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Originally published In Press as doi:10.1074/jbc.M501564200 on April 4, 2005

J. Biol. Chem., Vol. 280, Issue 23, 21713-21719, June 10, 2005
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Hepatitis B Virus DNA-negative Dane Particles Lack Core Protein but Contain a 22-kDa Precore Protein without C-terminal Arginine-rich Domain*

Tatsuji Kimura{ddagger}§, Nobuhiko Ohno¶, Nobuo Terada¶, Akinori Rokuhara||, Akihiro Matsumoto||, Shintaro Yagi{ddagger}, Eiji Tanaka||, Kendo Kiyosawa||, Shinichi Ohno¶, and Noboru Maki{ddagger}

From the {ddagger}Research and Development Division, Advanced Life Science Institute, Inc., Wako, Saitama 351-0112, Japan, the Department of Anatomy, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Tamaho-cho, Yamanashi 409-3898, Japan, and the ||Department of Internal Medicine, Shinshu University School of Medicine, Matsumoto, Nagano 390-8621, Japan

Received for publication, February 10, 2005 , and in revised form, March 31, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA-negative Dane particles have been observed in hepatitis B virus (HBV)-infected sera. The capsids of the empty particles are thought to be composed of core protein but have not been studied in detail. In the present study, the protein composition of the particles was examined using new enzyme immunoassays for the HBV core antigen (HBcAg) and for the HBV precore/core proteins (core-related antigens, HBcrAg). HBcrAg were abundant in fractions slightly less dense than HBcAg and HBV DNA. Three times more Dane-like particles were observed in the HBcrAg-rich fraction than in the HBV DNA-rich fraction by electron microscopy. Western blots and mass spectrometry identified the HBcrAg as a 22-kDa precore protein (p22cr) containing the uncleaved signal peptide and lacking the arginine-rich domain that is involved in binding the RNA pregenome or the DNA genome. In sera from 30 HBV-infected patients, HBcAg represented only a median 10.5% of the precore/core proteins in enveloped particles. These data suggest that most of the Dane particles lack viral DNA and core capsid but contain p22cr. This study provides a model for the formation of the DNA-negative Dane particles. The precore proteins, which lack the arginine-rich nucleotide-binding domain, form viral RNA/DNA-negative capsid-like particles and are enveloped and released as empty particles.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatitis B virus (HBV)1 infects more than 300 million people and is a major cause of liver diseases. The HBV belongs to the Hepadnavirus family and is a small (42 nm) enveloped DNA virus, which possesses a 27-nm icosahedral nucleocapsid composed of core protein and a 3.2-kb partially double-stranded, circular genome (1). Although the term "Dane particles" refers to the 42-nm HBV particles (2) and is often used in reference to the complete HBV particles, electron microscopic studies have suggested that the DNA-negative "empty" Dane particles are predominant in sera (3–6). The capsids of the empty particles are thought to be composed of core protein but have not been studied in detail.

The HBV genome encodes two core-related open reading frames, precore and core genes (Fig. 1). These are expressed because of two in-frame ATG initiation codons located at the 5' end of the genes. The first ATG encodes a 25-kDa protein (p25) containing the 29-amino acid (aa) precore sequence fused to the N terminus of the HBV core antigen (HBcAg). The p25 is directed toward the secretory pathway by a 19-aa signal sequence that is cleaved during translocation into the lumen of the endoplasmic reticulum (ER), producing a 22-kDa protein. Subsequent proteolytic cleavages within the arginine-rich C-terminal region (34 aa) generate a 17-kDa protein that is secreted as hepatitis B e antigen (HBeAg) (7–10). A heterogeneous population of these precore derivatives has been observed in the sera of patients and is serologically defined as HBeAg (9, 11, 12). Conversely, the second ATG specifies the 21.5-kDa HBcAg, which assembles into dimers that form the virus capsid (7, 9, 13–15). HBcAg is a 183-residue protein with two domains, the assembly domain that forms the capsid and the C-terminal arginine-rich domain that is responsible for RNA packaging (Fig. 1). The assembly domain, lacking the C-terminal domain, is sufficient for self-assembly into capsid particles. The arginine-rich C-terminal domain is involved in binding to the HBV RNA pregenome or the HBV DNA genome but is dispensable for HBV capsid assembly in Escherichia coli (16–19) and insect cells (20). The capsid is enclosed within an envelope containing the viral glycoprotein surface antigen (HBsAg) and released to the circulation as Dane particles.

We previously developed enzyme immunoassays (EIAs) for HBcAg (21) and HBV core-related antigens (HBcrAg) (22, 23). Serum specimens were pretreated with SDS to release and denature antigens and to inactivate antibodies. The HBcAg assay specifically measures core protein (21), and the HBcrAg assay measures precore/core proteins, including core protein and HBeAg (22, 23).

The present study investigated precore/core proteins in HBV-infected human sera using the new assays. The results suggest that most of the Dane particles were DNA-negative and were composed of a 22-kDa precore protein containing the uncleaved signal peptide and lacking the C-terminal arginine-rich domain. We present a new model for the formation of HBV DNA-negative particles.



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  		FIG. 1.
Schematics of precore/core genes and their products. At its 5' end, the precore/core gene contains two closely spaced ATGs (black dots) enclosing the precore region, which encodes a 29-aa precore sequence. Translation of the core mRNA results in the production of the cytoplasmic core protein, which assembles into icosahedral capsids enclosing the RNA pregenome, and then the nucleocapsids are enveloped and released as complete particles. Precore protein p25 is directed to the ER by a 19-aa-long signal sequence (black boxes) located at its N terminus. This signal sequence is removed during translocation into the ER, and then the C-terminal 34-aa-long arginine-rich domain (hatched boxes) is eliminated. Mature HBeAg is then secreted. p22cr is a novel precore protein identified with HBV DNA-negative particles.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Serum/Plasma Samples—Hepatitis B plasma panels were purchased from Boston Biomedica, Inc. (BBI, West Bridgewater, MA), or ProMedDx (Norton, MA). Clinical sera were collected between 1997 and 2001 at the Shinshu University Hospital (Matsumoto, Japan) from patients with persistent HBV infection. Thirteen of these serum samples containing ≥0.05 ng/ml HBcAg were immunoprecipitated to examine HBcAg/HBcrAg ratios. Of the 30 total serum samples (from 23 males and 7 females), 22 were HBeAg-positive, and 7 were HBeAb-positive. The remaining sample was positive for both HBeAg and HBeAb. None of the 30 patients was treated with anti-viral agents such as interferon or lamivudine. All sera were stored at –30 °C or below until testing. The study design conformed to the 1975 Declaration of Helsinki and was approved by the Ethics Committees of the institutions involved in this study. A written informed consent was obtained from each patient.

Recombinant HBV Core-related Antigens—Recombinant HBcAg (rH-BcAg, aa 1–183) and HBeAg (rHBeAg, aa –10 –149) were expressed and purified as described (21, 22). The concentration of these antigens was determined using the BCA protein assay kit (Pierce) and bovine serum albumin standards according to the manufacturer's instructions.

Monoclonal Antibodies and EIAs for HBcAg or HBcrAg—Anti-HB-cAg and anti-HBcrAg monoclonal antibodies were established as reported previously (21, 22). The HBcAg-specific monoclonal antibody, HB50, recognizes SPRRR repeats in the arginine-rich domain of HBcAg (21), whereas the anti-HBcrAg monoclonal antibody, HB91, recognizes aa 1–19 of HBcAg and thus reacts to denatured HBcAg, HBeAg, and other precore/core proteins (22).

HBcAg and HBcrAg were measured by EIA as described previously (21–23). The assays contain a sample pretreatment step that inactivates antibodies and dissociates antigens in samples. The assays can thus detect antigens within the viral envelope or complexed with antibodies in addition to free antigens.

HBV Markers and HBV DNA Measurement—HBeAg and HBsAg were measured by radioimmunoassay or by chemiluminescent immunoassay (Abbott, Tokyo), respectively. HBV DNA was detected by PCR using the Amplicor HBV monitor test (Roche Applied Science). Samples showing values over the detection range were remeasured after dilution to obtain quantitative results.

Sucrose Density Gradient Ultracentrifugation—Aliquots (1.7 ml) of 10, 20, 30, 40, 50, and 60% (w/w) sucrose in a solution containing 10 mM Tris-HCl, 150 mM NaCl, and 1 mM EDTA (pH 7.5) were carefully layered in a 12-ml Ultracentrifuge tube and left at room temperature for 6 h. HBeAg-positive plasma (0.1–1.0 ml) was layered on this sucrose gradient, and ultracentrifugation was performed at 200,000 x g for 15 h at 4 °C in a Beckman Sw40Ti rotor. Fractions were collected from the top to the bottom of the gradient. The density of each fraction was calculated from the weight and volume. Each fraction was diluted 10-fold and tested for HBcAg and HBcrAg as well as for HBsAg, HBeAg, and HBV DNA.

Immunoprecipitation—Immunoprecipitation was carried out using magnetic beads coated with polyclonal anti-HBsAg from the "HBV-Direct Mag kit" (JSR Corp., Tokyo) (24). A 200-µl aliquot of sample was mixed with 50 µl of reaction buffer from the kit and 50 µl of a magnetic bead suspension. The mixture was incubated for 30 min at room temperature with gentle agitation and then magnetically separated. HBcAg and HBcrAg in supernatant and precipitate were measured by EIA. Because some samples contain a large amount of HBsAg, which exceeds the capacity of anti-HBsAg beads, if the precipitated HBcAg ratio was less than 90%, the sample was diluted 10- or 100-fold and then reimmunoprecipitated.

Electron Microscopy—A 500-µl aliquot of HBV-positive plasma was subjected to ultracentrifugation on linear 10–50% (w/w) sucrose density gradients. The high density HBcrAg peak fractions (corresponding to Fig. 3A, fractions 23 and 24) and HBcAg peak fractions (corresponding to Fig. 3A, fractions 25 and 26) were separated by the second ultracentrifugation through linear 35–50% (w/w) sucrose density gradients. The fractions were fixed by adding paraformaldehyde solution to a final concentration of 4%. A 4-µl aliquot of each fraction was diluted in 90 µl of distilled water in 5-mm diameter polyallomer centrifugation tubes (Beckman Instruments), and copper grids filmed with Formvar membranes and treated additionally with poly-L-lysine were placed on the bottom of the tubes in the solution. Ultracentrifugation (200,000 x g, 4 °C, 2 h) was performed in a Beckman TLS-55 swinging bucket rotor to concentrate the virus particles and allow them to attach to the Formvar membranes on the copper grids. Afterward, the attached virus particles were negatively stained with 4% uranyl acetate and observed at an accelerating voltage of 80 kV in an electron microscope (H-7500, Hitachi, Tokyo). Fifteen electron micrographs of the virus particles from each fraction were taken randomly at a magnification of x80,000. The number of virus particles in the 3.76 µm2 area was then counted on each electron micrograph. The diameters of the virus particles in each fraction were also measured.

Western Blot Analysis—Samples were subjected to SDS-PAGE through a 15–25% polyacrylamide gel under reducing conditions. Proteins in the gel were electroblotted onto a polyvinylidene difluoride membrane (Immobilon-P, Millipore) at 15 V for 45 min. The membrane was blocked and probed using alkaline phosphatase-conjugated HB50 (for HBcAg) or HB91 (for HBcrAg) monoclonal antibody at room temperature for 1 h. The membrane was washed and incubated with 5-bro-mo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate solution (KPL, Gaithersburg, MD) for 15 min (for HBcrAg) or 90 min (for HBcAg), respectively.

N-terminal Amino Acid Sequence Analysis—A 6-ml aliquot of HBV-positive plasma was subjected to ultracentrifugation on linear 10–60% (w/w) sucrose density gradients, and subsequently the high density HBcrAg peak fractions (Fig. 3A, fractions 23 and 24) were separated by gel filtration through Superose 6 HR (Amersham Biosciences). Void fractions were collected and ultracentrifuged at 200,000 x g for 15 h at 4 °C using a Beckman SW 50.1 rotor. The precipitate was separated by SDS-PAGE and electroblotted onto a polyvinylidene difluoride membrane (Immobilon-P, Millipore) at 15 V for 45 min. Proteins on the membrane were stained using Coomassie Brilliant Blue-R250. The N-terminal amino acid sequence of the 22-kDa band was analyzed using the Procise 494 cLC protein sequencing system at the Apro Life Science Institute, Inc. (Tokushima, Japan).

Mass Spectrometry Analysis—The 22-kDa protein was purified as described above. The 22-kDa band was cut from the SDS-polyacrylamide gel and digested in-gel by trypsin at 35 °C for 20 h. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) of the digested sample was performed on a Voyager-DE STR (Applied Biosystems) in positive ion reflection mode. External mass calibration was performed using four points bracketing the mass range of interest. Results were analyzed using the NCBI non-redundant data base (molecular mass range 15–30 kDa) by the MS-Fit 3.1.1 ProteinProspector 3.2.1 program (University of California), taking into account probable post-translational modifications. LC-MS/MS was performed using a Q-Tof2 (Micromass, Manchester, UK) quadrupole time-of-flight electrospray ionization mass spectrometer in nanoflow LC ionization mode. The analyses were performed at the Apro Life Science Institute, Inc.



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  		FIG. 2.
Reactivity of the HBcAg assay and the HBcrAg assay. Recombinant HBcAg ({blacktriangleup}) and HBeAg ({square}) were diluted and tested for the HBcAg assay (A) and the HBcrAg assay (B). The assay reactivity is shown as log relative luminescence intensity (RLI).

 
Statistical Analyses—The virus particle numbers on each electron micrograph were statistically compared by Welch's t test. The diameters of the virus particles were statistically compared by Student's t test. Paired t tests were used to analyze differences between log concentrations of HBcrAg and those of HBcAg. Differences were considered significant at p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Specificity of HBcAg and HBcrAg EIAs—The specificity of the HBcAg and HBcrAg assays was confirmed by using rHB-cAg and rHBeAg. The HBcAg assay specifically reacted to rHBcAg but not to rHBeAg (Fig. 2A). The HBcrAg assay reacted equally to rHBcAg and rHBeAg (Fig. 2B).

Density Distribution of HBV Precore/Core Proteins—HBV DNA-positive plasma (ProMedDx 9990776, HBsAg-positive, HBeAg-positive, HBV DNA 9.1 log copies/ml) was subjected to ultracentrifugation through a 10–60% (w/w) sucrose density gradient. Fractions were tested for HBcAg, HBcrAg, HBsAg, HBeAg, and HBV DNA (Fig. 3A). HBcAg appeared in the high density fractions and peaked in the same fraction (fraction 25) as HBV DNA. HBsAg was distributed in fractions of lower density, and HBeAg was dispersed widely in fractions of much lower density. HBcrAg peaked in fraction 24, slightly lower in density than the HBV DNA and HBcAg peaks in addition to a peak corresponding to HBeAg at much lower density. The concentration of HBcrAg in fraction 24 was 13-fold higher than the concentration of HBcAg in fraction 25. The high density HBcrAg peak was therefore predominantly composed of precore proteins other than core protein.

High density HBcrAg fractions (Fig. 3A, fractions 23–26) were reanalyzed under gentler (30–50%) sucrose density gradient sedimentation (Fig. 3B). HBcrAg concentration peaked in lower density fractions than HBcAg and HBV DNA, indicating that high density HBcrAg clearly differs from HBcAg. HBsAg concentration exhibited a shoulder at the HBcrAg peak fraction.



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  		FIG. 3.
Sucrose gradient analysis of HBV-positive plasma. A, ProMedDx HBV plasma 9990776 was subjected to ultracentrifugation using a 10–60% (w/w) sucrose density gradient. B, fractions 23–26 were reanalyzed by ultracentrifugation in a 30–50% sucrose density gradient. Density of each fraction is shown as a broken line. Fractions were diluted 10-fold and tested for HBeAg ({square})(x10 signal/cutoff), HBsAg ({triangleup}) (x102 IU/ml) in A and (IU/ml) in B), HBcrAg ({blacksquare}) (ng/ml), HBcAg ({blacktriangleup}) (ng/ml), and HBV DNA ({circ}) (106 copies/ml).

 
Immunoprecipitation by Anti-HBsAg—Sucrose density fractions (Fig. 3A) were immunoprecipitated by the anti-envelope protein HBsAg. Most of the HBcAg (97.5, 97.8, 96.2, and 95.1% from fractions 23–26) was precipitated by anti-HBsAg. Although more than 94% (94.5, 94.1, and 94.3% from fractions 7, 10, 13) of low density HBcrAg was observed in the supernatant, more than 96% (96.2, 96.8, 96.9, and 96.5% from fractions 23–26) of high density HBcrAg was in the precipitate. These data suggest that similar to the core protein, the high density HBcrAg exists in enveloped particles.

Stability of HBcrAg Particles—The HBV core forms very stable capsid particles resistant to denaturing pH, temperature, or detergents (25). Particle fractions of HBV-positive plasma were treated with or without 3% Nonidet P-40 detergent at 37 °C for 30 min and then subjected to gel filtration through Superose 6 HR (exclusion limit = 4 x 107 Da). Fractions were tested for HBcrAg and HBcAg. Regardless of Nonidet P-40 treatment, HBcrAg and HBcAg appeared in the void fractions (Fig. 4), indicating that HBcrAg formed high molecular mass (> 107 Da) particles resistant to 3% Nonidet P-40 treatment, as did the HBcAg.



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  		FIG. 4.
Gel filtration analysis of the particle fractions. The particle fractions of a density gradient were treated without (A) or with 3% Nonidet P-40 detergent (B) and then subjected to gel filtration through Superose 6 HR. The elution from 6 to 18 ml were fractionated into 24 fractions and tested for HBcrAg ({blacksquare})(x102 pg/ml) and HBcAg ({blacktriangleup}) (x10 pg/ml).

 
Electron Microscopy—HBcAg and HBcrAg in plasma 9990776 were separated by sequential sucrose density ultracentrifugation. The resultant HBcrAg-rich fraction (fraction A) contained 6.06-fold more HBcrAg than the HBcAg-rich fraction (fraction B) but contained only 3 and 38% of the HBV DNA and HBcAg, respectively, found in fraction B (Table I). Virus particles in the two fractions were concentrated and attached to the copper grids by ultracentrifugation and then negatively stained and observed under the electron microscope. Although virus particles appearing similar to Dane particles were observed in fraction B, more such Dane-like particles were seen in fraction A (Fig. 5), which contained HBV DNA at only 3% of that in fraction B. Fraction A contained 17.9 ± 11.6/3.76 µm2 Dane-like particles, which was significantly more than in fraction B (5.6 ± 3.8/3.76 µm2) (n = 15, p < 0.001) (Table I). The Dane-like particles in fractions A and B were not morphologically distinguishable (Fig. 5) but were quite similar to those reported previously (2–4, 6). The mean diameters of the measured particles were 41.5 ± 2.2 nm in fraction A and 42.0 ± 2.2 nm in fraction B (Table I). The mean diameters were not significantly different from one another (n = 60, p = 0.27) and were similar to the sizes reported previously (2).


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  		TABLE I
HBcAg, HBcrAg, HBV DNA and Dane-like particles in fractions A and B

HBcAg and HBcrAg in plasma were separated by sequential sucrose density ultracentrifugation. The HBV-DNA, HBcAg, and HBcrAg concentrations in the resultant HBcrAg-rich fraction (fraction A) and HBcAg-rich fraction (fraction B) are shown. The numbers of virus particles in the 3.76 µm2 area were counted on each of 15 electron micrographs (Fig. 5).

 
Identification of Particle HBcrAg as a 22-kDa Precore Protein (p22cr) Lacking the C-terminal Domain—HBV DNA-positive plasma (BBI PHM935A-14) was subjected to a 10–60% sucrose density gradient and fractionated into 15 fractions. The fractions were then analyzed by Western blotting using monoclonal antibodies for HBcAg and HBcrAg (Fig. 6A). HBcAg was detected only in fraction 8 and the original plasma. Conversely, four bands were detected by anti-HBcrAg in plasma. HBeAg and two additional proteins, which were considered HBeAg precursors, were detected in low density fractions by anti-HBcrAg. A 22-kDa protein, which was termed p22cr, was also detected in fraction 8. To confirm whether p22cr was identical to HBcAg, the p22cr band was compared with the neighboring HBcAg band (Fig. 6B). The p22cr protein exhibited slightly higher molecular weight than HBcAg. A fainter HBcAg band was also detected by anti-HBcrAg. Because p22cr did not react with the HB50 anti-HBcAg antibody, SPRRR sequences (positioned at aa 155–174 as three repeats) were presumed absent. Furthermore, p22cr maintained its 22-kDa molecular mass without the N-glycosylation consensus site. These data suggest that p22cr contains a complete or nearly complete precore region, including the signal sequence.



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  		FIG. 5.
Electron micrographs of virus particles in the two density gradient fractions of HBV-positive plasma. The particles in equal volumes of each fraction (Table I) were collected on copper grids by ultracentrifugation and negatively stained and observed under an electron microscope. Dane-like particles can be seen on the electron micrographs of both fraction A (upper panel, arrows, and inset) and fraction B (lower panel, arrowheads, and inset). Bars: 100 nm.

 
The p22cr protein was purified, and the N-terminal amino acid sequence was analyzed. p22cr showed no significant amino acid signal (data not shown), suggesting that the N terminus of p22cr might be blocked.

We then applied mass spectrum analysis. Data from MALDI-TOF MS were analyzed by MS-Fit search using the NCBI non-redundant data base. The search selected 117 of 87,559 entries for the molecular mass range 15–30 kDa. The top 20 matches were all HBV core or precore proteins. Six of 50 input peptide masses matched five precore/core peptides (Table II) that spanned 40% (86 of 212 aa) of the sequence. The N-terminal precore tryptic peptide (peptide 1, aa –28 to aa –9) was found to be N-terminally acetylated and was, therefore, not directly accessible to Edman sequencing. p22cr lacked the first N-terminal methionine of the precore protein. Another peptide, peptide 5, was identified as a precore/core peptide comprising aa 128–150. LC-MS/MS analysis was also applied. Two peptide fractions corresponding to peptides 2 and 5 of Table II were recognized as HBV precore/core proteins. Thus, the p22cr protein was confirmed to be a precore protein from N-terminally acetylated aa –28 to at least aa 150.


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  		TABLE II
MALDI-TOF MS analysis of p22cr

The 22-kDa protein band was digested in-gel and analyzed by MALDI-TOF MS. The results were analyzed using the NCBI non-redundant data base, taking into account probable post-translational modifications. Five precore/core peptides matched to six of 50 input peptide masses are shown.

 



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  		FIG. 6.
Western blot of HBcrAg. HBV-positive plasma (BBI PHM935A-14) was subjected to ultracentrifugation through a 10–60% (w/w) sucrose density gradient and fractionated into 15 fractions. A, plasma and its sucrose density gradient fractions were analyzed by Western blotting using anti-HBcAg (HB50) or anti-HBcrAg (HB91) monoclonal antibodies. B, a membrane blot containing three lanes of fraction 8 was cut in half and probed by anti-HBcAg or anti-HBcrAg.

 
HBcAg and HBcrAg Levels in HBsAg-positive Particles from Chronic Hepatitis B Sera—The levels of precore/core proteins were investigated in HBV particles of chronic hepatitis B sera. Sera were immunoprecipitated by anti-HBsAg, and then levels of HBcAg and HBcrAg in the supernatant and precipitate were measured. More than 91% of the HBcAg was detected in precipitate fractions. HBcrAg in precipitate fractions included p22cr and HBcAg. In the precipitate fractions, HBcAg concentration ranged from 0.08 to 165 ng/ml, whereas HBcrAg ranged from 0.59 to 1,079 ng/ml (Fig. 7A). Log concentrations of HBcrAg were significantly higher than those of HBcAg (p < 0.001). HBcrAg predominated over HBcAg in precipitates from both HBeAg-positive and -negative sera. HBcAg represented only 3.1–37.4% (median 10.5%) of HBcrAg (Fig. 7B), indicating that the remaining p22cr was the dominant precore/core protein in HBsAg-positive particles. Similar results were also obtained from high density fractions of the sucrose gradient in six tested samples.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we demonstrated that HBV DNA-negative Dane particles are dominant in serum and are composed of a precore protein p22cr, which contains an uncleaved signal sequence and lacks a C-terminal arginine-rich domain. Early electron microscopic and radiolabeling studies have suggested that less than 10% of Dane particles include full cores with viral DNA (3–6). However, the particle formation mechanisms have not been thoroughly examined. Core protein lacking the arginine-rich C-terminal domain can still assemble into capsid particles but fails to bind nucleic acids (16). Our findings present a new model for the formation of DNA-negative particles. The precore proteins, which lack the nucleotide-binding domain, form viral DNA-negative capsid-like particles, and the particles are enveloped and released to blood circulation.

Our new assays for HBcAg and HBcrAg enabled us to study precore/core proteins in HBV particles. The assays include sample pretreatment with SDS, which releases core protein from the particles, inactivates antibodies, and denatures antigens. Thus the HBcAg assay is able to detect the core protein in virion (21), and the HBcrAg assay is able to detect free HBeAg, HBeAg-antibody complex, and precore/core proteins in particles (22, 23). Unexpectedly, the HBcrAg assay detected abundant high density protein in addition to HBeAg and HBcAg (Figs. 3 and 6). The protein formed Nonidet P-40-resistant particles (Fig. 4) that did not contain HBV DNA but were enveloped by HBsAg. The protein was detected together with HBV DNA-negative particles that were morphologically identical to the complete virion (Fig. 5). The unknown precore/core protein proved to be a 22-kDa precore protein species (p22cr) containing the uncleaved signal peptide (Table II) and lacking the C-terminal arginine-rich domain (Fig. 6). The HBcrAg particles appear at a slightly lower density than HBcAg or HBV DNA (Fig. 3), which is also consistent with the observation that HBcrAg particles lack high density DNA components. Collectively, these data strongly suggest that p22cr forms the core of HBV DNA-negative Dane particles.

Our findings indicate that p22cr particles are more abundant than HBcAg capsid in sera (Figs. 3, 5–7, and Table I). In chronic hepatitis B sera, HBcAg comprised only 10.5% of HBcrAg (containing p22cr and HBcAg) in HBsAg-positive particles (Fig. 7). In addition, electron microscopic study indicated that Dane-like particles were more abundant in the HBcrAg-rich fraction than in the HBcAg/HBV DNA-rich fraction (Fig. 5 and Table I). This coincides with the previously reported abundance of empty particles (3–6). Empty and complete Dane particles were differently stained with uranyl acetate (3, 4, 6), but we could not distinguish Dane particles containing HBV DNA from those not containing HBV DNA. This might be due to differences in fixation and/or the negative staining procedure. We used paraformaldehyde for fixation to avoid biohazards.

The present study demonstrated that p22cr is a precore protein from aa –28 to at least aa 150 (Table II). The assembly domains (residues 1 to 149) self-assemble into capsids (16–19). In addition, precore protein containing the assembly domain could form capsid-like particles (26–28), whereas precore proteins are secreted as soluble HBeAg (7–12, 29, 30). A precore protein similar to p22cr, but containing the first methionine, has been isolated as soluble HBeAg from pooled sera of HBV carriers (30). This could represent the soluble form of p22cr, which was secreted without processing of the signal peptide. Our findings indicate that the majority of p22cr exists in enveloped particles (Figs. 6 and 7).



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  		FIG. 7.
HBcAg and HBcrAg levels in HBsAg-positive particles of chronic hepatitis B serum. HBsAg-positive particles were immunoprecipitated by anti-HBsAg-coated magnetic beads from 30 samples of HBV-infected sera. Precipitated proteins were eluted by SDS solution. Levels of HBcrAg and HBcAg in precipitate were measured by EIA. A, data are presented as HBcAg and HBcrAg concentrations per ml of serum. •, HBeAg-positive; {circ}, HBeAg-negative sample. B, HBcAg percentage per HBcrAg in the precipitate. The box plots show the 10th, 25th, 50th, 75th, and 90th percentiles, and diamonds denote the outliers.

 
Mass spectrum study indicated that the protein band contained precore peptide (Table II). We believe that this peptide was not derived from minor precore protein contamination of the major core protein because: (a) core protein concentrations in the original plasma were much lower than concentrations of precore/core proteins; (b) the purified sample appeared as a single band on SDS-PAGE; (c) the 22-kDa protein band could not be sequenced by Edman degradation, indicating that the N-terminal end of the peptide was blocked; and (d) the antibody targeting the C-terminal SPRRR repeats did not react with the 22-kDa p22cr (Fig. 6), suggesting that p22cr possesses the nearly complete N-terminal precore sequence.

Although the median HBcAg to HBcrAg (HBcAg + p22cr) ratio of HBsAg-positive particles was 10.5%, the actual ratios ranged widely from 3.1 to 37.4% (Fig. 7B). Because precore protein expression is abolished by precore nonsense mutation (31), the precore mutation must influence the HBcAg/HBcrAg ratios. In addition, the particle HBcAg/HBcrAg ratios would depend on the amount of precore proteins that are secreted as HBeAg or form p22cr particles. The ratios of particle-forming p22cr to soluble HBeAg in serum ranged from ~10:1 to 1:100.2

The manner in which precore protein containing the signal peptide forms particles remains unclear, but inefficient translocation of the precore protein might lead to particle formation in the cytosol. As with most secreted proteins, translocation of the precore protein across the ER membrane is mediated by signal recognition particles (8). However, translocation of the precore proteins is inefficient (8, 32, 33). In Xenopus oocytes, precore protein (p25, aa –29 to +183) was produced but not translocated into the ER lumen without processing (33). If translated precore proteins were to evade translocation to the ER, disulfide bridges would not form in the reducing environment of the cytosol. An intramolecular disulfide bridge between Cys-7 and Cys-61 determines the structure of the HBeAg (34, 35). HBe protein without Cys-7 also assembles into particles (29, 34–36). Conversely, Cys residues are not essential for the assembly of viral core particles (37). We therefore hypothesize that precore proteins remaining in the cytosol, which do not form disulfide bridges between Cys-7 and Cys-61, cannot assume the HBeAg conformation but can assemble into capsid-like particles.

The mechanisms for cleaving the C-terminal domain are unclear. Maassen et al. (38) reported that an N-terminal fusion core protein (with foreign sequences comprising 14 aa) assembles into capsid-like particles, but the fusion is sensitive to proteolytic attack within the arginine-rich C terminus. The uncleaved precore region (aa –28 to –1) might thus promote cleavage of the C-terminal domain.

Based on numerous in vitro or animal studies (14–19, 27, 29, 35, 38), the HBV capsid is believed to be a construct of core protein alone. However, nonsecreted precore protein and core protein can assemble to form hybrid nucleocapsids (28). The p22cr displayed a shoulder in virion fractions from density gradients (Fig. 3B, fraction 26–27), and the concentration of p22cr protein greatly exceeded that of HBcAg. The nucleocapsid of complete HBV particles could therefore contain p22cr.

Although the functions of the DNA-negative particles are largely unknown, the particles have been suggested to play a role in the persistence of HBV infection (3, 5, 6). p22cr in the particles may be a disturbing antigen for the host reactions. Overexpression of the precore gene results in inhibition of HBV replication in culture cells or transgenic mice (28, 39). The p22cr might be a molecule that inhibits HBV replication in human hepatocytes during natural infection. Furthermore, the number of particles containing p22cr or the antibodies specific for p22cr could be clinical markers for hepatitis B.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Research and Development Division, Advanced Life Science Institute, Inc., 2-10-23 Maruyamadai, Wako, Saitama 351-0112, Japan. Tel.: 81-48-465-2761; Fax: 81-48-465-2765; E-mail: tkimura{at}alsi-i.co.jp.

1 The abbreviations used are: HBV, hepatitis B virus; HBcrAg, HBV core-related antigens; HBcAg, hepatitis B core antigen; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; EIA, enzyme immunoassay; aa, amino acid; ER, endoplasmic reticulum; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; HBeAb, hepatitis B e antibody; rHBcAg, recombinant HBcAg; rHBeAg, recombinant HBeAg; LC, liquid chromatography; MS/MS, tandem mass spectrometry. Back

2 T. Kimura, C. Ohue, A. Rokuhara, A. Matsumoto, E. Tanaka, K. Kiyosawa, and N. Maki, unpublished data. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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