The gene encoding the hepatitis delta virus structural antigen (HDAg) was linked to a .... Instrument Group) with a Bio-Sil TSK (7.5 mm à 75 mm) guard column (Bio-Rad). ..... severe delta agent infection in Venezuelan Indians. Hepatology 3,.
Journal of General Virology (1990), 71, 1339-1345. Printedin Great Britain
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Stable expression of hepatitis delta virus antigen in a eukaryotic cell line T. B. Macnaughton, 1 E. J. Gowans, 1. B. Reinboth, 2 A. R. Jilbert 1 and C. J. BurrelP 1Division o f Medical Virology, Institute o f Medical and Veterinary Science, Box 14, Rundle Mall Post Office, Adelaide 5000 and 2Department o f Pathology, University o f Adelaide, GPO Box 498, Adelaide 5000, South Australia
The gene encoding the hepatitis delta virus structural
g/ml by equilibrium centrifugation in caesium chloride,
antigen (HDAg) was linked to a neomycin resistance
and in rate zonal centrifugation it sedimented with a
gene in a retrovirus expression vector, and human HepG2 cells were transfected with the recombinant plasmid. A stable cell line was cloned that expressed HDAg in the nuclei of 100% of cells, in a pattern indicating a close relationship with cell nucleoli. Analysis of partially purified recombinant HDAg by H P L C showed an M~ in the range o f 7 x l 0 5 to 2 x 10 6, which appeared to contain conformation-dependent epitopes, whereas the density of the antigen was 1.19
value of 50S, close to that of particulate hepatitis B virus surface antigen. Immunoblotting demonstrated a single polypeptide with an Mr of 24K which corresponded to the smaller of the two HDAg-specific polypeptides present in infected sera. The recombinant HDAg polypeptide was shown to be a RNA-binding protein with specificity for both genomic and antigenomic species of hepatitis delta virus RNA.
Introduction
depending on the position of the stop codon (Wang et al., 1986), whereas a human-derived sequence predicts 214 aa (Makino et al., 1987) and a woodchuck-derived sequence predicts 195 aa (Kuo et al., 1988). The predicted Mr values are approximately 24K and 21K for the 214 aa and 195 aa proteins respectively. Examination of the polypeptides of HDAg from infected liver or serum has generally shown two coexisting major proteins with Mr values of 27K and 24K (Bergmann & Gerin, 1986) or 29K and 27K (Bonino et al., 1986; Roggendorf et al., 1987). These differences are likely to result from differences in Mr standards. Although these two major proteins react with a human monoclonal antibody to HDAg (Pohl et al., 1987), and therefore contain at least one similar antigenic epitope, the mechanism for the production of the two peptides is still unclear. However a chimpanzee-derived ORF-5 sequence has been shown to express both 24K and 27K proteins in transfected bacteria as a result of suppression of a termination codon (Weiner et al., 1988). Recently, transfection of COS7 cells with the human-derived HDAg sequence led to the production of a single polypeptide of 26K that was shown to be a nuclear phosphoprotein with an RNA-binding activity (Chang et al., 1988). In this study, we report that a chimpanzee-derived HDV cDNA clone with an amber stop codon at aa position 195 (Wang et al., 1986; Weiner et al., 1988) expressed recombinant HDAg (recHDAg) in the nuclei
Hepatitis delta antigen (HDAg) was first detected in hepatocyte nuclei in some hepatitis B virus (HBV) carriers (Rizzetto et al., 1977). This antigen was subsequently shown to be an internal antigen of hepatitis delta virus (HDV), a small defective R N A virus which is dependent on coinfection with HBV or with the closely related woodchuck hepatitis virus for its replication and expression (Ponzetto et al., 1984; Rizzetto et al., 1980). The HDV virion envelope, which is composed of the surface antigen of the helper hepadnavirus, can be removed by detergent treatment to expose the internal HDAg (Bonino et al., 1981; Ponzetto et al., 1984; Rizzetto et al., 1980). Electron microscopy and molecular cloning showed that the virus genome is a single-stranded circular R N A of approximately 1.7 kb with the ability to form a rodlike structure by extensive intramolecular base-pairing in a manner similar to viroids (Kos et al., 1986; Kuo et al., 1988; Makino et al., 1987; Wang et al., 1986). There are five conserved open reading frames (ORFs) distributed over both genomic and antigenomic strands and one of these (ORF-5) in the anti-genomic strand is the gene coding for HDAg (Kuo et al., 1988; Makino et al., 1987 ; Wang et al., 1986). At this time, only ORF-5 has been assigned a protein product. The sequence of different cDNA clones of chimpanzee-derived HDV predicts HDAg to be either 195 or 214 amino acids (aa) 0000-9373 © 1990SGM
1340
T. B. Macnaughton and others
of a continuous line of transfected hepatoma cells, and we define some characteristics of the antigen so produced.
Methods Plasmid construction. HDV cDNA clones BMB37 and BMB104 (Gowans et al., 1988) derived initially from HDV clones 64 and 6115 respectively (Wang et al., 1986), were generous gifts from Drs J. Gerin and B. Baroudy. The restriction enzyme Sinai (Bresa) was used to digest BMB37 partially and to digest BMB104 fully. The excised HDV fragment from BMB104 was then ligated to the linearized BMB37 fragment to produce a full-length HDV cDNA (subsequently named pTM63) in pGEM-3 (Promega Biotec). The region of nucleotides 966 to 1679 (Wang et al., 1986) representing ORF-5 (minus 10 bp at the 3' end) and including 81 bp upstream of ORF-5 was then excised from pTM63 by double digestion with HindlII and SalI, end-filled using the Klenow fragment of DNA polymerase I (Boehringer Mannheim), and then ligated into the unique Bam-I site of the retrovirus expression vector pRSV009 (to produce plasmid pTM5E), pRSV009, which contains a neomycin resistance gene, (Neo R) was modified by and kindly supplied by Dr A. J. Robins. The construction of plasmid pTM5E is outlined in Fig. 1. Cell transfection. Recombinant plasmids were amplified in Escherichia colt strain DH-5 essentially as described by Davis et al. (1986) and used to transfect HeLa or HepG2 cells (Aden et al., 1979), by a modification of a published method (Sells et al., 1987). Briefly, 1 ml of Dulbecco's modified Eagle's medium (DMEM) containing 5 gg polybrene (Sigma) and 5 gg plasmid DNA was added to a 25 cm 2 flask of cells, and incubated with gentle agitation for 6 h at 37 °C. The DNA solution was replaced with 5 ml of DMEM containing 10% foetal calf serum and 30% DMSO (Aldrich) for 4 min, the cells were washed, incubated in fresh DMEM for 48 h at 37 °C, then cloned in medium containing 350 gg/ml Geneticin (G418) into 24-well microtitre plates (Costar). During selection the wells were examined microscopically on a daily basis to ensure that colonies were derived from single cells. Full Geneticin selection was maintained for 3 months. Analyses o f recombinant HDAg. RecHDAg was detected in acetonefixed cell monolayers essentially as described (Jilbert et al., 1986). Immunofluorescence specificity was determined previously with a fluorescein isothiocyanate-conjugate supplied by Dr M. Rizzetto. Immunoblotting was performed as described (Bergmann & Gerin, 1986). Trypsinized test and control cells and serum-derived HDV centrifuged through 2 0 ~ sucrose were resuspended in loading buffer. In some experiments, 2-mercaptoethanol was omitted from the buffer. Human anti-HD from a chronic carrier of HDV constituted the primary antibody which was in turn detected by ~zs I-labelled Protein A (Gowans et al., 1989). The specificity of immunoblots was verified by substitution of the anti-HD positive serum with normal human serum, and by the use of control homogenates. Preparation and purification o f recombinant HDAg. In some experiments, the cells were removed by trypsinization and recHDAg was released by 6 M-urea followed by freezing and thawing three times. This preparation was then clarified and analysed by HPLC. In other experiments, the recHDAg was partially purified and concentrated prior to further analysis as follows. Trypsinized packed cells were resuspended in four volumes of phosphate-buffered saline (PBS) containing 50 mM-Tris-HCl pH 8-0, Triton X-100 was added to 1%, and the preparation was vortexed, frozen and thawed twice and sonicated for 60 s on ice before a further two cycles of freezing and
S
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~
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Fig. 1. Strategy for construction of recombinant plasmid pTM5E, which expresses HDAg under the control of the Rous sarcoma virus promoter.
Recombinant hepatit& delta antigen
1341
thawing. The preparation was centrifuged at 27000 g at 4 °C for 10 rain, the pellet re-extracted as above with twice the original volume of PBS, and both supernatants were pooled. The pool was cleared by the addition, with stirring, of ammonium sulphate powder to 25%, and the precipitate was removed by centrifugation (20000g at 20 °C for 10min). Ammonium sulphate was then added to 65% and the precipitate was recovered by centrifugation as above. The pellet reacting with human anti-HDAg redissolved in PBS and precipitated by the addition of acetone to 40% After centrifugation (9500 g at 4 °C for 10 rain) the pellet was washed in 90% acetone, the actone was then removed completely and the pellet was redissolved in PBS.
HPLC analysis. Two hundred p~l of crude or partially purified recHDAg was injected into an HPLC column and the flow rate adjusted to 0.8 ml/min. The HPLC was performed by gel permeation through Varian TSK G4000 SW in a 7.5 mm × 50 cm column (Varian Instrument Group) with a Bio-Sil TSK (7.5 mm × 75 mm) guard column (Bio-Rad). A Waters (Waters Associates) solvent delivery system equipped with a Wisp 710B injector fitted with a 200 ~tlloop and linked to a model 440 fixed wavelength detector was employed. High Mr protein standards (Pharmacia) were used to calibrate the column. Eight-hundred ~tl fractions were collected and tested for HDAg by radioimmunoassay (RIA; Gowans et aL, 1990). Fractions containing 4 M-guanidine hydrochloride (GuHCI) were diluted 1:4 prior to RIA. Ultracentrifugation analysis of recombinant HDAg. Recombinant HDAg was analysed on a preformed caesium chloride gradient (1.1 to 1-5 g/ml). A 200 ~tl sample of ammonium sulphate-precipitated recHDAg was overlaid on a 10 ml gradient and centrifuged (120000g for 25 h at 20 °C in an IEC ultracentrifuge with a Type 488 rotor), and 0-5 ml fractions were collected. Similarly, a 200 ~tl aliquot of the same recHDAg preparation was analysed in a 10 ml linear 5 to 20~ sucrose gradient centrifuged at 208000 g for 1 h at 4 °C. The fractions were tested for HDAg activity by RIA as above, and the density was estimated from the refractive index. In each case, analysis of the recHDAg was compared with the profile of the HBV surface antigen (HBsAg) from an HBV-infected serum sample, using Auszyme (Abbott Laboratories) to detect HBsAg. RNA binding assay. This was performed essentially as described (Petit & Pillot, 1985). HPLC-purified recHDAg was transferred to nitrocellulose after SDS PAGE, the nitrocellulose was incubated in blocking buffer (10 mM-Tris-HC1 pH 7.0, 50 mM-NaC1, 1 mM-EDTA, 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone, 0-02% Ficol1400) for 5 hours at room temperature and then incubated in the same buffer containing 5 × 105 c.p.m./ml 3zp-labelled HDV RNA for 2 h at room temperature. The nitrocellulose was then washed four times in the same buffer, dried and exposed to X-ray film. Both genomic and antigenomic HDV RNA transcribed from plasmids BMB37 and BMB104 (Gowans et al., 1988) which contain contiguous regions that collectively constitute the total HDV genome, were used. The binding assay was controlled using RNA transcribed from plasmid pV18 which contains a 2263 bp insert of human papillomavirus (HPV) type 18 DNA (G. D. Higgins, personal communication).
Results Immunofluorescence o f transfected cells S e v e r a l G e n e t i c i n - r e s i s t a n t c l o n e s w e r e e x a m i n e d for H D A g e x p r e s s i o n by i m m u n o f l u o r e s c e n c e w h e n t h e cells in i n d i v i d u a l wells r e a c h e d c o n f l u e n c e . S e v e r a l c l o n e s s h o w e d v a r y i n g p e r c e n t a g e s o f H D A g - p o s i t i v e cells b u t a p r o g r e s s i v e loss o f cells t h a t e x p r e s s e d h i g h l e v e l s o f
Fig. 2. Detection of HDAg by direct immunofluorescence in A3 cells (a) showing nucleolar localization of HDAg characteristic of this cell line. A control cell line (CI) is shown in (b). The cells were originally photographed at 400 × magnification. Bar represents 50 lam.
H D A g w a s n o t e d a n d t h e s e c o u l d n o t be c l o n a l l y a m p l i f i e d . T h i s w a s p a r t i c u l a r l y n o t i c e a b l e for t h e H e L a cells a n d w e w e r e u n a b l e to e x p a n d a n y r e c H D A g p o s i t i v e H e L a cells. T h e s e o b s e r v a t i o n s s u g g e s t t h a t H D A g m a y b e c y t o t o x i c . H o w e v e r , it w a s p o s s i b l e to r a i s e o n e H e p G 2 cell c l o n e (A3) w h i c h s h o w e d t h e p r e s e n c e o f H D A g in e v e r y cell e x a m i n e d ; a s e c o n d c l o n e (C1), w h i c h n e v e r s h o w e d H D A g e x p r e s s i o n , w a s also e x p a n d e d in G e n e t i c i n - s u p p l e m e n t e d m e d i u m , a n d this a n d t h e o r i g i n a l H e p G 2 cells w e r e u s e d as c o n t r o l cell lines. T h e b u l k o f t h e r e c H D A g w a s d e t e c t e d in t h e n u c l e u s o f A 3 cells, a n d w a s closely a s s o c i a t e d w i t h t h e n u c l e o l u s (Fig. 2). T h i s l o c a l i z a t i o n p a t t e r n o f r e c H D A g d i f f e r e d f r o m t h e diffusely n u c l e a r d i s t r i b u t i o n r e p o r t e d for H D A g in n a t u r a l l y i n f e c t e d h e p a t o c y t e s ( R i z z e t t o et al., 1977). E x p r e s s i o n o f r e c H D A g was i n d e p e n d e n t o f t h e d e g r e e o f cell c o n f l u e n c e o f t h e culture. A n a l y s i s b y R I A o f a cell c u l t u r e m e d i u m c o n c e n t r a t e d 20-fold f a i l e d to detect HDAg, suggesting that recHDAg was not secreted f r o m t h e cells ( d a t a n o t s h o w n ) .
T. B. Macnaughton and others
1342
o o
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30 10 15 20 25 30 Fractions (ml) Fig. 3. The detectionof HDAg by RIA in fractionscollectedafter HPLC analysisof recHDAg: (a) recHDAg freshlyextracted and analysedin 6 M-urea;(b) recHDAgpartiallypurifiedby ammoniumsulphatefollowedby acetoneprecipitationwhichwas either stored at - 20 °C for 24 h (O) or frozenand thawedfivetimes (ff])prior to HPLC; (c) and (d), recHDAgdenaturedand analysedin 4 M-GuHC1 either before (c) or after (d) dialysisof the fractions against PBS.
HPLC Either crude or partially purified recHDAg was analysed by exclusion HPLC. Crude recHDAg showed one discrete peak (Fig. 3a) which appeared just behind the void volume (exclusion limit of Mr 2 x 106); it is likely that this represents the intracellular form of recHDAg because this preparation was not only prepared in 6 M-urea by freeze-thaw cycles only but also analysed in 6 M-urea. However, owing to the low levels of HDAg present, further H P L C analysis of this type of preparation was not possible. Analysis of partially purified, concentrated recHDAg confirmed a small peak at 2 × 106 Mr, with a dominant peak at 5 x 105 M r (Fig. 3 b). In preparations frozen prior to chromatography, the peak at 2 x 106 M r w a s undetected (Fig. 3b). HDAg activity was never detected in fractions corresponding to 24K unless the preparations were previously denatured and analysed in 4 M-GuHC1. Nevertheless, when the
fractions were analysed by RIA in the presence of GuHC1 (Fig. 3 c), HDAg was detected only in high Mr fractions. In contrast, after dialysis to remove the GuHC1 (thus permitting refolding of the polypeptides) the bulk of HDAg activity was shown to be present in fractions corresponding to 24K (Fig. 3d). This indicates that recHDAg contains conformation-dependent epitopes which are recognized by human anti-HD.
lmmunoblotting Immunoblotting experiments were performed to determine the Mr of the recHDAg polypeptide, and to compare recHDAg with serum-derived HDAg. In several different experiments, a single band with an Mr of approximately 24K was detected in the A3, but not in the HepG2 or C1 cells (Fig. 4). The electrophoretic mobility of recHDAg coincided with that of the smaller
Recombinant hepatitis delta antigen
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1343
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0.15 Fig. 4. Immunoblot analysis of r e c H D A g and serum-derived H D A g . Lanes 1 to 5 were tested with normal h u m a n serum and lanes 6 to 10 were tested with h u m a n anti-HD as the primary antibody. Lanes 1 and 6 contain the A3 cell homogenate. Lanes 2 and 7 contain the H e p G 2 cell homogenate, lanes 3, 8, 4 and 9 contain H D V pelleted from two positive h u m a n serum samples respectively, and lanes 5 and 10
represent normalhumanserumtreatedin the sameway,The smeared bands in the upper part of the gel are likelyto correspond to human globulin. M~markers are indicatedon the left of the gel.
of the two polypeptides derived from serum containing HDV after centrifugation through sucrose. The Mr values of the serum-derived HDAg polypeptides were determined as 27K and 24K, as reported previously (Bergmann & Gerin, 1986). Our results also confirmed that the polypeptide profiles of both recHDAg and serum-derived HDAg were unchanged when 2mercaptoethanol was omitted from the denaturing buffer. The concentrated cell culture medium was negative for HDAg by immunoblotting, confirming the previous RIA data.
Measurement of density and sedimentation value of recHDAg The characteristics of the recHDAg were then examined by isopycnic and rate zonal centrifugation. After isopycnic centrifugation, the peak HDAg activity corresponded to a density of 1.19 g/ml in caesium chloride (Fig. 5a). A similar analysis of liver-derived HDAg which was extracted in GuHCI (Rizzetto et al., 1980) produced a similar result (1.20 g/ml). Thus although these figures are low compared with other reports of 1.28 g/ml (Bonino et al., 1984; Rizzetto et al., 1980) it was clear that the recHDAg and the liver-derived HDAg showed similar densities. In comparison, serum-derived
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Fraction n u m b e r Fig. 5. Isopycnic and rate zonal centrifugation analysis of recHDAg. Two-hundred ktl of a m m o n i u m sulphate-precipitated r e c H D A g was centrifuged at 120000 g for 25 h on a preformed CsC1 gradient (a) or at 208000 g for 45 m i n in a 5 to 20% sucrose gradient (b). In each case, H B s A g was analysed, and liver H D A g was also analysed by isopycnic centrifugation. H D A g and H B s A g was detected in the gradient fractions by RIA and enzyme i m m u n o a s s a y respectively. The density
of the CsC1was estimatedfromthe refractiveindex. (a) recHDAg(I-q) and liver HDAg (O); (b) recHDAg (D) and HBsAg(O).
HBsAg corresponded to a density of 1-22 g/ml (Fig. 5a), close to a previously reported value (Gerin et al., 1969). The buoyant density of recHDAg was unaltered after extraction with either GuHC1 or chloroform (data not shown) suggesting that the low density was not due to the presence of lipid. Further analysis by rate zonal centrifugation showed that the recHDAg sedimented through sucrose only slightly slower than serum-derived HBsAg 22 nm particles, suggesting that the recHDAg determinants were present on particles of similar size (Fig. 5b). Analysis of the sedimentation coefficient (S value) using a computer program (Young, 1978) determined the S values of recHDAg and HBsAg to range between 35 and 72 (peak 50S) and 46 and 83 (peak 58S) respectively; although a range of S values has been published for 22 nm HBsAg particles, the above value is close to that of 54S reported previously (Gerin et al., 1971).
1344
T. B. Macnaughton and others
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Fig. 6. RNA binding assay to demonstrate that HDAg specifically binds HDV RNA. HPLC-purifiedrecHDAg was denatured in loading buffer lacking 2-mercaptoethanol and, after electrophoresis, transferred to nitrocelluloseand incubated with 32p-labelled RNA. Lane 1 shows Coomassie blue staining for total protein and lane 2 represents an immunoblot to identify HDAg polypeptides, Lanes 3 and 4 were incubated with anti-genomic HDV RNA and lanes 5 and 6 with genomic HDV RNA transcribed from plasmids BMB104and BMB37 respectively.Lanes 7 and 8 were incubatedwith positive-and negativesense HPV RNA respectively,labelled to the same specific activity.
R e c H D A g binds H D V R N A specifically Although recombinant HDAg was previously shown to bind HDV R N A (Chang et al., 1988) the specificity of the reaction was not reported. We examined this specificity by incubating strips of nitrocellulose, to which HPLC-purified recHDAg had been bound, with 32p_ labelled RNA. Although the recHDAg preparation failed to show an absorbance peak at 280 nm (data not shown) the preparation contained a number of proteins which were detected by Coomassie blue staining; the strongest band and a band that comigrated with the dye front both reacted with anti-HD (Fig. 6, lanes I and 2). However, genomic and anti-genomic HDV R N A bound strongly to the recHDAg bands only. In contrast, positive and negative polarity HPV RNA, labelled to the same specific activity, bound very weakly, showing that the binding was specific for H D V R N A (Fig. 6). It is likely that the lower Mr HDAg-specific peptide (approx. 20K) was formed during storage of the recHDAg at - 7 0 °C, and degradation of liver-derived HDAg has been reported previously (Roggendorf et al., 1987).
Discussion In this paper, we report the expression of H D A g in a hepatocyte-derived cell line by placing the HDAg gene under the control of a Rous sarcoma virus promoter in a vector designed to integrate into the host cell chromosome. Although we developed a number of HDAgpositive HeLa clones, these cells were lost in culture, whereas a proportion of HDAg-positive HepG2 clones were expanded successfully. Our results suggest that HDAg cytotoxicity may contribute to the cytopathic
nature of H D V that was postulated previously (Popper et al., 1983) and experiments are currently under way to clarify this point. The recHDAg produced in the A3 cells possessed similar antigenic reactivity to H D A g derived from in vivo infected livers (Gowans et al., 1990). However, the A3 cell model differed from the in vivo production of HDAg in several ways: the recombinant HDAg was composed of the smaller (24K) polypeptide only; HDAg was not secreted from the cells; recombinant HDAg present in the A3 cells showed a nucleolar distribution compared to a nucleoplasmic distribution in infected hepatocytes; there was no H D V genome replication from native virus; finally, there was an absence of HBV superinfection. In our hands, the H D A g gene expressed a single polypeptide which corresponded to the smaller of the serum-derived H D A g polypeptides and it is still unknown how the second polypeptide is derived in natural infection. The data in this paper show that the single polypeptide formed a HDAg-reactive particle with a density of 1-19 g/ml and an approximate S value of 50. As we used gentle methods, avoiding any precipitation steps, to prepare the recHDAg for H P L C analysis, it is likely that the high Mr form of recHDAg occurred naturally within the nuclei of A3 cells in this form, and was not the result of aggregation during purification. A possible mechanism for the production of this particle could be the formation of a ribonucleoprotein by the interaction of HDAg with high Mr RNA, but the density of the particle in caesium chloride suggests that this is unlikely. We have also shown that recHDAg contains conformational epitopes which were detected with human anti-HD produced in response to infection, and thus it is likely that native H D A g also shows a degree of conformational specificity. The 214 aa form of HDAg was reported recently to be an RNA-binding phosphoprotein (Chang et al., 1988). We have extended this observation to the 195 aa form of HDAg and shown that this binding is specific to both the protein and the R N A components of the reaction and that HDAg binds both genomic and antigenomic R N A with equal efficiency. This finding is not surprising since HDV R N A shows a high degree of intramolecular basepairing (Kuo et al., 1988; Wang et al., 1986) and consequently, there must be regions in genomic and antigenomic R N A with similar (as well as complementary) base sequences. As we have used two contiguous sequences of R N A that constitute the whole genome, our experiments also suggest that at least two discrete regions of the genome are involved. However we have not been able to examine whether native HDAg also binds H D V RNA, because of our inability to obtain sufficient quantities of native antigen.
Recombinant hepatitis delta antigen
We thank Dr A. Robins, Department of Biochemistry, University of Adelaide for supplying plasmid pRSV009, and Drs J. L. Gerin and B. M. Baroudy, Division of Molecular Virology and Immunology, Georgetown University, for plasmids BMB37 and BMBI04. We also thank Mrs C. Bayley for typing and the staff of the Institute photographic unit. This work was supported by a grant from the National Health and Medical Research Council of Australia, and much of the expertise was gained during the tenure of a previous grant held with Professor B. P. Marmion.
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(Received 13 November 1989; Accepted 9 February 1990)
