1 Department of Virology, Division of Investigative Science, Imperial College Faculty of Medicine, St Mary's Campus, Norfolk Place, London W2 1PG, UK
2 Max von Pettenkofer Institut, Abteilung für Virologie, LMU-München, Germany
Correspondence
Richard F. Greaves
richard.greaves{at}imperial.ac.uk
Gabriele Hahn
ghahn{at}m3401.mpk.med.uni-muenchen.de
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ABSTRACT |
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Present address: Division of Hepatology and Gene Therapy, Department of Internal Medicine, University of Navarra, Pamplona, Spain.
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INTRODUCTION |
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Emphasizing the likely role of apoptosis in innate immunity, many viruses encode apoptosis inhibitors (Benedict et al., 2002; Cuconati & White, 2002
; Everett & McFadden, 2001
), which prevent the premature death of infected cells (Cuconati & White, 2002
; Pilder et al., 1984
). DNA viruses may have to stimulate the cell cycle to induce a nuclear environment that is conducive to genome replication (Nevins, 1994
; White, 2001
), but this inappropriate growth stimulus may trigger events such as p19ARF and p53 activation that potentiate apoptosis (Debbas & White, 1993
; de Stanchina et al., 1998
). Human cytomegalovirus (HCMV) infection accordingly drives quiescent fibroblasts into the cell cycle and towards a block in late G1 or S phase (Bresnahan et al., 1996
; Dittmer & Mocarski, 1997
; Jault et al., 1995
; Lu & Shenk, 1996
). HCMV-encoded functions can stimulate (Castillo et al., 2000
; Kalejta et al., 2003
; Murphy et al., 2000
; Poma et al., 1996
; Sinclair et al., 2000
; Wiebusch & Hagemeier, 1999
) or stall (Lu & Shenk, 1999
; Murphy et al., 2000
; Noris et al., 2002
; Wiebusch & Hagemeier, 1999
, 2001
) the cell cycle through interactions with cellular proteins (Fortunato et al., 1997
; Hagemeier et al., 1994
; Kalejta et al., 2003
; Margolis et al., 1995
; Pajovic et al., 1997
; Poma et al., 1996
) and the induction of cellular genes (Bresnahan et al., 1998
; Song & Stinski, 2002
). By subverting normal cell-cycle control, HCMV may optimize conditions for genome replication, but might concurrently stimulate apoptosis.
A functional screen identified several anti-apoptotic proteins that are encoded within the HCMV UL3638 gene region. The UL37 exon 1 protein (pUL37x1) (termed viral mitochondrion-localized inhibitor of apoptosis or vMIA) blocks apoptosis in response to death-receptor ligation, DNA damage or infection by an E1B-deficient adenovirus (Goldmacher, 2002; Goldmacher et al., 1999
). pUL37x1 localizes predominantly to mitochondria and inhibits cytoplasmic cytochrome c release. Related glycoproteins (gpUL37 and gpUL37M) that incorporate UL37x1-encoded residues localize partially to mitochondria and have similar, albeit weaker, anti-apoptotic activities (Colberg-Poley et al., 2000
; Goldmacher et al., 1999
). Two domains (aa 534 and 118147) within the 163 aa pUL37x1 are together both necessary and sufficient for its anti-apoptotic function (Fig. 1a
) (Hayajneh et al., 2001
). These domains are invariant in clinical strains (Hayajneh et al., 2001
) and conserved in functionally homologous primate CMV UL37 proteins (McCormick et al., 2003a
). pUL37x1 has no sequence similarity to the cellular apoptotic inhibitor Bcl-2, yet it sequesters the pro-apoptotic Bcl-2 relative Bax in an inactive form in mitochondria (Arnoult et al., 2004
). pUL37x1 also binds the adenine nucleotide translocator component of the mitochondrial permeability transition pore (Goldmacher et al., 1999
). Expression of pUL37x1 in fibroblasts or HCMV infection disrupts mitochondrial networks (McCormick et al., 2003b
). Another unrelated HCMV protein, pUL36 (the viral inhibitor of caspase activation or vICA), inhibits extrinsic apoptotic pathways in an analogous manner to that of viral FLICE-inhibitory proteins or v-FLIPs, preventing activation of caspase 8 in response to death-receptor ligation (Skaletskaya et al., 2001
). Immediate-early proteins IE1 p72 and IE2 p86 also have anti-apoptotic properties (Zhu et al., 1995
) and IE2 p86 binds and functionally inhibits p53 (Speir et al., 1994
).
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The UL37x1 ORF is well-conserved in all HCMV strains (Hayajneh et al., 2001). Intact gpUL37 is dispensable for virus replication in cultured fibroblasts, demonstrated by deletion of UL37x3 (Borst et al., 1999
). Furthermore, several laboratory-adapted strains of HCMV do not encode functional pUL36, revealing this anti-apoptotic function to be inessential (and possibly detrimental) for virus replication in cultured fibroblasts (Skaletskaya et al., 2001
). A preliminary report has stated that a virus with a deletion of UL37x1 cannot replicate without the provision of pUL37x1 protein in trans (Brune et al., 2003
). Recent genomic screens also indicate that UL37x1 is essential for virus growth (Dunn et al., 2003
; Yu et al., 2003a
).
Here, we have addressed the importance of UL37x1 for the ability of HCMV to replicate in tissue culture by genetic modification. Infection with UL37x1 mutants caused apoptosis of fibroblasts within 4 days, preventing the release of significant quantities of viral progeny.
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METHODS |
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Coding sequences for Bcl-XL and HCMV UL37x1 were PCR-amplified by using Pfu polymerase (Stratagene) and primer pairs 5'-GCCGAGATCTATGTCTCAGAGCACCGGGAGC-3' and 5'-GTACGTCGACTCATTTCCGACTGAAGAGTGAGC-3' or 5'-GCCGAGATCTATGTCTCCAGTCTACGTGAATC-3' and 5'-GTACGTCGACTTACTGGTGAGACTGCTGGGG-3', respectively. UL37x1 was amplified from AD169 cosmid clone pCM1017 (Fleckenstein et al., 1982). A Bcl-XL cDNA template was provided by Graham Packham (Cancer Sciences Division, University of Southampton, UK). BglIISalI fragments ligated into pBabe-Puro (Morgenstern & Land, 1990
) produced vectors pRG379 (BclXL) and pRG380 (UL37x1). A BamHIEcoRIXhoISnaBIMunIBglIISalI polylinker added to pBabe-Puro produced vector pRG299. An EcoRIHindIII fragment of plasmid pCMVE1B19K (White et al., 1992
) ligated into pRG299 produced vector pRG325. Plasmid pBabe-Puro-bcl2 was a gift from Martin Bennett (Department of Medicine, University of Cambridge, UK).
Retroviral vectors pBabe-bcl2, pRG379, pRG380, pRG325 and pBabe-Puro were packaged in NX-A packaging cells (http://www.stanford.edu/group/nolan/protocols/pro_helper_dep.html), filtered and applied to GM03468A fibroblasts with 5 ng polybrene ml1. Polyclonally transduced lines were established after 2 weeks puromycin selection (1 µg ml1). Apoptosis inhibition was confirmed by comparing the responses of transduced populations following 8 h exposure to 1 µM staurosporine with pBabe-Puro-transduced control cells, using terminal transferase dUTP nick end-labelling (TUNEL). Expression of E1B19K was confirmed by using antibody DP07L (Oncogene Research Products).
Mutant bacterial artificial chromosome (BAC) generation and virus culture.
BAC DNAs were propagated as described previously (Borst et al., 1999). AD169-BAC carries the full AD169 coding complement and BAC sequences are removed by Cre recombinase during reconstitution, leaving a residual loxP site between ORFs US1 and US2 (Hobom et al., 2000
). In HB5-BAC, BAC sequences replacing ORFs US2US6 are retained in reconstituted viruses (Borst et al., 1999
). Mutants K8E3, K7H10, K14B1 and K13G11 were isolated from the Tn1721-based TnMax8 transposon-insertion mutant library of AD169-BAC (Hobom et al., 2000
). Insertion coordinates were sequenced by using universal primer sequences within the TnMax8 transposon (Kahrs et al., 1995
) and mapped (Chee et al., 1990
) at nt 52711 (K8E3), 52688 (K7H10), 52526 (K14B1) and 52522 (K13G11) (Fig. 1a
). To construct delUL37x1 mutants by site-directed PCR-based mutagenesis, oligonucleotides were used to amplify pACYC177 DNA (New England Biolabs). The following primers were used: UL37x1for(fko) 5'-GACTGCTGGGGGCCGTTGTGCTGCAGCATCCGAGCTCGTTGCCGCCGTTGCCACAGGAACCGGTGTCTCGATTTATTCAACAAAGCCACG-3' and UL37x1rev(fko) 5'-CATCTTCATGTATATAAGACGGTGTTTCAAGACGACGTGAGACCCACACGCGGGTTTCACTTCTTTCGCCAGTGTTACAACCAATTAACC-3' (HCMV homologous sequences are shown in bold and kanR homologous sequences in italics). The resulting PCR fragment contained kanR flanked by short regions of HCMV homology and was electroporated into DH10B Escherichia coli cells containing either AD169-BAC or HB5-BAC and expressing the recombination functions red
/
/
(Datsenko & Wanner, 2000
; Hahn et al., 2003
; Wagner et al., 2002
). Colonies were selected on plates containing both kanamycin and chloramphenicol. Viral sequences replaced by kanR extended from nt 52293 to 52713 (Chee et al., 1990
). Mutant BAC structures were verified by Southern blot analysis.
To reconstitute recombinant viruses, MRC5 cells or GM03468A fibroblasts expressing E1B19K (3468A-E1B19K cells), Bcl-2 or Bcl-XL were plated into a six-well plate at 7080 % confluence. The next day, cells were washed in Dulbecco's modified Eagles' medium (DMEM) and incubated in DMEM for 30 min prior to transfection. BAC DNA (12 µg) was added to 10 µl SuperFect transfection reagent (Qiagen) and adjusted to 100 µl final volume with DMEM. For AD169-BAC-based mutants, a Cre recombinase-expressing plasmid was added to the transfection (Hobom et al., 2000). After incubation for 30 min at room temperature, 0·9 ml DMEM plus 5 % fetal calf serum (FCS) was added to the transfection mixture. DMEM was removed from the cells and replaced with 1 ml DMEM plus 5 % FCS prior to adding the transfection mix. After incubation for 4 h at 37 °C, the transfection mix was removed and replaced with DMEM plus 5 % FCS for 1 week. Cells were subsequently split into a T25 flask and cultured until appearance of a cytopathic effect (CPE) after approximately 710 days. Cultures where no CPE appeared were split and the culture was continued for up to 1 month. For each virus, three to six such independent transfections were performed.
Growth-curve analysis.
3468A-E1B19K or GM03468A cells (5x105) were infected with parental virus or UL37x1 mutant viruses (derived from 3468A-E1B19K cells) at an m.o.i. of 0·1 or 3. At the indicated days after infection, viral titres from supernatant media were determined by a standard plaque assay (Greaves & Mocarski, 1998) on 3468A-E1B19K cells.
Protein samples.
GM03468A cells (2x105) were infected with wild-type parental or UL37x1 mutant viruses at an m.o.i. of 3. At 90 h post-infection (p.i.), viable and detached cells were collected, washed in PBS and lysed directly into 125 mM Tris/HCl, pH 6·8, 2 % SDS. After determination of protein concentration by using the Bio-Rad DC assay, the remaining components of standard SDS-PAGE sample buffer were added. Total protein (10 µg) was subsequently analysed by Western blotting.
TUNEL/fluorescence-activated cell sorter (FACS) assays.
GM03468A fibroblasts, 3468A-E1B19K fibroblasts or GM03468A fibroblasts (4x105) transduced with the control retroviral vector pBabe-Puro were infected at an m.o.i. of 3 with viruses harvested from 3468A-E1B19K cells. At 4896 h p.i., viable and detached cells were pooled, paraformaldehyde-fixed and TUNEL-stained by using an In Situ Cell Death Detection kit with fluorescein as indicated by the manufacturer (Roche/Boehringer Mannheim). TUNEL staining was quantified by using a Becton Dickinson FACSort machine. Data were collected, analysed and presented by using CellQuest Pro software. Coverslip-grown cells were similarly fixed and TUNEL-stained in situ and then stained with antibody CH167 as described below.
Immunofluorescence analysis.
GM03468A fibroblasts (1x105) grown on glass coverslips were infected at an m.o.i. of 1 and fixed and permeabilized as described previously (Greaves & Mocarski, 1998). Monolayers were incubated with mAbs at room temperature for 1 h [p63.27 for IE1, CCH2 for ppUL44, M23 for pUL112113,
-UL6966 for pUL69, CH167 for pUL57 and 3A12 for pp65; supplied as specified by Gawn & Greaves (2002)
] and diluted in PBS plus 10 % FCS as described previously (Gawn & Greaves, 2002
), except for CCH2 (DAKO; diluted 1 : 25) and 3A12 (Insight; diluted 1 : 50). PBS-washed cells were then incubated for 1 h with Texas red (Serotec)-conjugated goat anti-mouse Ig for 30 min with PBS/10 % mouse serum and for 1 h with fluorescein isothiocyanate (FITC)-conjugated E13 antibody (Argene-Biosoft) diluted 1 : 50 in PBS/10 % mouse serum. Monolayers were counterstained with Hoechst dye. For the results shown in Table 1
, three to four representative fields of 80130 stained cells were photographed and quantified visually/manually for Hoechst (total cells), FITC (IE protein-positive cells) and Texas red (query antigen) staining.
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RESULTS |
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To reconstitute UL37x1 mutant viruses, human fibroblast lines designed to resist apoptosis were generated by retroviral transduction, using the cellular genes encoding Bcl-2 and Bcl-XL or the viral genes encoding adenovirus type 5 E1B19K protein or HCMV pUL37x1. Freshly transduced fibroblast cultures resisted apoptosis after staurosporine treatment, showing negligible TUNEL staining, whilst 3050 % of control fibroblasts became TUNEL-positive. Only E1B19K-expressing fibroblasts retained staurosporine resistance effectively following long-term culture and displayed the greatest growth potential of these anti-apoptotic lines. These cells were designated 3468A-E1B19K. pUL37x1-expressing cells were particularly difficult to maintain over significant periods in cell culture and were not further utilized. Following antibody staining, 95 % of 3468A-E1B19K cells expressed E1B19K in a perinuclear ring and sometimes also in a fibrous cytoplasmic pattern.
Transfection of fibroblast lines expressing Bcl-2, Bcl-XL or E1B19K readily resulted in reconstitution of infectious virus from BACs carrying mutations in UL37x1. Infection spread slowly, with 3468A-E1B19K cells yielding the highest-titre stocks. Viral strains reconstituted in 3468A-E1B19K cells were selected for further propagation. Reconstituted virus strains were assigned their BAC designation with the prefix RV, e.g. RVK7H10 or RVAD169delUL37x1. 3468A-E1B19K cells were used for amplification and titration. Mutant stocks were generally 10100 p.f.u. ml1 lower in titre than corresponding wild-type stocks.
UL37x1 mutant virus strains grow defectively in normal fibroblasts
Multiple-step growth-curve experiments using an input m.o.i. of 0·1 were undertaken for all UL37x1 mutant strains and compared with the corresponding parental virus strains. Growth in 3468A-E1B19K fibroblasts was compared with growth in GM03468A fibroblasts and progeny were plaque-assayed in 3468A-E1B19K cells. Growth of UL37x1 mutants was defective in both cell types (Fig. 2). Mutant strain RVK8E3, which retains an intact UL37x1 ORF, was the least defective of the mutants tested, but still displayed a 10-fold growth deficiency in both cell types (Fig. 2a and b
). Transposon mutants RVK7H10, RVK14B1 and RVK13G11 grew weakly in 3468A-E1B19K cells, but did reach output titres slightly higher than the input (Fig. 2a
). In contrast, minimal replication was observed for mutants RVK7H10, RVK14B1 and RVK13G11 in GM03468A fibroblasts (Fig. 2b
), with progeny titres not approaching input titres, consistent with our failure to reconstitute these virus strains after transfection of normal fibroblasts with the cognate BACs. The deletion mutants RVAD169delUL37x1 and RVHB5delUL37x1 also grew weakly in 3468A-E1B19K cells (Fig. 2c
), but did not replicate significantly in GM03468A cultures (Fig. 2d
). A single-cycle growth-curve experiment in GM03468A cultures compared RVAD169 with RVK8E3 and RVK7H10 (Fig. 2e
). RVK8E3 grew with normal kinetics up to 4 days after infection, but revealed a 10-fold defect in growth at later time points. RVK7H10 replicated extremely poorly, but some virus release was detected after the eclipse phase.
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Apoptosis was measured independently by Western blotting of the 89 kDa fragment of cleaved poly(ADP-ribose) polymerase (PARP) (Fig. 5). In cells infected for 90 h, abundant amounts of cleaved PARP were detected for each of the transposon mutants and for the RVHB5delUL37x1 mutant, compared with mock-infected fibroblasts. Control staurosporine-treated fibroblasts showed a smaller increase in cleaved PARP. Wild-type viruses RVAD169 and RVHB5 showed a very modest increase in cleaved PARP, relative to mock-infected cells. This PARP cleavage assay confirmed that fibroblasts infected by the UL37x1 mutant viruses underwent apoptosis. However, by using this measure, the behaviour of the RVK8E3 mutant was indistinguishable from that of other, more severely growth-compromised mutants.
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DISCUSSION |
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Mutant RVK8E3 had a modest replication defect, but demonstrably induced apoptosis. We propose that timing of apoptosis is key to these differing growth phenotypes. Fully defective UL37x1 mutants apparently induced apoptosis immediately prior to late-phase progression and virus release. A slight delay in the onset of apoptosis in RVK8E3-infected cells, due to residual pUL37x1 expression, could open a short window for virus release and spread, resulting in the observed growth phenotype. In support of this model, E1B19K expression in trans delayed, but did not prevent, apoptosis, yet permitted propagation of UL37x1 mutants. Single-cycle growth kinetics in normal fibroblasts revealed that RVK8E3 reached lower peak titres at an earlier time than wild-type virus, consistent with a limited window of virus release. The modestly reduced levels of apoptosis induced by RVK8E3 in a TUNEL assay also supported this model, but such marginal differences in apoptotic potential have not been discernible by other means. A possible discordance between apoptosis induction and replication inhibition queries the significance of apoptosis in the growth phenotype of UL37x1 mutants. However, the caspase inhibitor ZVAD-FMK substantially restored replication of RVK7H10, confirming virus-induced apoptosis as the primary limitation to growth of replication-defective UL37x1 mutant viruses. Partial complementation of the growth of UL37x1 mutants by three independent apoptosis inhibitors (E1B19K, Bcl-2 and Bcl-XL) also argues that the anti-apoptotic functions of UL37x1-encoded proteins are vital for sustainable virus replication. In contrast, the gammaherpesviruses EpsteinBarr virus (EBV), human herpesvirus 8, murine gammaherpesvirus 68 and herpesvirus saimiri all encode Bcl-2 sequence homologues (Benedict et al., 2002; Cuconati & White, 2002
) with related function to pUL37x1, but which for EBV is inessential for latent or lytic virus infection in vitro (Marchini et al., 1991
).
We studied laboratory strain AD169, whose UL36 inhibitor of caspase activation is non-functional (Skaletskaya et al., 2001). Whilst responses of virus-infected cells to death-receptor ligation will be altered as a result of the absence of UL36 function, AD169 certainly requires an intact UL37x1 gene to protect against infection-initiated apoptosis and to allow sustainable virus replication. It will be informative to analyse UL37x1 mutants constructed against a UL36 wild-type background. pUL36 (vICA) may protect against virus-induced apoptosis in the absence of UL37x1 function; stimulation of death-receptor pathways during the virus late phase could play a role in infection-induced apoptosis. However, we anticipate that the trigger for infection-induced apoptosis is intracellular and that such mutants would also induce apoptosis.
The timing of HCMV-induced apoptosis corresponds with the onset of the late phase of viral gene expression, rather than with virus-induced cell-cycle advance and block. For example, peak cyclin E-associated kinase levels and late G1/S block have been observed at 24 h after HCMV infection (Bresnahan et al., 1996; Jault et al., 1995
; Lu & Shenk, 1996
). Induction of apoptosis by adenovirus was formerly thought to occur mainly via E1A-mediated activation of the p19ARFmdm2p53 pathway and probably via functional induction of the Bcl-2-related Bax protein. However, recent studies question this model. The BH3 protein Bak mediates apoptosis induced by an adenovirus E1B19K mutant in the absence of Bax (Cuconati et al., 2002
). Bak is normally sequestered by the MCL-1 protein, which is degraded during adenovirus infection and during the cellular DNA damage response. In the absence of E1B19K, Bak then forms pro-apoptotic complexes with Bax (Cuconati et al., 2003
). Other indications suggest that DNA damage could be the primary trigger for apoptosis, either through E1A-induced cellular dysfunction or via viral DNA replication and packaging (Cuconati et al., 2003
). Similarly, DNA damage could also be a primary trigger for apoptosis during HCMV infection. As viral polymerase inhibitors blocked HCMV-induced apoptosis, a pro-apoptotic DNA damage signal may result from viral genome replication or packaging. A genetic analysis of host factors required for HCMV-induced apoptosis should determine whether a Bak/MCL-1 mechanism operates similarly to adenovirus or whether other mechanisms such as p53-mediated synthesis of Bax or BH3-only proteins (Yu et al., 2003b
) are important.
HCMV late-phase functions might induce apoptosis. Pro-apoptotic late proteins may be involved: some viral proteins implicated in HCMV cell-cycle control accumulate to their greatest levels at late times of infection (Kalejta et al., 2003; Lu & Shenk, 1999
) and thus might trigger apoptosis. Alternatively, stress induced by accumulation of late-phase viral proteins could trigger apoptosis. Replication of bovine viral diarrhea virus and respiratory syncytial virus both trigger caspase 12-mediated apoptosis via the endoplasmic reticulum (ER) stress pathway (Bitko & Barik, 2001
; Jordan et al., 2002
) and pUL37x1 can protect against ER stress (Boya et al., 2002
).
As UL37 proteins regulate gene expression (Colberg-Poley et al., 1992, 1998
), we investigated viral gene expression during infection by UL37x1 mutants. We detected no gross defects in the accumulation of immediate-early or delayed-early proteins, although more sensitive techniques might detect smaller modulations. Our results contrast with observations that UL3638 products facilitate viral DNA replication (Smith & Pari, 1995
), probably due to stimulation of the promoters of viral delayed-early genes (Iskenderian et al., 1996
). Specifically, defined UL3638 transcriptional targets (ppUL44 and pUL57) accumulated normally during UL37x1 mutant infection and viral DNA replication compartments were established normally. However, fewer mutant-infected cells accumulated the viral late protein pp65 at 72 h p.i., probably due to apoptotic loss of cells entering late phase, as accompanied by significant cell death. However, absence of UL37x1 function could also affect late gene expression directly. Indeed, UL37x1 mutant growth was still modestly deficient when apoptosis was inhibited by ZVAD-FMK, suggesting an additional minor growth defect that is not attributable to apoptosis induction.
Our results indicate that UL37x1 expression protects the host cell from apoptosis triggered by virus infection itself. Adequate UL37x1 expression levels are probably essential for maximal apoptosis inhibition. Our preliminary unpublished work indicates that HCMV strains carrying mutations in the major immediate-early gene affect both pUL37x1 expression and the survival time of infected cells. Modulation of UL37x1 may exert sensitive control over the timing of cell death, and regulation of UL37x1 expression may prove to be complex and environmentally responsive. Intriguingly, HCMV may control when its host cell dies.
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ACKNOWLEDGEMENTS |
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Received 18 June 2004;
accepted 25 August 2004.