Institute of Microbiology and Immunology, School of Life Science, National Yang-Ming University, Taipei, Taiwan 112, Republic of China 1
Author for correspondence: Szecheng J. Lo.Fax +886 2 2821 2880. e-mail losj{at}ym.edu.tw
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Abstract |
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Introduction |
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A model of the three-dimensional structure of HBV nucleocapsid has been revealed to a resolution of 35·07·4 by cryoelectron microscopy (Crowther et al., 1994
; Conway et al., 1997
, 1998a
; Zlotnick et al., 1996
, 1997
; Bottcher et al., 1997
, 1998
). The proposed models agree that three regions of core protein, aa 7882, 127130 and 145153, are exposed on the shell of nucleocapsids. Neither the N nor the C terminus of the core protein is on the external surface. The N-terminal portion (aa 1150) of core protein is capable of self- assembling into nucleocapsids even in the absence of the C terminus (Birnbaum & Nassal, 1990
; Nassal, 1992
; Halton et al., 1992
). In contrast, core mutants bearing a small insertion, substitution or deletion in the N-terminal domain of HBV core protein (Beames & Lanford, 1995
; Metzger & Bringas, 1998
; Konig et al., 1998
) or woodchuck hepatitis virus (WHV) core protein (Yu et al., 1996
) fail to form nucleocapsids. However, studies on duck HBV show that the core protein N-terminal additions have various effects on capsid formation depending on the nature of the extension peptides (von Weizsacker et al., 1996
; Kock et al., 1998
). Therefore, the influence of the core protein N terminus on nucleocapsid assembly needs to be further investigated.
Conway et al. (1998b) used an extraneous octapeptide to demonstrate that the core protein N terminus is localized at the spike near the entrance of the shell. In this study, we constructed and expressed N-terminal extension core proteins which were either rich in a positive charge of histidine (designated HisC183) or rich in a negative charge of glutamic acid (designated FlagC183) to test their effect on nucleocapsid assembly in either a prokaryotic or a eukaryotic system. We also tested similar constructs for their influence on virion formation by trans-supplementing a core- negative HBV clone in HuH-7 hepatoma cells. We have demonstrated that the N-terminal extension of core proteins does not interfere with core protein dimerization (Zheng et al., 1992
; Zhou & Standring, 1992
) and nucleocapsid assembly. Since protein kinase activity has been demonstrated in the HBV nucleocapsid (Albin & Robinson, 1980
; Gerlich et al., 1982
), in which the serine residues located at the C terminus of core protein are the substrate of the HBV-associated kinase (Roossinck & Siddiqui, 1987
; Yeh & Ou, 1991
), we have also shown that the N-terminal core mutants do not interfere with kinase encapsidation. However, the result of blocking nucleocapsids from envelopment by surface antigens supported the hypothesis that the N terminus of the core protein is localized near the surface of the capsid shell.
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Methods |
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Purification of core protein.
E. coli lysates from the 800 ml culture described above were incubated with Ni2+-chelated HisBind resin (Novagen) in binding buffer (20 mM TrisHCl, pH 7·9, 0·5 M NaCl and 5 mM imidazole) at room temperature for 1 h. After three washes with binding buffer and washing buffer (20 mM TrisHCl, pH 7·9, 0·5 M NaCl and 60 mM imidazole), the proteins were eluted with elute buffer (20 mM TrisHCl, pH 7·9, 0·5 M NaCl and 1 M imidazole) at room temperature for 30 min. The eluate (5 ml) was collected (concentration 0·17 mg/ml) and aliquots (10 µl) were run on 15% SDSPAGE (Laemmli, 1970 ) and stained with Coomassie brilliant blue for analysis of protein purity.
CsCl gradient centrifugation.
E. coli lysates or nucleocapsids from media or cell lysate (see below) were resuspended in a high-salt TNE buffer (10 mM TrisHCl, pH 7·4, 150 mM NaCl, 1 mM EDTA and 0·1% sodium azide) and subjected to CsCl centrifugation (average density 1·24 g/ml) at 35000 r.p.m. in an SW41 rotor for 44 h. The gradients (final density 1·11·5 g/ml) were fractionated into 0·5 ml samples from the top to bottom and each fraction was subjected to ELISA for detection of core antigen and/or surface antigen (General Biologicals). The density of individual fractions was determined by the refractive index using a refractometer.
Electron microscopy.
Core particles prepared from Ni2+-chelated HisBind resin and CsCl fractions were collected and spotted onto Formvar-coated grids, then negatively stained with saturated uranyl acetate and visualized in a JEOL JEM-2000ExII transmission electron microscope as described previously (Chang et al., 1987 ).
Cell culture and transfection.
Human hepatoma cells (HuH-7) were grown in Dulbecco's modified Eagle's medium supplemented with 10 % foetal bovine serum, 1 mM glutamine, 10 U/ml penicillin and 100 µg/ml streptomycin. HuH-7 cells were transfected with an appropriate amount of plasmid, either singly or in combinations, using the calcium phosphate co-precipitation procedure (Graham & van der Eb, 1973 ; Sambrook et al., 1989
). At 3, 6 and 9 days post-transfection, media were harvested for viral particle analysis. At 9 days post-transfection, cells were harvested for analysis of nucleocapsid and viral DNA.
Viral nucleocapsid isolation.
To collect nucleocapsids from intracellular extracts, transfected cells were detached from plates and incubated in PBS with 1% NP-40 at 4 °C overnight (Beames & Lanford, 1995 ). After a low-speed centrifugation to remove nuclei and NP-40-insoluble cell debris, the cellular lysate was ready for a further isolation step. To collect nucleocapsids and virion-like particles from culture fluids, the media were incubated with or without detergent (NP-40 to a final concentration of 1%) at room temperature for 2 h before centrifugation. Both samples were clarified by centrifugation at 13000 r.p.m. for 30 min in a JA-20 rotor (Beckman Instruments). The nucleocapsids were then concentrated by further centrifugation at 45000 r.p.m. in a Ti55.2 rotor for 2·5 h while the Dane-like particles were enriched under the same conditions for 5 h. The isolated viral particles were subsequently resuspended in a low-salt TNE buffer (10 mM TrisHCl, pH 7·5, 100 mM NaCl and 1 mM EDTA).
Immunoprecipitation and Western blotting analysis.
The precleared cell lysate was incubated with primary antibodies bound with Sepharoseprotein A or G. Antibodies of rabbit polyclonal anti-core (Dako), mouse monoclonal anti-6xHis (Clontech) and anti-Flag (Kodak) were used. The antigenantibody complexes were precipitated and washed three times with NET buffer (50 mM Tris, pH 7·5, 150 mM NaCl, 0·5 mM EDTA and 0·5% NP-40) and then boiled in sample buffer (Laemmli, 1970 ) and analysed by 15% SDSPAGE under reducing (with DTT) or non-reducing (without DTT) conditions. Western blotting was performed by reacting with anti-core, anti-6xHis or anti-Flag antibodies following by reacting with secondary antibodies, conjugated with horseradish peroxidase (Organ Teknika). The blot was developed with an enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia).
In vitro kinase assay.
The immunoprecipitated nucleocapsids were washed three times and then incubated in a kinase reaction buffer (50 mM TrisHCl, pH 7·4, 10 mM MgCl2 and 0·4% NP-40) containing 10 pmol [-32P]ATP (7000 Ci/mmol; Amersham) as described previously (Jeng et al., 1991
; Lin & Lo, 1992
). After incubation at 37 °C for 1 h, the complex was washed five times with NET buffer and then subjected to 15% SDSPAGE separation and autoradiographed.
Endogenous DNA polymerase assay.
Endogenous DNA polymerase activity was assayed as described previously (Junker et al., 1987 ) with some modification (Chiang et al., 1990
; Lin & Lo, 1992
). Briefly, one-fifth of the partially purified viral particle samples was incubated with a pol-mix buffer (50 mM TrisHCl pH 7·4, 40 mM NH4Cl, 5 mM MgCl2, 0·5% NP-40, 0·2% 2- mercaptoethanol and 25 µM each of dATP, dGTP and dTTP) at 37 °C for 2 h in the presence of [
-32 P]dCTP (5000 mCi/mmol; Amersham). Subsequently, a chase was performed for 2 h by adding unlabelled dCTP (25 µM final concentration) at 37 °C. Unwanted nucleic acids present outside of particles were digested by micrococcal nuclease (5 U) for 1 h at 37 °C. Particles were then digested by 50 µg/ml proteinase K treatment in the presence of 1% SDS for 1 h at 37 °C. Glycogen was added (0·8 mg/ml final concentration) and samples were extracted with an equal volume of phenol/chloroform. The labelled DNAs were precipitated with 2·5 vol. ethanol which contained 0·3 M sodium acetate (pH 4·8), separated on 1% agarose gels by electrophoresis, and then autoradiographed.
PCR and Southern hybridization.
In addition to using an endogenous DNA polymerase assay to analyse HBV nucleic acids in secreted viral particles, Dane-like particles collected from CsCl density gradients were subjected to PCR amplification followed by Southern hybridization. Particles were first digested with DNase (10 U) in a DNA buffer (10 mM TrisHCl, pH 8·3, 50 mM KCl and 1·5 mM MgCl2 ) at 37 °C for 1 h and then disrupted by boiling for 10 min (Bottcher et al., 1998 ). Viral DNAs were amplified by PCR using the forward primer C401F (5' ATGGACATCGACCCTTATAAAG 3') and the reverse primer S1764R (5' TGTTCCTGAACTGGAGCCACCAGCA 3') for 40 cycles of 96 °C for 1 min, 57 °C for 1 min and 74 °C for 20 s and an additional extension period at 72 °C for 5 min. The amplified DNA products were resolved by electrophoresis on 2% agarose gel and subjected to Southern blot analysis (Southern, 1975
) using a probe containing the 279 bp HBV BamHI/EcoRI fragment (see also Fig. 7 a
).
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Results |
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To clarify whether or not the His-tag of HisC183 is exposed at the surface of nucleocapsids, we used an affinity column to purify HisC183. Results of Ni2+-affinity binding resin showed a single band in a 15% SDSPAGE gel stained with Coomassie brilliant blue (Fig. 2c), indicating that the His-tag of HisC183 was accessible by nickel resins. Protein concentration analyses revealed that this band comprised approximately 8·5 mg HisC183, which was recovered from the 800 ml overnight culture. However, HisC183 bound on nickel resins could be in monomeric, dimeric or particulate form. Electron microscopic examination revealed that at least some HisC183 proteins, if not all of them, retained the integrity of nucleocapsids as those separated by CsCl gradient (Fig. 2d
).
Dimerization of HBV core proteins with an N-terminal extension in hepatoma cells
Since the core-like particles obtained from E. coli lack several features of nucleocapsids, e.g. encapsidation of viral pregenome and cellular kinases, pHBVHisC183 (Fig. 1a, line 3) was constructed for expression of HisC183 in HuH-7 hepatoma cells. Another plasmid, pHBVFlagC183 (Fig. 1a
, line 4), was also constructed for expression of FlagC183, which has 10 extra amino acid residues, including five negatively charged glutamic acid residues, at the N terminus of the core protein (see Fig. 1b
). In addition to having a different tag sequence to pHBVC183, pHBVFlagC183 is driven by the cytomegalovirus (CMV) promoter to express FlagC183 and the HBV sequence within it lacks the polyadenlyation signal. To characterize and test the expression of the N-terminal extension mutants, pHBVHisC183 or pHBVFlagC183 were co-transfected with a core-negative plasmid (pHBV
C; Fig. 1a
, line 6) (Hui et al., 1999
) into HuH-7 hepatoma cells. In addition, a full-length HBV-containing plasmid, pMH 3/3097 (Fig. 1a
, line 1), co-transfected with a vector plasmid, pUC-MT (Fig. 1a
, line 7), was used as a positive control. Western blot analyses showed that wild-type and two mutant cores were detected by anti-core antibodies (Fig. 3a
). Migration of wild-type C183, and mutants HisC183 and FlagC183 was as expected, i.e. molecular masses of 21·5, ~24 and ~22 kDa, respectively. With a His-tag or Flag-epitope at the N terminus, HisC183 and FlagC183 could be recognized by anti-His or anti-Flag antibodies, respectively, while wild-type HisC183 could not (Fig. 3a
,
His and
Flag panels). To test whether the His-tagging and Flag- tagging could affect dimer formation of core proteins (Zheng et al. , 1992
; Zhou & Standring, 1992
), samples were analysed under non-reducing conditions. Results showed that a dimer was present in the wild-type C183 as well as in HisC183 and FlagC183 (Fig. 3 b
), indicating that formation of intermolecular disulfide bonds was not inhibited by the extensions of amino acid residues located at the N terminus.
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To further confirm that the observation of envelopment impairment occurred in mutant nucleocapsids, a more sensitive assay was carried out. Cell culture media precleared by anti-core antibodies as described above were fractionated by CsCl gradient centrifugation and divided into three tubes: <D, lighter density than Dane particles; D, equal density to Dane particles; and >>D, heavier density than Dane particles. Each tube was subjected to PCR amplification followed by Southern blot analysis for detection of HBV DNA present in the fraction. ELISA analyses of CsCl fractions showed that no naked nucleocapsids were present in any preparations (Fig. 7b ). PCR and Southern blot results showed two bands of 1363 bp and 1046 bp in the sample co-transfected with pHBV
C and pHBV
PSX and a single band of 1363 bp in the sample co- transfected with pMH 3/3097 and pUC-MT (Fig. 7b
), indicating that they were indeed derived from Dane-like particles instead of nucleocapsids in media. Lighter bands present in both <D and >>D tubes of positive control groups might result from the incomplete separation and the oversensitive detection by PCR amplification plus Southern hybridization. Consistent with the DNA repairing assay results, no PCR product was detected in the tubes of <D, D and >>D from samples which were co-transfected with mutant core plasmids and pHBV
C (Fig. 7b
, lanes 712); this strongly suggests that no detectable virion was secreted by these transfected cells. Taking together all results from this study, we conclude that HBV core mutants with an N-terminal extension, HisC183 and FlagC183, can form a functional nucleocapsid intracellularly but are incapable of forming a mature and secretable Dane-like particle.
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Discussion |
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In the past, HBV core proteins have been successfully used to express heterologous epitopes in many prokaryotic and eukaryotic systems because of their ability to form particles (see review by Pumpens & Grens, 1999 ). In addition, HBV nucleocapsids are exceptionally potent antigens that induce both T-cell-dependent and T-cell-independent responses (see review by Schodel et al., 1996
). Several insertion sites have been tested to improve the immunogenicity of foreign peptides (Clarke et al., 1990
; Pumpens et al., 1995
; Borisova et al., 1996
; Ulrich et al., 1998
). Three insertions, located at aa 13, 7493 and 141183 of core proteins, are found to be regions that are dispensable for nucleocapsid assembly (Pumpens & Grens, 1999
). In this study, the nucleocapsid assembled by HisC183 is consistent with previous studies and gives a new example that additional amino acids at the N terminus do not disturb formation of the nucleocapsid. Furthermore, it shows that the His-tag of HisC183 is possibly located at the surface of nucleocapsids, since the particles can be purified on a nickel column (Fig. 2c
and d
). It is noted that three bands of HisC183 were observed on the Western blot as compared with one single band of purified HisC183 on the gel stained by Coomassie brilliant blue (Fig. 2a
, lane 8 vs c, lane 2). This could be because (i) the HisC183 protein inside the bacteria is not completely reduced by DTT while the purified HisC183 is fully reduced, or (ii) degradation of HisC183 occurs inside bacteria while those degraded forms cannot be recovered from the nickel column.
In addition to demonstrating that nucleocapsids are assembled from HisC183 in E. coli, we also showed that nucleocapsids are able to assemble using HisC183 in human hepatoma cells and that they retained kinase and DNA polymerase activities. To date, the role of cellular kinase encapsidated by HBV core particles remains poorly defined. Our current finding provides evidence that the N-terminal additions to the core protein do not interfere in cellular kinase incorporation. Although we did not produce FlagC183 in E. coli to test for nucleocapsid formation, we showed that FlagC183 has all the characteristics of HisC183 in hepatoma cells. Basically, the additional amino acids present in HisC183 or FlagC183 do not disrupt the formation of core dimer, which provides grounds for further polymerization to form particles (Zhou & Standring, 1992 ). If the intermolecular Cys-61Cys-61 disulfide bond of core proteins is disrupted, no particles are produced (Conway et al., 1998b
).
In this study, we provide additional information on the envelopment of mutant nucleocapsids, since envelopment is one of the critical steps in HBV maturation. Three surface proteins (L-HBsAg, M-HBsAg and S- HBsAg) on the envelope are important for virus maturation and infection (Ueda et al., 1991 ; Bruss & Ganem, 1991
; Le Seyec et al., 1998
). The molecular nature of the HBV envelopment signal is still unknown. However, previous reports have suggested that triggering the envelopment signal is linked to genomic replication in the interior of nucleocapsids (Gerelsaikhan et al., 1996
; Wei et al., 1996
). Such a signal may result in a conformational change in the core proteins and allow interaction between the core proteins and surface proteins to occur. Bottcher et al. (1998)
have demonstrated that L-HBsAg may bind to Glu-77 and Asp- 78 of the core protein. Therefore, the impairment of envelopment in nucleocapsids assembled from HisC183 and FlagC183 (Fig. 7
) is possibly influenced by the six histidines or the five glutamic acids at the N terminus of the core protein (Fig. 1b
). These N-terminal extensions could either block the envelopment signal or interfere with surface antigen binding to core particles. Based on results of the accessibility of the His-tag and Flag-tag by antibodies (data not shown) or nickel resins (Fig. 2c
, d
), we favour the second hypothesis. Nevertheless, further cryoelectron microscopy is required to reveal the structure of HisC183 particles isolated from E. coli , which are easily obtained from the pHisC183-harbouring cells. The structure of HisC183 particles will definitely provide a better conclusion.
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Acknowledgments |
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References |
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Received 18 May 1999;
accepted 24 June 1999.