THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1993 hy The American Society for Biochemistry and Molecular Biology, Inc Vol. 268, No. 2, Issue of January 15, pp. 1332-1337, 1993

Printed m U. S. A.

Structure of Hepatitis B Virus Core and e-Antigen A SINGLE PRECORE AMINO ACID PREVENTS NUCLEOCAPSID ASSEMBLY*

(Received for publication, August 20, 1992)

Florian Schiidel$#, Darrell PetersonB, Jian Zhenfl, Joyce E. Jones//, Janice L. Hughes11 , and David R. MilichII From the $Max-Planck-Institut fur Biochemie, 8033 Martinsried, Germany, the nDepartment of Biochemistry, Virginia Commonwealth University, Richmond, Virginia, 23298-0614 and the 1IDepartment of Molecular Biology, The Scripp.9 Research Institute, La Jolla, C a ~ i f o r n ~ 92037

The hepatitis B virus core gene codes for two poly- peptides: the core protein, which assembles to form particles (HBcAg), and the secreted precore protein (HBeAg). Expression vectors directing the synthesis in Escherichia coli of a recombinant HBeAg correspond- ing in sequence to serum-derived HBeAg encompassing the 10 precore amino acids remaining after cleavage of the precursor and residues 1-149 of HBcAg (PC- HBeAg) were constructed. Recombinant PC-HBeAg, HBcAg, and C-terminally truncated HBcAg were iso- lated from E. coli and analyzed by sucrose velocity sedimentation, electron microscopy, anti-HBc/e spe- cific monoclonal antibody analysis, and for immuno- genicity. HBcAg and truncated HBcAg formed 27-nm particles and displayed HBc antigenicity. In contrast, PC-HBeAg was nonparticulate and did not band in sucrose gradients. PC-HBeAg was recognized effi- ciently by HBeAg-specific antibodies and displayed little HBc antigenicity. Immunogenicity studies includ- ing T and B cell recognition confirmed that PC-HBeAg demonstrates HBe antigenicity. The presence of the 10 precore amino acids therefore prevented particle for- mation. To analyze which precore amino acids might be responsible for the prevention of particle formation a cysteine to glutamine substitution at amino acid po- sition -7 was introduced into PC-HBeAg (-7cdQ)PC- HBeAg. This single amino acid change at position -7 restored particle formation and HBc antigenicity. The evolutionarily conserved cysteine at position -7 thus appears responsible for the prevention of particle as- sembly in the HBeAg biosynthesis pathway.

Hepatitis B virus (HBV),’ a small enveloped DNA virus, is the prototype member of the family of hepadnaviruses (for a review see Ref. 1). The HBV core gene codes for two distinct protein products: a 21.5-kDa protein that assembles to form nucleocapsid particles designated HBcAg, which in mature virions contains the viral DNA as well as the viral polymerase

* This work was supported by grants of the Walter Schulz Stiftung and the Wilhelm Sander Stiftung (to F. S.) and by National Institutes of Health Grant AI20720 (to D. R. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adoertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed Dept. of Bacterial Diseases, Walter Reed Army Institute of Research, Washington, D. C. 20307-5100. Tel.: 202-576-3756; Fax: 202-576-07~8.

The abbreviations used are: HBV, hepatitis B virus; ER, endo- plasmic reticulum; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody; ELISA, enzyme-linked immunosorbent assay; MHC, major ist to compatibility complex.

and RNase H, and a precore protein, which is directed to the endoplasmic reticulum (ER), and is N- and C-terminally processed and secreted as non-particulate e-antigen (HBeAg). The precore protein has an extra 29 N-terminal amino acids, which in part serve as a signal peptide directing the nascent polypeptide chain into the secretory pathway (2-7). Mature HBeAg is C-terminally truncated at HBcAg amino acid po- sition 149 (8) and retains 10 precore amino acids (9). HBcAg and HBeAg are distinct~y recognized by antibo~es (lo) but are highly cross-reactive at the T cell level (11, 12).

The function of HBeAg is not completely clear. It is not a structural component of the virus, and viruses that lack the precore AUG are replication-com~etent and apart from the lack of HBeAg production do not appear to differ from wild- type hepadnaviruses (13, 14). However, the precore sequence and HBeAg secretion are conserved in all hepadnaviruses which argues for a function of the protein product. In this regard, secreted HBeAg may modify the host immune re- sponse. For example, exposure to HBeAg in utero establishes T cell tolerance to HBeAg and HBcAg in a murine transgenic model and may predispose neonates born to HBV-infected mothers to persistent infection by a similar mechanism (15).

HBcAg can be produced in recombinant Escherichia coli (16, 17) and even in this prokaryotic host assembles to form core particles indistinguishable from those found in virions. C-terminally truncated HBcAg preparations remain particu- late but expose HBeAg epitopes in addition to HBcAg epitopes (12). A number of other modifications to HBcAg including additions to the N or C terminus and internal insertions have been performed, and none of these modifications prevents particle assembly (18-21). In order to use truncated HBcAg as a ligand for anti-HBe antibody detection its core antige- nicity must be reduced by denaturation (12, 22, 23). In view of these observations, what prevents the assembly of precore proteins into particles in the lumen of the ER? The precore signal sequence directs the polypeptide to the ER where the concentration of HBeAg may be insufficient for particle as- sembly. However, this seems unlikely since HBeAg secreted under the control of the hemagglutinin signal sequence forms core particles (24). Alternatively, the residual 10 precore amino acids may preclude particle assembly. An E. coli- derived recombinant HBeAg with the amino acid composition of serum-derived HBeAg (ie . -10 to 149) has not been pre- viously described. The present paper describes the production and characterization of an E. coli-derived recombinant HBeAg containing the 10 precore amino acids present in the native HBeAg (PC-HBeAg). Analysis of PC-HBeAg revealed that the residual 10 precore amino acids do indeed prevent particle formation and furthermore that the cysteine at position -7 plays a critical role in preventing assembly.

1332

A Precore Cysteine Prevents Hepatitis B Capsid Assembly 1333

EXPERIMENTAL PROCEDURES

Mice-C57BL/10 (BlO), BlO.S, BlO.D’, BlO.M, BALB/c, and BALB/c athymic (nu/nu) mice were obtained from the breeding colony of the Scripps Research Institute. Female mice 6-8 weeks of age at the initiation of the experiments were used.

Construction of Expression Vectors for HBeAg-Manipulation of bacterial strains and plasmids followed routine protocols (25). Syn- thetic oligonucleotides and restriction enzyme recognition sites were calculated on a Vax computer (Digital) with University of Wisconsin Genetic Computer Group software (26). Peptide sequences were translated into the genetic code with the program “backtranslate” with codons used by highly expressed genes in E. coli (EcoHighCod) unless insertion of restriction sites required otherwise. A plasmid expressing the gene coding for HBeAg in E. coli was constructed in three steps. The vector pFS14 expressing native HBcAg under tac promoter control (27,28) was modified by polymerase chain reaction- assisted, oligonucleotide-directed mutagenesis as described (29) using the synthetic oligonucleotides: 5’-GGG CCA TGG ATA TCG ATC CTT ATA AAG AAT TTG GAG CTA CTG-3‘ and 5‘-GGG CCA T G G T T T T T T C C T C C T T A T G T G A A A T T G T T A T C C G C T CAC AATT-3’. The resulting vector pFS14nsd contains a synthetic Shine-Dalgarno sequence 8 bases from the HBcAg AUG and three unique restriction sites at the 5’-end of the HBcAg coding sequence (Fig. 1). As estimated on Coomassie Brilliant Blue-stained SDS- PAGE and by Western blotting pFS14nsd synthesizes a level of HBcAg 4-8-fold elevated in overnight cultures of E. coli JM105 without isopropyl-1-thio-0-D-galactopyranoside induction compared with the parent plasmid pFS14 (Fig. 2). Plasmid pFS14nsd was subsequently modified to include the gene coding for 10 HBV precore

pair of synthetic oligonucleotides (5’-CAT GTC CAA GCT TTG amino acids (subtype uyw) at the amine terminus by insertion of a

CCT GGG TTG GCT GTG GGG TAT GGA T-3’ and 5’-ATC CAT ACC CCA CAG CCA ACC CAG GCA AAG CTT GGA-3’) between the unique NcoI-EcoRV restriction sites. The resulting plasmid pFS14PC directs the synthesis of an e-reactive polypeptide with the expected retarded gel electrophoretic behavior in SDS-PAGE when expressed in JM105 (Fig. 2). Finally, the precore-core gene in pFS14PC was truncated after the triplet coding for HBcAg amino acid 149, again employing polymerase chain reaction-mediated oli-

oligonucleotides used were: 5’-GGG AAT TCC TGT TTT GGC GGA gonucleotide-directed mutagenesis to introduce a stop codon. The

TGA GAG AAG-3‘ and 5’-GGG AAT TCT AAA CAA CAG TAG TCT CCG GAA GTG-3’). The resultant vector pFS14e directs the synthesis of a 17.5-kDa e-reactive polypeptide when expressed in JM105 (Fig. 2). Oligonucleotide-directed mutagenesis as previously described was used to change the triplet coding for HBeAg cysteine -7 to one coding for glutamine. The coding region of all plasmids and the promoter region in pFSlNSD were verified by dideoxy DNA sequencing.

Western Blotting-Bacteria were grown overnight under aeration at 37 “C on a gyratory shaker. The bacteria were spun down and the bacterial pellet lysed by boiling in 2 X sample buffer (4% SDS, 125 mM Tris-HC1, pH 6.8, 10% 0-mercaptoethanol, 10% glycerol, and 0.002% bromphenol blue). The proteins were separated by 15% SDS- PAGE, electrophoretically transferred to nitrocellulose, incubated with HBeAg-specific monoclonal antibodies kindly provided by \’a- dim Bichko,’ followed by horseradish peroxidase-conjugated goat anti-mouse IgG (H+L) (Medac, Hamburg, Germany), and visualized by enhanced chemiluminescence (ECL, Amersham Corp.) on x-ray film (Kodak). The immunoblotting conditions and blocking reagents were previously described in more detail (29).

Protein Purification and Synthetic Peptides-Recombinant HBcAg and (-7C’Q)PC-HBeAg were purified as previously described (31). For the purification of PC-HBeAg, the protocol was modified in that a precipitation step with 10% polyethylene glycol (PEG 8000, Sigma) replaced the ammonium sulfate precipitation employed for the other proteins after lysis of E. coli. A HBcAg deletion mutant produced in E. coli lacking the C-terminal39 amino acids (17) was provided by S. Stahl (Biogen S.A., Geneva). The HBcAg mutant was produced as a high M, protein complex and was purified using a combination of differential centrifugation, size exclusion chromatography, and su- crose gradient centrifugation. The high M , form of HBeAg (S = 54) was dissociated to a low M, by denaturation with 5 M urea and

’ Bichko, V., Schodel, F., Nassai, M., Grene, E., Berzinsh, I., Borisova, G., Miska, S., Peterson, D. L., Gren, E., Pushko, P., and Will, H. (1993) Mol. Jmrnunol., in press.

titration to pH 9.6 followed by size exclusion chromatography on Sephacryl S200 at pH 9.6 in the absence of urea. The chromatography served to dissociate any undissociated protein. Because the low M , HBeAg (designated as HBeAgg.,) reversibly associated to the high M , form by back titration to neutral pH, HBeAg,., was maintained at pH 9.6 (12).

Synthetic peptides were synthesized by the Merrifield solid-phase method and were subjected to high pressure liquid chromatography on a C18 reverse phase column. All peptides used eluted as a single major peak (>go%). Synthetic peptides were produced in the pepti.de laboratory of R. W. Johnson Pharmaceutical Research Institute, La Jolla, CA, and were provided by B. Thornton. The HBeAglayw- derived peptides utilized were e73-87 (i.e. residues 73-87 of HBeAg), and the peptides derived from the precore (PC) sequence were pc15- 29, pc20-29, pc15-25, and pc17-27.

Antibody Reagents and AntigenlAntibody Assays-HBcAg-specific monoclonal antibodies (mAb) 3105 and 3120 and HBeAg-specific mAb 904 were kindly supplied by M. Mayumi (10). Polyclonai human anti-HBc/HBe and polyclonal human anti-HBc were obtained com- mercially (Abbott Laboratories, Abbott Park, IL). Rabbit anti-e73- 87 was produced by immunizing rabbits with keyhole limpet hemo- cyanin-coupled peptide. Antisera were collected and IgG fractions enriched by ion exchange chromatography (Bio-Rad Econo-Pac 10 DG) .

The recombinant antigen preparations were analyzed by liquid- phase, sandwich ELISA. The liquid phase, sandwich ELISA consisted of solid-phase HBcAg or HBeAg-specific mAb as the capture reagents and either polyclonal rabbit anti-peptide (e73-87) or polyclonal hu- man anti-HBc/HBe or human anti-HBc as the second antibody. Anti-HBc antibodies were measured in pooled murine sera by indirect solid-phase ELISA using solid-phase HBcAg (100 ng/well) and goat anti-mouse Ig as second antibody and were developed with a peroxi- dase-labeled, affinity-purified swine anti-goat Ig. The data are ex- pressed as antibody titers representing the highest dilution to yield 3 X the A492nm of preimmunization sera. Anti-HBe antibodies were measured in an identical manner on solid-phase HBeAg,., (100 ng/ well, coated at pH 9.6). Anti-peptide antibodies were measured on solid-phase peptides (500 ng/well). Mice were immunized for deter- mination of in vivo antibody production by intraperitoneal injection of 10 pg of recombinant proteins 3r 100 wg of synthetic peptides emulsified in complete Freund’s adjuvant. Sera were collected before and 10 and 24 days after primary immunization.

T Cell Proliferatiue Assay-Groups of four mice each were primed with either 10 pg of HBcAg or PC-HBeAg emulsified in complete Freund’s adjuvant by hind footpad injection. At 8 days after immu- nization draining popliteal lymph node cells were harvested, and 5 X lo5 cells in 0.1 ml of Click’s medium (32) were cultured with 0.1 ml of medium containing HBcAg, PC-HBcAg, various precore-derived synthetic peptides, or medium alone. Cells were cultured for 96 h at 37 “C in a humidified 5% CO, atmosphere. During the final 16 h, 1 pCi of [‘HJTdR (6.7 Ci/mmol; Du Pont-New England Nuclear) was added. The cells were then harvested onto filter strips for determi- nation of [3H]TdR incorporation. The data are expressed as counts/ min corrected for background proliferation in the absence of antigen ( A counts/min).

Velocity Sedimentation-Recombinant preparations were sedi- mented through 5 ml of 5-20% (w/v) sucrose gradients prepared in 10 mM Tris-HCI (pH 7.2 or 9.6). Samples were centrifuged for 45 min at 50,000 rpm in an SW 60 rotor (Sorvall) at 20 “C. Fractions were collected and analyzed for HBe and HBc antigenicity by liquid phase, sandwich ELISA.

RESULTS

Expression of Recombinant HBeAg

A vector expressing the HBcAg gene under tac promoter control (pFS14) was modified to introduce a synthetic Shine- Dalgarno sequence and unique restriction enzyme recognition sites at the 5’-end of the coding region (Fig. 1A). Based on this construct, a vector (pFS14PC) was constructed express- ing in E. coli the gene coding for HBcAg with the 10 N- terminal amino acids of the precore region that are present in mature serum-derived HBeAg. The expression product was recognized by HBeAg-specific monoclonal antibodies as a polypeptide with the expected molecular mass of approxi-

1334 A Precore Cysteine Prevents Hepatitis B Capsid Assembly

B!&LV.L Proteln D e S M a U m

1 183

pFS I 4 N S D HBCAg

10 1 I 8 3

DFS I 4 P C PC-HBCAg

-IO I 149

p r s I 4e PC-tlBeAg

- 1 9

pFS I4e-7 ( - 7 C+Q)PC-HBeAg

-7c-1.

FIG. 1. A , structure of the promoter region and 5'-end of the core in plasmid pFS14NSD. Unique restriction enzyme recognition sites are indicated above the DNA sequence. SD, Shine-Dalgarno sequence. The amino-terminal HBcAg amino acids are indicated in the one- letter code underneath the coding region. B, structure of HBcAg and HBeAg polypeptides. The expression vector names are indicated in the left panel; the protein designations in the right panel. Numbers above the boxes indicate amino acid positions (the HBcAg subtype uyw methionine is amino acid position 1; precore amino acids are indicated by negative numbers). Asterisk indicates exchange of cys- teine a t position -7 with a glutamine in mutant (-7-)PC-HBeAg.

A B 1 2 3 4 5 6 M M I

C 1 2 3 4 5 6 2 3 4

- 30

Fmlion

FIG. 3. Sucrose gradient analysis of recombinant HBcAg ( A ) , HBeAg0.e ( B ) , PC-HBeAg (C) , and (-7'-)PC-HBeAg ( D ) . HBc antigenicity (0) and HBe antigenicity (0) were determined by liquid phase sandwich ELISA using peroxidase-labeled human anti-HBc or anti-HBc/HBe, respectively, as the probes. Gradients were fractionated from the bottom (fraction 1).

PC-HBeAg (-7C-a)PC-HBeAg

FIG. 2. Expression of recombinant HBcAg and PC-HBeAg in E. coli. Whole cell bacterial extracts were separated on 15% SDS- PAGE and stained with Coomassie Brilliant Blue (A) or visualized by immunoblotting with monoclonal antibody 14E.11 ( B and C)? C is a longer exposure of a segment of B. 1, E. coli JM105; 2, JM105(pFS14); 3, JM105(pFS14NSD); 4, JM105(pFS14PC); 5, JMlOB(pFSl4e); 6, purified PC-HBeAg (1 pg).

mately 22.3 kDa in an immunoblot (Fig. 2). The vector was then further modified to terminate translation after the pre- C/HBc amino acid position 149 in vector pFS14e. This vector directed the synthesis of a polypeptide with HBe antigenicity migrating a t a position corresponding to the expected molec- ular mass of approximately 17 kDa in SDS-PAGE visualized by Coomassie Brilliant Blue staining and immunoblotting (Fig. 2). The product designated PC-HBeAg (see Fig. 1B) for the presence of the 10 additional precore amino acids found in serum-derived HBeAg was purified from recombinant E. coli and its amine terminus verified by partial Edman degra- dation and amino acid analysis. The sequence obtained was SKLCS as expected. When analyzed by sucrose gradient centrifugation, PC-HBeAg was found in the low molecular weight fractions, well separated from particulate HBcAg (Fig. 3, A and C), and sedimented similarly to HBeAg,, (Fig. 3B). In contrast to HBeAg9.6, PC-HBeAg sedimented a t either pH 7.2 or 9.6 yielded similar results, whereas HBeAgg,6 had to be maintained at pH 9.6 during sedimentation to prevent particle

HBcAg (upper left), HBeAgs.e (upper right), PC-HBeAg r.Ic. 4. Electron micrographs of recombinant proteins:

(lower left) , and (-7-)PC-HBeAg (lower right). Recombi- nant proteins (100 pg/ml) were applied to grids and fixed with 1.5% glutaraldehyde + 1.0% paraformaldehyde in cacodylate buffer, pH 7.4, for 10 min. The samples were then negatively stained with aqueous uranyl formate.

reassembly (12). Electron microscopy of PC-HBeAg revealed no particulate structures (Fig. 4). These data demonstrate that the N-terminal addition of the 10 residual precore amino acids to HBcAg prevents capsid assembly and the non-partic- ulate material expresses HBe antigenicity.

Mutation of a Cysteine at Position -7 Restores Particle Assembly

To determine the influence of the cysteine at position -7 on HBeAg and HBcAg folding and assembly we exchanged it for a glutamine. The purified polyprotein designated (-7- Q)PC-HBeAg sedimented in sucrose gradients similar to na- tive HBcAg, displayed HBcAg antigenicity (Fig. 30), and was particulate by electron microscopy (Fig. 4). Therefore, ex- change of a single amino acid in the precore region can restore particle formation, and it can be inferred that the precore cysteine a t position -7 in HBeAg is critical in the prevention of particle assembly.

A Precore Cysteine Prevents

Antigenicity of Recombinant PC-HBeAg as Determined by Monoclonal Analysis

Monoclonal antibodies specific for HBeAg (904) and HBcAg (3105, 3120) were used in quantitative liquid-phase sandwich ELISAs to analyze the antigenicity of HBcAg, PC- HBeAg, and (-7‘-)PC-HBeAg (Fig. 5 ) . While HBcAg was recognized by the HBcAg-specific monoclonal antibodies 3120 and 3105, PC-HBeAg showed little reactivity with 3120 and reduced binding to 3105 as compared with HBcAg. Mono- clonal antibody 3105, while reduced in titer, still recognized PC-HBeAg indicating that some HBcAg epitopes may be partially conserved on PC-HBeAg. Native HBcAg was only marginally recognized by the HBeAg-specific monoclonal an- tibody 904 a t high protein concentrations. In contrast, mono- clonal antibody 904 efficiently recognized PC-HBeAg similar to its reaction with serum-derived HBeAg. The exchange of a single cysteine a t position -7 in the precore portion of PC- HBeAg reduced binding of monoclonal antibody 904 to the low reactivity seen with HBcAg, while binding to the core- specific monoclonal antibodies 3120 and 3105 was restored. Therefore, exchange of the -7 cysteine results in a molecule with antigenic characteristics similar to native HBcAg.

Immunogenicity of PC-HBeAg In Vivo Antibody Production-In order to investigate the

immunogenicity of PC-HBeAg and the specificity of the an- tibodies elicited by immunization with PC-HBeAg, groups of BALB/c mice were injected with 10 pg of either HBcAg or PC-HBeAg. As shown in Table I, HBcAg immunization re- sulted in predominantly anti-HBc antibody production at day 10, and by day 24 antibodies specific for both HBcAg and HBeAg were present, although the anti-HBc response was predominate. In contrast, immunization of BALB/c mice with PC-HBeAg resulted in an antibody response predominated by anti-HBe antibodies. Note, however, that immunization with PC-HBeAg did elicit some anti-HBc reactivity, which may correspond to the partial preservation of the HBcAg epitope recognized by mAb 3105. Immunization with (-7‘‘ “)PC-HBeAg particles resulted in efficient HBcAg-specific antibody production and minimal anti-HBe antibody produc- tion, which confirms the HBcAg-like nature of this material. I n general, the immunogenicity data correlate with the mono- clonal analysis of PC-HBeAg and (-7‘-)PC-HBeAg.

HBcAg is a T cell-independent as well as a T cell-dependent antigen (33). To investigate whether PC-HBeAg could also stimulate antibody production in a T cell-independent man-

t.4, I (A) HBcAg , (E) PC-HBeAg 1 (C) (.7‘ “)PC-HBeAg

1 ’l! .O

0.4 I ,0003 .W8 0.2 5 0 ,0003 ,008 0.2 5.0 Mo3 ,008 0.2 5.0

Antigen (pgiml)

FIG. 5. Monoclonal antibody analysis. The three indicated re- combinant proteins were analyzed in a liquid phase sandwich ELISA. HBcAg-specific mAbs 3105 and 3120 and HBeAg-specific mAb 904 were used as solid-phase capture reagents. Varying concentrations of the recombinant proteins were added, and the second antibody was rabbit anti-e73-87, detected with a peroxidase-labeled goat a-rabbit Ig. Data are expressed as A 4 9 2 nm values corrected for background.

Hepatitis B Capsid Assembly 1335

TABLE I I n contrast to HBcAg, PC-HBeAg is strictly a T cell-dependent

antigen Groups of 4 BALB/c euthymic (+/+) or athymic (nu/nu) mice

were immunized with 10 pg of HBcAg, PC-HBeAg or (-7‘+9PC- HBeAg. Sera were collected before, and 10 and 24 days after immu- nization, and anti-HBc and anti-HBe antibody levels were deter- mined by solid-phase ELISA. The data are expressed as a reciprocal of the dilution required to yield 3X the A192nm value of preimmuniza- tion sera (l/titer).

Strain Antibody (l/titer)

HBcAg HBeAg9.6 Immunogen Sera

days BALB/c +/+ HBcAg 10

24 BALB/c +/+ PC-HBeAg 10

24 BALB/c +/+ (-7-)PC-HBeAg 10

24 BALB/c nu/nu HBcAg 10

24 BALB/c nu/nu PC-HBeAg 10

24 -

40,960 2,560 2.6 X lo6 163,840

640 10,240 40,960 327,680 40,960 2,560

2.6 X IO6 327,680 10,240 640 40,960 640

0 0 0 0

TABLE I1 Precore amino acids -20 to -29 do not appear to represent a B cell

recognition site Groups of four mice of the indicated strains were immunized with

PC-HBeAg (10 pg in CFA), and sera collected 24 days after the

by solid-phase ELISA. Additionally, BALB/c mice were immunized immunization were analyzed for reactivity with the panel of antigens

with a precore peptide representing residues 15-29 (100 pg in CFA). Data are expressed as the reciprocal of the dilution required to yield 3X the A492nm of preimmunization sera (l/titer).

Strain Immunogen Antibody (l/titer)

PC-HBeAn ~ 1 5 - 2 9 1x20-29 ec15-25 ec17-27

BALB/c PC-HBeAg 655,360 0 0 0 0 B10 PC-HBeAg 655,360 0 0 0 0 B1O.S PC-HBeAg 167,840 0 0 0 0 BALB/c pc15-29 0 163,840 2,560 163,840 640

ner we immunized athymic nude BALB/c mice with HBcAg and PC-HBeAg and analyzed serum antibodies specific for HBcAg and HBeAg. As shown in Table I, PC-HBeAg was unable to elicit antibody production in athymic BALB/c nu/ nu mice in contrast to HBcAg. Therefore, PC-HBeAg requires the presence of T cells for immunogenicity and is a strictly T cell-dependent antigen.

To investigate whether the 10 precore amino acids present in PC-HBeAg were immunogenic, we immunized BALB/c, B10, and B1O.S mice with PC-HBeAg or a synthetic peptide representing the 14 C-terminal precore residues (pc15-29). Immunization of mice with PC-HBeAg resulted in efficient antibody production to PC-HBeAg in all three strains, but no detectable peptide-specific antibodies against the precore re- gion were produced (Table 11). Note also that BALB/c anti- pc15-29 antibodies did not recognize the precore region on PC-HBeAg. However, the majority of antibodies generated against the precore peptide were directed against precore amino acids 15-25, with amino acids 15 and 16 appearing to be critical to antibody binding. Therefore, we cannot conclude from the anti-pc15-29 data that the 10 precore amino acids in PC-HBeAg (i.e. 20-29) are not surface accessible. However, antibodies raised against PC-HBeAg do not recognize any of the precore synthetic peptides suggesting that the precore sequence of PC-HBeAg, even if surface accessible, appears not to be immunogenic in the native protein at least in these three murine strains (Table 11).

1336 A Precore Cysteine Prevents Hepatitis B Capsid Assembly

T Cell Recognition-To investigate the immunogenicity of PC-HBeAg at the T cell level and to determine whether the precore sequence in PC-HBeAg contains a T cell recognition site, we immunized B10, BlO.S, B10.D2, and B1O.M H-2 congenic mice with PC-HBeAg and analyzed proliferative T cell responses in draining lymph nodes (Table 111). Immuni- zation with PC-HBeAg effectively primed T cells reactive with PC-HBeAg and cross-reactive with HBcAg. The PC- HBeAg-specific T cells did not proliferate in response to the precore peptides pc15-29 and pc20-29. Both the high degree of cross-reactivity with HBcAg, which lacks the precore amino acid sequence, and the lack of reactivity with precore synthetic peptides suggest that the precore sequence does not contain an epitope recognized by HBeAg-specific T helper cells at least for these four murine strains representing four MHC haplotypes.

DISCUSSION

In an attempt to determine the role, if any, of the residual C-terminal precore amino acids on HBeAg biosynthesis a recombinant HBeAg corresponding in sequence to serum- derived HBeAg encompassing the 10 precore amino acids plus residues 1-149 of HBcAg (i.e. PC-HBeAg) was produced in E. coli. The PC-HBeAg was purified and analyzed by sucrose velocity sedimentation, electron microscopy, monoclonal analysis, and for T and B cell immunogenicity. This analysis revealed that the inclusion of the 10 precore amino acids at the N terminus of HBeAg in recombinant PC-HBeAg pre- cluded spontaneous assembly into particulate structures and resulted in a molecule that exhibits HBe antigenicity and minimal HBc antigenicity.

Previously produced recombinant HBeAg consisting of C- terminally truncated HBcAg (i.e. residues 1-149) minus pre- core sequences have been shown to spontaneously assemble into particulate structures exhibiting HBc and HBe antige- nicity, and required denaturation to convert the particles into non-particulate HBeAg (12, 23, 34). Furthermore, a recom- binant HBcAg encompassing the complete precore sequence (i.e. 29 residues) produced in yeast was also shown to assemble into particles (35). However, the particles were less stable in high salt concentration than HBcAg particles without the precore extension. It, therefore, appears that it is not the precore signal sequence per se but rather the segment of the precore region remaining on mature HBeAg ( i e . -20 to -29) that prevents nucleocapsid assembly. Further evidence for the sequence-specific influence of precore residues -20 to -29 comes from the observation that a number of N-terminal

TABLE I11 Precore amino acids -20 to -29 do not appear to represent a T cell

recognition site Groups of four mice each were immunized with PC-HBeAg (10

pg). Draining popliteal lymph node cells were cultured with the indicated antigens, and specific proliferation minus the proliferation induced by media alone (A counts/min) is shown. Conc., concentra- tion.

Strain H-2 Conc. Antigens (A counts/min)

PC-HBeAg HBcAg Conc. pc15-29 pc20-29

i d m l d m 1 B10 b 1.0 120,203 50,123 50 0 0

0.04 31,448 20,489 2 0 0 BIO.S s 1.0 130,989 86,293 50 0 0

0.04 48,246 53,026 2 0 0 B10.D2 d 1.0 154,539 85,283 50 0 0

0.04 43,371 47,728 2 0 0 BIO.M f 1.0 141,520 53,985 50 0 0

0.04 30,343 32,126 2 0 0

extensions fused to HBcAg for vaccine purposes have not prevented particle assembly (18, 21).

It was therefore reasonable to assume that one or more of the 10 residual precore amino acids was specifically inhibiting particle formation. Recent studies have revealed that the cysteine disulfide bridges within HBcAg particles stabilize but are not essential for particle formation (31, 36). Was it pos- sible that the conserved cysteine at position -7 in the precore region of PC-HBeAg actually prevented capsid assembly? To address this question a glutamine was substituted for the cysteine at amino acid position -7 in PC-HBeAg (i.e. (-7& Q)PC-HBeAg). The exchange of this single amino acid was sufficient to restore particle formation and HBc antigenicity with the concurrent loss of HBe antigenicity at the level of antibody recognition. Therefore, the cysteine at position -7 is essential to prevent particle assembly probably by forming disulfide bridges. However, it should be noted that preliminary results suggest that (-7-)PC-HBeAg particles are less sta- ble than HBcAg particles at low pH (data not shown). This suggests that additional precore sequence-specific residues may interfere with assembly. A series of HBeAg cysteine mutants has been prepared (31) and can now be analyzed in the context of (-7-)PC-HBeAg to determine which of the three HBeAg cysteines, if any, interacts with the -7 precore cysteine to prevent particle assembly. In view of the fact that the presence of the entire 29-amino acid precore sequence does not prevent particle formation (35), the cysteine at position -7 may only prevent assembly in mature HBeAg, in which the other 19 precore amino acids including three addi- tional cysteines are proteolytically removed. Conceivably the -7 cysteine may not be available for disulfide bridging with a cysteine(s) within HBeAg in the context of the three other precore cysteines.

Because the cysteine at position -7 is conserved in all HBV genomes it is reasonable to assume that secretion of non- particulate HBeAg is advantageous for the virus. In view of the fact that HBeAg is not a structural component of the virus and is not required for infection or replication (13, 14), the function of HBeAg may involve modification of the host immune response. In the context of the hypothesis that in utero exposure to HBeAg establishes T cell tolerance to both HBeAg and HBcAg (see below), it is essential that HBeAg remain non-particulate in order to traverse the placenta. Because the gene encoding the e-antigen is also conserved in avian hepadnaviruses in which placental transfer is not rele- vant, secreted HBeAg may modify the host immune response in a number of ways in addition to the induction of T cell tolerance. For example, circulating HBeAg may directly dampen the T cell-mediated viral clearance mechanism by inducing suppressor T cell function or by cytotoxic T-lym- phocyte blocking CTL recognition by directly interacting with MHC class I molecules, although no such function has yet been identified. Alternatively, secreted HBeAg may prefer- entially activate a Th2-like response in some individuals. An HBeAg-specific response predominated by Th2 cells would result in antibody production and the concurrent down-regu- lation of Thl cells which mediate inflammatory responses. This response pattern may favor persistent infection. We have observed the preferential induction of HBeAg-specific Th2-like responses in the murine system and among human HBV chronic hepatitis patients!

Although the recombinant PC-HBeAg and HBcAg proteins are serologically distinct as are their native counterparts, T cells do not appear to distinguish between PC-HBeAg and

3T. Maruyama, A. McLachlan, S. Iino, K. Koike, K. Kurokawa, and D. R. Milich, submitted for publication.

A Precore Cysteine Prevents Hepatitis B Capsid Assembly 1337

HBcAg. PC-HBeAg-primed T cells from four murine H-2 congenic strains recognized PC-HBeAg and HBcAg equiva- lently and did not recognize synthetic peptides derived from the precore sequence. Previous studies have revealed signifi- cant cross-reactivity between HBeAg and HBcAg by murine and human T cells (11,30,33); however, these studies did not include an analysis of T cell recognition of the precore region. The absence of T cell recognition of the 10 residual precore residues in this preliminary murine study suggests the possi- bility that exclusive T cell recognition of HBeAg and not HBcAg may not occur at least to any significant degree. Exclusive T cell recognition of HBcAg via recognition of the C-terminal 35 residues absent from HBeAg remains possible and has been suggested for the H-2d murine haplotype (11). We were also unable to detect precore-specific antibody pro- duction among a panel of murine strains immunized with PC- HBeAg. Therefore, the residual 10 precore amino acids pres- ent on HBeAg appear not to be immunogenic at the antibody or helper T cell levels at least in the murine model. This does not rule out the possibility that the precore sequence may contain MHC class I-restricted T cell recognition sites. In- deed, it has recently been postulated that secretion signal peptide domains may frequently contain class I-restricted cytotoxic T-lymphocyte epitopes by virtue of their accessibil- ity to the class I presentation pathway without the require- ment for cytosolic proteolysis and subsequent transport to the ER (37).

The production of a recombinant HBeAg protein that does not self-assemble into particles exhibiting HBc as well as HBe antigenicity also has practical implications. Serum-derived HBeAg is heterogeneous in size and difficult to purify in sufficient quantities for use in diagnostic immunoassays. Therefore, C-terminally truncated and denatured recombi- nant HBcAg has been used as a surrogate HBeAg-specific ligand to measure anti-HBe antibody in serum samples (12, 23). The availability of a more native-like recombinant HBeAg encompassing the 10 precore amino acids at the N terminus and truncated at the HBcAg position 149 may represent a superior reagent for use in the analysis of immune responses to HBeAg. Furthermore, the ability to express PC- HBeAg in E. coli facilitates large scale production.

Acknowledgments-We thank Professors M. Mayumi and V. Bichko for monoclonal antibodies, Stephen Stahl for truncated HBCAg, Ursula Morgenroth for expert technical assistance, and Rene Lang for preparation of the manuscript.

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