from hepatitis patients - NCBI

The EMBO Journal vol.4 no.5 pp.1287-1292, 1985

The HBV HBx gene expressed in E. coli is recognised by from hepatitis patients

A.Kay, E.Mandart, C.Trepo1 and F.Galibert Laboratoire d'Hematologie Experimentale, Centre Hayem, H6pital SaintLouis, 2 place du Dr. Fournier, 75475 Paris Cedex 10, and 1Unit6 de Recherche sur les Hepatites et le r6le des virus hepatotropes dans I'oncogdn6se, (INSERM U.271), Faculte de Medicine Alexis Carrel, 69372 Lyon Cedex 08, France Communicated by M.Yaniv

We have cloned the X gene (HBx) and the HBc antigen (HBc Ag) gene of human hepatitis B virus (HBV) in Escherichia coli as fusion products with g-galactosidase. Both HBV genes are expressed in E. coli strain CSR 603. Expression is detected by u.v. irradiation of the bacteria, metabolic labelling and electrophoresis of the labelled extracts on SDS-polyacrylamide gels. The HBc Ag protein produced in bacteria can be recognised by anti-HBc sera and peptides derived from the protein are also recognised by anti-HBe sera. The HBx protein is recognised by some, but not all, sera which are antiHBe positive. HBx Ag is also recognised by a woodchuck antibody similar to anti-HBe (anti-WHe). These results constitute the first proof that the open reading frame X is a true viral gene and is expressed during HBV (and WHV) infection and that an HBx/anti-HBx system, which may have important biological implications, can exist in parallel with the classic HBe/anti-HBe system. Key words: E. coliIHBc Ag/HBe Ag/HBV/HBx Ag

Introduction Because of the lack of a suitable in vitro model system, much of our knowledge about the biology of human hepatitis B virus (HBV) has come from two directions, serological studies and molecular cloning of the genome. The serological studies have identified three major classes of HBV-specific antigens, HBs Ag, HBc Ag and HBe Ag (for a review, see Hoofnagle, 1981). HBs Ag, or Australia antigen, is the major structural surface protein of the virus. It is the most commonly used marker of HBV infection. The presence of anti-HBs is a reliable marker of recovery from, and immunity to, type B hepatitis. HBc Ag is found in the core of infectious viral particles and in the nuclei of infected hepatocytes. Anti-HBc appears early in infection and can indicate either previous or ongoing infection. HBe Ag, as defined originally, appeared to be a complex family of soluble proteins (Magnius and Espmark, 1972) and at least three components el, e2, e3 have been distinguished (Williams and Le Bouvier, 1976; Trepo et al., 1978). HBe Ag is found early in HBV infection and is associated with high infectivity, numerous circulating mature virions and strong DNA polymerase activity. Seroconversion to anti-HBe signals the end of HBV replication and infectivity, and coincides with resolution of hepatitis. Molecular cloning and nucleotide sequencing of the genomes of HBV (Galibert et al., 1979; Valenzuela et al., 1981) and of the related woodchuck (WHV, Galibert et al., 1982) and ground squirrel (GSHV, Seeger et al., 1984) hepatitis viruses show that © IRL Press Limited, Oxford, England.

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all these viruses have conserved four open reading frames (ORFs) which have the potential to code for proteins. By various means (see Discussion for details), three of the ORFs can be assigned to known viral proteins, the polymerase, HBs Ag and HBc Ag. In addition, proteolytic treatment of HBc Ag cloned in E. coli converts HBc Ag into anti-HBe reactive material (MacKay et al., 1981) and the major form of HBe Ag found in serum (a peptide with a mol. wt. of 15 000 daltons) is derived from the N terminus of HBc Ag (Takahashi et al., 1983). No known protein or function has so far been formally assigned to the remaining ORF, originally called region 5 (Galibert et al., 1979) but now more commonly referred to as region X. Here, we show that the HBV X gene can be expressed in E. coli, and the product can be recognised by sera from HBV-infected patients or WHV-infected woodchuck. Furthermore, the X gene product is recognised only by some sera which are anti-Hbe positive but not by sera which are HBe Ag positive, indicating that the X gene product is a previously unrecognised antigen expressed during the HBe Ag positive phase of HBV infection, i.e., the phase of active viral replication. -

Results Construction of recombinant plasmidsfor expression of HBc and HBx genes The different steps in the construction of plasmids capable of expressing HBc or HBV X gene in E. coli are outlined in Figure 1. A double digestion of HBV DNA with BamHI and TaqI yields, amongst others, two fragments, one 506 bp long and the other 998 bp long. The smaller fragment arises from a BamHI site 28 nucleotides after the start of the X gene and a TaqI site 70 nucleotides after the end of the gene. The larger fragment arises from the same TaqI site, which is situated eight nucleotides after the start of the HBc gene and finishes with a BamHI site located 450 nucleotides after the stop codon for HBc. After isolation, the fragments were rendered blunt-ended by treatment with E. coli DNA polymerase I and EcoRI linkers (8-mers) were added. The plasmid expression vector system chosen was that developed by Charnay et al. (1978), consisting of pBR322 containing a fragment of X u.v. Lac 5 possessing the lac promoter, ribosome binding site and the beginning of the (3-galactosidase gene up to an EcoRI site. The EcoRI site has been manipulated so that within a series of plasmids, pPC4)1, 02 and )3, one can choose the vector appropriate for the expression of a fusion product between (3-galactosidase and the gene of interest. Based on our knowledge of the sequence of the fragments prepared from HBV and of the manipulations carried out, we inserted the HBc gene fragment into pPC43 and the X gene fragment into pPC42. After transfection into E. coli C600, colonies containing the inserts were isolated and the orientation of the insert was determined by restriction enzyme analysis. Plasmids containing the desired fragments in the correct orientation were chosen and the junctions between the inserts and the vector were sequenced to verify the constructions. While the pPC43-HBc construction was 1287

A.Kay et al. Pre- s

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Fig. 1. An outline of the major steps involved in the cloning of the HBx and HBc Ag genes into the beginning of the X u.v. Lac 5 gene contained in plasmids pPC4I and pPC43. The actual starting material was the HBV genome (subtype ayw) originally cloned in Xgt WES-XB (Charnay et al., 1979a) and subcloned through the EcoRI site into pBR322. The final structures of the recombinant genes, verified by sequencing, are shown. RI, B and T: respectively the EcoRI, BamHI and TaqI restriction enzyme sites.

expected, the pPC)2-X gene construction was not. Treatment of the original BamHI-TaqI fragment with E. coli DNA polymerase had apparently led to removal of the 5' overhangs rather than filling them in. The cloned fragment was excised from pPC42 and re-inserted into pPC41, which restores the correct reading frame. The final structure of the recombinants is shown in Figure 1. The X gene recombinant should yield a protein of 154 amino acids, the first 10 residues of the X gene product being replaced by 10 amino acids coded by the vector and EcoRI linker. The theoretical mol. wt. of the fusion product is 16 600 daltons. The HBc recombinant has the first three amino acids of HBc replaced by 12 amino acids from the vector, giving a mol. wt. of 21 900 daltons for the recombinant product. Expression in maxicells To study the expression of the cloned genes, the plasmids were used to transform E. coli CSR 603. These cells are u.v.-sensitive, and after irradiation, damaged DNA is not repaired and is degraded, leading to inactivation of most of the bacterial chromosomal genes after several hours of incubation. However, those plasmid molecules which have escaped u.v. damage because of their small target size continue to replicate, and residual protein synthesis in the cells is due mostly to plasmid-encoded genes. Cells transformed either with vector (pPCc/d), or with X gene or HBc constructions were grown up, irradiated and labelled several as

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hours later with [35S]methionine. After labelling, the cells were harvested and lysed by boiling in the presence of SDS. Extracts were analysed on an SDS-polyacrylamide gel (Figure 2). Extracts containing the vector alone show two major labelled bands migrating as proteins of 30-40 K, which are not found in nontransformed cells, and presumably arise from the genes of pBR322. Extracts containing the X gene or HBc constructions both show an additional labelled band, at 17 K for X gene and at 23 K for HBc, in good agreement with the calculated mol. wts. of the expected fused proteins. The intensity of these bands is increased somewhat by the addition of IPTG during labelling, indicating that the proteins produced are under the control of the lac promoter (Figure 2). We would not expect a large effect with IPTG in this system since the synthesis of lac repressor is presumably already affected by the irradiation and the presence of the lac operator on the multi-copy plasmids probably titrates out most of the repressor left. Immunoprecipitation If patients are exposed to X gene product during the course of infection, then we would expect them to mount an immune response to the protein. We therefore attempted to immunoprecipitate the protein using different sera. Labelled extracts of bacteria harbouring the vector alone (vector extracts), the X gene construction (HBx extracts) or the HBc construction (HBc ex-

Expression of HBV HBx gene in E. coli

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Fig. 2. Expression of the HBx and HBc Ag genes in the maxicell system. Samples are [35S]methionine-labelled and have been electrophoresed on a 5-20% gradient polyacrylamide-SDS gel. 1, 2: extract from bacteria transformed with pPC4I. 3, 4: extract from bacteria transformed with the pPC4I-HBx plasmid. 5, 6: extracts from bacteria transformed with the pCO43-HBc Ag plasmid. Samples 2, 4 and 6 were supplemented with isopropyl ,B-D-thiogalactopyranoside (IPTG, Sigma, 10 mM) during the labelling period. Each sample represents 1 ml of irradiated bacteria. M: 14C- labelled protein mol. wt. markers. From the bottom, the mol. wts. are 14 300, 30 000, 46 000 and 69 000 daltons.

tracts) were prepared by lysozyme-Triton lysis and reacted with normal, anti-HBe or anti-HBc sera. None of the sera precipitated specific proteins from the vector extracts (not shown). Normal sera do not precipitate protein from HBc extracts (Figure 3A). As expected, the anti-Hbc sera precipitate a protein migrating at the position of HBc. The anti-HBe sera, which are also anti-HBc positive, precipitate not only the intact HBc but also precipitate faster migrating material. These faster migrating species, which are precipitated neither from vector extracts with any of the sera tested nor from the HBc extracts with anti-HBc sera are therefore specifically recognised by the antiHBe sera. They presumably represent incomplete or degraded HBc Ag molecules and confirm that at least part of the HBe response is due to altered forms of HBc Ag (MacKay et al., 1981; Takahashi et al., 1983). When immunoprecipitation of HBx extracts is carried out using the same antisera, only the anti-HBe sera used react, precipitating the X gene product (Figure 3B). The anti-HBe sera are also strongly anti-HBc positive, but since the anti-HBc sera used do not react with HBx, the recognition of the protein is due either to the anti-HBe activity of the sera or to a separate unknown anti-HBx activity which is not found in the anti-HBc sera. Such an anti-HBx activity appears as a minor activity eventually present in anti-HBe sera and which varies from patient to patient. This can be see in Figure 4, where several strong anti-HBe sera have been tested on HBx and HBc Ag extracts. With the HBc Ag extracts, anti-HBe activity (i.e., the immunoprecipitation of HBc Ag-derived fragments) is prominent in all three human sera, designated BR., 334 and MO., with no major differences between them. With HBx extracts however, the situation is different. While the serum BR. clearly immunoprecipitates HBx, the protein can only be seen in the 334 immunoprecipitate upon long exposure, and may not represent specific

Fig. 3. (A) Immunoprecipitation of [35S]cysteine-labelled extracts of bacteria transformed with the pPC43-HBc Ag plasmid. Samples were electrophoresed on a 20% polyacrylamide-SDS gel. T, 5: Triton X-100 or SDS lysates of labelled bacteria (from 0.5 and 0.15 m~l culture respectively). 1, 2: two different normal human sera. 3, 4: two different human anti-HBe plus anti-HBc sera (AB. and BR.). 5, 6: two different human anti-HBc sera (LC. and LV.). The immunoprecipitations

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preparation purified from WHe Ag-positive blood (Hantz et al., 1983). This serum recognises HBx in our test, strongly indicating that the equivalent woodchuck gene, WHx, is expressed during infection with WHV. In addition, the serum reacts relatively poorly with HBc Ag fragments recognised by human anti-HBe sera. It is therefore possible that the cross-rection of such woodchuck sera with human HBe Ag preparations can be used to predict the presence of HBx.

Discussion Although there is no suitable in vitro culture system or simple laboratory animals capable of supplying ample amounts of pure virus, molecular cloning of the genome in E. coli has permitted the determination of the nucleotide sequence of several subtypes of HBV (Galibert et al., 1979; Valenzuela et al., 1981) as well as that of the related viruses WHV (Galibert et al., 1982), DHBV (Mandart et al., 1984) and GSHV (Seeger et al., 1984). These viruses possess the smallest known genomes of animal DNA viruses, ranging from 3311 nucleotides for GSHV to 3021 nucleotides for DHBV. Comparison of the sequence of HBV with that of WHV (Galibert et al., 1982) and of GSHV (Seeger et al., 1984) shows that these viruses have conserved four ORFs, all on the same strand of the genome, plus a short non-coding stretch of nucleotides which is thought to be part of the viral origin of replication (Galibert et al., 1982; Molnar-Kimber et al., 1984). ORF 6, covering > 80% of the genome probably codes for the virus-associated polymerase (Galibert et al., 1979; Toh et al., 1983; Mandart et al., 1984; Kamer and Argos, 1984). ORF 7 has been identified as the coding region for HBs Ag (Chamay et al., 1979b). This ORF gives rise to other larger proteins including one coded for by the entire ORF (Heennann et al., 1984). ORF 8 codes for HBc Ag (Galibert et al., 1979; Pasek et al., 1979). This ORF is also involved in the production of at least part of what is known as HBe Ag. The major component of HBe Ag found in serum, a polypeptide with a mol. wt. of 15 500 daltons, is derived from the N terminus of HBc Ag (Takahashi et al., 1983). HBc Ag, either in serum or produced in E. coli, can be converted into HBe Ag by denaturation or by partial proteolysis (Budknowska et al., 1979; Ohori et al., 1980; MacKay et al. 1981, see also this work). HBe Ag is a complex group of antigens (Magnius and Espmark, 1972; Williams and Le Bouvier, 1976; Trepo et al., 1978) which appears early in infection and is associated with the replicative or infectious phase of illness (Hoofnagle, 1981). Seroconversion to anti-HBe positivity is closely, though not completely, associated with resolution of the illness and loss of infectivity (Stevens et al., 1979; Hadziyannis et al., 1983). No function or known protein product has been identified for the fourth ORF, but nucleotide sequence studies have shown that animal DNA viruses are extremely economical in their use of coding potential. We were intrigued therefore by this fourth ORF of HBV, designated region 5 or X gene, which overlaps the end of the polymerase gene and precedes the HBc Ag gene (Figure 1). The X gene is conserved as an ORF, and therefore as a potential coding region, in all of the sequenced subtypes of HBV, in WHV and GSHV. Although there is relatively little homology at the nucleotide or amino acid level between the X genes of HBV and WHV, except at their N termini which are well conserved, there is striking homology between the two proteins when one compares the predicted secondary structures (Schaffer and Sninsky, 1984). However, it should be noted the DHBV does not appear to contain an independent X gene (Mandart et al., 1984). To show that the HBV X gene is expressed during viral infec-

4. Immunoprecipitation of [35S]methonine-labelled extracts with various anti-HBe positive sera. (A) X: SDS lysate of labelled bacteria (from 0.3 ml of culture) transformed with pPCOI-HBx plasmid. The position of the recombinant HBx protein is indicated by the arrow. 1-4: immunoprecipitates from Triton extracts of the bacteria using the sera W90, BR., 334 and MO. respectively. The equivalent of 2.5 ml of culture were used per immunoprecipitation, and 10 1l of antiserum. (B) C: SDS lysate of labelled bacteria transformed with pPC43-HBc Ag plasmid. The position of the recombinant HBc protein is indicated by the arrow. 1-4: as in A. Fig.

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Expression of HBV HBx gene in E. coli

tion, and that it therefore codes for a genuine viral protein, we have cloned the X gene ORF in E. coli in phase with the beginning of the lacA gene (,B-galactosidase, Figure 1). The recombinant protein product was then used as a marker to detect antibodies in patients sera which recognise HBx. We have also made a similar construction using the HBc Ag gene. Since we know that HBc Ag can be expressed in E. coli and that welldefined anti-HBc are available, this construction serves as a positive control for expression and for immuno-detection. In addition, we considered that the antisera most likely to recognise HBx would be anti-HBe sera, which have been shown to recognise several different antigens (Trepo et al., 1978). Since altered forms of HBc-Ag are implicated in this response, the HBc Ag construction could help us to delimit the role played by HBc Ag and the possible role played by HBx. To detect expression of the genes in E. coli we have used the maxicell system, which offers the advantage of greatly lowering the background of endogenous bacterial protein synthesis, allowing clear visualisation of plasmid encoded proteins (Sancar et al., 1979). We find that both HBx and HBc Ag are expressed in this system (Figure 2). For reasons that we do not understand, while expression of HBc Ag in this system is never a problem, expression of HBx is variable. Although weaker, the expression of HBx is sufficient to show that several sera from patients infected by HBV recognise the protein (Figures 3 and 4). Some sera from patients who are anti-HBe positive (and anti-HBc positive) recognise HBx, HBc Ag and degraded or incomplete forms of HBc Ag. Antisera which are positive only for anti-HBc (but which are HBe Ag-positive) recognize HBc Ag but not HBx (Figure 3). Normal sera do not specifically recognise any of our HBV-derived products. The X gene is, therefore, expressed during infection with HBV and this expression appears to be correlated with the HBe antigen phase of infection, the phase in which the virus is actively replicating. Our results also show that the reactivity of anti-HBe sera with HBx varies from patient to patient, and that even patients with a high anti-HBe titre such as patient 334 who react very well with altered forms of HBc Ag react very poorly, if at all, with HBx. High anti-HBc titres probably reflect activity directed against HBc Ag-derived antigens and are not predictive for anti-HBx activity. HBx is presumably either a poor immunogen or is present in relatively small amounts during infection. Another possibility that we cannot rule out is that the replacement of the N terminus of genuine HBx with the beginning of,B-galactosidase alters the conformation of the protein in some way which affects its recognition by the sera. During the preparation of this manuscript, we learnt that M.Meyer, L.Vitvitski, N.Nath and J.J.Sninsky (personal communication), using an approach similar to ours, have found French and American anti-HBe sera which recognise recombinant-derived HBx Ag. The serum W90 (Figure 4) was obtained from a woodchuck carrier ofWHV boosted with purified WHe Ag. It shows crossreactivity with the HBe Ag/anti-HBe system (Hantz et al., 1983). The serum recognises HBx (Figure 4) indicating that the X gene ofWHV is also expressed during infection of woodchucks. Also, the serum reacts relatively poorly with the HBc Ag-derived peptides. The reactivity of anti-WHe sera with HBe Ag preparations may therefore provide indirect evidence for the presence of HBx Ag. More animals will have to be tested to establish this point. The reactivity of anti-WHe sera with HBx is somewhat surprising since the amino acid sequences of HBx and WHx are not very well conserved (Galibert et al., 1982) and the most conserved region, the N terminus of HBx, has largely been eliminated

from our construction. However, our recombinant protein does retain regions which, predicted from the amino acid sequence, would permit the constitution of common epitopes between HBx and WHx. The fact that HBx and WHx possess similar secondary structure (Schaeffer and Sninsky, 1984) can also account for the observation that a woodchuck serum can recognise HBx. In view of these results, it is surprising that DHBV apparently does not contain an identifiable X gene (Mandart et al., 1984). However, the position and the size of the DHBV core gene ORF is consistent with this ORF representing a fusion between an Xtype gene and the core gene. The expression during infection of the X genes of HBV, WHV and, in all probability, GSHV implies that the protein has a function, and we know of no fundamental differences between the life cycle of DHBV and the other viruses that can be explained on the basis of DHBV lacking a specific protein. Perhaps the question to ask is rather how DHBV has conserved both the X-gene function and the coregene function within a single ORF. What can be the function of HBx? One obvious candidate is that of the role of the 5'-terminal protein covalently associated with the minus strand of viral DNA (Gerlich and Robinson, 1980; Molnar-Kimber et al., 1983). This would correspond well with the fact that we find HBx within the group of antigens closely associated with replication of the virus. Such a protein would presumably be present during infection in small amounts compared with the major structural proteins HBs Ag and HBc Ag. The presence of HBx in small amounts in serum, the variability of levels of antibody directed against it, and its size, 16 000 daltons, close to that of the major HBc Ag-derived peptide present in HBe Ag positive sera can explain why HBx so far has gone unrecognised. The presence of an HBx Ag/anti-HBx system functioning almost in parallel with the classic HBe Ag/anti-HBe system may also explain some previous confusing results. Although seroconversion to anti-HBe positivity is strongly correlated with the disappearance of markers of viral replication (viral DNA in the serum, DNA polymerase activity, etc.) and the loss of infectivity of the blood, the correlation is not absolute (Stevens et al., 1979; Hadziyannis et al., 1983). It is possible that patients who are anti-HBe positive but remain ir-ctious have manufactured little or no anti-HBx. Another phenomenon which may be related to HBx Ag expression is the recently described therapeutic effect of anti-HBe sera (Stephan et al., 1984). These authors have shown that anti-HBe sera, but not anti-HBc sera, protect chimpanzees from subsequent HBV infection. However, injection of very large doses was necessary to produce the effect, indicating that a minor component might be responsible. This component may be anti-HBx. -

Materials and methods All enzymes used were from commercial sources (Amersham, BRL, Boehringer Mannheim, New England Biolabs, Sigma). EcoRI linker (dGGAATTCC) was obtained from Biologicals (Toronto). The starting material for obtaining HBV (subtype ayw) DNA fragments was the HBV genome, originally cloned in Xgt WES-XB (Chamay et al., 1979a), and subsequently subcloned through the EcoRI site into pBR322. Containment conditions were as recommended by the French National Control Committee. The different methods used in cloning HBV DNA fragments into plasmids were basically as described in the book by Maniatis et al. (1982). Large-scale plasmid preparations were carried out according to the method of Birnboim and Doly (1979). For small-scale preparations we used the method of Holmes and Quigley (1981). The sequences of clones were verified by the method of Maxam and Gilbert (1980). Expression of cloned genes was monitored by the 'maxicell system' (Sancar

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A.Kay et al. et al., 1979). The u.v.-sensitive E. coli strain CSR 603 (RecA, uvrA, phrl) was first transformed by the appropriate plasmid. Cells were then grown at 37°C to a density of 0.4-0.6 A640 units, irradiated with a u.v. germicidal lamp (20 JIm2), and incubated for 1 h at 37°C. D-Cycloserine (Sigma, 150 'y/ml) was then added to kill survivors and incubation was continued overnight at 37°C. Cells were then harvested, washed and resuspended in sulphate-free Hershey medium. After incubation for 1 h at 37°C, [35S]methionine or -cysteine was added (5-20 tsCi/ml), as well as IPTG (10 mM) where appropriate, and incubation was continued for 1 h at 370C. Cells were then chilled, harvested, and washed with cold medium. For direct analysis on SDS-polyacrylamide gels (Laemmli, 1970), bacterial pellets were lysed directly in Laemmli sample buffer (30 jl buffer/I ml original volume) followed by boiling for 3 min before application to gels. For immunoprecipitation, bacterial extracts were prepared by lysozyme-EDTATriton X-100 lysis. Usually, 10 ml of labelled bacteria were centrifuged, washed in cold medium, resuspended in 0.45 ml of Tris pH 8 (50 mM) sucrose (20%) and then 50 1d of lysozyme (Sigma, 10 mg/ml in Tris-sucrose buffer) was added for 5 min, followed by 0.1 ml ethylene diamine tetraacetate (EDTA, 0.2 M) for a further 5 min. Phenyl methyl sulphonyl fluoride (PMSF, Sigma, 1 mM final) and aprotinine (Sigma, 0.1% final) were also added at this time. After an additional 5 min at 4°C, lysis of the fragilised cells was completed by the addition of 0.5 ml of Tris pH 8, 50 mM; EDTA, 25 mM; Triton X-100, 0.2%. After 15-30 min of incubation at 4°C, the lysates were clarified by centrifugation at 12 000 g (Eppendorf centrifuge) for 5 min. Immunoprecipitation was carried out following the method of Schwyzer (1977). 5-10 ,ul of antiserum was added to bacterial extracts (in Eppendorf tubes) which were then incubated at 40C for 20-30 min. 5-10 1l of settled protein ASepharose 4B (Pharmacia, suspended in lysis buffer) were then added and the extracts were shaken vigorously overnight at 4°C. The protein A-Sepharose beads were then sedimented by centrifugation and washed with 5 x 1 ml of Tris pH 8, 0.1 M; 2-mercaptoethanol, 1%; LiCl, 0.5 M. The beads were transferred to minicolumns [ 1 ml Eppendorf cones plugged with a disk of porous polyethylene (Bel Art)] with a further 1 ml of washing solution, centrifuged to eliminate traces of salt, and then the protein A-bound antibodies and protein were eluted with 3 x 25 1d of Laemmli sample buffer (with SDS increased to 1% and 2-mercaptoethanol to 2%). The samples were boiled for 3 min before application to polacrylamide-SDS gels. After electrophoresis, gels were prepared for fluorography (Bonner and Laskey, 1974) by treatment with EN3HANCE (New England Nuclear Inc.), drying, and exposure to X-ray film under intensifying screens. HBV serology for HBs Ag, HBc Ag, HBe Ag, anti-HBs, anti-HBc and antiHBe was carried out by radioimmune assay using Abbott kits. The antisera named in this study have the following characteristics. LC. and LV. Sera with high titres of anti-HBc (1/104) which are also positive for HBs Ag and HBc Ag as well as for HBV DNA (by dot blot hybridisation) and for DNA polymerase activity. AB., BR., 334 and MO. Sera from asymptomatic HBV carners which are positive for HBs Ag; for anti-HBc and anti-HBe by radioimmune assay. They are negative for HBV DNA and for DNA polymerase activity, and have normal transaminase levels. These healthy carriers had no known previous clinical history of hepatitis and were detected through routine blood bank screening. These sera were further selected for this study because they had precipitating levels of anti-HBe detectable by immunodiffusion and exhibited both anti-HBel and anti-HBe3 reactivities (Trepo et al., 1978). Normal sera. Sera obtained from the blood bank of our hospital and which are devoid of any serological markers of HBV infection. W90. An anti-WHs positive woodchuck boosted with a preparation from a WHV carrier animal positive for WHs and WHe Ags and showing high DNA polymerase activity (Hantz et al., 1983). The anti-WHc titre is low, which is the rule for a woodchuck having recovered from WHV infection and no longer exposed to WHc Ag.

Acknowledgements We are grateful to O.Hantz who provided the W90 serum, C.Pichoud and L.Vitvitski who carried out the serological determinations, and M.Springer who introduced us to the maxicell system. This work was supported by INSERM (SC. 15), the CNRS (LP. 101) as well as by a grant No. 960056 from the 'Programme Mobilisateur Biotechnologies' (CNRS-MRT).

References Birnboim,H.C. and Doly,J. (1979) Nucleic Acids Res., 7, 1513-1520. Bonner,W.M. and Laskey,R.A. (1974) Eur. J. Biochem., 46, 83-88. Budkowska,A., Kalinowska,B. and Nowaslawski,A. (1979) J. Immunol., 123, 39-46.

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Note added in proof

Recently, Moriarty,A.M., Alexander,H., Lerner,R.A. and Thornton,G.B. (1985) Science (Wash.), 227, 429433, have also presented evidence that some sera from human hepatitis patients can recognise the HBx gene product.