Large Surface Proteins of Hepatitis B Virus ... - Journal of Virology

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The sequence of hepatitis B virus DNA contains an open reading frame which ... protein, P39, and its glycosylated form, GP42, in hepatitisB virus particles and ...
JOURNAL OF VIROLOGY, Nov. 1984, p. 396-402 0022-538X/84/110396-07$02.00/0 Copyright C 1984, American Society for Microbiology

Vol. 52, No. 2

Large Surface Proteins of Hepatitis B Virus Containing the Pre-s Sequence KLAUS H. HEERMANN,' UDO GOLDMANN,1 WOLFGANG SCHWARTZ,' TORSTEN SEYFFARTH,l HORST BAUMGARTEN,2 AND WOLFRAM H. GERLICHl* Departments of Medical Microbiologyl and Immunology,2 University of Gottingen, Gottingen, Federal Republic of

Germany Received 6 June 1984/Accepted 25 July 1984

The sequence of hepatitis B virus DNA contains an open reading frame which codes for a not-yet-identified protein of at least 389 amino acids. Only the products starting at the third (GP33/GP36) or the fourth (P24/ GP27) initiation signal have been characterized as components of the viral surface antigen. We found a larger protein, P39, and its glycosylated form, GP42, in hepatitis B virus particles and viral surface antigen filaments. Immunological cross-reactions showed that P39/GP42 is partially homologous to P24/GP27 and GP33/GP36. The unique portion of its sequence bound monoclonal antibodies which had been induced by immunization with hepatitis B virus particles. Proteolytic cleavage patternsand subtype-specific size differences suggested that the sequence of P39 starts with the first initiation signal of the open reading frame. Its amino-terminal part (pre-s coded) is exposed at the viral surface and, probably, is highly immunogenic. A model is presented of how the open reading frame for the viral envelope leads to defined amounts of three different proteins.

A typical consequence of acute or chronic infection with hepatitis B virus (HBV) is secretion of the viral surface antigen (HBsAg) into the blood of infected persons. Even in the presence of efficient viral replication, only a very minor part of the total HBsAg forms the viral envelope. A larger amount is found on filaments of 20-nm diameter and variable length. By far the most HBsAg is present on small, noninfectious, 20-nm particles (20). These particles are used in purified form as a vaccine against HBV. There are two types of chronic carriers of HBsAg. A smaller fraction of the carriers has high titers of HBV, a high concentration of excess HBsAg, and soluble viral core protein (e-antigen) in the blood. Most adult HBsAg carriers have, however, very little or no infectious HBV or filaments in the blood and only moderate concentrations of HBsAg 20nm particles and antibodies against e antigen. The mechanism for the suppression of viremia in these carriers is unknown. Immune reactions against unidentified proteins may be involved (1, 5). HBsAg from both types of carriers consists of a major protein, P24, and its glycosylated form, GP27 (19). Purified HBsAg 20-nm particles from viremic carriers have, in addition, a further glycoprotein, GP33, and its twofold-glycosylated form, GP36 (24, 25). The amino-terminal protein sequence of P24 has been analyzed (19) and aligned with the sequence of cloned HBV-DNA (6a, 17, 29). A coding sequence of 226 triplets ending with a stop codon was identified for P24 (gene s). Gene s begins only at the fourth possible start codon of a larger open reading frame (ORF), so it is preceded in phase by 163 or 174 codons, depending on the viral subtype (pre-s region 28). Recently, it was shown that GP33 consists of the P24 sequence and an aminoterminal part of ca. 55 amino acids (13a, 24). The finding of an amino-terminal methionine in GP33 (14) and the transcription data (2, 22a) suggest that the sequence of GP33 starts at the third initiation signal of the ORF, which is 55 codons upstream of the fourth signal (2). The translation products beginning at the first or second start codon have not yet been identified. The conservation of the pre-s region during evolution (15) and the in vitro transcription data (13) make it *

very likely that at least one larger surface protein exists. In the search for this hypothetical protein, we analyzed purified HBV particles and filaments in addition to 20-nm particles. Immunization of mice with HBV particles led to monoclonal antibodies, which selectively detected two large viral surface proteins. Cross-reactions with P24 and GP33, partial proteolysis patterns, glycosylation data, and subtype heterogeneities suggest strongly that these two large surface proteins are products of the total ORF and that the monoclonal antibodies are directed against epitopes coded by the pres region. We present models of how the six envelope proteins of HBV are derived from one continuous ORF and of how their expression may be regulated. Being immunogenic components of the viral envelope, the large surface proteins may be of great medical importance. MATERIALS AND METHODS Purification of HBV and HBsAg particles. Plasma units (250 ml) from one HBV carrier were passed through a column (10 by 120 cm) of Bio-Gel A5M (Bio-Rad Laboratories) with TNE (0.13 M NaCl, 0.01 M Tris-hydrochloride (pH 7.4), 0.001 M disodium EDTA). Samples of the fractions close to the void volume were mixed with 0.3% ,B-mercaptoethanol and 0.5% Nonidet P-40, and the HBV core antigen was determined by an enzyme immune assay (7). Positive fractions were combined and centrifuged in an angle rotor (6 by 90 ml) through a layer of 20% sucrose-TNE for 20 h at 25,000 rpm and 10°C. The pellets were suspended in 0.5 ml of TNE and layered on an S-shaped sucrose gradient with best resolution between 35 and 45% (wt/wt) sucrose. After 20 h at 34,000 rpm in an SW42 rotor, fractions were assayed for HBsAg and viral core antigen. In electron microscopy, peak fractions of viral core antigen consisted of HBV particles with less than 10% filaments. Peak fractions of HBsAg consisted of >95% long filaments. HBsAg 20-nm particles with much GP33/GP36 were purified from Bio-Gel ASM fractions containing the highest HBsAg activity by banding in CsCl (9). These preparations were free of HBV or long

filaments. Antisera. For raising anti-P24, purified HBsAg without minor proteins (27) was treated for 30 min with 1% dithiothreitol (DTT) and 1% sodium dodecyl sulfate (SDS) and

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then dialyzed for 2 h against 0.01% DTT-0.01% SDS in 0.13 NaCl. For anti-GP33, purified HBsAg with much GP33 but with undetectable GP42 was used without denaturation. Portions (100 pg) of HBsAg subtype ad were mixed with Freund complete adjuvant and injected intramuscularly into guinea pigs. The injections were repeated with incomplete adjuvant after 4 weeks. Sera were taken 10 days later. Monoclonal antibody. Ten micrograms of purified HBV was mixed with complete Freund adjuvant and injected intraperitoneally into BALB/c mice. After 4 weeks the injections were repeated with incomplete adjuvant, and 10 days later spleens were obtained for fusion with myeloma cell line P3-X63-Ag8.653 as described previously (10). Supernatants of immunoglobulin-producing hybridomas were placed on microtiter plates which were coated with 40 ng of HBV and filaments per well. Binding of mouse immunoglobulin was detected by the addition of peroxidase-labeled antimouse immunoglobulin (Dako, P161). Clone A18/7 (IgG,K) was recloned and injected into BALB/c mice for growth as ascites tumors. Immunoglobulin G from ascites liquid was purified by precipitation with 18% (wt/vol) sodium sulfate. Gel electrophoresis and immune staining (26). Purified HBV or HBsAg was denatured by 2.5% SDS and 5% DTT for 5 min at 100°C. Proteins were separated in 12 or 15% polyacrylamide-N,N'-methylenebisacrylamide gels with the Laemmli buffer system and 2 to 5 V/cm (23). After electrophoresis, the separated proteins were transferred to a porous membrane (GVHP; Millipore Corp.) by transversal electrophoresis at 40 V in 0.025 M Tris (pH 8.3), 0.192 M glycine, and 15% (vol/vol) methanol. Nonspecific protein binding was saturated with 20% fetal calf serum-TNE for 1 h. Thereafter, a suitable amount of antibody was added for 1 h under agitation. After a thorough washing, 1251_ or peroxidase-labeled second antibody against the first antibody in 20% fetal calf serum was added for 1 h. The presence of antibody was visualized either by autoradiography or by enzymatic staining with 0.01% diamino benzidine-0.06% H202-0.05 M Tris-hydrochloride (pH 7.4). RESULTS Protein composition of HBV particles, filaments, and 20-nm particles. All three morphological forms of HBsAg consistently contained six protein bands: P24, GP27, GP33, GP36, a 39-kilodalton (kDa) protein, and a 42-kDa protein. Experiments to be described later showed that the 42-kDa protein was glycosylated, but the 39-kDa protein was not. Thus, they are referred to as P39 and GP42 (Fig. 1). HBV particles contained, in addition, the P22 band of the viral core protein (7) and several weaker protein bands of >45 kDa (Fig. 1, lane 1) Mock preparations of HBV particles from negative human plasma also showed several high-molecular-weight bands, but there were no bands at the position of the viral

proteins. The relative staining intensities of P24, GP27, GP33, and GP36 were very similar in the three morphological forms. The intensities of P39 and GP42 differed significantly, however, among the three forms. In HBV particles and filaments, the GP42 band was more intense than the GP33 band; in 20-nm particles, the GP42 band was weaker than the GP33 band. P39 and GP42 occurred in all isolates as pairs; GP42 was always more intense than P39. This typical staining behavior of the three morphological forms was confirmed with samples from six further HBV carriers. The intensity of the protein bands after silver staining suggests that HBV particles may contain up to 20 times more P39 and GP42 than do 20-nm particles from the same plasma source. According to the particle mass of 3 x 106 daltons, 20-nm particles

PRE-s PROTEINS OF HEPATITIS B VIRUS

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FIG. 1. Protein composition of HBV particles (lane 1), HBsAg filaments (lane 2), and 20-nm particles (lane 3). The particles were purified from the plasma of a chronic HBV carrier (subtype ay). Samples with similar protein contents were electrophoresed through 12% polyacrylamide, and the proteins were stained with silver (16). Size markers were lysozyme (14.3 kDa), trypsin inhibitor (20 kDa), carbonic anhydrase (30 kDa), ovalbumin (45 kDa), serum albumin (68 kDa), and phosphorylase B (94 kDa).

consist of ca. 100 protein subunits. The weak staining suggests that only one or occasionally two GP42/P39 molecules may be present in 20-nm particles. In contrast, HBV, with its four-times-larger surface, may contain 40 to 80 P39 or GP42 molecules per virion. Monoclonal antibody against HBV. Five mice were immunized with purified HBV particles, and all developed high serum antibody titers against HBsAg. Several thousand hybridoma clones were derived from the spleen cells of the mice, but only two of the clones produced antibodies against HBV. Neither antibody bound to mock preparations of HBV. The antibodies were apparently directed against the same or very closely related epitopes, since mixtures of the antibodies did not produce stronger binding than single antibodies. One of the monoclonal antibodies (MA18/7) was used for further experiments. In a two-site enzyme immune assay with MA18/7 at the solid phase and peroxidase conjugated as labeled antibody, HBV particles gave the strongest signal of the three morphological forms. A 2-fold-higher protein concentration of purified filaments or a 20-fold-larger amount of 20-nm particles was necessary to bind the same amount of labeled antibody to the solid phase (Fig. 2). The HBV particles used for immunization had been of HBsAg subtype ayw. MA18/7 reacted, however, with subtype adw as well. The part of the viral envelope which bound MA18/7 was sensitive to proteolysis by trypsin. A limited digestion of HBsAg 20-nm particles completely abolished the binding of MA18/7. In contrast, the binding of a conventional anti-P24 antibody increased slightly after the digestion (Fig. 3A).

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idoo 160 FIG. 2. Binding of labeled monoclonal antibody MA18/7 to increasing amounts of HBV (U), HBsAg filaments (0), or 20-nm particles (A). Wells of microtiter plates were first coated with 0.5 ng of MA18/7 each and then incubated with the indicated dilutions of purified particles for 1 h at 37°C. After being washed, peroxidaselabeled (8) MA18/7 in 1% bovine serum albumin-phosphate-buffered saline was added for 1 h, and after further washings, orthophenylenediamine-H202 as substrate was added. The colored reaction product was assayed at 493 nm (E493).

Reduction of the HBsAg particles did not change the binding of MA18/7, but most of the conventional anti-P24 did not bind any longer to the particles (Fig. 3B). Immune reactivity of isolated HBsAg proteins. When the immune blot technique was used after gel electrophoresis, MA18/7 bound only to P39 and GP42 (Fig. 4, lane C). This finding showed that P39 and GP42 shared an epitope which was not present on the smaller HBsAg proteins. The finding also excluded the possibility that the P39 and GP42 bands were mere aggregation artifacts consisting of P24 or GP27. The epitope of MA18/7 was completely resistant to the combined effects of reduction, detergent, and heat, which were employed before electrophoresis. The sizes of P39 and GP42 were consistent with the assumption that they might be products of the whole ORF for HBsAg. According to that hypothesis, P39 and GP42 would have a sequence unique to them of 108 or 119 amino acids, and MA18/7 would bind to this region. In addition, they would have all sequences and the denaturation-resistant epitopes of P24 or GP33. Two conventional guinea pig antisera were used to test this hypothesis, because monoclonal antibodies against denatured P24 or the pre-s part of GP33 were not available. An antiserum against P24 and GP27 was raised by the injection of reduced and denatured particles which did not have detectable GP33, GP36, P39, or GP42. This anti-P24 antibody bound equally well to all six HBsAg proteins, including P39 and GP42 (Fig. 4, lane A). A second antiserum was produced by the injection of 20-nm particles which contained much GP33 and GP36 but no detectable P39 or GP42 (23). The injected animals produced more antibody against the pre-s part of GP33 than against denatured P24, although the inoculum contained more P24 than GP33. At the dilution used for the experiment shown in Fig. 4, lane B, immunoglobulin G from this antiserum bound well to GP33 and GP36 but weakly to P24 and GP27. P39 and GP42 were also well immune stained by this antiserum. In summary, GP42 and P39 are indistinguishable by immu-

nological methods. The two proteins have three antigenic regions: one is defined by the HBV-specific antibody MA18/ 7, the second is coded by the pre-s part of GP33, and the third is coded by gene s. Proteolytic cleavage between the pre-s and gene s coded sequences. Evidence on the primary structure of P39 and

GP42 was obtained by digestion with the glutamic acidspecific protease from Staphylococcus aureus V8. Gene s has only two glutamic acids at positions 2 and 164; the pre-s sequence does not have any glutamic acid. Previously, we have shown that GP33 or GP36 is slowly cleaved by V8 protease into P24 or GP27 and an 11-kDa fragment which consists of the 55 pre-s-derived amino acids and of N-linked glycan (24). If P39 and GP42 contain the whole pre-s sequence linked to the gene s sequence, V8 protease should generate a fragment of 164 or 175 amino acids which binds MA18/7. Figure 5, lanes B, shows that such a fragment of 18 kDa was generated. Since both GP42 and P39 were cleaved by the protease, the production of only one fragment reacting with MA18/7 showed that P39 and GP42 were completely identical in their pre-s part. Subtype heterogeneity of the pre-s sequence. All known DNA sequences of different HBsAg subtypes specify a constant size of gene s. The pre-s sequences of the two sequenced ayw subtypes have 163 codons (6a, 17). The adw2 subtype is largely a homolog of subtype ayw, but it has an additional 11 codons at the 5' end of the ORF (29). In agreement with the DNA sequence data, P39 and GP42 from four adw subtype isolates were consistently larger than those from two ayw subtypes (Fig. 6, lanes B). The difference was ca. 1.0 to 1.5 kDa. A subtype-independent microheterogeneity of the HBsAg proteins has been described previously. This heterogeneity resides in the gene s part (23) and is also visible in P24/GP27 of the isolates shown here. A smaller GP33 or GP36 from ayw subtypes was not noted (Fig. 6, panel A), and so the observed subtype-specific size heterogeneity is not due to the sequences present in the gene s part. The subtype differences between adw and ayw suggest that the sequence of P39 and GP42 starts with the very first codons of the ORF for HBsAg. Glycosylation of the HBsAg proteins. A sensitive, glycopro-

A

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jig/ml b 6o * 1i0 - + FIG. 3. Effects of trypsin (A) and DTT (B) on the binding of MA18/7 (0) and anti-P24 (0). Purified HBsAg 20-nm particles (0.8 ,ug in 100 ,ul) with relatively much GP42 were adsorbed to microtiter

wells (Nunc II) for 4 h at 20°C. The wells were washed, and 100 p.l of

different trypsin dilutions or of 0.1 M DTT was added for 30 min at 37°C. Reduced SH groups were blocked with 0.1 M iodacetamide for 16 h at 4°C. Wells were washed, and a 1:8 dilution of hybridoma supernatant MA18/7 or a 1:8,000 dilution of anti-P24 guinea pig serum in 1% bovine serum albumin-TNE was added for 1 h at 37°C. After a thorough washing, binding of the antibodies was quantitated by the addition of peroxidase-labeled second antibody (1:1,000 Dako) and by the measurement of bound peroxidase at 493 nm (E493)-

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major proteins (p22 and p25). They suggested "repeating antigenic determinants" in all HBsAg proteins (21). These findings are consistent with our data. Stibbe and Gerlich (24) showed that limited proteolysis of GP33 or GP36 generated the same fragments as proteolysis of P24 or GP27, but the two larger proteins had an amino-terminal extension of 50 to 60 amino acids, with three proteolytic cleavage sites predicted by the pre-s(2) sequence (see Fig. 7 for definition). Machida et al. (14) also found two minor glycoproteins, p31 and p35, to be coterminal with P24. They showed by amino acid analysis that these proteins contained a cyanogen bromide fragment which was coded by the pre-s(2) region. The fragment carries the HBV-associated receptor for crosslinked human albumin (13a). Neurath et al. synthesized a peptide containing the 26 amino-terminal amino acids of the pre-s(2) region. Antibodies against this peptide bound to GP33 and GP36 (16a). The major mRNA of HBV in infected liver starts (2, 22a) closely upstream of the third initiation codon of the ORF (17, 29), and so it is very likely that GP33 and GP36 also start at

A

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tein-specific stain (3) showed that GP42 was glycosylated, but P39 was completely glycoside free (Fig. 5, lane C). In agreement with the conclusion drawn before, the 18-kDa fragment of the glu-specific cleavage also did not contain glycan. Thus, the glycan of GP42 is bound to the gene s part, most probably at the same site as in GP27 (18) or GP36 (24). The amino-terminal (24) mannose-rich glycan (22, 23) of GP33 or GP36 is apparently nonexistent in the products of the whole ORF. The nature of the glycan in GP42 was studied further by digestion with endoglycosidase F. This enzyme removes asparagine-linked glycans from their protein part (4). The electrophoretic mobility of glycoproteins is shifted by 3 kDa for every glycan removed. Figure 5, lanes D, show that GP42 decreased after digestion and P39 increased. GP36 disappeared completely, GP33 decreased strongly, and an intense P30 band appeared. This finding supports the conclusions that GP42 contains one asparagine-linked glycan group and that this group is the only difference from P39. The results shown in Fig. 5, lanes D, directly confirm our previous report (24) that GP33 has one glycan group and GP36 has two glycans. DISCUSSION The protein composition of HBsAg has been the subject of numerous studies. Due to the variability of the HBsAg proteins (23), discrepant results on the minor proteins were reported. The electrophoretic components larger than GP42 are now understood as dimers (11, 23), and in this study no such proteins could be reliably identified as HBsAg or HBV components. Feitelson et al. found the minor HBsAg proteins p43, p35, and p32, which probably correspond to GP42, GP36, and GP33. These proteins shared many tryptic peptides with P24 or GP27, but they also had unique peptides (6). Sanchez et al. demonstrated serological cross-reactions between the minor proteins (p27, p31, p35, and p40) and the

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FIG. 5. Generation of an 18-kDa pre-s-coded protein fragment by V8 protease (lanes B), concanavalin A binding of HBV surface glycoproteins (lane C), and removal of glycan by endoglycosidase F (lanes D). (A) Silver stain. (B) HBsAg filaments (adw2) were digested with 6 ,ug of V8 protease (Bio-Rad) in 25 ,ul of 0.1% SDS1% DTT-0.1 M Tris-hydrochloride (pH 7.4) for 72 h at 37°C. Each 24 h, new protease was added. The digested proteins were separated by SDS-gel electrophoresis, transferred to a membrane, reacted with MA18/7 and 125I-labeled anti-mouse immunoglobulin G, and autoradiographed. Lane Bi, Control incubation without protease; lane B2, protease digest. (C) Before treatment with MA18/7, the membrane with the separated proteins was incubated with 100 u.g of concanavalin A per ml and 10 mg of bovine serum albumin per ml in 0.13 M NaCI. After 30 min at 37°C, the washed membrane was agitated with 30 ,ug of horseradish peroxidase (type VI; Sigma Chemical Co.) per ml in 0.13 M NaCl. After a further 30 min and subsequent washings, the bound peroxidase was detected as described in the text. Lane C shows the staining of lane B2. Note the absence of staining in P39 and the 18-kDa fragment (-). (D) In a parallel experiment, filaments were digested with 5 ,u1 of endoglycosidase F (Bio-Rad) in 35 ,ul of 0.1 M sodium phosphate (pH 6.1)-S50 mM disodium EDTA-0.1% SDS-1% Nonidet P-40-1% mercaptoethanol for 16 h at 37°C. The proteins were separated, transferred to a membrane, and immune stained with anti-P24 serum. Lane Dl, Control incubation; lane D2, endoglycosidase F digest. Note the increase of P39 and the formation of P30 in the digest.

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cells transformed with the appropriate DNA fragments (13). Since the first initiation signal is strong, the smaller HBsAg proteins are probably not derived from this larger mRNA. The second AUG in the ORF of some HBV isolates is not conserved and is probably not an initiation signal. The large mRNA has a promoter with a TATA box (13), but the promoter of the small mRNA is more like the late simian virus 40 promoter (2). Thus, expression of P39/GP42 may be completely independent of expression of the smaller HBsAg

proteins.

Since designations based on electrophoretic sizes are

GP 27_-inI P 24-

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FIG. 6. Different sizes of GP42 and P39 in HBsAg samples of different subtypes. Proteins of purified 20-nm particles were separated in duplicate by SDS-gel electrophoresis. Panel A of the gel was stained with silver. Panel B was transferred to a membrane and immune stained with MA18/7. Lanes 1 and 3, Subtype ay; lanes 2, 4, 5, and 6, subtype adw. Note the smaller distance between GP33/ GP36 and P39/GP42 in panel A and the faster migration of P39/GP42 in panel B with the ay subtypes.

this translation signal (Fig. 7). The first AUG codon of this mRNA, however, lacks the typical flanking bases of a strong initiation codon (12), so protein synthesis will probably more often begin at the start of P24 or GP27 with its typical initiation codon. By such a mechanism the two coterminal proteins with different amino termini may be translated in defined proportions from one mRNA. All results of this study confirm the hypothesis that P39 and GP42 are the translation products of the whole ORF. Thus, at least small amounts of a mRNA starting upstream of the first initiation codon of the ORF must be present in HBV-producing cells, although it has not yet been found in HBsAg-positive liver samples (2). Such a mRNA was, however, present in COS 947

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ambiguous for many reasons, it may be preferable to distinguish the three translation products of the ORF for HBsAg as large, middle, and small (or major) surface proteins. The sequence of the ORF may be divided into three independently expressed parts: pre-s(1), present only in the large surface protein; pre-s(2), present also in the middle protein; and gene s, as suggested previously (28). The expression of three different envelope proteins by the variable use of initiation codons in one ORF certainly saves many hundreds of coding capacity triplets and regulating signals. The genetic organization of this ORF is another example-in addition to the use of overlapping ORFs-that the HBV genome is evolutionarily selected to minimum size. The glycosylation patterns of the three surface proteins show interesting differences. Approximately 40% of the small protein is glycosylated at the carboxy-terminal binding site for N-linked glycans. As suggested by the staining intensity of the protein bands, the same site is used less often in the middle protein, but probably more often in the large protein. The single binding site of the middle protein in the pre-s(2) region is always occupied by a mannose-rich glycan, and no glycoside-free middle protein P30 is found in serum. The same site, however, is not used at all in the large protein. This observation demonstrates the strong influence of an amino-terminal sequence on the processing of the

following sequences. Our findings with seven different isolates suggest that the large surface proteins are essential components of complete 2104 bases

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FIG. 7. Regulated expression of surface proteins from HBV DNA and structure of the HBsAg proteins in relation to their coding ORF. The DNA sequence of the ORF (top line) is from Pasek et al. (17). The position of the four 5'-proximal initiation sites (strong [>] and weak [>]) for translation and the numbering of codons in relation to gene s are shown in the second line. The second signal is not conserved in other HBV DNAs (6a, 29). Mapping of the two postulated mRNAs is explained in the text. The binding sites of N-linked mannose-rich glycans (Gm) and complex glycans (Gj) were mapped previously (24). The exact terminal map positions of P39/GP42 in the HBsAg particles or HBV are not yet known, but they are probably identical to or close to the proposed sites.

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HBV or of filaments, but not of HBsAg 20-nm particles. The binding of MA187 to native HBV or HBsAg in an enzyme immune assay shows that at least parts of the pre-s(1) sequence are located at the surface. The rapid inactivation of the MA18/7 epitope by trypsin is consistent with its exposed position, and it is also in agreement with the large number of basic amino acids in the pre-s(1) region. Previously, it was shown that the pre-s(2) region of GP33/GP36 is also at the outer side of HBsAg particles (24, 25), and the data on the albumin receptor coded by the pre-s(2) region confirm this observation (13a). As seen in the immune blots, both the epitope of MA18/7 and at least one major epitope of pre-s(2) are resistant to denaturation by heat, SDS, and DTT. These epitopes are apparently more immunogenic than are the epitopes of denatured P24. It is likely that the denaturation-resistent epitopes are defined by the primary sequence, irrespective of the protein conformation. This is in sharp contrast to the antigenicity of P24, which is highly dependent on the conformation specified by its synthesis in eucaryotic cells. In the search for immunogenic and protective poly- or oligopeptides as an alternate vaccine against HBV, sequences of the pre-s region may be of interest. Recently, Neurath et al. (16a) showed that the 26 amino-terminal amino acids in the pre-s(2) region act as a highly efficient immunogen. Testing cellular(5) and humoral (1) immunity in hepatitis B convalescents, it was noted that HBV had an antigenic component(s) which was absent or rare in HBsAg 20-nm particles. Most interesting, the immune reaction against the HBV-specific antigen was low or absent in viremic HBsAg carriers, but it was present

in nonviremic carriers. According to our results,

the pre-s(1) part of the large surface protein could be that antigenic component. ACKNOWLEDGMENTS We thank R. Thomssen and 0. Gotze for generous support, K. Lechte for technical asistance, and R. Stute for supplying HBVcontaining plasma. This work was partially supported by the Deutsche Forschungsgemeinschaft. LITERATURE CITED

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