this paper, we investigate the iap genes identified on the EppoMNPV genome and their ability to function as inhibitors of apoptosis. The ability of the IAPs to block multiple apoptosis pathways and the requirement of additional genes to produce an active IAP were investigated.
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Methods |
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CpGV iap and Bombyx mori NPV p35.
The iap gene from CpGV was kindly supplied as plasmid pSB490 by L. Miller (Departments of Genetics and Entomology, The University of Georgia, Athens, GA, USA) and the Bombyx mori NPV p35 gene (Kamita et al., 1993 ) was kindly supplied by S. Maeda (Department of Entomology, University of California, Davis, CA, USA).
Cloning and sequencing.
Screening of restriction enzyme libraries of EppoMNPV DNA was performed as described by Hyink et al. (1998 ). Sequencing of the termini of HindIII and EcoRI restriction fragment clones was used to identify homologues of iap-2 and iap-4 genes by BLAST searching of the GenBank database (Altschul et al., 1997
). Based upon comparison of the genetic maps of EppoMNPV (Hyink et al., 1998
) and OpMNPV (Ahrens et al., 1997
), the presence of an iap-1 gene and an iap-3 gene was predicted on HindIII fragment A. The HindIII-A fragment was subcloned in pBluescriptII SK+ and screened by sequencing for the presence of iap homologues. All four iap-containing clones were fully sequenced using an ABI 377 automated sequencer at the Centre for Gene Research, University of Otago. Analysis of derived sequences was performed using the Lasergene suite of DNA analysis programs (DNAStar). Alignment of IAP sequences was performed using the Clustal V algorithm (Higgins & Sharp, 1988
) and the phylogeny was determined using PHYLIP (Felsenstein, 1995
). The reliability of the phylogeny was estimated by BOOTSTRAP analysis using 100 datasets.
Induction of apoptosis.
Sf-21 cells were treated with actinomycin D at a final concentration of 2 µg/ml, anisomycin (50 µg/ml), cycloheximide (100 µg/ml), tumour necrosis factor- (TNF
; 0·1 µg/ml) plus cycloheximide (5 µg/ml), or 5,6-dichloro-1-
-D-ribofuranosylbenzimidazole (DRB; 50 µg/ml) for 18 h at 28 °C. Apoptosis induction by cycloheximide was also performed in Sf900-II medium that had been supplemented with 10% foetal calf serum (GIBCO BRL Life Technologies). All chemicals were obtained from Sigma. Apoptosis was induced by exposure of Sf-21 cells to UV irradiation by inverting 6-well tissue culture plates, from which the medium had been removed, onto a standard laboratory transilluminator (Ultra Violet Products) for 15 s. The medium was then replaced.
Analysis of apoptosis.
Sf-21 cell monolayers in 35 mm dishes were treated with 200 µl of lysis buffer (10 mM Tris pH 7·5, 25 mM EDTA, 0·2% Triton X-100) (Cartier et al., 1994 ) for 1 h at room temperature. The lysate was extracted once with phenol, once with phenol/chloroform, twice with chloroform, then alcohol-precipitated. The precipitate was centrifuged at 14000 r.p.m. for 5 min then washed with 70% alcohol and dried at 37 °C. The DNA was redissolved in 20 µl RNase A (50 µg/ml in water) and the total volume was electrophoresed on 1% agarose LE (Roche) gels in TAE buffer. DNA was visualized by ethidium bromide staining and UV illumination. To quantify the anti-apoptotic effect of anti-apoptotic genes, cell monolayers were stained with 0·04% trypan blue and cell viability was scored using an inverted light microscope. For each treatment, five fields of view were scored.
Inhibition of chemical- and UV-induced apoptosis.
The iap genes were obtained as clones in the plasmid pBluescriptII SK+ as shown in Fig. 1. Plasmid DNA was prepared for transfection using anion resin-exchange columns (Qiagen). Sf-21 cells (1·5x106 cells per 35 mm diameter well) were transfected with 2 µg of plasmid DNA using Cellfectin (GIBCO BRL Life Technologies) or FuGENE 6 (Roche) following the manufacturers protocols and the plates were incubated for 1824 h at 28 °C. The inducing chemical was then added to each well, or the cells were UV-irradiated to induce apoptosis, and the plates were incubated for 18 h or 30 h. Total cytoplasmic DNA was then extracted as described above and tested for oligonucleosome ladder formation by electrophoresis in 1% agarose.
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A further construct, designated orf69(RF-), was made in which the 3' end of clone 898iap-2 was removed to eliminate the RING finger motif from the iap-2 gene, leaving the orf69 homologue as the only complete gene in the clone. Clone 898iap-2 was digested with BglII, which has a recognition site 223 bp inside the 3' end of the iap-2 gene, and BamHI, which cuts in the multiple cloning site of pBluescriptII SK+. The plasmid was recircularized by ligation with T4 DNA ligase.
The iap-2 gene was subcloned into the expression vector pCMV·SPORT (GIBCO BRL Life Technologies). The gene was amplified by PCR using the universal forward primer and a primer complementary to the 5' end of the iap-2 gene (5' GAATGGATCCCTAAGCATGGATTTGCAAAAGT 3'). This primer contained a BamHI restriction site (underlined) and the ATG start codon (bold). The PCR product was digested with BamHI and HindIII and then ligated into BamHI- and HindIII-digested pCMV·SPORT.
All clones were transformed into E. coli strain DH5 and screened by standard protocols. All PCR-derived clones were sequenced to ensure that no PCR errors had occurred. The ability of each construct to inhibit apoptosis was determined as described above using 2 µg of plasmid per transfection.
Confirmation of iap-1 as an apoptosis inhibitor gene.
An analogous approach was taken with the iap-1-containing clone iap-1/42, which contained a homologue of OpMNPV orf42. Primer TM59 (5' TTCAACTCGGTCAAATGCGCG 3') was paired with the universal reverse primer to generate the iap-1 gene plus 302 bp of upstream sequence from the clone iap-1/42 by PCR. The PCR product was cloned into pBluescript SK- and designated as 302iap-1. The clone contained the iap-1 gene as the only complete gene and was tested as described above.
Nucleotide sequence accession numbers.
The GenBank accession numbers are: iap-1,AF119227; iap-2,AF037358; iap-3, AF180757; iap-4, AF119228.
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Results |
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The HindIII-T clone contained the iap-2 gene and three other open reading frames (ORFs; Fig. 1a). A partial homologue of lef3 was identified, as was a complete ORF with 69% amino acid identity to OpMNPV orf73 (61% identity to AcMNPV orf68). A complete gene with 57% identity to AcMNPV orf69 was also present. There is no homologue of AcMNPV orf69 in OpMNPV. The gene order in this region of the genome was identical to AcMNPV but not to OpMNPV.
The 2·1 kb XbaIKpnI subclone (clone iap-1/42) contained three ORFs in addition to the iap-1 gene (Fig. 1b). A homologue to baculovirus lef6 genes was located downstream of the iap-1 gene. BLAST analysis of the 70 amino acids present in the clone showed the closest similarity to the first 70 amino acids of the OpMNPV lef6 gene. A complete ORF of 127 amino acids located upstream of the iap-1 gene showed 62% and 52% identity to orf42 of OpMNPV and orf26 of AcMNPV respectively. A partial gene sequence representing 237 amino acids of a homologue of OpMNPV orf43 (AcMNPV orf25) was present upstream of the orf42 homologue. The order of the genes surrounding iap-1 is identical to that found in OpMNPV (Ahrens et al., 1997
).
The iap-3 ORF was the only complete ORF on the 1·787 kb MluI subclone (Fig. 1c). Flanking the iap-3 gene were two partial ORFs with similarity to OpMNPV fgf (119 amino acids) and OpMNPV orf36 (155 amino acids). The gene content and arrangement of this region of the EppoMNPV genome is different to that of OpMNPV (Ahrens et al., 1997
).
The iap-4 gene was the only complete ORF on the EcoRI-N clone. A 168 amino acid partial ORF upstream of the iap-4 gene was similar to OpMNPV orf107.
IAPs fall into distinct homology groups
The IAP-1 protein of EppoMNPV is a 284 amino acid protein that has a predicted molecular mass of 32550 Da, with 71% and 59% identity to OpMNPV IAP-1 and AcMNPV IAP-1 respectively. The IAP-2 protein contains 239 amino acids (27363 Da), with 63% and 57% identity to OpMNPV IAP-2 and AcMNPV IAP-2 respectively. The IAP-3 protein is a 261 amino acid protein (29981 Da) with a sequence identity of 56% to OpMNPV IAP-3. IAP-4 is a 141 amino acid protein (15932 Da) with 51% identity to the IAP-4 of OpMNPV. An alignment and phylogenetic analysis of the IAP-1, IAP-2 and IAP-3 proteins of baculoviruses is presented in Fig. 2. The IAPs fall into distinct groups (Fig. 2b
). The only exception to the clustering of IAP types is Buzura suppressaria NPV IAP-1, which was named by order on the viral genome rather than sequence homology (Hu et al., 1998
). IAP-4s were not included because none have been shown to be active, so there is no evidence, other than sequence homology, that these are indeed inhibitors of apoptosis. Two BIRs are present in the IAP-1, IAP-2 and IAP-3 proteins (Fig. 2a
). Comparison with other IAPs showed a conserved BIR motif of GX911CX2CX810E/DX5HX36C. A characteristic C3HC4 RING finger motif is also present in the IAP-1, IAP-2 and IAP-3 proteins (Fig. 2a
). Analysis of the IAP-4 protein showed only one BIR region and the RING finger motif.
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EppoMNPV IAP-3 and IAP-4 are not functional
IAP-3 and IAP-4 were not found to block apoptosis induced by actinomycin D (Fig. 4d, e
). The use of a CMV promoter, which had been shown to work for the iap-2 gene (see above), did not produce an active iap-3 gene. The iap-4 gene was not tested with a CMV promoter. Subsequent analysis showed no inhibition of apoptosis for any of the inducing agents used in this study for either the iap-3 gene or the iap-4 gene (Table 1
).
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Discussion |
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When only 330 bp of sequence was present upstream of the iap-2 gene, the iap-2 gene was not functional. The iap-2 gene was functional when the orf69 gene homologue and a possible promoter region were located upstream. Removal of the RING finger motif from iap-2 abolished anti-apoptotic activity, confirming both the role of EppoMNPV iap-2 and the essential requirement for a RING finger motif in baculoviral IAPs. The use of a CMV promoter to express the iap-2 gene confirmed the functionality of IAP-2 and suggests that the orf69 homologue has a role in expression of iap-2. A recent study by Li et al. (1999 ) has shown that orf69 of AcMNPV can function as an activator of gene expression. An alternative explanation is that iap-2 promoter sequences lie in the region between 898 and 330 bp upstream of the iap-2 gene, within orf69.
Comparison of the iap-1, iap-2 and iap-3 genes showed that they fall into distinct homology classes and that the naming of new IAPs should be based upon gene homologies rather than order within a viral genome. The homology groupings and the presence of multiple iaps on NPV genomes suggest a requirement for different iaps. As far as the authors know, this is the first report of anti-apoptosis activity for iap-1 and iap-2 genes. That these genes do possess anti-apoptosis activity suggests that this is likely to be their primary function. Given the broad range of anti-apoptotic activity demonstrated in this paper, the different iaps are more likely to be needed for different cells, tissues and hosts than for the inhibition of different stimuli. The iap genes that have been demonstrated as active from baculoviruses other than EppoMNPV cluster in the iap-3 group of iap genes. It is likely that the apparent lack of an active iap-3 gene in EppoMNPV is compensated by the activity of the iap-1 and iap-2 genes. EppoMNPV does not grow in Sf-21 cells (K. Caradoc-Davies, personal communication) and this study indicates that an active iap is unlikely to be a host range determinant for EppoMNPV infection of Sf-21 cells.
The induction of Sf-21 cell apoptosis by the RNA synthesis inhibitors actinomycin D, DRB and anisomycin has been reported previously (Clem & Miller, 1994 ). These transcription inhibitors have distinct modes of action (White & Phillips, 1988
; Zandomeni et al., 1983
), but would be expected to initiate the same or a very similar apoptotic signalling pathway. What was surprising was the induction by cycloheximide and TNF
. Clem & Miller (1994
) reported that cycloheximide does not induce apoptosis in Sf-21 cells. The study of Clem & Miller (1994
) employed cells in serum-containing medium. The addition of 10% serum to the Sf900-II medium prevented the induction of apoptosis in Sf-21 cells by cycloheximide. Cycloheximide-induced apoptosis has also been observed for Choristoneura fumiferana (CF-203) cells (Palli et al., 1996
). The induction by TNF
suggests the presence of a TNFR-like protein on the surface of the Sf-21 cell and the presence of a signalling pathway that may be analogous to that involving the death domain proteins such as RIP, TRAFF and caspase-8 in mammalian cells. The human cellular IAPs have been reported to interact with these death domain proteins (Rothe et al., 1995
; Uren et al., 1996
); however, it appears that mammalian IAPs are distinct from invertebrate cellular and viral IAPs. In particular, mammalian IAPs do not require a RING finger motif for activity and interact directly with caspase-3 to prevent apoptosis (Deveraux et al., 1997
; Roy et al., 1997
), rather than preventing terminal caspase activation as shown for invertebrate IAPs (Huang et al., 2000
; Manji et al., 1997
; Seshagiri & Miller, 1997
; Seshagiri et al., 1999
). Despite these differences, some invertebrate IAPs are active in mammalian cells. Hawkins et al. (1996
) have shown that OpMNPV IAP-3 is active in mammalian cells and the recent study of Huang et al. (2000
) showed that invertebrate IAPs can inhibit mammalian caspase-9, which is involved in the activation of terminal caspases. Huang et al. (2000
) predicted that SfIAP and CpIAP target a caspase-9-like protease in S. frugiperda cells to prevent the activation of Sf-caspase-1. These data support the conservation that is evident between the apoptosis systems of metazoans, allowing comparison with mammalian systems for elucidating the function of baculovirus IAPs.
It has been suggested that caspases are an amplification system that accelerates the cell death response (Li et al., 1997 ; Liu et al., 1996
). In mammalian cells, most apoptosis signals converge to cause the release of cytochrome c from mitochondria, which in turn contributes to apoptosome formation and the activation of caspase-9, followed by the subsequent activation of caspase-3 (Green & Kroemer, 1998
). The ability of EppoMNPV IAP-2 to block apoptosis induced by an array of induction signals that in mammalian cells converge at the mitochondrion, suggests that IAP-2 functions after the signals have converged. If an analogy is made to mammalian cells then the target of IAP-2 must be associated with the mitochondria, the apoptosome, or the direct activation of effector caspases. Seshagiri & Miller (1997
) have shown that at least one baculoviral IAP functions upstream of Sf-caspase-1 and Duckett et al. (1998
) have proposed that the human IAP-like protein regulates apoptosis downstream of the mitochondrion. The paper by Huang et al. (2000
) supports this theory.
One proposed theory of caspase amplification is that caspases interact with the mitochondrion in a cyclical amplification (Green & Kroemer, 1998 ). A consequence of this theory would be that direct inhibition of caspases that interact with the mitochondrion would lead to a delay in apoptosis but not a prevention of apoptosis. The delay in apoptosis caused by IAP-1 is such a response. IAP-1 may interfere with a caspase/mitochondrion amplification loop, although care must be taken when comparing invertebrate and mammalian cells.
This paper has presented the first evidence that iap-1 and iap-2 genes of baculoviruses can function as inhibitors of apoptosis. The paper also shows that these inhibitors can function against a wide array of apoptosis inducers that can be expected to trigger multiple initiator pathways of apoptosis, and provides insights into the mode of action of IAPs.
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Acknowledgments |
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Received 17 April 2000;
accepted 27 July 2000.