INTRODUCTION

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Hepatitis B virus (HBV), the causative agent of acute and chronic B-type hepatitis in humans (for a review, see reference 5), is a small enveloped DNA virus that replicates via reverse transcription of an RNA intermediate (for reviews, see references 24 and 25). Its 3.2-kb genome encodes a reverse transcriptase (P), a transactivator (X), three envelope proteins, and a single capsid, or core protein (C) of 183 amino acids (aa); a secreted, processed, and nonparticulate form of the protein is known as HBeAg. Authentic nucleocapsids are generated, in a highly specific reaction, by assembly of the capsid protein around a complex of P protein bound to a structured RNA element on one of the viral transcripts; subsequent conversion of the RNA into DNA yields mature core particles that acquire their outer envelope during export from the cell (for a review, see reference 26).

Heterologously expressed core protein, in the absence of further viral products, still forms particles resembling authentic liver-derived HBV capsids (10, 18). C-terminal truncations up to aa 144 or 140 rendered the protein assembly competent, while no particles were detected with the further truncated and poorly expressed variants ending with aa 138 or 139 (4, 43). These results defined the first 140 aa as the assembly domain of the protein (Fig. 1). Functional studies in the context of the complete viral genome demonstrated that the highly basic, R-rich C terminus, starting at aa 150, acts as a nucleic acid binding domain that is required for RNA encapsidation and proper reverse transcription (17, 22). Further genetic and biochemical analyses showed that the protein forms dimers that can be disulfide linked via the C-61 residues in the two monomers (23, 40) and that dimers are the only detectable assembly intermediates (41). While full-length capsids are extraordinarily stable, probably owing to the additional interactions between the basic C terminus and (nonspecifically) encapsidated RNA (4), capsids from truncated variants can be dissociated into dimers that spontaneously reassemble in vitro (39).

View larger version (24K): [in this window] [in a new window]   FIG. 1.   Structural organization of the HBV core protein. (A) Functional domains. The bar represents the primary sequence. The assembly domain is located in the first 144 aa and is followed by a nucleic acid binding region containing four clusters of R residues (indicated by +). The schematic representations of the core protein dimer shown below are based on cryo-electron microscopic reconstructions of capsids. The dimer interface forms the spikes visible on the capsid surface, and the R-rich C termini face its interior. (B) Architecture of the T=4 HBV capsid. A total of 120 dimers are arranged on an icosahedrally symmetric surface lattice; two-, three- and fivefold symmetry axes are indicated (adapted from reference 18). (C) Schematic representation of the arrangement of dimers around a local sixfold (strict twofold) axis of symmetry. The view is the same as in panel B.

After mapping studies of antibody-binding (27) and protease-sensitive (reviewed in reference 26) sites, more detailed structural information came from cryoelectron microscopic analyses of capsids (12, 18, 43). They showed two classes of icosahedrally symmetric particles, containing 90 (triangulation number T=3) and 120 (T=4) hammerhead-shaped, dyad-symmetry related dimers (Fig. 1A). Each dimer forms a prominent spike on the particle surface (Fig. 1B). Very recent data at resolutions below 10 Å revealed that the dimer interface is formed by a four-helix bundle (6, 11), in accord with a theoretical predicition (7). From these data, Böttcher et al. (6) proposed a largely -helical model for the fold of the core protein, including a tentative main chain trace, that is compatible with the available biochemical data.

In this study, we used a combination of interaction screening techniques and functional analyses of mutant core proteins to identify, at the primary sequence level, the regions in the protein that are involved in mediating the contacts between subunits; the rationale was that each monomer must have at least two interfaces, one mediating the dimerization of monomers and the other mediating the multimerization of dimers. The screening data revealed two prominent interaction regions, one between aa 78 to 117 (region I) and the other between aa 113 to 143 (region II). To functionally test the relevance of these regions, a series of core protein variants, designed according to the interaction data, were expressed in Escherichia coli and assayed for assembly competence. Surprisingly, mutations in region I had no detectable effect, although from the electron microscopy-based model (6), region I would overlap with two of the helices forming the four-helix bundle in the dimer. By contrast, several mutations in region II markedly affected particle stability, strongly suggesting region II to be one of the elements, if not the major element, in multimerization of the dimers. Since the dimer contacts are clearly revealed at the fivefold and quasi-sixfold vertices in the electron microscopy-based reconstructions, primary sequences between aa 113 and 143 are located at each of the two basal tips of the dimer.

	MATERIALS AND METHODS

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Plasmids. Plasmids pGAD-HBc1/183 and pGBT-HBc1/183 contain a synthetic full-length HBV C gene from plasmid pPLC4-1 (21) cloned into the polylinker sequences of pGBT9 and pGAD424 (Clontech, Heidelberg, Germany); these, as well as the corresponding HBV X gene control constructs, were kindly provided by L. Runkel.

Bacterial and yeast strains. For plasmid preparations, E. coli Top10 (Invitrogen, NV Leek, The Netherlands) was used. Expression experiments were performed either by thermoinduction (42°C) of appropriately transformed E. coli NF1 (29) cells (this strain carries an integrated copy of the temperature-sensitive lambda cI857 repressor) or by tryptophan induction of the corresponding GI724 or GI698 (Invitrogen) cells (in these strains, synthesis of the lambda cI repressor is suppressed by the addition of tryptophan); transformation and protein induction were performed as specified by the manufacturer recommendations; usually, the cells were grown to an optical density of 0.5 (600 nm) and induced for 3 to 4 h at 32°C; the P138G variant could be expressed in soluble form only at 23°C.

Protein purification. Protein c1-149 capsids were purified as previously described (4). Briefly, bacterial cells from 1 liter of induced culture were lysed by sonication and core protein was precipitated with 40% saturated ammonium sulfate. The precipitate was resuspended in phosphate-buffered saline (PBS) (140 mM NaCl, 2 mM NaH₂PO₄, 8 mM Na₂HPO₄ [pH 7.4]) and subjected to sedimentation in 10 to 60% (wt/vol) sucrose gradients in PBS (SW-40 rotor; 30,000 rpm for 15 h at 4°C). Fourteen 0.9-ml fractions were collected, and the presence of core protein was assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (0.1% SDS, 15% polyacrylamide) in the Laemmli system (19) with Coomassie blue staining. The same procedure was used for variants mutated at aa T91, K96, and P138.

Small-scale sucrose gradients. Cells from 30-ml cultures were collected by low-speed centrifugation, and the pellets were sonicated in 0.3 ml of PBS. The lysates were cleared by centrifugation, and 0.2 ml of the soluble fraction was loaded onto 10 to 60% (wt/wt) sucrose gradients (six steps of 0.2 ml each) in PBS (TLS55 rotor; 55,000 rpm for 40 min at 20°C), essentially as previously described (42). Fourteen fractions of 0.1 ml were collected from the top, and 10 to 20 µl of each fraction was monitored for core proteins by SDS-PAGE as described above or by Western blotting. Dimeric core protein, together with the bulk of E. coli proteins, was typically present in fractions 1 to 3, and capsids were present in fractions 6 to 8. For protein P138G, the sedimentation analysis was repeated on gradients containing TAE buffer (40 mM Tris-acetic acid [pH 8.1], 0.1 mM EDTA). The same gradient system was also used for analytical purposes.

Agarose gel electrophoresis of core particles. Agarose gel assays were performed as previously described (4). In brief, fractions containing between 1 and 10 µg of purified or enriched core protein were loaded on 1% (wt/vol) agarose (SeaKem; FMC Bioproducts, Natick, Maine) gels in TAE buffer and electrophoresed for about 3 h at 10 V/cm. In some experiments, encapsidated RNA was visualized by staining with 0.5 µg of ethidium bromide per ml and illumination at 302 nm. Proteins were stained with Coomassie blue. Core protein-specific bands were detected, after blotting the contents of the gel by capillary transfer in 20× SSC (0.3 M sodium citrate [pH 7.0], 3 M NaCl) to a nylon membrane (Porablot NY; Macherey-Nagel, Düren, Germany), with the monoclonal antibodies mc275 and mc312 (31, 32). Differential capsid stabilities were assayed by including 1 M urea in the gel.

Pepscan analysis. The pepscan filters (Jerini Bio Tools, Berlin, Germany) contained all possible 15-mer peptides spanning the core protein sequence from positions 1 to 161, each shifted by 2 positions. Pretreatment of the filter, incubation with core protein, and detection were performed essentially as recommended by the supplier. In brief, after blocking with 5% nonfat milk powder in Tris-buffered saline (TBS) (100 mM Tris-HCl [pH 8.0], 150 mM NaCl) containing 5% sucrose, the filter was incubated at 4°C overnight with the respective core protein (about 30 µg in 10 ml of the same buffer without milk powder). After two washes with TBS, four sequential electrophoretic transfers of the protein to a polyvinylidene difluoride (PVDF) membrane (Millipore, Eschborn, Germany) were performed. Core protein on the membranes was detected immunologically with the enhanced chemiluminescence system (Amersham, Braunschweig, Germany).

Dissociation and reassociation of capsids. Dissociation of gradient-purified capsids was performed by overnight dialysis against 25 mM sodium phosphate (pH 8.0) containing 2 M urea. Dimer formation was monitored by using analytical sucrose gradients and size exclusion chromatography in PBS as described above. For reassociation, isolated dimers, stored in phosphate buffer containing 2 M urea, were dialyzed overnight at 37°C against 25 mM sodium phosphate (pH 7.0) containing 100 or 500 mM NaCl.

Immunological techniques. For Western blotting experiments, the core proteins present on polyacrylamide or agarose gels were transferred to PVDF or nylon membranes and analyzed with monoclonal antibodies mc312, recognizing aa 76 to 84 as a linear epitope (31, 32), and mc275, recognizing only particulate core protein (32). Both antibodies were kindly provided as horseradish peroxidase conjugates by Behringwerke (Marburg, Germany). Detection was performed with the enhanced chemiluminescence substrate.

	RESULTS

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A two-hybrid-system screen suggests the presence of a central (region I) and an N-proximal (region III) interaction site in the HBV core protein. The yeast two-hybrid system (14, 20, 38) is an increasingly popular tool for identifying protein-protein interactions. Here we used a fusion of the complete HBV core gene with the Gal4 DNA binding domain (encoded by plasmid pGBT/c1-183) to screen a series of constructs encoding N-terminal and internal fragments of the core gene fused to the Gal4 activation domain (pGAD derivatives) (Fig. 2). An interaction between the fusion proteins should allow yeast cells containing both plasmids to grow on His medium and to produce -gal, giving rise to blue colonies on X-Gal-containing medium. As a positive control, a combination of plasmids pGBT/c1-183 and pGAD/c1-183, both containing the complete core gene, was used and produced the expected phenotypes. Individually or in combination with the respective HBV X gene plasmids, both constructs were negative in both assays, confirming the specificity of the test system. Of the constructs encoding C-terminally truncated core protein fusions, all except c1-81 scored positive in the -gal and His growth test in Hf7 cells (Fig. 2), although differences in plating efficiency that probably relate to the strength of the interactions were seen (16). The smallest fragment contains residues 1 to 36, suggesting the presence of an interaction site(s) in the N-terminal region (region III). In SFY526 cells, this construct did not give rise to blue colonies on X-Gal plates. By contrast, plasmid pGAD/c1-117, like pGAD/c1-183 and pGAD/c1-149, was clearly also positive in the SFY526 strain, suggesting the presence of one or more additional stronger interaction sites.

View larger version (30K): [in this window] [in a new window]   FIG. 2.   Homologous interactions detected with the yeast two-hybrid system. (Left) Overview of the pGAD/core fusion constructs. The bars below the schematic representation of the core protein indicate the core protein fragments present in individual constructs with respect to the primary sequence (central line). The short thick lines at the ends symbolize the presence of two to four vector-derived amino acids. Shaded areas correspond to the detected interaction regions I and III. (Right) Interaction data obtained with double transformants containing pGBT/c1-183 and the corresponding pGAD construct. The numbers indicate the first and last core-specific amino acids present in each construct. All constructs were tested for growth on His medium and for -gal activity in HF7c cells; selected constructs were also tested for -Gal activity in SFY526 cells. nd, not determined.

The pepscan technique confirms region I as a strong interaction site and suggests the presence of an additional C-proximal interacting region (region II). In the pepscan technique, originally developed by Geysen et al. (15), the sequence of a protein is represented as a series of relatively short overlapping peptides that are immobilized on a solid support, e.g., cellulose filters (for a review, see reference 28). The filter is then probed with the protein of interest. Detection of the protein on the filter, directly or after transfer to a second membrane, identifies interacting peptides.

View larger version (43K): [in this window] [in a new window]   FIG. 3.   Pepscan analysis of homologous interactions. (A) Dimeric protein c1-149 as a probe. The core protein sequence from aa 1 to 161 was represented on the filter by 15-mer peptides, each shifted by two positions. The sequences of the first and last peptide in each row are given on the left and the right, respectively. Protein bound to individual spots on the filter was electrophoretically transferred to a PVDF membrane and detected immunologically. Three N-proximal peptides that might correspond to region III in the two-hybrid screen are marked by asterisks. The strongly interacting regions I and II, with subregions a and b, are boxed. (B) Core protein c1-124 as a probe. The pepscan filter was treated as in panel A, except that the assembly-deficient variant c1-124 was used as the probe. (C) Correlation of regions I and II with the primary sequence of the core protein. The indicated borders of complete regions I and II and their subregions a and b correspond to the first amino acid of the first and the last amino acid of the last interacting peptide. Residues that are common to all peptides of a subregion are shaded in the primary sequence.

Analysis of the assembly competence of core protein variants mutated in regions I and II. If the above-defined candidate regions were indeed important for assembly, changes in their primary sequences would be expected to influence capsid formation and/or stability. We therefore expressed, in E. coli, several core protein variants mutated in the respective regions and characterized their quaternary structure by sedimentation in sucrose gradients, by electrophoresis on native agarose gels, and by gel filtration.

View larger version (32K): [in this window] [in a new window]   FIG. 4.   Quaternary structure of core protein c1-124. (A) Sedimentation analysis. E. coli-derived protein c1-124 (upper panel) was sedimented on an analytical sucrose gradient. Aliquots from each fraction were analyzed by SDS-PAGE and staining with Coomassie blue. Essentially all of the protein was present in the four top fractions (arrow), cosedimenting with the lysozyme (L) used during preparation of the cell lysate. The molecular masses of the marker proteins (lane M) are indicated on the right. Protein c1-149 was analyzed in parallel (lower panel) and was present mainly in fractions 6 to 8. (B) Size exclusion chromatography. Bacterial lysates containing protein c1-124 or c1-149 were analyzed on a Superdex 75 column equilibrated in PBS. The elution profiles and the positions of the two proteins as determined by SDS-PAGE are indicated. The dashed line shows the elution profile for a set of protein markers with the indicated molecular masses. AU280 nm, absorbance units at 280 nm.

View larger version (26K): [in this window] [in a new window]   FIG. 5.   Point mutations in regions I and II. In the context of core protein c1-149, T91 and K96 in region I and R127 and P138 in region II were replaced by the indicated amino acids. P138 is located in a P-rich motif; P residues are underlined.

View larger version (62K): [in this window] [in a new window]   FIG. 6.   Agarose gel electrophoresis assay of region I and II mutants. Capsid fractions from small-scale sucrose gradients of the indicated core proteins, all in the context of c1-149, were subjected to electrophoresis in agarose gels, in either the absence (left) or the presence (right) of 1 M urea. The amino acids present at positions 91, 96, and 127 in the wild-type protein and the individual variants are indicated at the top. The upper panels show Coomassie blue-stained gels; the lower panels show immunoblots with monoclonal antibodies mc275 and mc312. Only the sharp-edged bands visible in the Coomassie blue stain correspond to particles. Note that variant R127L formed particles only in the absence of urea (arrow), while no particles at all could be detected for variant R127Q.

View larger version (53K): [in this window] [in a new window]   FIG. 7.   Sedimentation analysis of R127 variants. (A) Analytical sucrose gradients of crude E. coli lysates. Aliquots of crude lysates from bacteria transformed with the appropriate expression plasmids encoding proteins c1-149 and its R127 variants were subjected to sedimentation in 10 to 60% sucrose gradients without prior ammonium sulfate precipitation. Aliquots of each fraction were analyzed by SDS-PAGE and Coomassie blue staining. R127L sedimented essentially to the same central fractions as did wild-type protein c1-149, while most of protein R127Q was present in the top fractions. (B) Reassociation of isolated dimers. Variants R127L and R127Q, purified as dimers by size exclusion chromatography, were subjected to reassociation conditions, in parallel with isolated c1-149 dimers (see Materials and Methods for details), and the reaction products were analyzed on analytical sucrose gradients. These conditions led to efficient reassociation of the wild-type protein (top), while only small amounts of the variant proteins were detected in particle-specific fractions.

View larger version (57K): [in this window] [in a new window]   FIG. 8.   Particle formation by variant P138G. (A) Sedimentation analysis. E. coli-derived variant P138G particles were sedimented, in parallel with protein c1-149, on 10 to 60% sucrose gradients after overnight incubation at room temperature (left) or at 42°C (right). Proteins in individual fractions were detected by SDS-PAGE and Coomassie blue staining. The molecular masses of the marker proteins (lanes M) are indicated in the middle; L, lysozyme. Note that both core proteins are present mainly in the central fractions while most of the contaminating E. coli proteins formed fast-sedimenting material (fraction 11) at 42°C. (B) Agarose gel assay. Purified particles from protein c1-149 and its variant P138G, as well as the assembly-deficient variant c1-124, were subjected to electrophoresis in agarose gels containing no urea or 1 M urea (see the legend to Fig. 6). The proteins were stained with Coomassie blue. While c1-149 particles remained stable at 1 M urea (white arrows), no particle-specific bands could be detected for the variants.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

Until recently, direct structural information about the HBV core protein remained scarce and theoretical predictions yielded controversial results (1, 7). Here we used a combination of screening procedures to experimentally define primary sequences within the protein that are involved in mediating the contacts between the 240 or 180 subunits forming the complete shell of the HBV capsid. Below we discuss the results in the context of the structural model proposed for the core protein by very recent high-resolution electron microscopy-based reconstructions (6, 11). Our data provide experimental evidence that the electron density visible at the fivefold and local sixfold symmetry axes between the core protein dimers in the capsid is provided mainly by residues from the sequence 113 to 143, i.e., region II, in accord with proposed model. The data for residues 83 to 115, i.e., region I from the two-hybrid and the pepscan screening, however, are not immediately understood in terms of the model.

Comparison of the two-hybrid system and the pepscan results. Since the HBV capsid is assembled from multiple core protein dimers, one subunit must have at least one interface mediating the dimerization of monomers and one mediating the multimerization of dimers. Both the two-hybrid system (14, 20, 38) and the pepscan technique (28) appeared able to define specific sequences involved in these interactions. In the two-hybrid screen, the interaction between full-length core protein fused to the GAD and GBT domains was clearly detectable in both the His growth assay and the -Gal assay. Within a series of terminal deletion constructs (Fig. 2), c78-117 was the smallest core protein fragment that gave the same phenotype, suggesting the presence of a strong interaction site in this region (region I). These results are compatible with the electron microscopy-based (6) as well as the theoretical (7) structural models, since region I overlaps largely with residues predicted to be part of the central helices at the dimer interface. Evidence for an additional interaction site (region III) within the first 36 aa was provided by fragment c1-36, which scored positive in both tests in HF7c cells but displayed a decreased plating efficiency on His medium and no detectable -Gal activity in the SFY526 strain, suggesting a weaker interaction (13, 14). That N-terminal residues are important for capsid formation is supported by the known detrimental effect of even short N-terminal deletions or insertions on assembly (3, 37).

Functional analysis of the relevance of regions I and II in HBV capsid assembly. As surprising as the reactivity of region I peptides with dimeric c1-149 protein was the lack of a detectable assembly defect of region I variants at T91 and/or K96 when analyzed in the context of c1-149. The proteins formed particles in E. coli, and these capsids were stable in the agarose gel assay even in the presence of 1 M urea. Unless one doubts the electron microscopy-based assignment of the corresponding residues to the dimer interface (6) this suggests that the mutations were not radical enough to significantly perturb the monomer-monomer interaction. In keeping with such a very robust nature of the dimer interface, which is also predicted by the four-helix-bundle model, is our observation that mutants with C61 replaced by W or R form stable particles (data not shown) although C61 is clearly located in the dimer interface, since it readily forms a homologous disulfide bridge with the same residue in a second subunit (23, 40). While these questions will probably be resolved only by direct higher-resolution structural analyses, a coherent picture for the role of region II emerged from our data.

View larger version (21K): [in this window] [in a new window]   FIG. 9.   Location of regions I and II in the core protein dimer. (A) Top and side view of the dimer. Region II is represented as a dark-shaded area at each of the two basal tips of the dimer. Region I most probably corresponds to residues forming the dimer interface. (B) Arrangement of dimers around a local sixfold axis. According to the mapping of region II (A), the contacts between individual dimers rely mainly on residues located between positions 113 and 143, forming a network around the local sixfold axes. Only one of the two contact sites in each dimer is shown. (C) Arrangement of dimers around a fivefold axis. Based on its icosahedral symmetry, the T=4 capsid must contain 12 pentameric arrangements of dimers. The contacts mediated by region II are similar to those at the local sixfold axes but not identical, implying a certain structural flexibility.