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Journal of Virology, January 2000, p. 110-116, Vol. 74, No. 1
0022-538X/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
An 80-Kilodalton Protein That Binds to the Pre-S1
Domain of Hepatitis B Virus
Chun Jeih
Ryu,1
Dae-Yeon
Cho,1
Philippe
Gripon,2
Hee Sun
Kim,1
Christiane
Guguen-Guillouzo,2 and
Hyo Jeong
Hong1,*
Antibody Engineering Research Unit, Korea
Research Institute of Bioscience and Biotechnology, Yuseong, Taejon
305-600, Korea,1 and Hepatologique U49,
Institut National de la Santé et de la Recherche
Médicale, Hôpital de Pontchaillou, 35033 Rennes Cedex,
France2
Received 8 June 1999/Accepted 22 September 1999
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ABSTRACT |
It has been suggested that hepatitis B virus (HBV) binds to a
receptor on the plasma membrane of human hepatocytes via the pre-S1
domain of the large envelope protein as an initial step in HBV
infection. However, the nature of the receptor remains controversial.
In an attempt to identify a cell surface receptor for HBV, purified
recombinant fusion protein of the pre-S1 domain of HBV with glutathione
S-transferase (GST), expressed in Escherichia coli, was used as a ligand. The surface of human hepatocytes or HepG2 cells was biotinylated, and the cell lysate (precleared lysate)
which did not bind to GST and glutathione-Sepharose beads was used as a
source of receptor molecules. The precleared lysate of the biotinylated
cells was incubated with the GST-pre-S1 fusion protein, and the bound
proteins were visualized by Western blotting and enhanced
chemiluminescence. An approximately 80-kDa protein (p80) was shown to
bind specifically to the pre-S1 domain of the fusion protein. The
receptor binding assay using serially or internally deleted segments of
pre-S1 showed that amino acid residues 12 to 20 and 82 to 90 are
essential for the binding of pre-S1 to p80. p80 also bound specifically
to the pre-S1 of native HBV particles. Analysis of the tissue and
species specificity of p80 expression in several available human
primary cultures and cell lines of different tissue origin showed that
p80 expression is not restricted to human hepatocytes. Taken together
the results suggest that p80 may be a component of the viral entry machinery.
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INTRODUCTION |
Hepatitis B virus (HBV) causes acute
and chronic hepatitis and is an etiological agent for hepatocellular
carcinoma (6). HBV infection is highly species specific;
only humans and closely related species are known to be susceptible to
HBV infection (32). The tissue and species specificity of
HBV infection suggests the preferential attachment and entry of HBV
into hepatocytes as an initial step in HBV infection and/or the
existence of host- and liver-specific regulatory elements for viral
replication. However, little is known about the molecular mechanism of
the initiation of HBV infection.
The HBV envelope proteins have been considered crucial molecules in
recognizing a possible receptor on the plasma membrane of human
hepatocytes (22). The envelope consists of three distinct related proteins, designated small (S), middle (M), and large (L)
(24). All of these proteins have a common carboxyl-terminal 226-amino-acid sequence (S protein). M protein has an additional 55-amino-acid (pre-S2) sequence located at the amino terminus; beyond
this segment, L protein contains an additional 108- or 119-amino-acid
(pre-S1) sequence (depending on subtype ay or ad, respectively). Neurath et al. (23) postulated that HBV
particles bind to human hepatocytes and hepatoma HepG2 cells via
pre-S1(21-47) (a peptide consisting of amino acids [aa] 21 to 47 of
pre-S1). Pontisso et al. (28) also showed that L protein
bound to human liver membrane and that a monoclonal antibody specific
to pre-S1(21-47) inhibited this binding. However, the identity of the
putative receptor that binds to pre-S1(21-47) remains controversial.
Human immunoglobulin A (IgA) receptor (29),
glyceraldehyde-3-phosphate dehydrogenase (27), interleukin-6
(25), asialoglycoprotein receptor (34), a 31-kDa
protein (4), and a serum glycoprotein of 50 kDa
(3) have been proposed as possible receptors, but the nature
of the interaction of HBV with the putative receptors in HBV infection
has not been confirmed.
In this study, in an attempt to identify an HBV receptor on human
hepatocytes, a recombinant fusion protein of the pre-S1 or pre-S
(preS1/preS2) domain with glutathione S-transferase (GST) was produced from Escherichia coli and used as a ligand, the
surface of human hepatocytes or hepatoma HepG2 cells was biotinylated, and the cell lysate was used as a source of receptor molecules. Through
the ligand-receptor binding experiments, we have identified an 80-kDa
protein (p80) on the cell surface that specifically binds to the pre-S1
domain of HBV. The p80 binding sites on the pre-S1 domain were mapped,
and the tissue and species specificity of p80 expression was analyzed.
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MATERIALS AND METHODS |
Cell culture.
Adult human hepatocytes were prepared by
enzymatic dissociation of a noncancerous liver fragment
(10). All experimental procedures were in compliance with
French laws and regulations and were approved by the National Ethics
Committee. Rat hepatocytes were prepared by the same method. Freshly
isolated human and rat hepatocytes were cultured in medium for human
hepatocytes (H medium) (75% minimal essential medium [Gibco], 25%
medium 199 [Gibco], insulin [5 µg/ml], bovine serum albumin
[BSA; 1 mg/ml], antibiotic-antimycotic [Gibco]) supplemented with
5 × 10
6 M hydrocortisone hemisuccinate, 2%
dimethyl sulfoxide, and 10% porcine serum (9). HepG2 cells
were cultured in H medium supplemented with 5 × 107
M hydrocortisone hemisuccinate and 10% fetal bovine serum. Human oral
epidermal and dermal fibroblasts primary cultures were prepared and
maintained as described previously (5, 8). Human peripheral blood mononuclear cells (PBMC) were obtained by Ficoll-Hypaque density
gradient centrifugation from healthy human plasma. Human embryonic
kidney (HEK) and HeLa cell lines were grown in Dulbecco modified Eagle
medium (Gibco) supplemented with 10% fetal bovine serum.
Construction and expression of GST fusion proteins containing
pre-S1, pre-S, or partial pre-S1.
The coding sequence of pre-S,
pre-S1, or partial pre-S1 was synthesized from pHBV315 (adr
subtype [15]) or ayw subtype plasmid (14) by PCR using 5' and 3' primers and subcloned into the
BamHI or BamHI-EcoRI site of plasmid
pGEX-2T (Pharmacia), respectively. To construct the pre-S1 mutants with
internal deletion, the 5'- and 3'-end fragments, which do not contain
the internally deleted segment, were synthesized by PCR from their
wild-type plasmid counterparts and recombined by subsequent recombinant
PCR (20). The 5' primer contained a BamHI site,
and the 3' primer contained a BamHI or EcoRI
site. The GST fusion proteins were expressed and purified according to
the protocol provided by the supplier (Pharmacia). The fusion proteins
were expressed in E. coli DH5
by induction with 0.1 mM
isopropyl-
-D-thiogalactopyranoside for 3 h. The
intracellular soluble proteins were applied to glutathione-Sepharose beads, and the bound proteins were eluted with 5 mM reduced glutathione and subsequently dialyzed against phosphate-buffered saline (PBS) buffer.
Cell surface biotinylation.
Human and rat hepatocytes,
HepG2, HEK, and HeLa cells, human oral epidermal and dermal fibroblast
primary cultures, and PBMC were biotinylated by using an ECL (enhanced
chemiluminescence) protein biotinylation system (Amersham). The
cultured cells in a 75-cm2 flask were washed twice with
ice-cold PBS, mixed with 4 ml of bicarbonate buffer containing 160 µl
of biotinylation reagent (biotinamidocaproate
N-hydroxysuccinamide ester in dimethylformamide) at 4°C
for 30 min, and then washed again twice with ice-cold PBS. The cells
were treated with 4 ml of lysis buffer (25 mM Tris-HCl, [pH 7.5], 250 mM NaCl, 5 mM EDTA [pH 8.0], 1% Nonidet P-40, aprotinin [2
µg/ml], phenylmethylsulfonyl fluoride [100 µg/ml], leupeptin [5
µg/ml]) at 4°C for 20 min. Nuclei were removed by centrifugation, and the cell lysates were stored at 
70°C before use. The protein concentration in the cell lysates was quantitated by using a
bicinchoninic acid protein assay kit (Pierce).
Detection of pre-S1-binding protein.
To remove the cellular
proteins which nonspecifically bind to GST protein and
glutathione-Sepharose beads, 1 ml of the biotinylated cell lysate was
incubated with 20 µg of GST protein at 4°C for 5 h, which was
then added to 20 µl of 50% glutathione-Sepharose beads. After
incubation overnight, the precleared lysate was recovered and the beads
were extensively washed with lysis buffer for use as negative controls
for the binding experiment. The precleared lysate was incubated with 20 µg of GST-pre-S, GST-pre-S1, or mutant GST-pre-S1 fusion protein
at 4°C for 5 h and further incubated with the
glutathione-Sepharose beads as described above. To inhibit the
interaction between the pre-S1 and cellular proteins, various amounts
of purified pre-S1 (13), pre-S1 peptide, or pre-S1-specific monoclonal antibodies (KR127 and KR359) (C. J. Ryu, Y. K. Kim, H. S. Kim, H. Hur, Y. J. Kang, and H. J. Hong,
submitted for publication) were individually added to the mixture
before incubation with the beads. The beads were extensively washed
with lysis buffer, and the bound proteins were eluted from the beads by
heating to 100°C for 5 min. The precleared lysate or eluted proteins
were fractionated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) on a 10% polyacrylamide gel under
denaturing conditions and transferred to a nitrocellulose membrane. The
membrane was immersed in PBST (PBS containing 0.1% [vol/vol] Tween
20)-5% skim milk at room temperature for 1 h. After two rinses
with PBST, the membrane was incubated with streptavidin-horseradish
peroxidase (HRP) conjugate (1:1,500 [vol/vol]; Amersham) at room
temperature for 1 h. After washing, the biotinylated proteins were
visualized by ECL Western blotting detection reagent (Amersham).
Preparation of HBV particles.
HBV particles were purified
from HBsAg- and HBeAg-positive human sera as described by Robinson and
Greenman (31), with some modifications. Briefly, the sera
were layered onto a 10 to 30% discontinuous sucrose gradient, prepared
in TS-BSA buffer (0.01 M Tris, 0.5 M NaCl, 0.1% NaN3, 1%
BSA [pH 8.0]), and centrifuged in a Beckman SW41 rotor at 36,000 rpm
for 4 h. The pellet was suspended in TS-BSA buffer and centrifuged
again under the same conditions. The new pellet was resuspended in
one-half of the starting volume in TS-BSA buffer and stored at 70°C
before use. Viral genomic equivalents (vge) of the prepared viral
particles were quantitated by DNA dot blot assay with HBV genome
(15) as the standard.
Binding of p80 to HBV particles.
One milliliter of the
biotinylated HepG2 cell lysate was incubated with 30 µl of 10%
protein A-Sepharose (Amersham) at 4°C overnight. The precleared
lysate was recovered and incubated with 100 µl of mock preparation
(TS-BSA) or HBV particles (1011 vge) at room temperature
for 2 h. In a separate experiment, the HepG2 cell lysate was
preincubated with 100 µg of pre-S1 peptides (aa 12 to 20 and 73 to
90) at room temperature for 20 min prior to HBV addition. The mixture
was immunoprecipitated with 10 µg of anti-pre-S1 antibody KR127 and
30 µl of 10% protein A-Sepharose. After extensive washing with PBS,
the immunocomplex was subjected to SDS-PAGE (10% gel) and Western
blotting. The HBV-binding protein was visualized by ECL.
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RESULTS |
Identification of a protein (p80) on human hepatocytes that binds
specifically to the pre-S1 domain of HBV.
GST-pre-S1
(adr subtype, 119 aa) and GST as a negative control were
expressed, purified from E. coli, and used as a ligand to
search for HBV cell receptors. As a source of receptor, the surface of
human hepatocytes was biotinylated and the cell lysate was prepared. To
identify the pre-S1-binding protein, the purified GST protein was
incubated with the biotinylated cell lysate and further incubated with
glutathione-Sepharose beads. The unbound proteins (precleared lysate)
were then incubated with GST-pre-S1 followed by incubation with
glutathione-Sepharose beads. Subsequently, the bound proteins were
eluted from the beads by heating to 100°C and subjected to SDS-PAGE
(10% gel) followed by Western blot analysis using streptavidin-HRP
conjugate. The pre-S1-binding protein was visualized by ECL. As shown
in Fig. 1A, an approximately 80-kDa protein (p80) bound to the GST-pre-S1 beads (lane 2), but not to the
GST-glutathione-Sepharose beads (lane 1). In the same binding experiment, the lysate of unbiotinylated human hepatocytes was used as
a negative control. As expected, p80 was not detected in the eluent of
the GST (lane 3)- or GST-pre-S1 (lane 4)-immobilized beads. The
GST-pre-S1 protein used as a ligand was detected (lanes 2 and 4),
possibly due to the nonspecific binding of streptavidin-HRP conjugate
to the pre-S1 of the GST-pre-S1 protein on the membrane.
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FIG. 1.
An 80-kDa cell surface protein binds to the pre-S1
domain of the GST-pre-S1 fusion protein. Cultured human hepatocytes
(A) and HepG2 cells (B) were biotinylated. The cell lysates were
incubated with GST and further incubated with glutathione-Sepharose
beads. After the Sepharose beads were extensively washed with lysis
buffer, the bound proteins were eluted by heating to 100°C for 5 min,
subjected to SDS-PAGE (10% gel) and Western analysis using
streptavidin-HRP conjugate, and visualized by ECL (lane 1). The unbound
proteins (precleared lysate) were incubated with GST-pre-S1 fusion
protein followed by glutathione-Sepharose beads. The bound proteins
were then analyzed as described above (lane 2). As a negative control,
cell lysates of unbiotinylated human hepatocyte were incubated with GST
(lane 3) or GST-pre-S1 (lane 4) and then analyzed as described above.
Molecular size markers (M; in kilodaltons) are shown on the left, and
the position of p80 is indicated on the right.
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Since human hepatocytes are not easily available, human hepatoma cell
line HepG2, which was shown to have the biosynthetic
capabilities of
normal liver parenchymal cells (
1,
16) and
binds to HBV
(
23), was used for subsequent binding study. HepG2
cells
were grown in H medium to support the maintenance of
hepatocyte-specific
differentiated function because these cells tend to
be dedifferentiated
with extended time in culture in ordinary culture
medium (
30);
the biotinylated cell lysate was incubated with
GST or GST-pre-S1
protein. As shown in Fig.
1B, HepG2 cells also
expressed p80 that
bound to the pre-S1 domain (lane 2) but not to GST
(lane
1).
To further confirm the specificity of the binding of p80 to pre-S1, the
precleared lysate of HepG2 cells was incubated with
GST-pre-S in the
presence of increasing amounts of free pre-S1.
The result showed that
the interaction between GST-pre-S and p80
was competitively inhibited
by pre-S1 in a dose-dependent manner
and completely inhibited by 100 µg of pre-S1 (Fig.
2). This result
demonstrates that p80 binds to the pre-S1 domain specifically
but not
to the pre-S2 domain.
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FIG. 2.
Competitive inhibition of p80 binding of GST-pre-S1 by
pre-S1. The precleared lysate of biotinylated HepG2 cells was incubated
with GST-pre-S fusion protein in the presence of 0 (lane 1), 1 (lane
2), 2 (lane 3), 5 (lane 4), 10 (lane 5), 25 (lane 6), 50 (lane 7), or
100 (lane 8) µg of purified pre-S1 polypeptide, and the binding
experiment was done as described for Fig. 1. Molecular size markers (M;
in kilodaltons) are shown on the left, and the position of p80 is
indicated on the right.
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The biotinylation reagent used in this study is reported to be too
bulky to penetrate the surface of viable cells (
19).
Since
we biotinylated the hepatocytes attached to dishes after
removal of
dead cells by extensive washes, p80 is likely to be
expressed on the
cell surface. Nevertheless, specificity of the
cell surface
biotinylation was assessed by using a known intracellular
protein,
human Cdc2 (34 kDa), as an internal control. The biotinylated
HepG2
cell lysate was immunoprecipitated with rabbit anti-human
Cdc2 antibody
and protein A-Sepharose and subjected to Western
analysis using
streptavidin-HRP (Fig.
3, lane 3) or
rabbit anti-human
Cdc2 antibody and anti-rabbit IgG-HRP (lane 5). Cdc2
was immunoprecipitated
with anti-Cdc2 antibody (lane 5) but not with
streptavidin-HRP
(lane 3), while p80 was detected with streptavidin-HRP
(lane 1).
The result indicates that Cdc2 was not accessible to the cell
surface biotinylation reagent, providing indirect evidence that
the
cell surface biotinylation is potentially cell surface specific.
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FIG. 3.
Cell surface location of p80. Biotinylated HepG2 cell
lysates were divided into three fractions; one was subjected to the
pre-S1 binding assay (lane 1), and the other two were incubated with
protein A-Sepharose. The precleared lysate was immunoprecipitated with
rabbit anti-human Cdc2 antibody (Santa Cruz Biotechnology) and protein
A-Sepharose. The precleared supernatant (lane 2 and 4) and
immunoprecipitates (lane 3 and 5) were subjected to Western analysis
using streptavidin-HRP (lanes 1 to 3) or rabbit anti-human Cdc2
antibody and anti-rabbit IgG-HRP (lanes 4 and 5). The bands
corresponding to 50 and 25 kDa are likely the heavy and light chains,
respectively, of rabbit anti-human Cdc2 antibody (lane 5). Molecular
size markers (M; in kilodaltons) are shown in the middle, and the
positions of p80 and Cdc2 are indicated.
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Recently, it was shown that cytosolic heat shock protein Hsc70 binds to
the pre-S1 region (aa 70 to 94 of the
ayw subtype),
which is
likely to be responsible for the repression of cotranslational
translocation of the pre-S domain (
21). To test whether p80
is Hsc70, the biotinylated proteins bound to the GST-pre-S1 were
eluted and subjected to Western blot analysis using streptavidin-HRP
or
mouse anti-Hsp/Hsc70 monoclonal antibody and anti-mouse IgG-HRP.
As
shown in Fig.
4, p80 did not react with
anti-Hsp/Hsc70 antibody
(lane 4), while a 70-kDa protein from HepG2
cell lysates reacted
with the antibody (lane 3). This result
demonstrates that p80
is not Hsp/Hsc70.
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FIG. 4.
p80 is not Hsc70. Twenty-microliter aliquots of
unbiotinylated HepG2 lysates (lanes 1 and 3) and the biotinylated
proteins bound to GST-pre-S1 (lanes 2 and 4) were subjected to Western
analysis using streptavidin-HRP (lanes 1 and 2) or mouse anti-Hsp/Hsc70
antibody (Santa Cruz Biotechnology) and anti-mouse IgG-HRP (lanes 3 and
4). Molecular size markers (M; in kilodaltons) are shown in the middle,
and the positions of p80 and Hsc70 are indicated.
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To rule out the possibility that p80 is a serum protein that bound
tightly to the surface of cultured hepatocytes, biotinylated
fetal
bovine serum was incubated with GST-pre-S1, and the bound
protein was
eluted and visualized by Western blot analysis. p80
was not detected
(data not shown). Also, the possibility of p80
being the serum protein
lactoferrin, whose molecular mass is approximately
80 kDa, was checked.
Bovine lactoferrin that was biotinylated
and subjected to the
GST-pre-S1 binding experiment failed to bind
to pre-S1 (data not
shown).
Binding of p80 to the two sites of pre-S1.
To locate the
binding sites for p80 on the pre-S1 domain, pre-S1 (adr
subtype) was cleaved into two fragments and the N-terminal fragment was
serially deleted from the C terminus. Each fragment was expressed as a
fusion protein with GST in E. coli, purified (Fig.
5A), and then incubated with the
precleared lysate of the biotinylated HepG2 cells to test its ability
to bind to p80. As shown in Fig. 5B, GST and GST-pre-S1(1-11) did not
bind to p80 (lanes 1 and 2), while GST-pre-S1(1-20, 1-28, 1-35, 1-42, 1-49, 1-56, 57-119, or 1-119) and GST-pre-S bound to p80 (lanes 3 to 11). The results indicate that pre-S1(12-20) is essential for the
binding and that pre-S1(57-119) also has the binding site for p80. To
map the binding site more precisely, GST-pre-S1(57-72, 57-90, 57-104, and 57-119) were expressed and purified (Fig. 5C), and the receptor
binding experiment was performed. As shown in Fig. 5D, p80 did not bind
to GST-pre-S1(57-72) (lane 2) but did bind to GST-pre-S1(57-90,
57-104, and 57-119) (lanes 3 to 5), indicating that pre-S1(73-90) is
involved in the binding of pre-S1 to p80. To further dissect this p80
binding site, GST-pre-S1(57-81) was additionally prepared and
subjected to the receptor binding assay, which showed that p80 did not
bind to pre-S1(57-81) (Fig. 5E, lane 4). Taken together, the results
led to the conclusion that pre-S1(12-20) and pre-S1(82-90) are
essential for p80 binding.
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FIG. 5.
Mapping of the p80 binding site on the pre-S1 domain.
The pre-S1 domain and serially truncated segments thereof were
expressed as fusion proteins with GST in E. coli and
purified (A and C). These proteins were separately incubated with the
precleared lysates of biotinylated HepG2 cells, and the receptor
binding assay was carried out (B, D, and E). (A and B) Lane 1, intact
GST; lane 2, pre-S1(1-11); lane 3, pre-S1(1-20); lane 4, pre-S1(1-28);
lane 5, pre-S1(1-35); lane 6, pre-S1(1-42); lane 7, pre-S1(1-49); lane
8, pre-S1(1-56); lane 9, pre-S1(57-119); lane 10, pre-S1(1-119); lane
11, pre-S. (C and D) Lane 1, GST; lane 2, pre-S1(57-72); lane 3, pre-S1(57-90); lane 4, pre-S1(57-104); lane 5, pre-S1(57-119). (E)
Lanes 1 and 3, GST; lane 2, pre-S1(1-119); lane 4, pre-S1(57-81).
Positions of molecular size markers (lanes M; in kilodaltons) and p80
are indicated.
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To further confirm the two p80 binding sites, pre-S1(12-20),
pre-S1(73-90), or both were deleted from GST-pre-S1(1-56),
GST-pre-S1(57-119),
or GST-pre-S1(1-119), respectively. These
mutant proteins were
prepared (Fig.
6A) and tested for p80 binding. As shown
in Fig.
6B, deletion of aa 12 to 20 from pre-S1(1-56) (lane 3), aa 73
to 90 from pre-S1(57-119) (lane 5), or both segments from pre-S1(1-119)
(lane 7) completely abolished the ability of pre-S1 to bind to
p80,
while the intact pre-S1, pre-S1(1-56), and pre-S1(57-119)
bound to p80
(lanes 2, 4, and 6). These results clearly demonstrate
that p80 binds
to the two sites of pre-S1.
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FIG. 6.
p80 binding of pre-S1 mutants with internally deleted
segments. (A) Amino acid residues 12 to 20 (lane 3), 73 to 90 (lane 5),
or both (lane 7) were deleted from GST-pre-S1(1-56) (lane 2),
GST-pre-S1(57-119) (lane 4), or GST-pre-S1(1-119) (lane 6),
respectively, and these wild-type and mutant proteins as well as GST
(lane 1) were expressed and purified. (B) Each protein was analyzed for
p80 binding. Molecular size markers (in kilodaltons) are shown in lane
M, and the position of p80 is indicated on the right.
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Our data are not consistent with the previous postulate that HBV binds
to human hepatocyte via pre-S1(21-47) (
23). To explore
this
issue further, we examined the binding of p80 to GST-pre-S1
in the
presence of different pre-S1 peptides (Fig.
7). The binding
of GST-pre-S1 to p80 was
strongly inhibited by either pre-S1(12-20)
or pre-S1(73-90) or both
peptides but not by pre-S1(21-47). The
inhibitory effect of anti-pre-S1
monoclonal antibodies on the
binding of pre-S1 to p80 was also examined
in assays using two
monoclonal antibodies, KR359 (epitope, aa 20 to 28)
and KR127
(epitope, aa 42 to 49) (Ryu et al., submitted). The binding
of
GST-pre-S to p80 was partially inhibited by KR359 in a
dose-dependent
manner (Fig.
8A) but was
not inhibited by KR127 (Fig.
8B). The
difference may be due to the fact
that the epitope of KR359 is
immediately adjacent to the first binding
site (aa 12 to 20),
while that of KR127 is distant from the both
binding sites. These
results clearly shows that p80 binds to two sites
other than pre-S1(21-47).
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FIG. 7.
Effect of pre-S1 peptides on the binding of p80 to
pre-S1. Biotinylated HepG2 lysates were incubated with GST (lane 1),
and precleared lysates were incubated with GST-pre-S1 fusion protein
in the presence of PBS (lane 2), 100 µg of peptide pre-S1(21-47)
(lane 3), 100 µg of peptide pre-S1(12-20) (lane 4), 100 µg of
peptide pre-S1(73-90) (lane 5), 50 µg of peptide pre-S1(12-20) plus
50 µg of peptide pre-S1(73-90) (lane 6), or 100 µg of peptide
pre-S1(12-20) plus 100 µg of peptide pre-S1(73-90) (lane 7), and the
binding experiment was done as described for Fig. 1. Molecular size
markers (M; in kilodaltons) are shown on the left, and the position of
p80 is indicated on the right.
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FIG. 8.
Effect of anti-pre-S1 monoclonal antibodies KR359 (A)
and KR127 (B) on the p80 binding of pre-S1. Precleared lysates of
biotinylated HepG2 cells were incubated with GST-pre-S1 fusion protein
in the absence (lane 1) or presence of 5 (lane 2), 10 (lane 3), 50 (lane 4), 100 (lane 5), or 200 (lane 6) µg of KR359 or KR127.
Positions of molecular size markers (M; in kilodaltons) and p80 are
indicated.
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The pre-S1 sequence of the
ay subtype of HBV is shorter than
that of the
ad subtype in that the first 11 aa of the
ad subtype
are not present in the
ay subtype. To
examine whether the pre-S1
domain of the
ay subtype also
binds to p80, a GST fusion protein
with pre-S1(1-45), pre-S1(46-108),
or pre-S1(1-108) of the
ayw subtype was prepared (Fig.
9A) and tested for p80 binding. As
shown
in Fig.
9B, p80 bound to pre-S1(1-45) (lane 2), pre-S1(46-108)
(lane
3), and pre-S1(1-108) (lane 4) of the
ayw subtype, but
binding
of pre-S1(1-45) to p80 was weaker than that of pre-S1(46-108).
Comparison of the strength of p80 binding between pre-S1(1-56)
of the
adr subtype (lane 2) and pre-S1(1-45) of the
ayw
subtype
(lane 3) showed that pre-S1(1-45) was weaker than pre-S1(1-56).
Taken together, these results indicate that p80 may bind to all
subtypes of HBV.
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FIG. 9.
p80 binding of pre-S1 of the ayw subtype of
HBV. GST (lane 1) and GST fusion proteins with pre-S1(1-45) (lane 2),
pre-S1(46-108) (lane 3), or pre-S1(1-108) (lane 4) of the
ayw subtype were expressed and purified (A). The
biotinylated HepG2 cell lysates were incubated with GST (lane 1), and
the precleared lysates were individually incubated with each of the
GST-pre-S1 fusions for p80 binding (B). For comparison, GST (lane 1),
GST-pre-S1(1-56, adr subtype) (lane 2), and
GST-pre-S1(1-45, ayw subtype) (lane 3) were expressed (C),
and the binding experiment was carried out (D). Positions of molecular
size markers (M; in kilodaltons) and p80 are indicated.
|
|
Binding of p80 to HBV particles.
To examine whether p80 binds
to native HBV particles, the biotinylated HepG2 cell lysate was
incubated with 1011 vge of HBV particles (adr
subtype) purified from patient sera, and the mixture was
immunoprecipitated with anti-pre-S1 monoclonal antibody KR127. As shown
in Fig. 10, p80 indeed bound to HBV
particles (lane 2) as well as GST-pre-S1 (lane 5), while p80 was not
detected from the immunoprecipitation without HBV particles (lane 1) or with an irrelevant antibody with specificity for 4-1BB of human T cells
(lane 3). In addition, the binding of p80 to HBV particles was
completely inhibited by peptides pre-S1(12-20) and pre-S1(73-90) (lane
4). The results indicate that p80 binds specifically to the pre-S1
domain of native HBV particles.
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FIG. 10.
Binding of p80 to HBV particles. Biotinylated HepG2
lysates were incubated with a mock preparation (lane 1) or HBV
particles (1011 vge) in the absence (lane 2) or presence
(lane 4) of 100 µg of peptides pre-S1(12-20) and pre-S1(73-90) at
room temperature for 2 h and then immunoprecipitated with
anti-pre-S1 antibody KR127 and protein A-Sepharose. As a negative
control, an irrelevant antibody (anti-human 4-1BB monoclonal antibody)
instead of KR127 was used (lane 3). The immunoprecipitates were
subjected to Western analysis using streptavidin-HRP. The biotinylated
HepG2 cell lysates were subjected to the GST-pre-S1 binding assay in
parallel (lane 5). Molecular size markers (M; in kilodaltons) are shown
on the left, and the position of p80 is indicated on the right.
|
|
Tissue and species specificity of p80 expression.
To address
the tissue specificity of p80 expression, several available human
primary cultures and cell lines of different tissue origin, including
extrahepatic tissues such as skin, kidney, and PBMC that are
susceptible to HBV infection (7), were separately biotinylated, and the same amounts of total cellular proteins of the
cells were used to carry out the GST-pre-S1 binding assay. As shown in
Fig. 11, p80 was detected in HepG2
cells (lane 2), human oral epidermal primary culture (lane 4), PBMC
(lane 8), HEK cells (lane 10), and HeLa (cervix adenocarcinoma) cells
(lane 12) but not human dermal fibroblast primary culture (lane 6). Thus, p80 was found in all human tissues and cell lines examined except
fibroblasts. In an attempt to check the species specificity of p80
expression, rat hepatocyte primary culture was biotinylated and
subjected to the pre-S1 binding assay. The result showed that rat
hepatocytes also expressed p80 (lane 14).
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|
FIG. 11.
Tissue and species specificity of p80 expression. HepG2
(lanes 1 and 2), human oral epidermal primary culture (lanes 3 and 4),
human dermal fibroblast primary culture (lanes 5 and 6), PBMC (lanes 7 and 8), HEK cells (lanes 9 and 10), HeLa cells (lanes 11 and 12), and
rat hepatocyte primary culture (lanes 13 and 14) were biotinylated and
precleared with GST and glutathione-Sepharose beads (lanes 1, 3, 5, 7, 9, 11, and 13). The precleared lysates were then incubated with
GST-pre-S1 fusion protein (lanes 2, 4, 6, 8, 10, 12, and 14), and the
binding assay was performed. Molecular size markers (M; in kilodaltons)
are shown on the left, and the position of p80 is indicated on the
right.
|
|
| |
DISCUSSION |
The molecular mechanism of the attachment and penetration of HBV
into human hepatocytes is not known. This study shows that an
approximately 80-kDa protein (p80) on human hepatocytes binds specifically to the pre-S1 domain of HBV of both adr and
ayw subtypes. The p80 binding sites on pre-S1 are located at
aa 12 to 20 and 82 to 90. Tissue and species specificity of p80
expression was not observed, however. Taken together, the data suggest
that p80 may be involved in HBV entry.
Since Neurath et al. (23) proposed that the attachment of
HBV to HepG2 cells is mediated via pre-S1(21-47), the putative receptors that bind to pre-S1(21-47) had been identified. Neurath et
al. (25) reported that interleukin-6 contained recognition sites for pre-S1(21-47). Petit et al. (27) showed that
35-kDa glyceraldehyde-3-phosphate dehydrogenase strongly bound to the anti-idiotypic antibody against pre-S1(21-47). Dash et al.
(4) showed that a 31-kDa protein in the lysate of HepG2
cells bound to peptide 21-47. Also, human IgA receptor (29),
asialoglycoprotein receptor (34), and a serum glycoprotein
of 50 kDa (3) have been proposed as HBV receptors. However,
the nature of the interaction of HBV with the putative receptors has
not been confirmed. On comparing the assay systems to identify an HBV
receptor, we noted that the previous studies used synthetic peptide
21-47 or HBsAg (or HBV) as a ligand and the lysate of HepG2 cells or
liver membrane proteins as a source of receptor. In contrast, in this
study, purified pre-S1 domain was used as a ligand and cell surface
proteins of human hepatocytes or HepG2 cells were used as a source of
receptor. Our assay system may have several advantages. The whole
pre-S1 domain may exhibit more specific interaction with the receptor molecule than the synthetic peptides because its three-dimensional structure is closer in configuration to that of the native protein. In
addition, the purified pre-S1 protein is less likely to react nonspecifically with cellular proteins compared to HBsAg, which contains a small percentage of the L protein and consists mostly of
major S protein. Regarding the source of receptor, human hepatocytes and HepG2 cells, grown under conditions designed to maintain the hepatocyte-specific differentiated function, are thought to express HBV
receptor at maximum levels. In addition, biotinylation of only the cell
surface proteins will render undetectable intracellular proteins that
could bind to the pre-S1 domain. Using the assay system, we indeed
identified a 80-kDa protein (p80) that binds specifically to the pre-S1
domain (Fig. 1 and 2). So far, no other 80-kDa protein that binds to
HBV has been found.
Although we detected p80 by cell surface biotinylation, it might be
also located within the cell, as is the case of the cellular receptor
gp180 for duck HBV (DHBV), which is a Golgi-resident protein
(2). p80 was not detected on Coomassie blue- or
silver-stained gels after the total lysates of unbiotinylated HepG2
cells were subjected to the receptor-ligand binding assay (data not
shown), suggesting that it is a relatively minor cellular component.
More detailed analysis is needed to determine the subcellular
distribution of p80.
Initial mapping of the p80 binding site on the pre-S1 domain revealed
that p80 binds to aa 12 to 20 and 73 to 90 (Fig. 5). We further
confirmed the p80 binding sites by using the pre-S1 mutants lacking the
segments (Fig. 6) or synthetic peptides of the segments (Fig. 7). In
addition, these peptides were used to confirm the specificity of the
binding of p80 to HBV particles (Fig. 10). Recently, however,
Loffler-Mary et al. reported that Hsc70 bound to aa 70 to 94 of pre-S1
(ayw subtype), corresponding to aa 81 to 105 of pre-S1 of
the adr subtype (21). To examine whether the p80
binding site overlaps the Hsc70 binding site, GST-pre-S1(57-81) was
additionally produced and subjected to the receptor binding assay (Fig.
5E). The result showed that p80 did not bind to pre-S1(57-81),
indicating that aa 82 to 90 of pre-S1 are essential for p80 binding.
Since the p80 binding site overlapped the Hsc70 binding site, we tested
whether p80 is Hsc70. The result showed that p80 did not react with an
anti-Hsp/Hsc70 monoclonal antibody, demonstrating that p80 is other
than Hsp/Hsc70 (Fig. 4).
Our data showed that aa 12 to 20 and 82 to 90 of pre-S1 are essential
for p80 binding and that each site is responsible for the binding (Fig.
5, 6, and 9). These two sites correspond to aa 1 to 9 and 71 to 79 of
the ayw subtype. However, p80 binding by the first binding
site (aa 1 to 9) of the ayw subtype was weaker than that of
the adr subtype, while the second binding sites of the two
subtypes showed equally strong binding (Fig. 9). Comparison of amino
acid sequences of the first binding sites between these two subtypes
showed that the sequence of the adr subtype is
MGTNLSVPN, while that of the ayw subtype is
MGTNLSTSN. The adjacent sequences (aa 21 to 34) of these
two subtypes are identical. Therefore, the difference in binding
strength between these two subtypes may be due to the two divergent
residues (VP of the adr subtype and TS of the ayw
subtype). However, the first binding site of viral particles of the
ay subtype may not be functional in p80 binding because it
is myristylated and anchored to the membrane (26). In the
case of the second binding sites, the two subtypes have homologous
sequences (adr subtype,
GLLGWSPQAQGILTTVPA; ayw subtype,
underlined T and V residues were changed to Q and L, respectively). The
mode of interaction between p80 and the pre-S1 domain of the viral
particle remains to be elucidated.
It has been suggested that tissue and species specificity of HBV
infection may be due to the preferential attachment and penetration of
HBV into hepatocytes. We observed that p80 expression is not restricted
to human hepatocytes (Fig. 11); it was also detected in human oral
epidermal cells, PBMC, HEK cells, HeLa cells, and rat hepatocytes but
not human dermal fibroblasts. This result suggests that p80 may be
expressed in most human tissues and also other mammals, probably in
epithelial cells. With respect to its role in viral infection, p80
might be a component of the viral entry machinery, and some other
protein(s) which is specifically expressed on human hepatocytes may
also play a roles in these processes. For example, Hertogs et al.
(11, 12) found that endonexin II, present on human but not
rat liver plasma membranes, specifically binds to the phospholipid of
small HBsAg, and they postulated that endonexin II may play an
important role in the initiation of HBV infection, likely by inducing
membrane fusion. Therefore, it is conceivable that initiation of HBV
infection of hepatocytes is a multistep process involving interactions
of viral surface proteins with two or more cellular components.
Recently, gp180 (carboxypeptidase D), a transmembrane protein, was
reported to be a cellular receptor for DHBV (17, 18, 33).
gp180 interacts with a distinct pre-S subdomain of DHBV (35). However, gp180 is found not only in duck liver but
also in other tissues which are not susceptible to DHBV infection
(17). Also, its expression did not render transfected
heterologous cells permissive for productive infection (2).
The observations led the authors to conclude that gp180 possibly
functions as a primary virus attachment site and that a
species-specific coreceptor is needed to complete viral entry. Taken
together, the available data suggest that p80 may be analogous to gp180.
| |
ACKNOWLEDGMENTS |
We gratefully acknowledge Young Sook Son for providing human
epidermal and dermal fibroblast primary cultures.
This work was supported by grants FG1110 and FG1220 from the Ministry
of Science and Technology of Korea and by INSERM and the Association
pour la Recherche contre le Cancer, France.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Antibody
Engineering Research Unit, Korea Research Institute of Bioscience and
Biotechnology, P.O. Box 115, Yuseong, Taejon 305-600, Korea. Phone:
82-42-860-4122. Fax: 82-42-860-4597. E-mail:
hjhong{at}kribb4680.kribb.re.kr.
| |
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Journal of Virology, January 2000, p. 110-116, Vol. 74, No. 1
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Top
Abstract
Introduction
