Role of glycosaminoglycans for binding and infection of hepatitis B virus

Article (PDF Available)inCellular Microbiology 10(1):122-33 · February 2008with34 Reads
DOI: 10.1111/j.1462-5822.2007.01023.x · Source: PubMed
Abstract
Many parts of the life cycle of hepatitis B virus (HBV) infection of hepatocytes have been unravelled, but the attachment and entry process leading to infection is largely unknown. Using primary Tupaia hepatocyte cultures as an in vitro infection system, we determined that HBV uses cell-surface heparan sulfate proteoglycans as low-affinity receptor, because HBV infection was inhibited by heparin (IC50: 5 microg ml(-1)) or other higher-sulfated polymers, but not by lower-sulfated glycosaminoglycans, such as chondroitin sulfate. Pretreatment of primary hepatocytes with heparinase decreased viral binding and inhibited HBV infection completely. Interestingly, after preS1-dependent viral binding at 16 degrees C to the cell surface, subsequent infection could still be inhibited by HBV preS1-lipopeptides, but not by heparin any more, suggesting a shift of the virus to a high-affinity receptor. In summary, we suggest following multistep attachment process: in vivo, HBV is initially trapped within the liver in the space of Dissé by heparan sulfate proteoglycans. Thereafter, HBV binds via its preS1 attachment site and the N-terminal myristic acid to a yet unknown, high-affinity receptor that confers uptake in a yet unknown compartment.
Role of glycosaminoglycans for binding and infection
of hepatitis B virus
Corinna M. Leistner, Stefanie Gruen-Bernhard and
Dieter Glebe*
Institute of Medical Virology, Justus Liebig University,
Frankfurter Str. 107, 35392 Giessen, Germany.
Summary
Many parts of the life cycle of hepatitis B virus (HBV)
infection of hepatocytes have been unravelled, but
the attachment and entry process leading to infection
is largely unknown. Using primary Tupaia hepatocyte
cultures as an in vitro infection system, we deter-
mined that HBV uses cell-surface heparan sulfate pro-
teoglycans as low-affinity receptor, because HBV
infection was inhibited by heparin (IC50: 5 mgml
-1
)or
other higher-sulfated polymers, but not by lower-
sulfated glycosaminoglycans, such as chondroitin
sulfate. Pretreatment of primary hepatocytes with
heparinase decreased viral binding and inhibited
HBV infection completely. Interestingly, after preS1-
dependent viral binding at 16°C to the cell surface,
subsequent infection could still be inhibited by HBV
preS1-lipopeptides, but not by heparin any more, sug-
gesting a shift of the virus to a high-affinity receptor.
In summary, we suggest following multistep attach-
ment process: in vivo, HBV is initially trapped within
the liver in the space of Dissé by heparan sulfate
proteoglycans. Thereafter, HBV binds via its preS1
attachment site and the N-terminal myristic acid to a
yet unknown, high-affinity receptor that confers
uptake in a yet unknown compartment.
Introduction
Hepatitis B virus (HBV) belongs to the family of Orthohe-
padnaviruses that causes acute and chronic infections of
the liver. With over 370 million people chronically infected,
chronic HBV infections are a major cause of liver cirrhosis
and hepatocellular carcinoma in many regions of the
world (Baumert et al., 2007). Although our knowledge of
the molecular biology of this virus has increased over the
past years, resulting in an effective vaccine and new
therapeutics for chronic HBV carriers (Tillmann, 2007),
the viral and cellular determinants of viral binding and
entry of this virus are still enigmatic (Glebe and Urban,
2007). This was mainly due to the lack of a suitable and
practicable infection system for HBV in the past. In vivo,
HBV infects only humans and some primates (e.g. chim-
panzees) and till now, no easy small animal system is
available for the study of HBV infection. For in vitro
studies, primary human hepatocyte cultures (PHHs),
obtained after perfusion of liver pieces after surgical
resection, were for many years the only system to study
the early steps of this highly liver-specific infection (Glebe,
2006). However, these cultures are, for obvious reasons,
very limited in availability. Furthermore, PHHs are not
easy to handle and were reported to differ in susceptibility
due to the very heterogeneous quality of different liver cell
preparations (Gripon et al., 1988). These limitations were
partly overcome by the observation that primary hepato-
cyte cultures from Asia tree shrews, Tupaia belangeri
(primary Tupaia hepatocytes; PTH), are susceptible to
HBV (Walter et al., 1996; Köck et al., 2001). Interestingly,
this small mammal does not belong to the order primates
(Nishihara et al., 2002) but forms an own order called
Scandentia. Nevertheless, we (Glebe et al., 2003; 2005)
and others (Köck et al., 2001) were able to show that HBV
infection of PTH is homologous to infection of PHH and a
newly established HBV-susceptible cell line (Gripon et al.,
2002). HBV virions appear after negative staining in elec-
tron microscopy as spheres of 45 nm in diameter. Besides
the virions, HBV-infected hepatocytes constitutively
secrete also nucleocapsid-free subviral particles (SVPs),
composed of the hepadnaviral surface proteins. HBV-
SVPs exist in a spherical (22 nm in diameter) or filamen-
tous (same diameter, but variable length) form. The three
co-carboxyterminal HBV surface (HBs) proteins (L-, M-,
S-HBs) are distinguished by three domains: preS1 only in
LHBs, preS2 in LHBs and MHBs, and S in all three HBs
proteins. The SHBs is the major component of the virion
envelope and the SVPs; however, virions and filamentous
SVPs contain more LHBs than spheres (Heermann et al.,
1984). Till now, a still-growing list of potential binding
partners for all three HBV surface proteins in human
serum and on cellular membranes has been proposed;
however, none have even been proven to act as a func-
tional HBV receptor facilitating HBV infection (Glebe and
Urban, 2007). Recently, one group described binding and
hence purification of HBV and hepatitis C virus (HCV)
Received 8 May, 2007; revised 25 June, 2007; accepted 5 July, 2007.
*For correspondence. E-mail dieter.glebe@viro.med.uni-giessen.de;
Tel. (+49) 641 99 41203; Fax (+49) 641 99 41209.
Cellular Microbiology (2008) 10(1), 122–133 doi:10.1111/j.1462-5822.2007.01023.x
First published online 10 August 2007
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
from human plasma samples by heparin columns (Zahn
and Allain, 2005). Therefore, the question arises, whether
HBV uses sulfated glycosaminoglycans (GAGs) for
attachment to hepatocytes. GAGs are long, unbranched
polysaccharides, consisting of repeating disaccharides,
usually an amino sugar and an uronic acid. While
N-acetylglucosamine and glucuronic acid form heparan
sulfate (HS), the backbone of chondroitin sulfate (CS)
consists of N-acetylgalactosamine and glucuronic acid
(Esko and Selleck, 2002). GAG chain synthesis starts
with the addition of an amino sugar to a tetrasaccharide
that is attached via O-glycosylation to a serine residue,
followed by a glycine. Mature GAG chains undergo dis-
tinct modifications, like N- and O-sulfation and epimeriza-
tions, leading to the great variety of GAGs. Especially HS
chains are found mainly on membrane proteoglycans, like
glypicans and syndecans. Glypicans are heparan sulfate
proteoglycans (HSPGs) and linked via a glycosylphos-
phatidylinositol (GPI) anchor to the cell membrane. Syn-
decans contain also mainly HS chains, but are type I
transmembrane proteins (Hacker et al., 2005). Heparin is
known as a highly sulfated soluble subtype of HS. In vivo,
HS is an extracellular GAG and attached to a core protein,
while heparin is an intracellular GAG, synthesized in
granulated cells and is cleaved from its serglycin core
protein (Horner, 1986; Kresse et al., 1993). Although HS
are found on virtually every cell, highly sulfated liver-
specific HS provide binding sites for diverse ligands and
receptors involved in regulating growth control, lipid
metabolism, haemostasis, signal transduction and cell
adhesion (Esko and Lindahl, 2001; Kreuger et al., 2006).
Besides its physiological role, e.g. mediating binding of
apolipoprotein E to hepatocytes (Dong et al., 2001),
liver-HS provides binding sites for various liver-targeting
pathogens, like malaria circumsporozoite (Rathore et al.,
2001), dengue virus (Chen et al., 1997) and HCV (Barth
et al., 2006; Koutsoudakis et al., 2007). In this study, we
investigated the role of GAGs during the attachment and
infection of HBV to susceptible primary hepatocytes.
Results
Binding of HBV to heparin
Recently, one group reported an interaction of HBV with
heparin columns (Zahn and Allain, 2005). Heparin is a
complex polysaccharide consisting of repeated disaccha-
rides of uronic acid and glucosamine. The disaccharide
chains are modified by N- and O-sulfation that contribute
to a specific pattern of negative charge. To determine
specificity of binding of HBV surface proteins to heparin,
we coated 96-well plates with heparin and determined
binding of purified HBV particles (Fig. 1). Using increasing
concentrations of different sulfated polymers as competi-
tor, we determined that the highly sulfated heparin could
inhibit binding to heparin-coated plates completely at
10 mgml
-1
. CS that has a lower sulfation grade, resulted in
a dose-dependent reduction of binding, but no complete
inhibition, even at high concentrations (1000 mgml
-1
). To
investigate the role of sulfation of heparin for the binding,
we used also specifically de-N- and de-O-sulfated hep-
arins for competition of binding to heparin plates. In de-N-
sulfated heparin, N-sulfated glucosamine residues of
heparin are removed, while in de-O-sulfated heparin,
O-sulfate esters of heparin are removed. Interestingly,
complete inhibition of HBV binding to heparin could
neither be achieved with de-O-sulfated heparin, nor de-N-
sulfated heparin. However, use of de-O-sulfated heparin
resulted in 50% reduction of HBV binding, if used at very
high concentrations (1000 mgml
-1
). This suggests that the
overall negative charge, given by the degree of sulfation
of the polyanions, is important for viral binding and not
primarily a specific pattern of sulfation.
Binding and infectivity of HBV to primary hepatocytes
and different cell lines
In order to measure binding and infection of HBV to cells
from different origins, we incubated purified HBV or SVPs
with freshly isolated PTH, primary rat hepatocytes (PRH)
and established human cell lines (HepG2 and Hela). To
differentiate between binding and uptake, we incubated
the virus with the cells for 1 h at 16°C (Fig. 2). Incubation
Fig. 1. Binding of HBV to heparin-coated wells. Highly purified
HBV SVPs (100 ng ml
-1
) from chronic HBV carriers were incubated
with indicated concentrations of polyanions for 1 h at 16°C and
added to heparin-coated (25 mgml
-1
) 96-well plates. Plates were
incubated for 2 h at 37°C, and heparin-bound SVPs were detected
using an conformational-dependent mAb to SHBs in a colorimetric
reaction as described in Experimental procedures,no
polyanions. The dotted line indicates the cut-off.
Glycosaminoglycan-dependent HBV infection 123
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 10, 122–133
at this temperature inhibited cellular endosomal uptake as
detected by the absence of bulk-flow endocytosis below
18°C by a soluble peroxidase assay (Shurety et al., 1998)
(Fig. 2A, picture h). Nevertheless, this temperature
allowed binding of HBV, detected as a ring-like staining of
HBV particles in immunocytochemistry (Fig. 2A, picture c)
in contrast to the staining at 37°C, suggesting uptake of
HBsAg (Fig. 2A, picture a). We observed a specific stain-
ing for HBsAg not only to susceptible PTH (Fig. 2A,
picture a) but also to HepG2 and Hela cells that are
non-susceptible for HBV infection (Fig. 2A, pictures d and
f). To evaluate the amount of binding of the purified virus
to the different cells, we measured viral binding by real-
time polymerase chain reaction (PCR) (Fig. 2B). Interest-
ingly, only about one-tenth of the viral inoculum was
bound by susceptible PTH irrespective of the concentra-
tion ranging from 10
7
to 10
3
genome-equivalents ml
-1
.
However, non-susceptible HepG2 and Hela cells bound
purified HBV with the same magnitude like susceptible
PTH. Next, we performed binding and infection experi-
ments using freshly isolated PRH and PTH (Fig. 3). Sur-
prisingly, both rat and Tupaia hepatocytes bound
approximately 10% of the viral inoculum, ranging from 10
5
to 10
7
genomes ml
-1
(Fig. 3A), while only PTH got infected
in a dose-dependent manner as detected by secretion of
HBeAg 9–12 days after infection (Fig. 3B). Interestingly,
when we added highly sulfated polyanions during infec-
tion, e.g. dextran sulfate (100 mgml
-1
), infection was
Fig. 2. Binding of HBV to PTH and different
cell lines.
A. Immune staining of Tupaia hepatocytes
after incubation with purified HBV SVPs from
human plasma (2 mgml
-1
each) for 1 h at
37°C with a monoclonal antibody (mAb)
against the S-domain (red) and
counterstained with Meyer’s hemalaun (blue).
Binding of plasma-derived particles is
detected as seen for PTH (a), HepG2 cells (d)
and Hela cells (f) after incubation for 1 h at
37°C, while (c) indicates incubation of Tupaia
hepatocytes with purified HBV SVPs for
1 h at 16°C. Control slides without HBsAg
incubation: PTH (b), HepG2 (e) and Hela cells
(g). All panels have same magnification
(bar = 50 mm). The inhibition of cellular uptake
below 18°C was measured by the absence of
bulk-flow endocytosis of soluble peroxidase
(h) as described in Experimental procedures.
The dotted line indicates the cut-off.
B. Cellular binding assay for HBV. Highly
purified HBV from chronic carriers in the
indicated concentrations was incubated with
different confluent cell cultures in 12-well
plates for 4 h at 16°C. Bound HBV particles
were determined by real-time PCR as
described in Experimental procedures
(log copies ml
-1
).
124 C. M. Leistner, S. Gruen-Bernhard and D. Glebe
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 10, 122–133
completely blocked (Fig. 3B), while binding was only
decreased by a factor of 3. Pretreatment of hepatocytes
with dextran sulfate and subsequent removal before addi-
tion of virus had no effect on binding or infection of HBV
(data not shown). As a control for specificity of HBV infec-
tion of PTH, we added myristoylated HBV preS1 peptide
of amino acids 2–48 that has been shown to inhibit HBV
infection of PHHs and PTH in nanomolar concentration
(Glebe et al., 2005; Gripon et al., 2005). However, when
applied at 100 n
M, the peptide was unable to inhibit
binding of virus to PTH and PRH, although infection of
PTH could be completely inhibited (Fig. 3B). These data
indicate that at least in vitro, HBV binds to susceptible and
non-susceptible cells within the same magnitude (one-
tenth of a given HBV inoculum). The observation that
highly sulfated polyanions are able to inhibit HBV infection
completely, but could not inhibit HBV binding to PTH and
PRH, suggests that interaction of HBV with negatively
charged cell-surface structures is not species-specific.
Moreover, the inability of infection-interfering HBV myr-
preS1 lipopeptides to inhibit HBV binding at nanomolar
concentrations suggests a post-entry role of this kind of
inhibitor.
Infection of PTH in the presence of polyanions
To determine the role of various polyanions for HBV infec-
tion, we pre-incubated different concentrations of polyan-
ions, heparin, CS, dextran sulfate and the uncharged
dextran with purified virus from HBV carriers before addi-
tion to susceptible PTH (Fig. 4A). After binding at 16°C,
cells were washed extensively, and internalization of
bound viral particles was achieved by shifting the tem-
perature to 37°C for 12 h. Outcome of infection was deter-
mined by measuring secretion of newly synthesized
HBsAg of infected PTH 9–12 days after infection as
described. It turned out that heparin was a much more
efficient inhibitor of HBV infection (IC50: approximately
5 mgml
-1
) than CS (IC50: approximately 800 mgml
-1
)
(Fig. 4A). Dextran sulfate could inhibit HBV infec-
tion in nearly the same magnitude as heparin
(IC50: < 25 mgml
-1
), while pre-incubation of purified virus
with the uncharged dextran was unable to inhibit HBV
infection. As a control, myristoylated preS1 peptides aa
2–48 containing the essential binding domain (aa 9–18,
genotype D) inhibited HBV infection, while a myristoylated
preS1 peptide lacking this domain (myr-preS1 aa 19–48)
failed to inhibit infection as described (Glebe et al., 2005).
Treatment of cells with different enzymes that cleave
cell-surface bound glycans
These observations suggested that charged HSPGs
might be a primary binding factor for HBV on PTH. To
prove this hypothesis, we pretreated PTH with hepari-
nase, known to efficiently cleave sulfated HS chains from
the cell surface. The enzyme was removed by extensive
washing, and purified HBV was added at 16°C for 4 h.
During these conditions, appearance of newly synthe-
Fig. 3. Binding and infection of HBV to primary hepatocytes from
Tupaia and rat.
A. Highly purified HBV from chronic carriers in the indicated
concentrations was incubated with different cell cultures for 4 h at
16°C without competitor or after pre-incubation of virus with dextran
sulfate (100 mgml
-1
) or myristoylated HBV preS1 peptides 2–48
(100 nM) for 1 h at 16°C. Bound particles were determined by
real-time PCR as described in Experimental procedures.
B. Highly purified HBV from chronic carriers in the indicated
concentrations was incubated with different cell cultures for 4 h at
16°C without competitor or after pre-incubation of virus with dextran
sulfate (100 mgml
-1
) or myristoylated HBV preS1 peptides 2–48
(100 nM) for 1 h at 16°C. After extensive washing at 4°C, cells
were shifted to 37°C for 12 h to allow HBV uptake. Medium was
changed every 3 days. HBeAg production of infected cultures was
determined from day 9 to day 12 post infection. The dotted line
indicates the cut-off (multiples of cut-off signal).
Glycosaminoglycan-dependent HBV infection 125
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 10, 122–133
sized, or recycled re-sulfated cellular HS was unlikely,
because the estimated half-life of HS proteoglycanes at
the plasma membrane of cultured primary hepatocytes
was reported to be 3–6 h at 37°C (Oldberg et al., 1977;
Egeberg et al., 2001). Afterwards, the cultures were
washed again at 4°C and shifted to 37°C to allow uptake
of bound virus. As shown in Fig. 5A, treatment with hepa-
rinase reduced HBV infection in a dose-dependent
manner and resulted in complete loss of susceptibility at
6Uml
-1
. This result confirms the importance of HSPGs
for the infection process of HBV. To rule out the possibility
that other glycans than HS might contribute to infection,
Fig. 4. Influence of different polyanions on HBV infection of PTH
during or after viral binding.
A. Highly purified HBV (100 genomes per hepatocyte) from chronic
carriers were incubated with PTH for4hat16°C after pre-incubation
of virus with different concentrations of indicated polyanions for 1 h
at 16°C. Incubation with 100 nM infection-interfering myristoylated
HBV preS1 peptides 2–48, or inactive peptide (preS1 domains
19–48) for1hat16°C served as a control. After extensive washing
at 4°C, cells were shifted to 37°C for 12 h to allow HBV uptake.
Medium was changed every 3 days. HBsAg production of infected
cultures was determined from day 9 to day 12 post infection and is
displayed in percentage of the uncompeted control infection (Ø).
The dotted line indicates the cut-off.
B. Highly purified HBV from chronic carriers were incubated with
PTH (100 genomes per hepatocyte) for4hat16°C. After extensive
washing at 4°C, cells were incubated with different concentrations of
indicated polyanions and HBV peptides (100 nM) for1hat16°C.
After further washing steps at 4°C, cells were shifted to 37°C for
12 h to allow HBV uptake. Medium was changed every 3 days.
HBsAg production of infected cultures was determined from day 9 to
day 12 post infection and is displayed in percentage of the uncom-
peted control infection (Ø). The dotted line indicates the cut-off.
Fig. 5. Influence of different cellular glycans on HBV binding and
infection of PTH.
A. Primary hepatocytes were incubated with indicated
concentrations of heparinase III, PNGase F, or sialidase or
myristoylated preS1 peptides (100 nM) for 4 h at 16°C. After
washing at 4°C, highly purified HBV from chronic carriers were
incubated for 4 h at 16°C in the absence of enzymes, while in the
case of sialidase, enzyme was also present in one panel during the
whole infection period. After extensive washing at 4°C, cells were
shifted to 37°C for 12 h to allow HBV uptake. Medium was changed
every 3 days. HBsAg production of infected cultures was
determined from day 9 to day 12 post infection and is displayed in
percentage of the uncompeted control infection (Ø). The dotted line
indicates the cut-off.
B. Primary hepatocytes were incubated with indicated
concentrations of heparinase, for 4 h at 16°C. After washing at 4°C,
highly purified HBV from chronic carriers were incubated
(100 genomes per hepatocyte) for 4 h at 16°C in the absence of
enzymes. After extensive washing at 4°C, bound particles were
determined by real-time PCR as described in Experimental
procedures. Ø; no enzyme.
126 C. M. Leistner, S. Gruen-Bernhard and D. Glebe
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 10, 122–133
we also pretreated the cells either with peptido
N-glycanase F (PNGase F), resulting in cleavage of
N-linked sugars, or with sialidase, known to cleave nega-
tively charged sialic acid from N- and O-glycans.
However, none of these treatments had any effect on HBV
infection (Fig. 5A). The removal of the distinct sugars from
cell-surface proteins was tested by shifting of the heavily
N-glycosylated and sialylated asialoglycoprotein receptor
(ASGPR) in SDS-PAGE immunoblots from plasma-
membrane preparations of PTH (data not shown).
Because the ASGPR is a liver-specific lectin, it had been
speculated that it might serve as a specific receptor
for HBV, especially during treatment of hepatocytes with
sialidase (Owada et al., 2006). However, neither pre-
incubation with sialidase, nor incubation in the presence
of sialidase, resulted in significant increase or decrease of
infection (Fig. 5A). Furthermore, no effect of sialidase
treatment on HBV infection was detected when non-
susceptible rat hepatocytes or HepG2 cells were used
(data not shown). To determine the effect of heparinase
treatment of PTH on HBV binding, we treated PTH at
16°C with heparinase and determined binding of purified
HBV at 16°C for 4 h. As shown before (Fig. 3A), approxi-
mately 90% of the viral input did not bind to HBV and
remained within the supernatant of the cells (Fig. 5B).
However, even heparinase treatment of PTH that resulted
in complete inhibition of infection, caused only a threefold
decreased viral binding.
Role of sulfation of hepatocellular proteoglycans
for HBV infection
Heparan sulfate proteoglycans are heavily charged due to
the addition of sulfate groups to their sugar backbones.
Cultivation of cells in sulfate-free medium and addition of
sodium chlorate inhibits effective sulfation of CSPGs and
HSPGs in a dose-dependent manner. It has been
reported that low concentrations of chlorate (3–5 mM)
reduced mainly sulfation of CSPG, while higher chlorate
concentrations (10 mM) are needed for inhibition of
HSPG sulfation (Fjeldstad et al., 2002). Furthermore, inhi-
bition of sulfation of HSPGs was shown to significantly
decrease infection with vaccinia virus (Chung et al., 1998)
and inhibits adeno-associated virus 2 infection (Qiu et al.,
2000) in vitro. Therefore, we pre-incubated PTH 48 h
before infection with increasing concentrations of sodium
chlorate in sulfate-free medium, and added purified virus
in either the presence or absence of the drug (Fig. 6).
Pretreatment of PTH with sodium chlorate resulted in a
decrease of susceptibility towards HBV. Infection in the
presence of sodium chlorate showed slightly stronger
inhibitory potential (IC50: approximately 10 mM) than in
the absence of the drug (IC50: 25 mM) during infection.
Unfortunately, higher concentrations of sodium chlorate
were toxic to PTH when applied for 48 h. To rule out any
drug-related effects on the virus itself, we infected PTH in
the presence of sodium chlorate with indicated concen-
trations, but omitted pre-incubation of the cell cultures
with sodium chlorate. However, this had no significant
effect on HBV infection (Fig. 6). These observations indi-
cate that although cleavage of HS on hepatocytes can
decrease HBV binding only by a factor of 3, HBV infec-
tion is absolutely dependent on the presence of highly
sulfated HSPGs on the surface of susceptible primary
hepatocytes.
Effect of polyanions after viral binding to PTH
To further elucidate the effect of polyanions after interac-
tion of HBV with hepatocyte membranes, we first let the
virus bind to the cells at 16°C for 4 h, washed away the
unbound virus at 4°C and thereafter added the same
concentration of polyanions as used for pre-incubation
(Fig. 4B). After an additional hour at 16°C, the cells were
shifted to 37°C for 12 h to allow infection. Interestingly,
after viral binding, addition of polyanions had only minor
effect on HBV infection (Fig. 4B). This suggests that HBV
has switched from low-affinity binding to high-affinity
binding on the surface of hepatocytes at 16°C. However,
this process might not be completed after 4 h because,
with high concentrations of heparin (1000 mgml
-1
) and
dextran sulfate (100 mgml
-1
), a decrease in infectivity by
50% and 20% respectively was observed. Nevertheless,
Fig. 6. Influence of sulfation of GAGs on HBV infection of PTH.
Primary hepatocytes were incubated with indicated concentrations of
sodium chlorate in sulfate-free medium for 48 h at 37°C before (pre-
incubation) or for 12 h at 37°C during infection with HBV. Incubation
with 100 nM infection-interfering myristoylated HBV preS1 peptides
2–48, or inactive peptide (preS1 domains 19–48) during infection
served as a control. Medium was changed every 3 days. HBsAg pro-
duction of infected cultures was determined from day 9 to day 12
post infection and is displayed in percentage of the uncompeted
control infection (Ø). The dotted line indicates the cut-off.
Glycosaminoglycan-dependent HBV infection 127
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 10, 122–133
HBV infection could be efficiently inhibited by adding
myristoylated HBV preS1 peptides 2–48 in nanomolar
concentration, even after HBV binding. This is in line
with previous results demonstrating that HBV preS1-
lipopeptides could inhibit HBV infection when applied 2, 4
or 12 h before addition of virus (Glebe et al., 2005).
Discussion
Previously, we showed that HBV surface proteins bind to
HBV-susceptible PTH (Glebe et al., 2003; 2005). In this
study, we analysed HBV binding in more detail. Using
highly purified HBV from the plasma of chronically
infected patients, only 10% of the viral input bound to
susceptible PTH, and this proportion could not be
increased by raising the concentration. Binding of duck
hepatitis B virus (DHBV) to primary duck hepatocytes
is also inefficient and does also not reach saturation
(Klingmuller and Schaller, 1993). Interestingly, the same
proportion of viral binding was observed with non-
susceptible cells, such as PRH cultures, a human
hepatoma cell line (HepG2), and even with Hela cells.
These data confirm previous observations on non-
productive binding of HBV from various sources to differ-
ent cell lines, but this binding was usually not related to
infection (for review, see Glebe and Urban, 2007). Given
the finding that HBV binds to heparin columns (Zahn and
Allain, 2005), we analysed the interaction of HBV with
heparin in more detail. We detected that purified HBV
SVPs bound very well to heparin-coated wells, and this
specific binding could only be inhibited completely by
heparin itself. Use of lower-sulfated forms of heparin or
CS resulted in either no inhibition (de-N-sulfated heparin)
or dose-dependent reduction of HBV binding (CS and
de-O-sulfated heparin). This is in line with previous pub-
lications using heparin-affinity chromatography for HBsAg
purification (Einarsson et al., 1978; Tajima et al., 1992).
Interestingly, binding of HBV to susceptible PTH and non-
susceptible PRH was only moderately inhibited by similar
concentrations of highly sulfated polymers, such as
heparin or dextran sulfate. This is in contrast to the data
obtained by Ying et al. (2002), who found nearly complete
reduction (97%) of HBV binding to PHH and non-
susceptible cell lines in the presence of 100 mgml
-1
heparin. However, in contrast to the present work, those
studies presented by Ying et al. were conducted with
crude supernatants of stably HBV-producing cell lines
(e.g. HepAd38). It is well known that these cell lines
secrete, besides HBV virions, a large amount of partially
and non-enveloped core particles (Sun and Nassal,
2006). The HBV core protein, however, contains an
arginine-rich carboxyterminal domain that has been
shown to mediate attachment of nucleocapsids to cell-
surface HS (Vanlandschoot et al., 2005; Cooper and
Shaul, 2006). On the other side, the viral particles used in
our study, derived from human plasma and purified
through a sucrose-gradient, contain fully enveloped viral
particles, which were not permeable for nucleotide triph-
osphates (Mest, 1997). Therefore, the effects detected by
Ying et al. may be due to contaminations of the inoculum
with partially or non-enveloped viral core particles. Fur-
thermore, Ying et al. did not study the effect of polyanions
on HBV infection. In our study, a complete inhibition of
HBV binding by highly sulfated polyanions could not be
achieved by the use of purified plasma-derived HBV and
primary hepatocytes; however, these polymers (heparin
and dextran sulfate) could inhibit HBV infection of PTH
completely when given together with the viral inoculum. To
determine the role of sulfation of cell-surface GAGs for
HBV infection, we inhibited sulfation of cellular GAGs
by sodium chlorate that is known to inhibit heparin-
dependent infection of different viruses (Chung et al.,
1998; Qiu et al., 2000). Indeed, sulfation of cell-surface
GAGs seems to be important for HBV infection, because
inhibition of sulfation decreases HBV infection in a dose-
dependent manner. In a similar approach, this has been
demonstrated for a HBV-susceptible human hepatoma
cell line with HBV derived from a recombinant source
(Schulze et al., 2007). HCV, a highly liver-specific RNA
virus, was reported to use distinct N-sulfated cell-surface
HS for primary attachment (Barth et al., 2006), but in our
study, neither HBV binding to heparin-coated wells, nor
infection of PTH (data not shown), could be inhibited by
de-N- or de-O-sulfated heparin. This argues against the
requirement of a distinct sulfation pattern of cell-surface
GAGs in the case of HBV. The ubiquity of HS on mam-
malian cells may be one reason why purified HBV binds
with the same degree to susceptible and non-susceptible
primary hepatocytes, and even non-liver cells. The finding
that pretreatment of cellular surface proteins with hepari-
nase inhibits viral infection while PNGase and sialidase
had no effect, further supports the involvement of highly
sulfated cell-surface HS in the early steps of HBV
infection. Interestingly, highly sulfated polymers like
heparin or dextran sulfate have no effect on DHBV infec-
tion in vivo and in vitro (Offensperger et al., 1991).
Although DHBV shares many molecular similarities with
HBV, DHBV probably uses other receptors for primary
attachment than GAGs (for a recent review, see Glebe
and Urban, 2007). In a recent publication, Owada et al.
(2006) reported that treatment of HBV with sialidase
during incubation with human hepatoma cell line HepG2
would result in infection via the ASGPR, a liver-specific
lectin that is responsible for uptake of desialylated glyco-
proteins from blood into hepatocytes (Spiess, 1990) and
has been proposed as a receptor for HBV (Treichel et al.,
1994; 1997). In previous studies, we could show that N-
and O-glycans of HBV surface glycoproteins could be
128 C. M. Leistner, S. Gruen-Bernhard and D. Glebe
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 10, 122–133
desialylated by treatment with sialidase (Schmitt et al.,
1999; 2004), resulting in increased binding to HepG2 cells
(Glebe and Gerlich, 2004). This binding, however, did not
lead to infection of HepG2 cells or increased susceptibility
of PTH (Glebe and Gerlich, 2004).
Recently, we demonstrated that HBV SVPs consisting
only of SHBs were unable to bind primary hepatocytes,
while SVPs formed by a fusion protein of preS1 and the
S-domain bound to PTH like serum-derived SVPs, com-
posed of all three HBV surface proteins (Glebe et al.,
2005). Because it is known that the first 77 amino acids of
the preS1 domain are important for infection of PHH with
HBV (Le Seyec et al., 1999), this domain might serve as
a binding partner for negatively charged HS on the cell-
surface of hepatocytes.
Given the ubiquitous abundance of GAG on cellular and
extracellular matrices, the questions arises, why experi-
mental inoculation of very low concentrations of HBV (10–
100 particles) could induce acute HBV infection in
chimpanzees (Ulrich et al., 1989; Hsia et al., 2006; Yugi
et al., 2006). One possibility might be that the preS
domains are covered in the blood by a serum protein
partially protecting HBV from non-specific attachment to
blood vessels. A number of serum proteins have been
proposed for this function, including albumin (Krone et al.,
1990) and apolipoprotein H (Mehdi et al., 1996; Stefas
et al., 2001). In a recent publication, Deng et al. (2007)
propose lipoproteinlipase (LPL) as a binding factor for the
preS1 domain. Because LPL itself binds to HS proteogly-
canes (Spillmann et al., 2006), this might facilitate binding
of HBV to hepatocytes in vivo.
Heparin and dextran sulfate have also been shown to
inhibit HIV infection in vitro (Mitsuya et al., 1988); there-
fore, attempts have been made to use dextran sulfate and
similar substances as a potential antiviral for HIV-infected
patients (McClure et al., 1992). However, direct parental
administration of those substances interferes with blood
clotting, while their molecular weight is usually too high to
be enterally absorbed. However, they provide a potential
for the use as a topically applied microbicide for preven-
tion of sexual transmission of HIV. Different candidate
agents of highly sulfated polymers have been proven to
provide up to 90% protection of SIV transmission in the
macaque model (Weber et al., 2001) and are currently in
human phase II and III trials (Mayer et al., 2003). Because
HBV is also a sexually transmitted disease, use of polya-
nions to block sexual transmission of HIV might also work
on HBV; however, this has to be validated.
Experimental procedures
Reagents
Heparin sodium salt, N-acetylated-de-O-sulfated heparin, de-N-
sulfated heparin, CS, dextran sulfate, dextran, heparinase III,
soluble peroxidase, sialidase and sodium chlorate were pur-
chased from Sigma, Taufkirchen, Germany. PNGase F was from
New England Biolabs.
Isolation and purification of HBV virions and SVPs from
plasma of HBV-infected patients
Hepatitis B virus and natural HBsAg (genotype D, HBsAg
subtype ayw2) was isolated from HBeAg-positive plasma of two
asymptomatic chronic HBV carriers with normal transaminase
levels in serum. One carrier (ID326) had 8 ¥ 10
9
HBV genom-
es ml
-1
and 120 mgml
-1
HBsAg, and the second (ID304) had
1.6 ¥ 10
9
HBV genomes ml
-1
and 55 mgml
-1
HBsAg. The purifi-
cation was performed as described (Glebe and Gerlich, 2004). In
brief, HBV and SVPs from 350 ml human plasma were pelleted
through 10% and 15% sucrose for 15 h at 25 000 r.p.m. in a
SW28 rotor (Beckman, Munich, Germany). The resuspended
pellets were pooled and ultracentrifuged into a discontinuous
sucrose density gradient (15%, 25%, 35%, 45% and 60%) as
described above. Virus-containing fractions at 40–45% sucrose
were identified by quantitative real-time PCR (LightCycler
system, Roche, Germany) using primers and hybridization
probes against the HBV X-region as described (Jursch et al.,
2002). The assay was calibrated using the Eurohep reference
plasma, which has been converted to a World Health Organiza-
tion international standard sample (Saldanha et al., 2001).
Peptide synthesis and purification
Peptides were synthesized and purified in the Department of
Biomolecular Chemistry at the Zentrum für Molekulare Biologie,
Heidelberg (ZMBH), Germany, and by Peptide Specialty Labora-
tories GmbH, Heidelberg, Germany.
Isolation and culture of PTH
Primary hepatocytes of T. belangeri (Asian tree shrews) were
isolated as described (Glebe et al., 2003). In brief, the livers were
perfused via the portal vein with HANKS solution (Invitrogen,
Karlsruhe, Germany) containing 5 mM EGTA, followed by perfu-
sion with DMEM (Invitrogen) containing 0.05% collagenase
(Sigma). Hepatocytes were selectively pelleted three times at
40 g for 6 min at 4°C. Hepatocytes were resuspended in Tupaia
hepatocyte medium (THM) and plated on collagen-coated
12-well plates (10
5
hepatocytes per well) as described. Plating
efficiency was measured prior to infection and at the end of the
experiment by a modified MTT assay as described (Glebe et al.,
2005). The MTT assay involves the metabolic conversion of the
water-soluble compound MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide, obtained from Roche] to an soluble
formazan only at the membranes of intact living cells. Variability
as determined by MTT assay for each preparation was found to
be 10% or lower (data not shown). The organ harvest from
Tupaias has been approved by the local animal protection
committee.
Cell lines
Human cervix carcinoma cell line Hela and human hepatoma
cell line HepG2 were obtained from the American Type Culture
Glycosaminoglycan-dependent HBV infection 129
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 10, 122–133
Collection. Cells were cultivated in DMEM (Invitrogen) with
addition of 10% fetal calf serum at 37°C in a humidified incu-
bator. For binding experiments, cells were trypsinized and cul-
tivated on collagen-coated cell culture wells or coverslips until
use.
Binding of HBV to heparin
Ninety-six-well ELISA plates pretreated by plasma polymerization
(EpranEx, Plasso Technology, Portobello, UK) were coated with
25 mgml
-1
of heparin in PBS at 4°C for 12 h. After extensive
washing (three times with PBS/0.2% Tween 20, two times with
PBS) and subsequent blocking with 1% BSA (crystallized,
Sigma) in PBS for 2 h at 37°C, highly purified HBV SVPs from
chronic HBV carriers (100 ng ml
-1
) were incubated with indicated
concentrations of polyanions for 1 h at 16°C and added to
heparin-coated plates. Plates were incubated for 2 h at 37°C, and
after removal of the supernatant, the plate was again washed
with PBS and PBS/0.2% Tween 20, and peroxidase-conjugated
anti-HBs from the Enzygnost HBsAg ELISA kit (Dade Behring,
Germany) was added to each well and incubated for 1 h at 37°C.
After washing as described above, an o-phenylenediamine/H
2
O
2
substrate (tablets from DAKO) was added for 15 min at room
temperature, and the amount of coloured product was measured
by OD
492
.
Cellular binding assay for HBV
Primary Tupaia hepatocytes or indicated cell lines were plated on
collagen-coated 12-well plates in THM (1 ¥ 10
5
cells per well) as
described above. Purified virions from plasma were diluted in
THM to yield the final concentrations given in the individual
experiments. Cells were incubated for the times and tempera-
tures indicated, and unbound virus was removed by extensive
washing with ice-cold THM. HBV DNA was purified from cellular
lysates using a DNA extraction kit (Highpure, Roche, Germany).
HBV DNA was quantified by real-time PCR (LightCycler system,
Roche, Germany) using primers and hybridization probes against
the HBV X-region as described (Jursch et al., 2002).
Binding and uptake of plasma-derived HBV SVPs
Primary Tupaia hepatocytes, PRH or indicated cell lines were
plated on collagen-coated coverslips in THM (1 ¥ 10
3
cells per
coverslip) as described above. Purified SVPs from plasma con-
taining all three HBV surface proteins were diluted in THM to yield
a final concentration of 2 mgml
-1
. Cells were incubated for the
times and temperatures indicated and washed several times with
ice-cold THM. Cells were fixed with 3% paraformaldehyde for
0.5 h at 4°C and permeabilized with 0.1% Triton X-100 in PBS for
0.5 h at room temperature. HBsAg staining was performed with
the APAAP staining kit (DAKO, Hamburg, Germany) as described
(Glebe et al., 2003) using monoclonal anti-HBs (Novocastra,
Newcastle, UK).
Soluble horseradish peroxidase (HRP) uptake assay
Uptake of HRP was determined as described (Shurety et al.,
1998) with minor modifications. In brief, primary hepatocytes
were plated on collagen-coated 12-well cell culture dishes as
described above. For HRP uptake, cells were incubated with
0.1mgml
-1
HRP in DMEM for4heitherat37°Cor18°C.After-
wards, excess HRP was removed by several washes with ice-
cold DMEM. Cells were scraped off the dishes into PBS
containing 0.1% Triton X-100. After 10 min at 4°C, unsoluble
cellular debris was removed by centrifugation at 5000 g for
10 min at 4°C. Soluble cellular extracts were assayed for HRP
activity by adding an o-phenylenediamine/H
2
O
2
substrate (tablets
from DAKO) for 15 min at room temperature, and the amount of
coloured product was measured by OD
492
. Resultant OD
492
values were normalized for protein concentration of the cellular
extracts.
Infection-inhibition of PTH cultures using polyanions or
HBV preS1-lipopeptides
Highly purified HBV (100 genomes per hepatocyte) from chronic
carriers were incubated with PTH for 4 h at 16°C after pre-
incubation of virus with different concentrations of indicated
polyanions for 1 h at 16°C. Incubation with 100 nM infection-
interfering myristoylated HBV preS1 peptides 2–48, or inactive
peptide (preS1 domains 19–48) for 1 h at 16°C served as a
control. After extensive washing at 4°C, cells were shifted to 37°C
for 12 h to allow HBV uptake. For post-incubation of polyanions,
highly purified HBV from chronic carriers were incubated with
PTH (100 genomes per hepatocyte) for 4 h at 16°C. After exten-
sive washing at 4°C, cells were incubated with different concen-
trations of indicated polyanions and HBV peptides (100 nM) for
1 h at 16°C. After further washing steps at 4°C, cells were shifted
to 37°C for 12 h to allow HBV uptake. Medium was changed
every 3 days, and supernatant from day 9 to day 12 was mea-
sured for the appearance of secreted HBV e antigen (HBeAg)
and HBsAg. All experiments were conducted at least in three
independent series, and the results of one representative experi-
ment are shown in each case.
Enzymatic removal of cell-surface associated GAGs
Heparinase III, sialidase or PNGase F was incubated with PTH in
the appropriate digestion buffer according to the protocol given
by the manufacturer. After several washings with THM, viral
binding was performed for 4 h at 16°C with indicated concentra-
tions of purified virus. Subsequently, cells were washed at 4°C,
and were either analysed for viral binding as described above, or
shifted to 37°C to allow infection.
Inhibition of cellular GAG sulfation
For inhibition of sulfation of cell-associated proteoglycans, PTH
were cultivated for the times indicated in sulfate-free medium
(Joklik modified Earls medium, Sigma) in the presence or
absence of different concentrations of sodium chlorate, an known
inhibitor of cellular ATP-sulfurylase. HBV infection was performed
afterwards as described either in the presence or absence of
sodium chlorate.
Assay for HBV-specific proteins
HBeAg was determined quantitatively by a commercially avail-
able ELISA (AxSym, Abbott Laboratories, Delkenheim, Germany)
130 C. M. Leistner, S. Gruen-Bernhard and D. Glebe
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 10, 122–133
that reacts specifically with HBeAg but not with HBV core par-
ticles as described (Glebe et al., 2005). HBsAg was measured by
an in-house sandwich ELISA as described (Glebe et al., 2003).
Results were obtained as ratios of signal to cut-off and were
converted to percentage of the non-inhibited control.
Acknowledgements
We thank S. Broehl for excellent technical assistance, C. Zartner
and K.P. Valerius for maintaining the Tupaia colony, and
T. Discher, G. Bein and J. Misterek for supply of HBV-positive
plasma. Supported by Grant SFB A2 (to D.G. and Wolfram H.
Gerlich) from the Deutsche Forschungsgemeinschaft (DFG). The
authors thank Wolfram H. Gerlich for continuous support of this
work.
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    • "For example, hepatitis C virus utilizes HSPG as a necessary initial attachment receptor (Baumert et al., 2014 ), and dengue virus uses HSPG as a binding and entry receptor on hepatocytes (Hilgard, 2000 ). HSPG-mediated binding of HBV and its subviral particles were observed even in HBV non-susceptible cells such as HeLa and Chinese hamster ovary cells (Leistner et al., 2008; Schulze et al., 2007). These results of the current study provide strong evidence that HBV-mimicking particles are internalized by HSPG-dependent and NTCP-independent endocytosis . "
    [Show abstract] [Hide abstract] ABSTRACT: Sodium taurocholate cotransporting polypeptide (NTCP) was recently discovered as a hepatitis B virus (HBV) receptor, however, the detailed mechanism of HBV entry is not yet fully understood. We investigated the cellular entry pathway of HBV using recombinant HBV surface antigen L protein particles (bio-nanocapsules, BNCs). After the modification of L protein in BNCs with myristoyl group, myristoylated BNCs (Myr-BNCs) were found to bind to NTCP in vitro, and inhibit in vitro HBV infection competitively, suggesting that Myr-BNCs share NTCP-dependent infection machinery with HBV. Nevertheless, the cellular entry rates of Myr-BNCs and plasma-derived HBV surface antigen (HBsAg) particles were the same as those of BNCs in NTCP-overexpressing HepG2 cells. Moreover, the cellular entry of these particles was mainly driven by heparan sulfate proteoglycan-mediated endocytosis regardless of NTCP expression. Taken together, cell-surface NTCP may not be involved in the cellular uptake of HBV, while presumably intracellular NTCP plays a critical role.
    Article · Oct 2016
    • "In the stationary phase—when the conversion potential of the starting material was exhausted—the specific infectivity dropped again, arguing for a gradual secondary loss of infectivity due to aging effects (Figure 5F). & Microbe 20, 1–11, July 13, 2016 5 non-hepatic cells of diverse mammalian species such as humans, tupaias, and rodents (Leistner et al., 2008; Schulze et al., 2007 ). We could corroborate this apparent nonselectivity in an in vivo experiment in which highly purified, [ 131 I]-labeled B-type virions were injected into uPA/SCID/beige mice with human liver xenografts—a well-established small animal model for HBV (Dandri et al., 2001; Lü tgehetmann et al., 2011)—in order to trace the particles' organ distribution (Figures 6A and 6B). "
    [Show abstract] [Hide abstract] ABSTRACT: Hepatitis B virus (HBV) replication is strictly limited to the liver. Virions attach to hepatocytes through interactions of the viral PreS envelope protein domain with heparan sulfate proteoglycans (HSPGs). However, HSPG is ubiquitously present on many cell types, suggesting that HBV employs mechanisms to avoid attachment at extrahepatic sites. We demonstrate that HBV particles are released from cells in an inactive form with PreS hidden in the interior. These HSPG-non-binding (N-type) particles develop receptor binding competence by translocating PreS across the envelope onto their surface. Conversion into HSPG-binding (B-type) particles occurs spontaneously and renders HBV infectious. Low-dose inoculation of mice with human liver xenografts demonstrates superiority of N-type particles in establishing infections, while mature B-type virions, generated via N-type conversion, are profoundly impaired, correlating with non-selective accumulation in extrahepatic tissues. This dynamic topology switch represents a maturation process utilized by HBV to most likely avoid non-productive docking outside the liver.
    Full-text · Article · Jun 2016
    • "It has been shown that known substrates of NTCP as well as HSPGs interfere with HBV and HDV infection. These substances can be subdivided in different groups: firstly, natural or artificial substrates of HSPG like heparin, highly sulphated dextrans, suramin or negatively charged polymers [71][72][73]. They shall not be further discussed here. "
    [Show abstract] [Hide abstract] ABSTRACT: For almost three decades following the discovery of the human Hepatitis B Virus (HBV) the early events of virus infection (attachment to hepatocytes, specific binding to a receptor on hepatocytes) remained enigmatic. The gradual improvement of tissue culture systems for HBV has enabled the identification of viral determinants for viral infectivity and facilitated the discovery of the human sodium taurocholate co-transporting polypeptide (hNTCP) as a liver specific receptor of HBV and its satellite, the human Hepatitis Delta Virus (HDV). These findings are currently leading basic and clinical research activities in new directions. (1) Stable hNTCP-expressing cell lines have become a valuable platform to study the full HBV replication cycle from its native template, the cccDNA. (2) The suitability of NTCP complemented cell culture systems for high throughput screening approaches will facilitate identification of novel host factors involved in HBV replication (including those that determine the peculiar host specificity of HBV infection) and will enable identification and development of novel drug candidates for improved therapeutics. (3) Since NTCP is a major host-specific restriction factor for HBV and HDV, hNTCP-expressing animals provide the basis for future susceptible in vivo models. (4) The concept obtained with the entry inhibitor Myrcludex B demonstrates that NTCP is a suitable target for clinical interference with viral entry. This will foster further clinical approaches aiming at curative combination therapies.
    Full-text · Article · Apr 2016
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