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J. Biol. Chem., Vol. 276, Issue 39, 36613-36623, September 28, 2001
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From the TECNOGEN S.C.p.A., Parco Scientifico, 81015 Piana di Monte
Verna (CE), Caserta 81015, Italy and
Received for publication, March 16, 2001, and in revised form, May 22, 2001
A direct involvement of the hepatitis B virus
(HBV) preS1-(21-47) sequence in virus attachment to cell
membrane receptor(s) and the presence on the plasma membranes of HepG2
cells of protein(s) with receptor activity for HBV have been suggested
by many previous experiments. In this study, by using a tetravalent
derivative of the preS1-(21-47) sequence, we have isolated by affinity
chromatography from detergent-solubilized HepG2 plasma membranes a
44-kDa protein (HBV-binding protein; HBV-BP), which was found to
closely correspond to the human squamous cell carcinoma antigen 1 (SCCA1), a member of the ovalbumin family of serine protease
inhibitors. Comparison of SCCA1 sequence with the sequence of the
corresponding HBV-BP cDNA, cloned by polymerase chain reaction
starting from RNA poly(A)+ fractions extracted from
HepG2 cells, indicated the presence of only four nucleotide
substitutions in the coding region, leading to three amino acid
changes. Intact recombinant HBV-BP lacked inhibitory activity for
serine proteases such as The hepatitis B virus
(HBV),1 a member of the
hepadnaviruses family, is able to infect only humans or higher primates
and has a strong organ tropism for hepatocytes (1). The mechanism of hepatocyte infection induced by this noncytolytic virus is not yet clear.
The infectious unit of HBV is an enveloped virion of 42-nm diameter
that contains an icosahedral nucleocapsid that encompasses a circular,
partly double-stranded DNA molecule with a single strand region of
variable length, a DNA-linked protein with functions critical for
packaging and DNA replication, including priming, RNA- and DNA-
dependent DNA polymerase, and RNAH activities. The HBV envelope that
determines the targeting to the host cells and the early entry steps is
composed of three proteins anchored in a lipid bilayer, occurring in
mature virions in both glycosylated and nonglycosylated forms. The
three proteins are called small (S), middle (M), and large (L) surface
HBV antigens (HBsAg). They are the translational products of three
overlapping open reading frames that start from different initiation
codons localized at the 5' end of the preS1, preS2, and S regions of
the env gene (2). The role of the preS1 region of the
L protein appears to be important in cell attachment and consequently
in viral infectivity, since preS1 synthetic peptides and corresponding
antibodies inhibit virus binding to HepG2 cells (3, 4).
A cellular receptor is required for HBV binding and penetration in
liver cells. Studies in rat hepatoma cells transfected with the HBV
genome (5) and transgenic mice with the genome integrated (6) indicate
that HBV is able to replicate in rodent cells once it bypasses the
attachment and entry steps of infection, so that the absence of
specific cellular receptor(s) constitutes the barrier of HBV infection
and replication in nonhuman hepatocytes. A number of putative human
cellular receptors for HBV have been proposed, including the receptor
for immunoglobulin A (IgA), which shows a partial sequence similarity
between the Fc region of the IgA HBV receptor identification is hampered mainly by the virus-restricted
host range and tissue tropism of the virus and the lack of a
reproducible in vitro infection systems. Primary human hepatocytes are reported to be susceptible to HBV infection but are
difficult to study because they are short lived and require primary
explanted liver, and reproducibility of infection requires treatment of
the cells with chemical agents that may induce unnatural mechanisms of
viral entry (21). However, human hepatoma HepG2 cells, grown under
conditions designed to maintain the hepatocyte-specific differentiated
function, are thought to express the HBV receptor at maximum levels
(22-24). HepG2 cells have characteristics similar to normal liver
parenchymal cells (22, 23) and can support HBV replication following
transfection by cloned HBV DNA (24). HBV particles bind to HepG2 cells
in a saturable manner but are refractive to virus replication, probably
due to the inability of the incoming virus to reach the nucleus in
order to gain access to the cellular transcriptional machinery (25).
The binding of viral particles to HepG2 may be prevented by HBV
preS1-(21-47) peptide or anti-preS1-(21-47) antibodies (3, 4, 8),
suggesting that this sequence could be directly responsible for HBV
attachment to the cellular surface (26).
To identify the protein responsible for HBV attachment to HepG2 cells,
in this study we have developed a tetrameric form of the preS1-(21-47)
peptide in order to increase its functional affinity. This peptide has
been used to determine optimal conditions for the detergent
solubilization of HepG2 plasma membranes (PM) and as a ligand to
fractionate solubilized HepG2 PM extracts by affinity chromatography.
By this procedure, a 44-kDa protein has been purified, which has been
then cloned and expressed in Escherichia coli to
characterize its functional activity. Its role as a cellular receptor
for HBV has been studied in transfected CHO and HepG2 cells with the
corresponding cDNA. We have analyzed the effect of overexpression
of this protein in mammalian cells on virus particle binding and the
inhibitory effects of the recombinant protein and corresponding
polyclonal antibodies on HBV binding and internalization in transfected
cells and primary human hepatocytes.
Peptide Synthesis--
Peptide synthesis was performed following
the Fmoc
(N-(9-fluorenyl)methoxycarbonyl)/dicyclohexylcarbodiimide/N-hydroxybenzotriazole methodology on a fully automated 431A peptide synthesizer (Applied Biosystems). The 4-preS1-(21-47) peptide was prepared in a tetrameric form starting from a polylysine core on a
4-(hydroxymethyl)phenoxyacetic resin. After completion of the synthesis
cycles, peptide resins were dried overnight under vacuum; peptides were
cleaved from resins using trifluoroacetic
acid/phenol/thioanisole/H2O/triisopropylsilane (83:6:5:4:2;
5 ml/200 mg of resin), incubating at room temperature for 3.0 h.
After synthesis and deblocking from the resins, peptide was purified by
extensive dialysis against acetic acid using a membrane with a cut-off
of 12,000 Da. The peptides were characterized by analytical HPLC, amino
acid analysis, and time-of-flight matrix-assisted laser
desorption-ionization mass spectrometry. Synthetic myristyl-preS1 (adw2
subtype) was prepared as previously described (27, 28).
Isolation of HepG2 Plasma Membranes--
HepG2 PM were prepared
by the method of Hubbard (29) with few modifications. The protein
concentration of PM preparations was determined using the Bio-Rad
protein assay, and bovine serum albumin was used as standard protein.
PM were characterized by their enzymatic activity by measuring
5'-nucleotidase activity, an enzyme marker for the plasma membranes
(Sigma Kit 265-UV, following the manufacturer's instructions), and
glucose-6-phosphatase activity (30), an enzyme marker of endoplasmic reticulum.
100 µl of HepG2 PM preparations (2 mg/ml) were incubated with
detergents such as Tween 20, Triton X-100, or SDS at concentrations ranging from 0.1 to 1% (v/v) for 1 h at 4 or 25 °C. After
incubation, the samples were diluted with PBS (150 mM NaCl,
50 mM NaH2PO4, pH 7.5) and
centrifuged for 30 min at 18,000 rpm, and the soluble fraction was
recovered. To test the effect of acidic conditions, a 100-µl sample
of Triton-solubilized HepG2 PM fraction (1.8 mg/ml) was incubated with
900 µl of acetic acid 0.1 M or glycine 0.1 M
for 1 h at 25 °C. Samples were then neutralized with NaOH and tested for functionality by RIAs with 125I-4-preS1-(21-47)
(see below).
RIAs--
HBV synthetic antigens preS1-(21-47) and
4-preS1-(21-47) were iodinated with Bolton and Hunter reagent,
N-succinimidyl 3-(4-hydroxy, 5-[125I]iodophenyl)propionate (Amersham Pharmacia
Biotech). 5-20 µg of polypeptides were mixed with 1 mCi of reagent
in 0.1 M sodium borate, pH 8.5, and incubated under gentle
agitation overnight at room temperature. The reaction was stopped by
the addition of glycine 0.2 M, and nonincorporated reagent
was removed by ultrafiltration on Microcon R300 (Amicon); the
125I incorporation was checked by trichloroacetic acid
precipitation. The specific activity of the labeled polypeptides was
8 × 106 cpm/µg for 4-preS1-(21-47) peptide and
1.5 × 106 cpm/µg for preS1-(21-47) peptide.
Polystyrene microtiter plates were coated with HepG2 or control PM in
PBS overnight at room temperature. The plates were washed with PBS
containing 0.05% Tween 20 (PBS-T) and then blocked with a 10%
solution of bovine serum albumin (BSA) in PBS for 6 h at room
temperature. Plates were washed again with PBS-T, labeled polypeptides
were added, and binding reactions were performed overnight at room
temperature. The unbound polypeptides were removed by washing with
PBS-T, and the radioactivity associated with the wells was measured
( Purification of HBV-BP from Detergent-solubilized HepG2 Plasma
Membranes--
Affinity column with immobilized 4-preS1-(21-47)
peptide was prepared by incubating 10 mg of peptide ligand dissolved in
10 ml of 0.1 M sodium bicarbonate buffer, pH 8.5, with
1.0 g of CH-Sepharose 4B (Amersham Pharmacia Biotech) under gentle
agitation for 24 h at room temperature. The extent of ligand
incorporation was monitored by reverse phase HPLC and was close to
95%. HepG2 PM have been solubilized with 0.1% Triton X-100 for 1 h at 4 °C, and soluble fraction was recovered by centrifugation at
20,000 × g for 30 min at 4 °C. The soluble fraction
was diluted in loading buffer (50 mM sodium phosphate, pH
7.0) to reach a final concentration of 500 µg/ml before loading on
the 4-preS1-(21-47)-Sepharose affinity column, equilibrated at a flow
rate of 1 ml/min with loading buffer. After elution of unretained
material, the eluent was changed to 0.1 M glycine, pH 2.2, to elute bound proteins. The extent of purification was followed by
RIAs and SDS-polyacrylamide gel electrophoresis. The bound fractions
were dialyzed against 50 mM sodium phosphate, pH 8.0, concentrated on Microcon R300 (Amicon), and then digested with
trypsin (1:500 (w/w) enzyme/protein ratio) for 1 h at room temperature. The extent of trypsin degradation was followed by reverse
phase HPLC analysis of the reaction mixture using a linear acetonitrile
gradient (0.1% trifluoroacetic acid) ranging from 5 to 60% in 50 min
on a RP-8 column (Aquapore) and monitored at 225 nm. Peaks eluted were
collected, lyophilized, and submitted to automated sequencing (model
477A connected to a PTH-amino acid analyzer model 120A; Applied Biosystems).
Cloning and Sequencing of HBV-BP--
Total RNA from HepG2 was
extracted by cell lysis with guanidinium isothiocyanate, and
mRNA fraction was purified using the oligotex kit (Qiagen).
First strand of cDNA was synthesized (Superscript II kit;
Life Technologies, Inc.) and used to isolate HBV-BP cDNA by nested
polymerase chain reactions (PCR) (all of the oligonucleotide primers were purchased from Life Technologies). PCR-1 was performed with the first primer pair (primer-1 (sense)
(5'-CACAGGAGTTCCAGATCACATCGAG) and primer-2 (antisense)
(5'-CTGGAAGAAAAAGTACATTTATATGTGGGC), complementary to Expression and Purification of Recombinant HBV-BP
(rHBV-BP)--
The HBV-BP cDNA was cloned in the expression vector
pET21a (Novagen). The vector was digested with NdeI, which
generates a 3' protruding extremity, digested with T4 DNA polymerase
(Roche Molecular Biochemicals) to obtain blunt ends, and then digested with BamHI endonucleases. To obtain the HBV-BP cDNA with
compatible ends for cloning and to place the ATG start codon of HBV-BP
cDNA in the right context with respect to the ribosome
binding site carried by the vector, the pUC18/HBV-BP plasmid was used
as template for a PCR performed with the following primer pair: primer
(sense), 5'-CATGAATTCACTCAGTGAAGCCAAC-3' (complementary to
A single transformed BL21 (DE3) bacterial colony was inoculated in 25 ml of LB broth containing ampicillin (100 µg/ml) and grown at
37 °C for 20 h to obtain a saturated culture. This culture was
added to 1 liter of LB containing ampicillin and incubated at 37 °C.
At A600 = 0.6, the expression of foreign protein
was induced with 1 mM
isopropyl-
The bacteria were recovered by centrifugation and washed three times
with PBS buffer. The recovered pellet was resuspended in PBS (4 ml/300
mg, wet weight) and sonicated with the Ultrasonic W-380, with the
immersion probe microtip 419A, f 4.8 mm (Heat Systems
Ultrasonics), with the following parameters: boost, 1'; duty cycle,
40%; output power, 4. The sonication was stopped at 95-98% of lysis
of the bacteria, as checked with an optical microscope. The crude
bacterial extract was centrifuged at 15,000 × g, at 4 °C, for 30 min, to separate the soluble from the insoluble
fraction. The soluble fraction was dialyzed against 20 mM
MES, pH 6 (buffer A), and the protein concentration was determined with
the Bio-Rad Protein Assay kit. The dialyzed soluble fraction was
diluted in buffer A to reach a final concentration of 500 µg/ml. A
glass column (25 × 0.5-cm inner diameter) (Omnifit) was filled
with anionic exchange Source 30S resin (Amersham Pharmacia Biotech); the prepared column was equilibrated at a flow rate of 3.0 ml/min with
buffer A. The diluted soluble fraction was loaded on the column, and
following the elution of unretained material, a gradient from 0 to
100% of buffer B (20 mM MES, pH 6.0, 0.5 M
NaCl) in 20 min was applied to elute bound proteins. The retained
fraction was recovered and dialyzed against PBS, pH 7.5. The purified
fraction of rHBV-BP was stored at Production of Rabbit Anti-HBV-BP Polyclonal
Antibodies--
Polyclonal antibodies were produced by immunizing
rabbits with 350 µg of rHBV-BP in complete Freund's adjuvant and
boosted every 3 weeks with the same dose of protein in incomplete
Freund's adjuvant. Serum was collected, and the IgG fraction was
purified by protein A-Sepharose chromatography. The polyclonal antibody anti-HBV-BP was then purified by affinity chromatography on
rHBV-BP-Sepharose columns. Preimmune serum, after protein A
purification, was used in control experiments.
Binding of Myristyl-PreS1 to rHBV-BP--
Polystirene microtiter
plates (Nunc) were coated with rHBV-BP at 10 µg/ml (100 µl/well) in
0.1 M NaHCO3, pH 8.8, and incubated overnight
at 4 °C. The plates were washed three times with PBS buffer, and the
wells were saturated with 200 µl of 3% dried milk in PBS and
incubated for 1 h at 37 °C to block uncoated plastic surface.
Plates were then washed three times with 0.5% dried milk in PBS buffer
(PBS-M); myristyl-preS1 was added at different concentrations (0.2-20
µg/ml in PBS-M, 100 µl/well) and incubated for 1 h at 37 °C. The plates were washed six times, and an anti-preS1
monoclonal antibody (T0606; Sorin Biomedica) was added at 1 µg/ml in
PBS-M, 100 µl/well. After 1 h of incubation at 37 °C and
subsequent washing, wells were filled with 100 µl of horseradish
peroxidase-labeled goat anti-mouse (Sigma) solution diluted 1000-fold
with PBS-M. The plates were left to stand for 1 h at 37 °C,
washed six times, and then filled with 120 µl of
o-phenylenediamine dihydrochloride (Sigma) chromogenic
substrate solution freshly prepared according to protocols from the
manufacturer. The color was allowed to develop for 20 min, and the
A450 was determined with an enzyme-linked immunosorbant assay plate reader.
Kinetic Analysis--
Photometric substrates,
N Mammalian Cell Transfection and Selection of Stable Cellular
Lines--
The HBV-BP cDNA was cloned in the pcDNA3
(Invitrogen) mammalian expression vector that allows the selection of
G-418 stable clones for the presence of the neomycin gene. The complete
insert of pUC18/HBV-BP plasmid was recovered by digestion with
KpnI and XbaI endonucleases and ligated in the
pcDNA3 vector restricted in the same manner. E. coli
XL1-blue-MRF' (Stratagene) CaCl2-competent cells were
transformed with the product of ligation reaction, and the DNA of the
resulting recombinant plasmid (pcDNA3/HBV-BP) was prepared
(Qiagen Maxi-prep kit). HepG2 and CHO transfections were performed with
pcDNA3/HBV-BP DNA using the calcium phosphate precipitation
technique. Two stable clones, HepG2/HBV-BP and CHO/HBV-BP, were
selected for the maximal amount of the HBV-BP expression as established
by Western blot and RIAs.
Immunofluorescence Analysis--
Cells were fixed with 3.7%
formaldehyde in PBS, washed with 0.1 M glycine in PBS,
blocked with 5% normal goat serum in PBS, and incubated with
polyclonal anti-HBV-BP antibodies at 5 µg/ml in PBS containing
1% BSA for 30 min at room temperature. After washing with PBS, cells
were incubated with the supernatant of hybridoma producing the
monoclonal antibody W6/32 (ATCC number HB-95) diluted 1:50 in PBS plus
1% BSA. After 30 min at room temperature, cells were washed again with
PBS, and immunoreactivity was visualized by incubation with a
mixture of secondary antibodies composed of goat anti-rabbit,
conjugated with FITC and goat anti-mouse conjugated with
tetramethylrhodamine isothiocyanate (Sigma), both diluted 1:100 in PBS
plus 1% BSA for 30 min at room temperature. Images were acquired for
the different fluorescent emissions and overlapped.
HBV Particle Binding to HepG2/HBV-BP Cell
Surface--
Infectious viral particles were obtained under informed
consent from serum of a chronically infected patient with HBsAg- and HBeAg-positive serology, in which HBV DNA levels ranged from 3.4 to 1.8 ng/ml (Digene Hybrid Capture HBV DNA Assay) in several occasions. Enriched viral particle preparations were obtained by
discontinuous sucrose gradient ultracentrifugation, and preS1 protein
content was assessed using a previously standardized enzyme-linked immunosorbent assay (7). All viral particle preparations showed a
strong preS1 reactivity, with OD values at HBV Particle Adsorption and Entry in Different Cell
Lines--
The following cell lines were used to study the interaction
with HBV particles: HepG2, HepG2/HBV-BP, CHO, CHO/HBV-BP, primary human
hepatocytes, and RAT1. Stable cell lines grown at semiconfluence in
75-cm2 flasks were incubated with EMEM plus 10% FBS
containing 23 × 107 viral genome equivalents. After a
30-min incubation at 37 °C, medium was collected, and cells were
washed three times with PBS containing 0.05% Tween 20 and three times
with PBS alone before cellular DNA extraction and HBV DNA
determination. In inhibition experiments, cells were incubated with
serial dilutions of anti-HBV-BP antiserum or with preimmune rabbit
serum, as control, for 30 min at 37 °C and extensively washed with
EMEM plus 10% FBS before incubation with the HBV preparation. HBV DNA
was evaluated in cellular DNA extracts by molecular hybridization and
by PCR analysis (31). Viral DNA was detected by a solution
hybridization assay (Digene Hybrid Capture HBV DNA Assay) with a linear
range of detection between 106 and 108 genome
equivalents/ml. Molecular amplification of HBV DNA was carried out
using primers derived from the core gene of HBV (primer sequence: C1,
5'-TTG CCT TCT GAC TTC TTT CC-3' (core positions 1955-1974), and C2,
5'-TCT GCG AGG CGA GGG AGT TCT-3' (core positions 2401-2381)) with a
roughly linear detection range from 103 to 107
genomes/ml (data not shown). In similar experiments, cells were incubated with EMEM plus 10% FBS containing 100 µl of hepatitis C
virus (HCV)-positive serum with a known amount of RNA molecules (6 × 106 genome equivalents/ml). After extensive washes,
cellular RNA was extracted by RNAZOL (Biogenesis), and HCV RNA was
measured using a branched DNA assay (Quantiplex 2.0; Bayer
Corp.) as previously described (33). All experiments were repeated at
least four times. To evaluate the relative amount of HBV particle
binding and penetration into cultured cells, the following experiments were carried out in parallel. HepG2/HBV-BP cells grown to
semiconfluence in 25-ml flasks were incubated with EMEM plus 10% FBS
containing 22 × 107 genome equivalents for 2 h
at 4 °C or at 37 °C. Cells were then washed twice with PBS before
treatment with 0.5 mg/ml trypsin, 0.5 mM EDTA. Trypsin was
removed, and cells were collected after washing with PBS plus 10% FBS,
centrifuged at 1000 rpm for 3 min, and transferred to Eppendorf tubes.
To extract DNA from cytoplasm and nuclear cellular fractions, the cell
pellet was resuspended in lysis buffer (0.14 M NaCl, 1.5 mM MgCl2, 10 mM Tris (pH 8), 0.5%
Triton X-100) and homogenized with a loose fitting pestle, and after 5 min of settling, the supernatant was stratified over an equal volume of
24% (w/v) sucrose in lysis buffer. After centrifugation at 10,000 × g for 20 min at 4 °C, the cytoplasmic fraction (upper fraction) and the nuclear fraction (pellet) were recovered, the pellet
was washed with cold PBS, and both cellular fractions were proteinase
K-digested before nucleic acid extraction. In inhibition experiments,
increasing concentrations of rHBV-BP ranging from 0.18 to 18 µg were
mixed with 100 µl of HBV particle suspensions (11 × 107 genome equivalents) and incubated at 37 °C for 30 min before the addition to cells grown in 24-well plates. After a 2-h
incubation at 37 °C, medium was removed, and plates were washed
before trypsinization and total cellular DNA extraction. For inhibition
experiments of HBV entry in primary human hepatocytes, supplemented WE
medium containing 12 × 107 genome equivalents
preincubated for 30 min at 37 °C with increasing concentrations of
rHBV-BP (0.18, 18, and 180 µg) or anti-HBV-BP antibodies (0.1-1.0
µg/ml) or PBS was added to individual wells of 24-well plates where
105 human hepatocytes were seeded. Cells were then washed
twice with PBS plus 10% FBS before trypsin treatment and nucleic acid extraction.
Isolation of Primary Human Hepatocytes--
Primary human
hepatocytes were obtained from the explanted liver of a patient
suffering from hypercystinuria, an inherited metabolism disorder, at
the time of his double renal and liver transplant. Liver histology
documented normal features. Approval for experimental use of the liver
was obtained from the Medical Ethical Committee. Hepatocytes were
isolated by the collagenase perfusion technique (34), and cell
viability was >90%, as determined by the trypan blue exclusion
method. Cells were seeded on culture plates precoated with human liver
biomatrix (35), in Williams' E medium (Life Technologies) supplemented
with 10% fetal calf serum containing 2 mM
L-glutamine, 50 mM dexamethasone, 20 milliunits ml Isolation of the HBV-BP from HepG2 Plasma Membranes--
Molecules
containing multiple repeats of the same ligand have enhanced the
functional affinity of these ligands in several systems. The repeating
ligands have also improved their function in affinity chromatography
applications (36). In an attempt to obtain an efficient ligand to
capture the HBV-BP from HepG2 PM, we synthesized a multimeric form of
the preS1-(21-47) peptide, called 4-preS1-(21-47) containing four
identical preS1-(21-47) chains linked to a central polylysine core
(Fig. 1A). The evaluation of
4-preS1-(21-47) binding activity to HepG2 PM was carried out by RIAs.
The tetrabranched peptide showed a selective binding to HepG2 PM
preparation (Fig. 1B), with negligible binding to membranes
from an unrelated cell line (Rat-1). Its functional affinity for HepG2
PM was higher than the monomeric peptide preS1-(21-47) (Fig.
1C). 4-preS1-(21-47)/HepG2 PM interaction was specific, since unlabeled 4-preS1-(21-47) or detergent-solubilized PM reduced binding in a dose-dependent manner (Fig. 1D).
The RIA was used to evaluate the effect of various detergents, such as
Tween 20, Triton X-100, and SDS, at different concentrations,
temperatures, and lengths of treatment, on HepG2 PM solubilization.
Treatment with 0.1% Triton X-100 for 1 h at 4 °C led to the
highest recovery of protein content (87.5%) and of binding activity
(50%). The HBV-BP protein was isolated by affinity
chromatography loading the Triton-solubilized HepG2 PM extract on
an affinity column prepared by linking covalently 4-preS1-(21-47) to
activated Sepharose. An acidic buffer was used to elute the bound
proteins. One major component with an apparent molecular weight
of 44 kDa unit of mass was detected in the acid-eluted fraction (Fig.
2A). Fractions from the
affinity purification step were also assayed by RIA, and almost all of
the binding activity to 125I-4-preS1-(21-47) was recovered
in the bound fraction, while only negligible activity was found in the
column flow-through fraction (Fig. 2B).
These results suggested that the affinity-purified 44-kDa protein is
responsible for the binding activity of Triton-solubilized HepG2
PM.
The affinity-purified protein was then treated with trypsin in order to
generate peptide fragments that were purified by reverse phase HPLC to
be subjected to microsequencing. Two fragments were purified in
adequate amounts to provide useful sequence data: peptide 1 (NTYK) and
peptide 2 (TYLFLQEYL).
Homology search analyses on the Swiss-Prot data bank (BLITZ system, EBI
network service) indicated that human SCCA1, a member of the ovalbumin
family of serine protease inhibitors (Ov-serpins) (37) contained both
sequences. In the SCCA1 protein, the two sequences matching the tryptic
fragments were preceded by a lysine, as expected from a trypsin
digestion. To confirm the occurrence of SCCA1 or related proteins in
hepatic cells, we carried on nested PCR from the first strand of
cDNA synthesized by reverse transcriptase reaction performed on the
HepG2 mRNA fraction. PCR experiments performed with sense primer 1 and antisense primer 2 that recognized, respectively, the regions
Expression of Recombinant HBV-BP--
To confirm that the cloned
cDNA coded for the same protein isolated from HepG2 PM by affinity
chromatography using the synthetic 4-preS1-(21-47) HBV antigen and to
preliminarily characterize the functional properties of HBV-BP, a
recombinant form of HBV-BP was expressed in E. coli by the
use of the pET expression vector system and purified by anionic
exchange chromatography (Fig.
4A). The purification step was
very effective, since rHBV-BP was recovered with a high degree of
purity (90-95%) as showed by SDS-polyacrylamide gel electrophoresis
analysis (Fig. 4B). The purity and identity of the protein
were also confirmed by reverse phase HPLC and matrix-assisted laser
desorption-ionization mass spectrometry analysis (not shown). By
following this protocol, up to 20 mg of purified rHBV-BP per liter of
bacterial culture were obtained. The purified protein was used then to
produce antibodies in rabbits, which were purified first on protein A
and then on rHBV-BP affinity columns. As shown in Fig. 4C,
the antibody stained equally well both the 44-kDa protein obtained from
the 4-preS1-(21-47) affinity purification of HBV-BP from solubilized
HepG2 PM and the recombinant protein from E. coli, thus
confirming the occurrence of HBV-BP in HepG2 detergent solubilized PM.
To further investigate the role of HBV-BP in HBV recognition, binding
experiments were performed with a synthetic preparation of
myristyl-preS1 (adw2 subtype) (27, 28). This protein was prepared by
stepwise solid phase chemical synthesis and found to interact
specifically with HepG2 PM. The interaction between myristyl-preS1 and
rHBV-BP was evaluated on microtiter plates by coating rHBV-BP to the
plastic surface and detecting the binding of myristyl-preS1 by using a
monoclonal antibody specific for preS1. As shown in Fig. 4D,
myristyl-preS1 bound to rHBV-BP in a saturable and
dose-dependent manner, with EC50 = 0.5 µg/ml.
Thus, in addition to preS1-(21-47) binding, rHBV-BP did recognize the
entire preS1 domain, the proposed virus attachment domain.
HBV-BP Inhibition of Cysteine Proteases--
SCCA1 and SCCA2 genes
are tandemly arranged on chromosome 18 and encode for proteins that are
92% similar at the amino acid level (38). Protein data base comparison
showed that SCCA1 and SCCA2 belong to the superfamily of serine
proteases inhibitors (serpins), which in most cases are tightly binding
inhibitors of serine proteinases (38). Despite the high sequence
homology, significant differences do exist between the reactive site
loop responsible for protease inhibition, especially those residues flanking the putative scissile bonds, suggesting that these serpins may
inhibit different types of proteinases. Previous works suggested that
SCCA1 inhibits the ability of chymotrypsin to degrade gelatin and
ovalbumin (39), but other investigations indicated activity also for
cathepsin L (40). Given the high homology at the amino acid level
between HBV-BP and SCCA1, enzyme inhibition assays were performed with
rHBV-BP in order to evaluate the protein's ability to act as an enzyme
inhibitor. As shown in Fig. 5, rHBV-BP did inhibit with high potency the cysteine proteases papain and cathepsin L. On the contrary, no inhibitory effect was seen with HBV Binding to HBV-BP-transfected Cells--
In order to
establish whether HBV-BP was effectively involved in HBV interaction,
HepG2 and nonpermissive hamster CHO cells were transfected with HBV-BP
cDNA, first to verify that HBV-BP was localized on the cell
surface, an essential requirement for virus binding, and second to
verify that HBV-BP transfection conferred HBV binding capability. Cells
were transfected with the expression vector pcDNA3/HBV-BP,
and two stable cell lines were selected for their high levels of HBV-BP
expression, denoted HepG2/HBV-BP and CHO/HBV-BP, respectively.
The cellular localization of HBV-BP was evaluated first by flow
cytometry experiments, detecting HBV-BP by using anti-HBV-BP polyclonal
antibodies. Western blot analysis performed on total protein extracts
obtained from HepG2/HBV-BP cell lines indicated that the
affinity-purified antibody was highly specific, since only the 44-kDa
HBV-BP protein was stained (not shown). As shown in Fig.
6A, HepG2/HBV-BP cells
displayed a significantly higher proportion of HBV-BP on the cell
surface than HepG2. Transfected HepG2 showed an enhanced ability to
bind 125I-4-preS1-(21-47) compared with nontransfected
cells, with a 2-fold increase in the amount of bound ligand (Fig.
6B). Additional evidences of HBV-BP cell surface
localization were obtained by co-localization experiments
performed on HepG2/HBV-BP cells, by using as surface markers antigens
of class I (HLA A, B, and C) of major histocompatibility complex. These
proteins were detected by using the monoclonal antibody W6/32 (ATCC
number HB-95) on the same sample probed with the polyclonal antibody
anti-HBV-BP. As shown in Fig. 7, the two fluorescent signals were overlapping and perfectly corresponding to the
cell surface. These results confirmed that in HepG2/HBV-BP cells the
protein encoded by HBV-BP cDNA is localized on the cell surface.
Experiments carried out on permeabilized cells indicated that the
protein is localized also in the cytosol (data not shown), confirming
previous findings (42).
The role of HBV-BP in viral particle binding was examined with
transfected cells using hepatitis B viral particles obtained from the
serum of an HBsAg- and HBeAg-positive patient with ongoing HBV
replication. The binding of HBV particles to untransfected HepG2 cells
was clearly measurable by flow cytometry experiments, detecting the
attachment of viral particles to the cells by using anti-HBs polyclonal
antibodies (Fig. 8A,
top). As expected, transfected HepG2 cells showed an
enhanced capacity to bind viral particles, as indicated by the much
higher MFI compared with untransfected HepG2 (Fig. 8A,
bottom). This result clearly indicated that overexpression of HBV-BP on the cell surface was accompanied by an enhancement of
cellular ability to bind viral particles. The HBV-BP role in HBV
binding was further confirmed by the effect of soluble rHBV-BP on
virus/cell interaction. As shown in Fig. 8A
(bottom), the presence of the rHBV-BP protein with HBV
particles resulted in a consistent reduction of virus attachment to
HepG2/HBV-BP cells. The observed shift in the fluorescence signal (MFI)
from 417 to 160, a value even lower than that obtained with
untransfected cells, suggested that rHBV-BP in solution binds to viral
particles inhibiting the attachment of HBV to the cells' surface.
A quantitative evaluation of the effect of HBV-BP overexpression on HBV
binding, determining the amount of viral DNA found in different types
of cells exposed to the same number of HBV particles, indicated that in
HepG2/HBV-BP-transfected cells, the total amount of viral DNA
found after incubation with HBV was about 100 times higher than that
observed in nontransfected HepG2 cells under the same conditions (Fig.
8B and Table I). Even
HBV-BP-transfected CHO cells acquired a remarkable virus binding
capability compared with untransfected cells, where virus binding was
negligible; however, the total amount of viral DNA was considerably
lower than that observed with transfected HepG2 cells. Unrelated cell lines used in control experiments, such as Rat-1, did not significantly bind HBV under the same conditions, while primary human hepatocytes, obtained from the explanted liver of a patient suffering from hypercystinuria, bound HBV much better than nontransfected HepG2 cells.
Viral specificity of HBV-BP was determined with HCV. Interaction of HCV
was assessed in HepG2 and HepG2/HBV-BP cell lines, and HCV RNA was
measured in cellular RNA extracts. Apparently, HBV-BP is an
HBV-specific receptor and does not enhance HCV binding activity to
transfected cells (Table I).
Further confirmation that the increase of viral DNA found in
HBV-BP-transfected cells was directly correlated to the enhanced amount of HBV-BP protein on the cell surface was provided by
competition experiments with polyclonal antibodies raised against
HBV-BP. As shown in Fig. 8C, the addition of anti-HBV-BP
antibodies suppressed in a dose-dependent manner viral
attachment to HepG2 and CHO transfected cells. At a concentration of 1 µg/ml in the culture medium, anti-HBV-BP antibodies reduced the
amount of viral DNA found in the cells to roughly 2-5% of the
original value. Similar results were obtained with primary human
hepatocytes, where, at a concentration of 1.0 µg/ml, anti-HBV-BP
antibodies completely blocked HBV binding. Preimmune rabbit serum,
after affinity purification on protein A, was ineffective in reducing
HBV binding to transfected HepG2 and CHO cells and to primary human
hepatocytes (Fig. 8C).
These data clearly indicate that when HBV-BP is masked by antibodies,
cells lose their ability to bind viral particles.
HBV-BP Is Responsible for HBV Entry in Transfected Cells and
Primary Human Hepatocytes--
The role of HBV-BP in virus entry into
hepatic cells was examined by measuring the ability of soluble rHBV-BP
to inhibit viral particle penetration into HepG2/HBV-BP cells and
primary human hepatocytes. Internalized viral DNA was detected
according to a well established procedure (4, 12, 17, 43, 44) by incubating the cells first with the virus suspension for 2 h at 37 °C and subsequently by treating the cells with trypsin. To prove
that trypsinization removes bound HBV from the cell surface, control
cells were incubated in parallel with virus suspension at 4 °C, not
allowing HBV penetration. Viral particles that were not internalized
during incubation were therefore removed as shown in Fig.
9A, and analysis of cellular
extracted DNA was representative only of the internal nucleic acid
content. By Southern blot hybridization, HBV DNA was recovered in the
cytoplasmic fraction of cells incubated with the virus at 37 °C, but
not in the nuclear fraction, and the amount of recovered HBV DNA was
higher in the cytoplasmic fraction of HepG2/HBV-BP than in
nontransfected cells (Fig. 9B), suggesting an increased
ability of HepG2/HBV-BP cells to internalize HBV. The electrophoretic
pattern of the trypsin wash was similar to that of the HBV inoculum,
with a major viral DNA form migrating at 3.6 kilobase pairs,
corresponding to relaxed circular viral DNA molecules, known to exist
in HBV-infected human liver and in virions isolated from plasma of
infected patients (45). In the cytoplasmic fractions, predominant
double-stranded linear molecules, migrating at 3.2 kilobase pairs, were
detected in both cell types, as previously observed for internalized
nonreplicating forms of HBV DNA (46). The addition of increasing
concentrations of soluble rHBV-BP up to a concentration of 1.8 µg/ml
(40 nM) to HBV particles before incubation with
HepG2/HBV-BP cells decreased virus penetration to almost undetectable
values (Fig. 10A). These data were further confirmed by experiments with primary human hepatocytes. As shown in Fig. 10B, the amount of viral DNA
found in human hepatocytes treated with HBV particles was reduced by the addition of recombinant HBV-BP in a dose-dependent
manner. Inhibition of virus entry in primary human hepatocytes was
efficiently achieved also by anti-HBV-BP antibodies at a concentration
of 1 µg/ml (Fig. 10C).
These data indicate that HBV-BP is responsible for HBV attachment and
entry in primary human hepatocytes.
Data obtained in this study indicate that HBV-BP is important for
HBV binding to HepG2 cells and primary human hepatocytes. This protein
has been isolated by using a derivative of the preS1-(21-47) peptide,
the proposed virus attachment site, and interacts specifically with the
entire HBV preS1 domain. FACS and immunofluorescence analysis suggest
that HBV-BP is present on the surface of transfected HepG2 cells, an
essential requirement to support the proposed role as an HBV-binding
protein. Further support for this contention was provided by the
remarkable enhancement of virus binding capability by transfected
cells, even in the case of CHO cells, where HBV binding is normally
negligible. Viral particle attachment to transfected cells is specific,
since the addition of soluble rHBV-BP or anti-HBV-BP polyclonal
antibodies inhibit virus binding in a dose-dependent manner. Cells overexpressing HBV-BP on their surface internalize more
viral DNA than untransfected cells, and virus entry is blocked by the
addition of soluble rHBV-BP. Primary human hepatocytes, the natural
target for HBV infection, are refractive to HBV penetration in the
presence of rHBV-BP or the corresponding antibodies.
HBV-BP results strictly correlated to human SCCA1 (37), with only
three deduced amino acid differences: Gly351 to Ala,
Ala357 to Thr, and Ser389 to Pro. Chymotrypsin
(39), cathepsin L, and papain (47) react with SCCA1, but SCCA1 function
is substantially unknown. Like other human serpins such as plasminogen
activator inhibitor type 2, placental thrombin inhibitor, and
elastase inhibitor, SCCA1 belongs to the ovalbumin family of
serine protease inhibitors (Ov-serpin) (48), a subfamily of the large
serpin superfamily (49). Serpins have a highly ordered tertiary
structure defined by the crystal structure of the prototype molecule
Like SCCA1, HBV-BP displays a pair of serine residues in the reactive
site P1-P1' peptide bond localized at positions 354 and 355. Bovine
Post-translational modification of HBV-BP could be fundamental for its
function. In addition to glycosylation sites, sequence analysis of
HBV-BP shows the presence of many putative phosphorylation sites.
Phosphorylation of cellular receptors for viral particles is often
essential for their functionality in the initial events of the
infective process, as shown for the receptors of Epstein-Barr virus
(54), mouse mammary tumor virus (55), and polio virus (56, 57).
The genes coding for many serpins of the ovalbumin family are localized
closely on chromosome 18. The SCCA1 and SCCA2 genes are closely linked,
tandemly arrayed, and flanked by two closely related members of the
Ov-serpin family, plasminogen activator inhibitor type 2 and maspin.
All four serpins are located within 300 kilobase pairs of 18q21.3 (38).
Apparently, HBV-BP is not simply an allelic variant of SCCA1. PCR
experiments performed on human genomic DNA using primers designed to
amplify the last exon of the SCCA gene, where all of the three
nucleotide substitutions responsible for the three amino acid changes
in the HBV-BP are located, indicate that the HBV-BP sequence is
amplified as well as the SCCA1 and SCCA2 exons. This suggests that a
specific gene coding for HBV-BP is present in the human genome or that
a sequence coding for the last exon of the SCCA gene is present and
used for alternative splicing processes. Studies are in progress to identify the genomic sequences coding for HBV-BP.
Previous works did not report expression of SCCA1 and of the high
homologous SCCA2 gene in cells derived from human liver. SCCA1 was
isolated as a component of T-4 antigen complex from a human metastatic
squamous cell carcinoma (SCC) (58) and has been used as circulating
tumor marker for the management and diagnosis of SCC of the cervix,
head and neck, lung, and esophagus (59). SCCA1 is detected in normal
squamous epithelium and produced at high levels by the corresponding
carcinoma cells. The lack of tissue specificity may resemble the
situation occurring with carboxypeptidase D (gp180), the proposed
cellular receptor for duck HBV. In this case, the protein is found not
only in duck liver but also in other tissues, which are not susceptible
to duck HBV infection (60). As in our case, gp180 expression increases
duck HBV attachment to and entry in transfected heterologous cells, but
productive virus infection does not occur, suggesting that gp180 may
function as a primary virus attachment site and that a species-specific co-receptor is needed to complete viral replication. Although our data
show that HBV-BP is the primary factor responsible for HBV binding to
and entry in primary human hepatocytes, the possibility cannot be
excluded that additional, still unknown cellular components might be
involved for the active replication cycle of the virus.
We are deeply grateful to Professor
Maurizio Muraca (University of Padova) who allowed all of the
experiments with primary human hepatocytes; to Dr. Livio Trentin and
Monica Facco (University of Padova) and Maria Tortora and Angela
Scarallo (TECNOGEN) for excellent technical assistance; and
to Professor Maria Luisa Melli (University of Bologna) for critical
reading of the manuscript.
*
This research was performed under a contract of the Italian
Ministero dell'Università della Ricerca Scientifica e
Tecnologica assigned to TECNOGEN s.c.p.a. within the "Programma
Nazionale di Ricerca sui Sistemi Neurobiologici-Tecnologie della
Trasduzione del Segnale."The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Tel.: 39-0823-612-214;
Fax: 39-0823-612-230; E-mail: fassina@tecnogen.it.
Published, JBC Papers in Press, June 1, 2001, DOI 10.1074/jbc.M102377200
The abbreviations used are:
HBV, hepatitis B
virus;
HBV-BP, HBV-binding protein;
rHBV-BP, recombinant HBV-BP;
HBs, surface HBV;
HBsAg, surface HBV antigen(s);
PM, plasma membrane;
RIA, radioimmunoassay;
BSA, bovine serum albumin;
PBS, phosphate-buffered
saline;
MES, 2-(N-morpholino)ethanesulfonic acid;
CHO, Chinese hamster ovary;
FITC, fluorescein isothiocyanate;
FACS, fluorescence-activated cell sorting;
MFI, mean log fluorescence
intensity;
PCR, polymerase chain reaction;
SCCA, squamous cell
carcinoma antigen;
EMEM, minimum essential medium with Earles'
salts;
HBeAg, hepatitis B e antigen.
Cloning and Expression of a Novel Hepatitis B
Virus-binding Protein from HepG2 Cells*
,,, and Università
degli Studi di Padova, Dipartimento di Medicina Clinica e Sperimentale,
Clinica Medica 5, 35128 Padova, Italy
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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-chymotrypsin and trypsin but inhibited
with high potency cysteine proteases such as papain and cathepsin L. Direct binding experiments confirmed the interaction of recombinant
HBV-BP with the HBV preS1 domain. HepG2 cells overexpressing HBV-BP
after transfection of corresponding cDNA showed a virus binding
capacity increased by 2 orders of magnitude compared with untransfected
cells, while Chinese hamster ovary cells, which normally do not bind to
HBV, acquired susceptibility to HBV binding after transfection. Native
HBV particle entry was enhanced in transfected cells. Both recombinant
HBV-BP and antibodies to recombinant HBV-BP blocked virus binding and
internalization in transfected cells as well as in primary human
hepatocytes in a dose-dependent manner. Our findings
suggest that this protein plays a major role in HBV infection.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
chain and the preS1 domain of the
virus (7, 8), and the receptor for interleukin-6, due to the
presence of a recognition site for the preS1 domain on interleukin-6
(9, 10). In addition, HBV particles bind isolated asialoglycoprotein
receptors through the HBV preS1 domain (11), whereas the preS2 domain
seems involved in the interaction with the transferrin receptor (12).
It is also known that the preS region of the duck HBV, another member of the hepadnaviruses family, binds to a 170-kilodalton cellular protein having significant similarities to human and animal
carboxypeptidases H, M, and N (13). Other proteins have been identified
that bind to different domains of the HBV envelope proteins with a
proposed role as mediators in HBV attachment to the hepatocyte.
Apolipoprotein H (14) binds to the S domain, whereas polymerized human
serum albumin (15) and a human soluble serum factor (16) bind to the
preS2 domain of HBsAg. Annexin V, previously known as endonexin II (17,
18), binds with low affinity to HBsAg, but interaction involves HBsAg
lipids and not HBV proteins (19). More recently, an 80-kDa protein that
binds to the preS1 domain of HBV has been isolated but not identified.
In addition, this protein has been detected in rat hepatocytes, which
are not susceptible to HBV infection (20). These results are
conflicting, and no conclusive and convincing biological data have been
obtained to fully demonstrate the functional role of these proteins in
HBV binding and internalization. None of these proteins has been
obtained in a recombinant form to show inhibition of HBV binding to and
internalization on primary human hepatocytes.
EXPERIMENTAL PROCEDURES
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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-counter Isomedic 10/600 ICN).
31/7 and
+1355/+1384 regions of squamous cell carcinoma antigen 1 (SCCA1)
mRNA, respectively) and Taq polymerase (PerkinElmer Life
Sciences). Purified PCR-1 products were used as template for a more
internal pair of primers in PCR-2 (primer-3 (sense)
(5'-GTTCACCATGAATTCACTCAGTGAAGCC); primer-4 (antisense) (5'-GCAATCAGTTTACCAGAACATCTGCAG) complementary to 7/+21 and
+1212/+1238 regions of SCCA1 mRNA, respectively) performed with
Pwo DNA polymerase (Roche Molecular Biochemicals) to obtain
blunt end amplified products that were used directly in ligation with
pUC18 vector digested with SmaI restriction endonuclease.
XL1-blue-MRF' (Stratagene) CaCl2-competent cells have been
transformed with ligated DNA and plated on LB plates containing
ampicillin,
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside, and
isopropyl--D-thiogalactopyranoside for selection of
recombinant white colonies. The sequence of the insert of the
constructed plasmid pUC18/HBV-BP was determined using the AmpliTaq FS
Prism ready reaction cycle sequencing kit (PerkinElmer Life Sciences) electrophoresed and assembled on a 373 DNA sequencer (PerkinElmer Life Sciences).
1/+24
HBV-BP cDNA) and the universal sequencing primer M13 as antisense
primer to amplify also the vector polylinker sequence that carries a recognition sequence for BamHI endonuclease located
immediately at the 3' side of the HBV-BP cDNA insert. The reaction
was performed with Pwo DNA polymerase to obtain blunt end
products. The amplified product was BamHI-digested and
ligated with the pET21a vector. The ligation product was then used to
transform the E. coli XL1-blue-MRF' strain (Stratagene). The
sequence of the insert of the constructed plasmid pET21a/HBV-BP was
determined as described above. The constructed expression vector
pET21a/HBV-BP was then used to transform the E. coli strain
BL21(DE3) (Novagen) suitable for expression.
-D-thiogalactopyranoside, and the culture was
incubated at 30 °C. The maximal level of recombinant protein
production was reached after 3 h from the induction.
80 °C.
-benzoyl-L-arginine and
N-benzoyl-L-tyrosine, were diluted in PBS buffer
(150 mM NaCl, 50 mM
NaH2PO4, pH 7.5) and were used with the enzymes
-chymotrypsin and trypsin, respectively. The fluorogenic substrate,
used with the enzymes cathepsin L and papain, was diluted in the enzyme
reaction buffer containing 50 mM sodium acetate (pH 5.5), 4 mM dithiothreitol, 1 mM EDTA. The fluorogenic
substrate was a bisamide derivative of rhodamine 110, bis-(CBZ-L-phenylalanyl-L-arginine amide),
dihydrochloride (Rh110). The amount of the monoamide liberated was
determined fluorometrically with excitation and emission wavelengths of
499 and 521 nm, respectively, using a Shimadzu spectrofluorimetric detector, model RF-551. Protease inhibition by rHBV-BP was determined by use of a continuous assay procedure. Proteases were mixed with different concentrations of rHBV-BP, and the samples were incubated for
30 min at 25 °C in the appropriate reaction buffer, as described. The reaction was started by the addition of protease-specific substrate. The amount of carboxylic acid liberated from the hydrolysis of the photometric substrates was continuously recorded at a fixed wavelength (253 nm for
N-benzoyl-L-arginine and 256 nm
for N-benzoyl-L-tyrosine) using a Beckman
spectrophotometer, model DU 640.
492 >3. Samples containing 106 HepG2 nontransfected cells or HepG2/HBV-BP
cells were incubated with enriched HBV particle preparations (5 × 106 genome equivalents/sample) for 90 min at 4 °C,
washed with phosphate-buffered saline containing 10% fetal calf serum
(PBS plus 10% FCS), and incubated with rabbit anti-HBs antiserum
(Behringwerke, Marburg, Germany) for 20 min. After an additional wash
with PBS plus 10% FCS, FITC-conjugated goat anti-rabbit antiserum
(Dako) was added at a 1:10 concentration for 20 min at 4 °C. Similar
conditions were used in inhibition experiments where HepG2/HBV-BP cells
were simultaneously incubated with HBV particles in the presence of 18 pg of rHBV-BP. Cells were successively washed before the analysis. As
control for the fluorescence-activated cell sorter (FACS) procedure cells were directly incubated with anti-HBs antiserum, washed, and then
incubated with the FITC-conjugated anti-rabbit antibody. Cells were
then scored using a FACScan analyzer (Becton Dickinson), and data were
processed using CELLQuest software program (Becton Dickinson). Mean log
fluorescence intensity (MFI) values were obtained by subtracting the
MFI of the control from the MFI of the samples. To evaluate whether the
differences between the different samples were statistically
significant, the Kolmogorov-Smirnov test for analysis of histograms was used.
1 insulin, 100 units ml1 penicillin, and
100 µg ml1 streptomycin.
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Fig. 1.
Characterization of 4-preS1-(21-47) binding
to HepG2 PM by RIAs. A, schematic structure of the
4-preS1-(21-47) peptide used for HBV-BP affinity purification.
B-D, characterization of 4-preS1-(21-47) binding to HepG2
PM by RIAs, performed with the synthetic HBV antigens labeled with
125I. Specific binding was determined by subtraction of
values obtained from binding to plates coated with 10% BSA or plasma
membrane preparations deriving from unrelated cell lines. The results
shown represent the means of three independent experiments.
B, binding of 125I-4-preS1-(21-47) (specific
activity, 8 × 106 cpm/µg) to HepG2 PM coated on
microtiter plates at different concentrations: 1 ( 
), 5 (
), 25 (
), and 100 (
) µg/ml and to control Rat-1 membranes (
) at
100 µg/ml. C, binding of synthetic antigens
125I-4-preS1-(21-47) () and
125I-preS1-(21-47) (specific activity, 1.5 × 106 cpm/µg) () to HepG2 PM (100 µg/ml) on microtiter
plates. D, inhibition of 125I-4-preS1-(21-47)
binding to HepG2 PM (10 µg/ml) on microtiter plates by
4-preS1-(21-47) (), HepG2 PM treated with Triton (), and 10%
BSA ().
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Fig. 2.
Affinity purification of HBV-BP.
A, SDS-polyacrylamide gel electrophoresis analysis of bound
fraction (lane 1) from the affinity
chromatography purification of HepG2 PM solubilized with Triton X-100
(10 µg; lane 2) on the
4-preS1-(21-47)-Sepharose column. The proteins were separated on
polyacrylamide gel (12%) and stained by the Silver Stain method.
B, HepG2 PM affinity purification monitored by RIA. Samples
corresponding to HepG2 PM, HepG2 PM solubilized with Triton X-100
(loaded), unretained and bound fraction from the affinity
purification step, were coated on microtiter plates at the same
concentration (10 µg/ml); 125I-4-preS1-(21-47) was added
(160,000 cpm/well), and the recovered cpm were determined counting
directly the wells in a -counter.
31/7 and +1355/+1384 of SCCA1 mRNA sequence produced on agarose
gel only a smear without any discrete DNA band. The purified products
of the PCR-1 reaction were then utilized to perform a second PCR made
with a more internal primer pair that recognized the regions 7/+21
(sense primer 3) and +1212/+1238 (antisense primer 4); the agarose gel
analysis of PCR-2 showed a single DNA fragment of the expected size
(not shown). In order to obtain blunt end products, the PCR-2 was
carried out with the Pwo DNA polymerase. The amplified DNA
fragment was purified and cloned in the pUC18 vector restricted with
SmaI endonuclease. The sequence of cloned cDNA (Fig.
3) showed an open reading frame of 1173 bases encoding for a protein of 390 amino acids with a predicted
molecular mass of 44 kDa, containing the sequences of the two tryptic
fragments previously determined by protein microsequencing. The
nucleotide sequence of HBV-BP cDNA was strictly correlated to the
SCCA1 nucleotide sequence found by Suminami (37), with only four
nucleotide substitutions in the coding region leading to three amino
acid substitutions, Gly351 to Ala, Ala357 to
Thr, and Ser389 to Pro. One of these substitutions,
Ala357 to Thr, has been reported previously for the SCCA1
sequence. To confirm that the observed nucleotide differences were not
due to incorrect incorporation during amplification cycles, PCR
experiments on human genomic DNA with proofreading DNA polymerase
Pwo were carried out. Since the nucleotide substitutions
determining the three amino acid changes were located in the last exon
of the SCCA1 gene, two primers amplifying a DNA fragment encompassing the nucleotide substitutions were used. The obtained amplification products were cloned using the primer sense corresponding to the +770/+798 region and the primer antisense corresponding to the +1187/+1212 region of coding sequence. Twelve independent recombinant clones were sequenced, showing that the three sequences corresponding to SCCA1, SCCA2, and HBV-BP were equally represented. Analysis of the
deduced amino acid sequence (www.expasy.ch) indicated that the sequence
encompassing residues 27-45 could constitute a transmembrane domain.
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Fig. 3.
Nucleotide sequence and predicted amino acid
sequence of HBV-BP. Nucleotide sequence of the cDNA for the
HBV-BP from the position 7 to +1238; uppercase
letters refer to the four nucleotide differences from SCCA1
cDNA. The boxed amino acids in
yellow refer to the two tryptic fragments sequenced, and the
three deduced amino acid differences from the SCCA1 sequence are
boxed in blue. The two regions, interhelical loop
(amino acids 82-103) and reactive center loop (amino acids 344-364)
are underlined. The putative reactive site P1-P1' is
underlined twice. Putative
N-glycosylation sites are circled. -helical
regions are boxed in green, while structures
are boxed in orange. The predicted transmembrane
region is boxed in black.
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Fig. 4.
Expression and characterization of
recombinant HBV-BP. A, ionic exchange chromatography
profile (anionic exchange Source 30S resin; 25 × 0.5-cm inner
diameter) of the fractionation of bacterial extracts. The column was
equilibrated with 20 mM MES, pH 6.0, at a flow rate of 3.0 ml/min. Following the elution of unretained material, a gradient from 0 to 100% of 20 mM MES, 0.5 M NaCl, pH 6.0, in
20 min was applied to elute bound proteins. B,
SDS-polyacrylamide gel electrophoresis analysis of loaded sample,
unretained material, and material eluted by the buffer change. The
protein was visualized by Coomassie staining. C, Western
blot analysis of bound fraction of the HBV-BP affinity purification
(Bound) (see Fig. 2A) and of the recombinant
HBV-BP obtained from E. coli. D, binding of
myristyl-preS1 to recombinant HBV-BP. Recombinant HBV-BP was coated on
microtiter plates at 10 µg/ml (100 µl/well) in 0.1 M NaHCO3, pH 8.8, overnight at 4 °C.
Synthetic myristyl-preS1 was added at different concentrations (0.2-20
µg/ml) and incubated for 1 h at 37 °C. Complex formation was
detected by adding an anti-preS1 monoclonal antibody (T0606; Sorin
Biomedica). Four replicate assays were carried out, and means ± S.D. are shown.
-chymotrypsin and trypsin (not shown). These data confirmed recent reinvestigations of SCCA1 activity, where it was found that unlike SCCA2, SCCA1 lacked inhibitory activity against any of the more common
types of serine proteinases but was a potent cross-class inhibitor of
the archetypal lysosomal cysteine proteinases cathepsins K, L, and S
(41).
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Fig. 5.
Kinetic analysis of HBV-BP interaction with
cysteine proteases. Proteases were incubated with different
concentrations of HBV-BP for 30 min at 25 °C in the reaction buffer
(50 mM sodium acetate, pH 5, 5/4 mM
dithiothreitol, 1 mM EDTA). The reaction was started by the
addition of protease-specific substrate Rh110 (0.5 µM),
and the progress of inactivation of the proteases was followed by
measuring the relative fluorescence (r.f.u.) over time.
A, inhibition of papain activity (200 nM) ( )
by HBV-BP at 25 (
), 50 (), 100 (
), 200 (*), and 400 nM () concentration. B, inhibition of
cathepsin L activity (36 nM) () by HBV-BP at 3.6 (),
9 (*), 36 (), and 360 nM () concentration.
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Fig. 6.
Detection of HBV-BP in transfected cell
lines. A, FACS analysis of HepG2/HBV-BP and HepG2 cells
by using anti-HBV-BP antibodies. B, enhancement of
I25I-4-preS1-(21-47) binding to HepG2/HBV-BP. 250,000 HepG2, Rat-1, and HepG2/HBV-BP cells were plated in 24-well plates.
48 h later, I25I-4-preS1-(21-47) peptide (300,000 cpm/well) diluted in binding buffer (culture medium specific for the
cell line containing 0.1% BSA and 50 mM Hepes, pH 7.4) was
added. After 1 h of incubation, cells were extensively washed with
PBS supplemented with 0.5 mM CaCl2 and 0.5 mM MgCl2 and lysated with 40 mM Hepes, pH 7.4, 10% glycerol, and 10% Triton X-100. The
cell lysates were recovered, and the amount of iodinated antigens bound
to the cells was determined.
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Fig. 7.
Co-localization of HBV-BP and HLA in
HepG2/HBV-BP cells. Shown are HBV-BP immunostaining with
polyclonal antibody anti-HBV-BP (Aa, Ba), HLA
immunostaining with monoclonal antibody W6/32 (Ab,
Bb), overlapping of signals obtained in Aa and Ab
(Ac) and Ba and Bb (Bc), and image of a cell
obtained in a normal inverted microscope (Ad).
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Fig. 8.
Recombinant HBV-BP inhibits HBV binding to
transfected cell lines and primary human hepatocytes.
A, adsorption of enriched HBV particle preparation (5 × 106 genome equivalents/sample) to HepG2 and to
HepG2/HBV-BP cells (106) as detected by FACS by incubation
with rabbit anti-HBs serum (Behringwerke) followed by treatment with
FITC-conjugated goat anti-rabbit antiserum (Dako). In inhibition
experiments, HepG2/HBV-BP cells were simultaneously incubated with HBV
particles in the presence of 18 pg of recombinant HBV-BP. Cells were
successively washed before the analysis. As control for the FACS
procedure, cells were directly incubated with anti-HBs antiserum,
washed, and then incubated with the FITC-conjugated anti-rabbit
antibody. B, quantitative determination of HBV DNA in
cellular extracts by a solution hybridization assay (Digene Hybrid
Capture HBV DNA assay). C, inhibition of HBV (23 × 107 viral genome equivalents) attachment to HepG2/HBV-BP
cells (black), CHO/HBV-BP cells (white), and
primary human hepatocytes (gray) by serial dilutions of
affinity-purified anti-HBV-BP polyclonal antibodies. The
dashed bar corresponds to the effect of control
antibodies derived from rabbit preimmune sera on HBV attachment to
HepG2/HBV-BP cells. No inhibition was observed also for CHO/HBV-BP
cells and primary human hepatocytes.
Enhancement of HBV binding to HBV-BP cDNA-transfected cells
View larger version (24K):
[in a new window]
Fig. 9.
Viral entry in HBV-BP transfected cells.
A, HBV DNA detected by PCR in cellular DNA extracts of
trypsinized (lane 1) and nontrypsinized
(lane 2) HepG2/HBV-BP cells previously incubated
with HBV at 4 °C and in the corresponding trypsin washes
(lanes 5 and 6, respectively).
Lanes 3 and 4 correspond to HBV DNA
amplification of DNA extracted from control cells treated
(lane 3) or not treated (lane
4) with trypsin without HBV addition, while lane
7 corresponds to PCR amplification of DNA extracted from
HBV-containing medium. B, detection of HBV DNA in the
cytoplasmic fraction of HepG2 and HepG2/HBV-BP cells by Southern blot
analysis. The lane labeled Trypsin
wash refers to DNA extracted from trypsin solution after
treatment of HepG2/HBV-BP cells incubated with HBV, and
lanes labeled PBS washes correspond to
two consecutive washes of the same cells after the trypsin
treatment.
View larger version (33K):
[in a new window]
Fig. 10.
Inhibition of viral entry in HBV-BP
transfected cells and primary human hepatocytes. Inhibition of HBV
(22 × 107 genome equivalents) entry in HepG2/HBV-BP
cells (A) and in primary human hepatocytes
(B) by recombinant HBV-BP and by anti-HBV-BP antibodies (C).
0 indicates HBV DNA detected in the control samples, where
HBV particles were incubated with PBS, instead of different
concentrations of rHBV-BP or anti-HBV-BP antibodies, before incubation
with cultured cells. 446 base pairs is the expected size of the
amplified PCR product.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-antitrypsin, consisting of nine -helices and three
-sheets arranged in a stressed configuration with the reactive
center, which has the unusual feature of being the most
variable region, located in an exposed loop. When serpins bind to their
protease target, the inhibition occurs by a profound conformational
change initiated by reaction of the protease active site with the
reactive center of the serpin (50). HBV-BP does inhibit cysteine
proteinases such as cathepsin L and papain, and studies are in progress
in order to further characterize this activity. This property could be
very important for the proposed biological role of this protein as an
HBV-binding protein, since it could be speculated that receptors involved in HBV binding might lose their ability to inhibit
proteases, thus creating an area on the cell surface where proteases
can act, thus facilitating cell infection. Very often, the entry of enveloped viruses in the cells requires a proteolytic digestion of
envelope protein close to a fusion domain; the cleaved form of the
envelope protein allows the fusion of virus with PM or, after
endocytosis, with the endosomal membrane (51, 52).
1-antichymotrypsin is the only other serpin known to
contain a Ser-Ser at the reactive site (53). HBV-BP represents the
first Ov-serpin differing from all the others for the presence of a
proline residue in position 389, which is one of the three amino acids
differing from the SCCA1 sequence. The other two amino acid differences
between SCCA1 and HBV-BP, Gly351 to Ala and
Ala357 to Thr, are both localized within the reactive
center, and the last is particularly interesting, since the
Thr357 residue could represent a new putative site of glycosylation.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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