Institute for Animal Science and Health (ID-Lelystad), Department of Avian Virology, PO Box 65, NL-8200 AB Lelystad, The Netherlands1
University of Utrecht, Department of Infection and Immunity, Utrecht, The Netherlands2
Author for correspondence: Mirriam Tacken. Fax +31 320 238 668. e-mail m.g.j.tacken{at}id.wag-ur.nl
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Abstract |
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Introduction |
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IBDV is an unenveloped, icosahedral virus about 60 nm in diameter (Hirai & Shimakura, 1974 ). Its genome is composed of two double-stranded (ds) RNA segments designated A and B (Dobos et al., 1979
; Müller et al., 1979
). The larger segment, A (3·3 kb), encodes a 110 kDa polyprotein (pVP2VP4VP3) in a large open reading frame (ORF) (Hudson et al., 1986
; Spies et al., 1989
), which is cleaved autocatalytically to give pVP2 (48 kDa), VP3 (32 kDa) and VP4 (28 kDa). The viral protease, VP4, is responsible for this self-processing of the polyprotein, but the exact locations of the cleavage sites are unknown (Azad et al., 1987
; Jagadish et al., 1988
). How further processing of the precursor pVP2 takes place to yield the structural protein VP2 (40 kDa) has not been defined, but cellular proteases are not required for this maturation (Kibenge et al., 1997
). Since VP2 does not accumulate intracellularly, as the other viral proteins do, post-translational modification of pVP2 into VP2 probably occurs during or after virus assembly (Müller & Becht, 1982
). VP4 has often been described as a minor virion component because it was detected in purified virions prepared by a variety of methods (Kibenge et al., 1988
). However, Granzow et al. (1997)
showed that VP4 is not a constituent of mature virions but that its presence in virion preparations was due to contaminating VP4-containing type II tubules. The major structural proteins of the virion are VP2 and VP3, both constituents of the proteinaceous capsid of IBDV. VP2 carries major neutralizing epitopes (Azad et al., 1987
; Becht et al., 1988
), suggesting that it is at least partly exposed on the outer surface of the capsid. VP3, the major antigenic component (Fahey et al., 1985
), contains a very basic carboxy-terminal region that might interact with the packaged RNA and is therefore expected to be on the inner surface of the capsid (Hudson et al., 1986
). In addition to the large ORF, segment A also contains a second ORF, preceding and partially overlapping the polyprotein gene, which encodes VP5 (17 kDa). This non-structural protein has only been detected in IBDV-infected cells (Mundt et al., 1995
). VP5 proved to be non-essential for IBDV replication (Mundt et al., 1997
) but plays a role in virus pathogenesis (Yao et al., 1998
), although its exact function is still unknown. The smaller RNA segment, B (2·9 kb), contains one ORF encoding VP1 (90 kDa), the putative RNA-dependent RNA polymerase (RdRp) (Morgan et al., 1988
; Spies et al., 1987
). This protein is present as a free polypeptide and as a genome-linked protein, called VPg, within virions (Müller & Nitschke, 1987
).
Viral proteins generally function by interactions with viral and/or host-cell proteins. Information about these interactions is thus essential for understanding the infection process. The yeast two-hybrid system is a technique that can be used to identify proteinprotein interactions in vivo (Fields & Song, 1989 ). The system is based on the juxtaposition, driven by proteinprotein interaction, of a yeast DNA-binding domain with a transcriptional-activation domain, which results in transcription of a reporter gene (Bartel et al., 1993
; Chien et al., 1991
). Our aim is to use the yeast two-hybrid system to determine specific proteinprotein interactions in vivo between the IBDV proteins themselves and between IBDV proteins and cellular proteins. Generating interaction maps in this way may be a valuable first tool for the analysis of protein interactions present within a virion or during infection. Here, we report the evaluation of the interactions between the viral proteins VP1, pVP2, VP3, VP4 and VP5. We found that several complexes can form in yeast cells, some homologous and one heterologous. The heterologous interaction (VP1VP3) was also detected in vivo in IBDV-infected cells. These results suggest that the different interactions observed may be relevant to the functions of the proteins in the virus replication cycle.
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Methods |
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QT35 cells (Moscovici et al., 1977 ) were cultured in QT35 medium (Fort-Dodge) supplemented with 5% foetal calf serum. Plasmids pHB36W (A segment) and pHB34Z (B segment), which contain full-length genomic cDNA of IBDV strain CEF94, were prepared by using full-length RTPCR fragments generated from purified dsRNA (H. J. Boot, unpublished results).
A rabbit polyclonal antibody against VP1 was obtained after immunizing rabbits with a gel-purified E. coli expression product consisting of amino acids 580881 of VP1 of CEF94 (E. Claassen, unpublished results). A monoclonal antibody against VP3 (MAb C3) was kindly provided by H. Müller (University of Leipzig, Germany).
Construction of two-hybrid expression plasmids.
cDNA coding sequences of VP1, pVP2, VP3, VP4 and VP5 of IBDV strain CEF94 were amplified by PCR by using the Expand high-fidelity PCR system (Boehringer Mannheim). The set of primers used was designed to introduce an EcoRI site at the upstream (5') end and a stop codon plus a SalI, XhoI or StuI site at the downstream (3') end of each coding sequence (Table 1). Plasmid pHB36W was used as the DNA template for amplification of the pVP2, VP3, VP4 and VP5 genes and plasmid pHB34Z was used for amplification of the VP1 gene. The PCR products were precipitated, digested with EcoRI/SalI (pVP2, VP3 and VP5), EcoRI/XhoI (VP1) or EcoRI/StuI (VP4), gel-purified by the QIAEX-II method (Qiagen) and ligated with T4 ligase (New England BioLabs) into the yeast expression vectors pLexABD and pB42AD (Clontech). These vectors had previously been digested either with EcoRI/XhoI or with XhoI followed by a treatment with the Klenow fragment of DNA polymerase I and subsequent digestion with EcoRI. The ligation mixture was transformed into E. coli DH5
cells (Life Technologies), which were subsequently grown under ampicillin selection. Plasmid DNA prepared from several independent transformants was screened for the presence of the insert and plasmids from positive clones were sequenced at the fusion junction by cycle sequencing with an ABI 310 sequencer (Perkin Elmer) to ensure correct reading frames.
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Radiolabelling of infected cells, immunoprecipitation and gel electrophoresis.
Confluent monolayers of QT35 cells (25 cm2) were infected with IBDV strain CEF94 at an m.o.i. of 10 or mock-infected. At 2·5 h post-infection (p.i.), cells were starved of methionine for 1·5 h in methionine-free EMEM medium (Gibco/BRL). At 4 h p.i., cells were either pulsed for 4 h or, in the case of the pulsechase radiolabelling, for 1 h, with 20 µCi/ml [35S]methionine (Amersham) in methionine-free EMEM medium. The latter were subsequently chased for different times in QT35 medium. After the 4 h pulse or the pulsechase period, the cell-culture medium was discarded (in the case of the 4 h pulse experiment) or collected (in the case of the pulsechase experiment) and brought to a concentration of 1xPBSTDS lysis buffer with a 5xPBSTDS lysis buffer stock solution (5% Triton X-100, 2·5% sodium deoxycholate, 0·5% SDS, 0·7 M NaCl, 14 mM KCl, 50 mM Na2HPO4, 7·5 mM KH2PO4). The cell monolayers were washed three times with ice-cold PBS and solubilized in 1xPBSTDS lysis buffer for 10 min at room temperature. Both the medium and cell lysates were then frozen and thawed twice and clarified by centrifugation for 20 min at 13000 r.p.m. in a microfuge. All lysates were precleared with Protein ASepharose (Amersham) before being immunoprecipitated with anti-VP1 or anti-VP3 antibodies. Protein ASepharose-bound immune complexes were washed three times in 1xPBSTDS lysis buffer and eluted in 20 µl SDS sample buffer [60 mM TrisHCl (pH 6·8), 2·5% SDS, 5% -mercaptoethanol, 10% glycerol, 0·1% bromophenol blue]. Proteins were resolved on 12% separating gels by SDSPAGE and visualized by phosphorimaging (STORM-840, Molecular Dynamics) and ImageQuaNT software (Molecular Dynamics).
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Results |
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All possible pairwise combinations of plasmids containing the LexABD and B42AD fusion proteins were co-transformed into S. cerevisiae strain EGY48. We observed strong homologous interactions of the viral proteins pVP2, VP3 and VP5 (Tables 2 and 3
). The strength of these interactions was judged by the intensity of the blue phenotype, which has been suggested to reflect semi-quantitatively the stability of the interaction between the candidate proteins (Estojak et al., 1995
; Li & Fields, 1993
; Yang et al., 1992
).
A possible homologous interaction was found for VP1. The yeast strain with LexABD-VP1 and B42AD-VP1 showed limited growth on leucine-selective medium but, however, remained negative for -galactosidase expression. Measurements of
-galactosidase activity were done after 1 day, when colonies of the positive control (LexABD-p53 with B42AD-SV40 T) were deep-blue, because at that time we could distinguish variations in intensity of the blue colour of positive colonies. Beyond 3 days, the true positive colonies all had the same colour intensity. Nevertheless, after 3 days of incubation, we observed some
-galactosidase activity for the homologous interaction of VP1 (data not shown). However, we did not consider this blue phenotype to represent a true positive interaction because at that time-point we also observed weak interactions between some of the B42AD fusions and LexABD-Bicoid or LexABD-Lamin C, although not between B42AD-VP1 and LexABD-Bicoid or LexABD-Lamin C (data not shown). Moreover, it is known that there is an increased risk of false-positive results after a prolonged incubation due to the sensitivity of the lacZ reporter system. Taken together, the yeast two-hybrid data indicate that VP1 might interact with itself, although this would be a very weak interaction according to our observation that only one of the two reporters was sensitive enough to detect this interaction.
One heterologous interaction was found, between VP1 and VP3. This interaction was found for both reciprocal combinations, although the combination of LexABD-VP1 with B42AD-VP3 proved to have a stronger lacZ reporter activity than the combination of LexABD-VP3 with B42AD-VP1 (Table 3).
The lack of any significant interaction of the LexABD-VP4 fusion may be a consequence of the protein not entering the yeast nucleus and binding to operators, since it led to almost no decrease in -galactosidase activity in a repression assay (data not shown). This repression assay exploits the fact that LexA when bound to its operator blocks activation of a constitutively expressed lacZ reporter gene (Brent & Ptashne, 1984
).
In summary, the yeast two-hybrid assay demonstrated homologous interactions of pVP2, VP3, VP5 and possibly VP1 and a heterologous interaction between VP1 and VP3.
VP1 interacts with VP3 in IBDV-infected cells
Since a complex between VP1 and VP3 may have an important function in the virus replication cycle (see Discussion), we employed a co-immunoprecipitation assay to obtain corroborating evidence for this interaction.
Both IBDV-infected and mock-infected cells were metabolically labelled at 4 h p.i. with [35S]methionine for 4 h. The proteins in the cleared cell lysates were subjected to immunoprecipitation with a rabbit polyclonal antibody against VP1 or a monoclonal antibody against VP3 and analysed by SDSPAGE. In addition to the protein against which the antibodies were directed, each antibody co-immunoprecipitated the other protein, indicating that an interaction had taken place between them (Fig. 2). The observation that only a small amount of VP1 was co-precipitated by the antibody against VP3 (lane 2) may be a consequence of the use of a limiting amount of anti-VP3 antibodies that was unable to precipitate all of the VP3. VP3 is probably synthesized in much larger quantities than VP1, since analysis of the protein composition of IBDV has shown that VP3 constitutes 40%, whereas VP1 constitutes only 3% of the protein in the infectious virus (Dobos et al., 1979
; Kibenge et al., 1988
). Therefore, precipitation of a fraction of VP3 will precipitate only that proportion of VP1 that is interacting with it, whereas precipitation of all of the VP3 will precipitate all of the interacting VP1.
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The other bands obtained in the precipitates with lower apparent molecular sizes were VP3 specific, as they were precipitated by anti-VP3 and also co-precipitated by anti-VP1 antibodies, whereas they were absent in the mock-infected control. Moreover, the VP3 specificity of these bands, as well as the VP1 specificity of the 90 and 95 kDa bands, was confirmed by Western blot analysis (data not shown).
To test for non-specific co-immunoprecipitation of VP1 and VP3, we synthesized and radiolabelled VP1 and VP3 separately in vitro in a rabbit reticulocyte lysate and tested both antibodies for cross-reactivity. We found that the anti-VP1 and anti-VP3 antibodies each precipitated only their cognate protein (data not shown). In addition, we analysed whether VP1 and VP3 could also interact in vitro by co-expression in a rabbit reticulocyte lysate. However, the results showed that neither VP1 nor VP3 co-precipitated in vitro (data not shown).
In summary, the heterologous interaction between VP1 and VP3 that was detected in the two-hybrid system could be confirmed by co-immunoprecipitation from IBDV-infected cells.
Kinetics of association between the viral proteins VP1 and VP3 in vivo
A pulsechase experiment was performed with IBDV-infected cells to assess the rate of complex formation between VP1 and VP3. Both IBDV-infected and mock-infected cells were metabolically labelled at 4 h p.i. for 1 h and chased for different times. Subsequently, the proteins in the cleared cell lysates as well as in the cell-culture media were immunoprecipitated with antibodies specific for either VP1 or VP3 and analysed by SDSPAGE. Complexes consisting of VP1 and VP3 were detected in cell lysates directly after the pulse (Fig. 3a, lanes 1 and 5). VP1 detected immediately after the pulse again appeared to be present in two forms, of 90 and 95 kDa, in nearly equal amounts (Fig. 3a
, lanes 1 and 5). However, during the chase, the amount of the 90 kDa protein in the cell lysate decreased more rapidly than the amount of the 95 kDa protein (lanes 14). Furthermore, after 5, 9 and 19 h of the chase, VP3 interacted only with the 95 kDa form of VP1 and not with the 90 kDa form (lanes 68). The same phenomenon was detected in the cell-culture media. Here, the complexes consisting of VP1 (95 kDa) and VP3 were detected from 5 h of chase onwards, that is 10 h p.i. (Fig. 3b
). This timing is consistent with the release of progeny virus particles into the culture medium (Petek et al., 1973
).
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Finally, after 5, 9 and 19 h of the chase, two additional bands were detected in the high molecular size region (Fig. 3a, b
). These two bands proved to be VP1 specific, as confirmed by Western blot analysis (data not shown).
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Discussion |
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In order to verify whether VP1 and VP3 can interact physically, their association was analysed further by co-immunoprecipitation studies. We found that VP1 and VP3 interacted in vivo in IBDV-infected cells but not in vitro in a rabbit reticulocyte lysate. In the in vitro experiment, the protein(s) may not be folded in their native conformation, which would hinder the interaction. Likewise, Black et al. (1998) detected interactions in a co-immunoprecipitation assay among the proteins G2R, A18R and H5R of vaccinia virus expressed during infection, whereas they failed to detect these interactions when these proteins were synthesized in vitro in a rabbit reticulocyte lysate. The interaction between VP1 and VP3 is specific, since the antibodies used showed no cross-reactivity in a co-immunoprecipitation assay of VP1 and VP3 synthesized separately in vitro. It should also be mentioned that all immunoprecipitations were performed in the presence of a small amount of SDS to disrupt virions. Since this detergent did not disrupt the VP1VP3 complex, this interaction proved to be relatively strong, which is consistent with the data from the yeast two-hybrid assay.
The interaction between VP1 and VP3 is intriguing, as the known or putative biochemical and biological properties of these proteins do not suggest the likelihood of such an interaction. The complexes consisting of VP1 and VP3 are formed immediately or shortly after translation in the cytoplasm of IBDV-infected cells and are eventually released into the cell-culture medium from 10 h p.i. onwards. Therefore, it is likely that VP1VP3 complexes are also present in mature virions, since this timing is consistent with the release of extracellular progeny virus particles into the culture medium (Petek et al., 1973 ).
In our co-immunoprecipitation studies, we detected two proteins in the molecular mass region of VP1, of 90 and 95 kDa. Müller & Becht (1982) and Jackwood et al. (1984)
have previously reported the existence of these two polypeptides in different IBDV strains. They indicated that these proteins may have a precursorproduct relationship. Since the VP1 specificity of these proteins has been confirmed by Western blot analysis (data not shown), we now have the first evidence that these are indeed two forms of VP1. We found that VP3 interacts with both forms of VP1 immediately or shortly after translation (Fig. 2
, lane 2; Fig. 3a
, lane 5), but that later during infection VP3 interacts only with the 95 kDa form of VP1 (Fig. 3a
and b
, lanes 68). Further experimentation is required to determine the exact nature of these two forms of VP1 and whether there is a precursorproduct relationship between them. The difference in size between these two forms is probably not a consequence of a VP1RNA complex, since there was no change in size of either the 90 or 95 kDa protein after RNase treatment (data not shown). However, it has been shown for infectious pancreatic necrosis virus that short VP1-linked oligonucleotides can survive RNase treatment, probably due to steric hindrance by the unusually large VP1 (Magyar et al., 1998
).
It was noteworthy that, after RNase treatment, the two additional VP1-specific bands detected in the high molecular size region after 5, 9 and 19 h of the chase (Fig. 3a, b
) had disappeared, meaning that these bands represented VP1RNA complexes.
It should also be mentioned that, in addition to the 32 kDa form of VP3, we also detected at least two smaller forms of VP3 (Figs 2 and 3
). Such forms are frequently seen in IBDV-infected cells but are usually ignored or confused with VP4. In infectious pancreatic necrosis virus, one such form has been described as VP3a (Dobos, 1995a
).
Recently, the interaction between VP1 and VP3 has also been described by Lombardo et al. (1999) , who observed the interaction by co-localization and co-immunoprecipitation studies of vaccinia virus-expressed VP1 and VP3. Their and our results raise several interesting possibilities regarding the function of the interaction between VP1 and VP3. The interaction may be involved in the regulation of viral RNA synthesis or may be a part of the replication apparatus, as has been proposed for the interaction between the RdRp and the virus coat protein of tobacco vein mottling virus (Hong et al., 1995
). An interaction between the RdRp and the virus coat protein has also been observed for alfalfa mosaic virus (AlMV) (Quadt et al., 1991
). In this case, minus-strand synthesis by the AlMV RdRp is inhibited by AlMV coat protein (Quadt et al., 1991
). In rotavirus, an interaction between the inner capsid protein, VP6, and the inner core polypeptide, VP3, is necessary for recovery of RNA polymerase activity (Sandino et al., 1994
). Alternatively, the interaction between VP1 and VP3 may be involved in virus assembly or encapsidation of the virus. It is known for hepatitis B viruses that an interaction between the viral polymerase and capsid protein is required for encapsidation of the pregenomic RNA (Ziermann & Ganem, 1996
). These and other possibilities await further experimental study to elucidate the exact function of the interaction between VP1 and VP3.
Of the homologous two-hybrid interactions found, we did not interpret the VP1VP1 interaction as a true positive interaction, since this interaction resulted in very weak reporter activity. The signal for this homologous interaction was so weak that lacZ expression was undetectable (Table 3). The weakness of this interaction may be related to an intrinsically weak interaction between VP1 polypeptide chains. However, as mentioned above, weak signals in the two-hybrid assay are not necessarily indicative of the strength of a specific proteinprotein interaction; poor or unstable expression, improper folding or steric hindrance of the fused LexABD or B42AD domains at the site of interaction may impair the interaction. Using the two-hybrid system, Xiang et al. (1998)
reported a very weak interaction between the proteins 3AB and 3CDpro of poliovirus, which was observed as a strong interaction when tested by far-Western blotting. It is conceivable that a homologous interaction of VP1 can occur. Xiang et al. (1998)
reported an interaction between VPg and the polymerase 3Dpol of poliovirus. The poliovirus protein VPg is covalently linked to the 5' ends of both genomic and antigenomic viral RNA and 3Dpol is the RdRp. These authors suggested that a direct interaction between these molecules is involved in the mechanism of initiation of viral RNA synthesis. VP1 of IBDV also exists as a genome-linked protein (VPg) (Müller & Nitschke, 1987
). Therefore, an interaction between VPg and VP1 of IBDV may have a similar function. Moreover, it has been suggested that the initiation of viral RNA synthesis of birnaviruses may involve two VP1 molecules, one serving as a primer and the other for polymerase chain elongation (Dobos, 1995b
).
We expected to find an interaction between VP2 and VP3, since these two proteins comprise the proteinaceous capsid of IBDV. Although we used pVP2 instead of mature VP2, this should not influence the results, since Kibenge et al. (1999) showed that processing of pVP2 to VP2 is not necessary for capsid assembly. However, no heterologous interaction between pVP2 and VP3 was detected, only strong homologous interactions of pVP2 and VP3. Of course, false-negative results from the yeast two-hybrid assay are not without precedent, as failure to identify other known proteinprotein interactions in the two-hybrid system has been reported (Cuconati et al., 1998
; Fields & Sternglanz, 1994
; Van Aelst et al., 1993
). On the basis of electron micrographs, the subunits of the IBDV capsid are predominantly clustered as trimers (Bottcher et al., 1997
). On the outer surface, the trimer units protrude from a continuous shell of density, and on the inner surface the trimers appear as Y-shaped units. Bottcher et al. (1997)
suggested that it is likely that the outer trimers correspond to the protein VP2, carrying the dominant neutralizing epitopes, and that the inner trimers correspond to protein VP3, which has a basic carboxy-terminal tail expected to interact with the packaged RNA. According to this study, it is not surprising to find strong homologous interactions for pVP2 and VP3. Therefore, it is also possible that (p)VP2 only interacts with VP3 when they are both present as a trimer subunit. If so, this cannot be detected in the yeast two-hybrid system. It is worth noting that, in the co-immunoprecipitation studies with anti-VP3 serum, we detected no interaction between VP2 and VP3, consistent with the data obtained with the yeast two-hybrid system. However, as mentioned above, all the immunoprecipitations were performed in the presence of a small amount of SDS, so the presumed VP2VP3 interaction could have been disrupted.
A homologous interaction was also detected for VP5. Since the exact function of this protein is still unknown, it is difficult to speculate about the functional significance of this interaction. A VP5-deficient virus can replicate in the bursa of inoculated chickens but will not induce bursal lesions (Yao et al., 1998 ). Whether VP5 must assemble into dimers or multimers to produce its effects is unknown.
One potential drawback of a two-hybrid system arises when the fusion protein fails to localize to the nucleus and to bind operators. This was possibly the case for the LexABD-VP4 fusion protein, since this fusion protein showed no significant interaction with other viral proteins. Moreover, we performed a repression assay in which VP4 caused hardly any repression of transcription of the lacZ reporter gene (data not shown). This means that nuclear localization or operator binding of this fusion protein is impaired. However, since the B42AD-VP4 fusion protein, which possesses a nuclear localization signal and does not have to bind operators, also showed no interaction with other viral proteins, VP4 is probably not able to form heterologous complexes with the distinct viral proteins. Whether VP4 is able to interact with itself therefore remains uncertain. A homologous interaction of VP4 is not inconceivable, since VP4 aggregates to type II tubules (Granzow et al., 1997 ).
All of the interactions found between the viral proteins of IBDV were detected in the classical attenuated strain CEF94. To check whether there were significant differences between this attenuated strain and a very virulent strain, we also determined the two-hybrid interactions between the viral proteins of the very virulent isolate D6948, with the exception of VP1. The interactions observed were the same as those found for the classical attenuated strain, indicating that there are probably no great differences between these strains in this respect (data not shown).
Taken together, the interactions observed between the viral proteins of IBDV described in the present study underscore the highly co-ordinated nature of the events in which these proteins must participate during genome expression, replication and assortment. However, extensive studies are still required to confirm the role of these proteins and the functional relevance of the interactions described.
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Acknowledgments |
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References |
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Received 29 July 1999;
accepted 22 September 1999.