hepatitis b virus infection initiates with a large surface protein–dependent binding to heparan...

Hepatitis B Virus Infection Initiates With a LargeSurface Protein–Dependent Binding to Heparan

Sulfate ProteoglycansAndreas Schulze,1 Philippe Gripon,2 and Stephan Urban1

Contrary to many other viruses, the initial steps of the hepatitis B virus (HBV) infection,including attachment to hepatocytes, specific receptor interactions, and membrane fusion,are unsolved. Using HepaRG cells as an in vitro cell culture system, we here report that HBVentry into hepatocytes depends on the interaction with the glycosaminoglycan (GAG) sidechains of cell-surface–associated heparan sulfate proteoglycans. Binding to GAGs requiresthe integrity of the pre-S domain as a part of the large (L-) viral envelope protein. HBVinfection was abrogated by incubation of virions with heparin, but not the structurallyrelated GAGs chondroitin sulfate A, B, and C. Infection was also abolished by suramin, aknown inhibitor of duck hepatitis B virus infection or highly sulfated dextran sulfate.Polycationic substances such as poly-L-lysine, polybrene, and protamine also preventedinfection, however, by addressing cellular components. Enzymatic removal of defined acidiccarbohydrate structures from the cell surface using heparinase I/III or the obstruction ofGAG synthesis by sodium chlorate inhibited HBV infection of HepaRG cells and, moreover,led to a reduction of HBV cell surface binding sites. The biochemical analysis showedselective binding of L-protein–enriched viral particles (virions or filaments) to heparin.GAG-dependent binding of HBV was improved by polyethylene glycol, a substance thatspecifically enhances HBV infection. Conclusion: HBV infection requires the initial attach-ment to the carbohydrate side chains of hepatocyte-associated heparan sulfate proteoglycansas attachment receptors. This interaction initializes the multistep entry process of HBV andcannot be bypassed by alternative routes. (HEPATOLOGY 2007;46:1759-1768.)

Hepatitis B virus (HBV) represents the medicallyimportant prototype of a family of small, envel-oped DNA viruses (hepadnaviridae), which are

widespread in mammals and birds. All members of thisfamily possess high species and, at least with respect to thepreferential site of replication, also tissue specificity.1 He-padnaviral infections cause transient or persistent liverinflammation, with approximately 360 million peopleworldwide being chronic HBV carriers and approxi-mately 650,000 deaths each year attributable to HBV-related progressive liver failure (cirrhosis or hepatocellularcarcinoma).2

For a long time, in vitro studies of the HBV life cycle,particularly the early infection events (attachment, recep-tor binding, and fusion), were only feasible using primaryhuman hepatocytes (PHH). The application of PHH was,however, limited through restrictions in accessibility andhigh variations in susceptibility to HBV infection.3 The es-tablishment of the highly differentiable human hepatomacell line HepaRG resolved this issue and facilitated the sys-tematic analysis of the early HBV infection events.4

Abbreviations: CHO, Chinese hamster ovary; DHBV, duck hepatitis B virus;ELISA, enzyme-linked immunosorbent assay; GAG, glycosaminoglycan; ge, genomeequivalent; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HSPG,heparan sulfate proteoglycan; IU, International Units; L-protein, hepatitis B virus mid-dle surface protein; LDH, lactate dehydrogenase; M-protein, hepatitis B virus mediumsurface protein; PCR, polymerase chain reaction; PEG, polyethylene glycol; PHH, pri-mary human hepatocytes; S-protein, hepatitis B virus small surface protein.

From the 1Department of Molecular Virology, Otto-Meyerhof-Zentrum (OMZ),University of Heidelberg, Heidelberg, Germany; and the 2Institut National de la Santeet de la Recherche Medicale (INSERM), Hopital de Pontchaillou, Rennes, France.

Received March 26, 2007; accepted June 26, 2007.Supported by grants from the Deutsche Forschungsgemeinschaft (DFG) UR72/4-1

and UR72/4-2 and the federal state of Baden-Wurttemberg in its research activityForschungsschwerpunktprogramm Baden-Wurttemberg, Infektionsstrategien.

Address reprint requests to: Stephan Urban, Molekulare Virologie, Otto-Meyer-hof-Zentrum (OMZ), Universitat Heidelberg, Im Neuenheimer Feld 350, 69120Heidelberg, Germany. E-mail: [email protected]; fax:�49-6221-561946.

Copyright © 2007 by the American Association for the Study of Liver Diseases.Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/hep.21896Potential conflict of interest: Nothing to report.Supplementary material for this article can be found on the HEPATOLOGY Web

site (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

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The HBV envelope consists of the large (L), middle(M), and small (S) surface proteins. They are encoded in asingle open reading frame containing 3 in-phase startcodons.1 L- and M-proteins share the 226 amino acids ofthe S-domain. The M-protein is characterized by an N-terminal extension of S by 55 amino acids (called preS2),whereas further extension with 108 (genotype D) or 119(genotypes A and C) amino acids (designated preS1) de-fine the L-protein.

Several observations indicate an essential role of thepreS1 domain within the viral L-protein for early infec-tion events: (1) DNA-containing virions, in contrast tothe huge excess of empty spherical subviral particles, areenriched in L-protein.5 (2) In vitro infection of PHH withHBV requires myristoylation of glycine-2 in the preS1domain.6,7 (3) The integrity of the N-terminal 77 aminoacids is essential for infectivity, whereas the preS2 domainis dispensable.8,9 (4) Acylated peptides comprising the N-terminal 47 amino acids of the preS1 domain abolishHBV infection in vitro10-13 and in vivo (Petersen et al.,unpublished observations, 2007).

For HBV, a variety of cellular binding factors havebeen described in the past3; however, none of them hasbeen experimentally proven to be involved in HBV entryin infection studies.

Glycoproteins, glycolipids, and proteoglycans are con-stitutive components of the extracellular matrix and theplasma membrane. They have been selected by differentpathogens, including protozoae (for example, leishma-nia14), bacteria (for example, Bordetella pertussis15), andviruses (such as herpes simplex viruses16 or human immu-nodeficiency virus17) as attachment sites. However, pro-ductive entry, resulting in the nucleocapsid release intothe cytosol, requires additional steps, such as high-affinityreceptor interactions and, for enveloped viruses, mem-brane fusion.

Glycosaminoglycans (GAGs) are unbranched polysac-charides composed of hexosamine/hexuronic acid repeats.They acquire negative charges through N- and O-sulfa-tion of the carbohydrate moieties in the Golgi appara-tus.18 GAGs are bound to core proteins or lipids to formglycoconjugates. GAG–ligand interactions are complexand characterized by either merely electrostatic forces orspecific interactions involving hydrogen bonding or hy-drophobic contacts.19 The strength and specificity ofthese contacts are determined by the cell type–specificstructural heterogeneity of GAGs, reflected in variationsin length and composition of the carbohydrate chains.Interestingly, hepatocytes constitute a unique GAG com-position.20,21 This, and the distinct architectural arrange-ment of hepatocytes in the liver,22 might contribute to the

hepatotropism of pathogens, although their primary in-teraction is not exclusively restricted to liver cells.

Here we investigated the role of GAGs in HBV entryinto hepatocytes. We provide evidence that HBV infec-tion depends on these cell surface structures as attachmentreceptors. We further demonstrate that binding to hepa-rin, a soluble GAG requires the integrity of the preferen-tially virion-associated L-protein, suggesting selectivity inthe binding of virions to hepatocytes.

Materials and Methods

Reagents. Heparin, chondroitin sulfate A, B, and C,heparan sulfate, hyaluronic acid, suramin, dextran, dex-tran sulfate, sodium chlorate, protamine, poly-L-lysine,hexadimethrine bromide (polybrene), de-N-sulfated hep-arin, de-N-sulfated acetylated heparin, heparinase I, andheparinase III were purchased from Sigma-Aldrich.

Cell Lines. HepaRG cells were cultivated as de-scribed.4 Chinese hamster ovary (CHO) cells K1 andpgsA74523 were grown in Dulbecco’s modified Eagle’smedium with the addition of 10% fetal bovine serum,100 U/mL penicillin,and 100 �g/mL streptomycin.

Infection of HepaRG Cells With HBV. As an infec-tious inoculum, we used an approximately 100-fold con-centrated culture supernatant of HepG2.2.15 cells.12 Ifnot stated otherwise, differentiated HepaRG cells wereincubated with a 1:20 dilution of this virus stock in me-dium supplemented with 4% PEG 8000 (Sigma-Aldrich)for 16 hours at 37°C. At the end of the incubation, thecells were washed and further cultivated. Medium waschanged every 3 days. To quantify the infection, hepatitisB surface antigen (HBsAg) and hepatitis B e antigen se-creted into the culture supernatant from day 7 to 11postinfection was determined by enzyme-linked immu-nosorbent assay (ELISA).

Infection competition experiments were performed inpresence of the corresponding drug during virus inocula-tion. For pre-incubation studies, HepaRG cells or viruswere mixed for 0.5 or 1 hour at 37°C with the substancefollowed by its removal before infection. All experimentswere performed in duplicate and repeated at least 2 timesindependently. To analyze possible cytotoxic effects ofthe reagents, we performed lactate dehydrogenase (LDH)release assays (Cytotoxicity Detection KitPLUS, Roche Ap-plied Science).

Inhibition of Cellular GAG Sulfation by SodiumChlorate. To prevent the sulfation of cell-associated pro-teoglycans, HepaRG cells were cultivated for 48 hours at37°C in the presence of sodium chlorate. Subsequent vi-rus inoculation was performed in the presence of the in-hibitor.

1760 SCHULZE, GRIPON, AND URBAN HEPATOLOGY, December 2007

Enzymatic Removal of GAGs From the Surface ofHepaRG Cells. The lyases were solved in their digestionbuffers according to the manufacturers’ protocol, addedto prewashed cells, and incubated for 1 hour at 37°C.Subsequently, the cells were washed, and binding (4hours, 37°C) or infection was performed in the absence ofthe enzymes. Enzyme concentrations are expressed in in-ternational units (IU) per milliliter (1 IU � 600 Sigmaunits).

Quantification of Cellular HBV Binding. Attach-ment of HBV to cells was determined by quantitativepolymerase chain reaction (PCR) detecting the viralDNA. Binding/uptake was performed for 4 hours at37°C. Unbound virus was removed by washing. ViralDNA was prepared from the cellular lysates with theNucleoSpin Blood Kit (Macherey-Nagel) according tothe manufacturers’ protocol. For the SybrGreen-based re-al-time PCR (Invitrogen), the primer set Taq-HBV-F(5�-TCCCAGAGTGAGAGGCCTGTA-3�) and Taq-HBV-R (5�-ATCCTCGAGAAGATTGACGATA-AGG-3�) was used. The reaction process started at 50°Cfor 2 minutes and 95°C for 15 minutes followed by 45cycles at 95°C for 15 seconds and 60°C for 1 minute. Allexperiments were performed at least twice.

Binding of HBV Particles to Immobilized Heparin.Concentrated culture supernatants of HepG2.2.15 cellswere applied to a 1 mL Hi-Trap Heparin HP column (GEHealthcare) equilibrated with 0.02 M Tris/Cl (pH 7.4),0.14 M NaCl. After loading, the column was washed with10 to 20 column volumes of equilibration buffer. Step-wise elution was performed with 0.24 M, 0.34 M, 0.54M, 1 M, and 2 M NaCl in 0.02 M Tris/Cl (pH 7.4).Fractions of 1 mL were collected and analyzed by westernblot, immune- and DNA-dot blot using antibodiesagainst the HBV L- (Ma18/75) and S- (goat-anti-HBsAg,Fitzgerald) protein or an HBV-specific [32P]-labeledDNA probe.

Trypsin Digestion of Viral Particles. Elution frac-tions from a heparin column were adjusted to 140 mMNaCl in 0.02 M Tris/Cl (pH 7.4). These fractions weretreated with 12.5 �g/�L trypsin for 5 hours at 37°C.After the incubation, the protease was inactivated by theaddition of aprotinin (Fluka).

Results

HBV Infection and Binding Is Specifically Inhib-ited by Heparin. To test whether soluble GAGs inhibitHBV infection, we performed infection competition ex-periments in the presence of increasing concentrations ofheparin, heparan sulfate, chondroitin sulfate A, chon-droitin sulfate C, or dermatan sulfate (chondroitin sulfate

B). The HBsAg secreted into the medium from day 7 to11 postinfection was determined. To affirm that the mea-sured HBsAg represents newly synthesized S-protein, weincluded a control inhibition experiment using the previ-ously described entry inhibitor HBVpreS/2-48myr,10

which led to a reduction of HBsAg secretion below thedetection limit (data not shown). As shown in Fig. 1A,heparin (approximately 2.4 sulfate groups/disaccharide)abolished HBV infection with a concentration that inhib-its 50% of approximately 9.4 �g/mL. Heparan sulfate, a

Fig. 1. Heparin specifically inhibits HBV binding to and infection ofHepaRG cells. (A) HepaRG cells were infected with HBV in the presenceof heparin, heparan sulfate, chondroitin sulfate A, chondroitin sulfate C,and dermatan sulfate at increasing concentrations (0-300 �g/mL).HBsAg secreted from day 7 to 11 postinfection was determined by anELISA. The values are given as percentage of the uncompeted controlinfection. The sensitivity of the ELISA (cutoff) is indicated by the dottedline. (B) HBV was preincubated with heparin (630 �g/mL) for 30minutes at room temperature. Unbound GAGs were removed by ultrafil-tration. Infection (left) was performed with the pretreated HBV particlesunder standard conditions. The amount of HBsAg in the supernatant ofday 7 to 11 postinfection was determined. In parallel, binding (4 hoursat 4°C) (right) of the heparin-treated HBV particles to HepaRG cells wasquantified by real-time PCR and presented as cell-associated genomeequivalents per well (106 cells).

HEPATOLOGY, Vol. 46, No. 6, 2007 SCHULZE, GRIPON, AND URBAN 1761

lower sulfated heparin derivative (0.8-1.4 sulfate groups/disaccharide) showed a reduction to 60% at 300 �g/mL.In contrast, GAGs with sulfate contents below 1.1 sulfategroups/disaccharide, such as chondroitin sulfate A and Cbut also dermatan sulfate with approximately 1.1 sulfategroup/disaccharide, exhibited no effect on infection. Totest whether heparin addresses viral structures, we prein-cubated HBV with heparin (630 �g/mL) and removedunbound GAGs before infection of HepaRG. The infec-tion was reduced to 8% in comparison with the control(Fig. 1B, left). At no concentration could significant cy-totoxic effects be observed (Supplementary Fig. 1). Toexamine whether this inhibition is attributable to an in-terference with cellular attachment, we analyzed the bind-ing of heparin pretreated viruses to HepaRG cells by aquantitative real-time PCR. The quantification showed adecreased viral binding of 54% of the control (Fig. 1B,right). To exclude an effect on replication steps after virusbinding, we added heparin to HepaRG cells at differenttimes during and after inoculation with the virus. Hepa-rin showed a time-dependent reduction in its ability tointerfere with HBV infection when applied during inoc-ulation (Supplementary Fig. 2A). At later times (48 and72 hours) after initiation of infection, heparin had noeffect. This emphasizes the exclusive action of heparin onan early step of HBV infection. The same held true for theinhibitors described later (Supplementary Fig. 2B).

Sulfation of Cellular Glycosaminoglycans Is a Pre-requisite for HBV Infection. To address the role ofsulfation for infection inhibition, differentially sulfatedheparin variants were analyzed. Completely de-N-sul-fated heparin did not interfere with infection (Fig. 2A). Incontrast, partially de-N-sulfated heparin exhibited an in-hibition potential of 70% at the highest concentrationtested (200 �g/mL), indicating that the degree of sulfa-tion within the carbohydrate structure is important.

To investigate the contribution of the carbohydratebackbone on the infection inhibition potential, we per-formed neutralization experiments using the syntheticpolyanions dextran sulfate (composed of glucose unitswith 2.3 sulfate groups/glucosyl residue) and suramin, aderivative of urea (6.0 sulfate groups/molecule), whichhas been reported to block duck hepatitis B virus(DHBV) and hepatitis delta virus infection.24 (Fig. 2B).

Although suramin (concentration that inhibits 50% ofapproximately 33 �g/mL) and dextran sulfate (concen-tration that inhibits 50% of approximately 9 �g/mL)neutralized HBV infection, dextran but also hyaluronicacid, an acidic, non-sulfated GAG, were inactive, even at300 �g/mL.

To determine whether the inhibitory effect of suraminis attributable to an interference with HBV attachment to

Fig. 2. The degree of sulfation correlates with the ability to interferewith infection. (A) HepaRG cells were infected in the presence ofunmodified, partially de–N-sulfated or completely de–N-sulfated heparin.The inoculum with the heparins was incubated overnight on the cells. At11 days postinfection, the culture supernatants from day 7 to 11postinfection were analyzed for HBsAg. (B) HepaRG cells were infectedwith HBV in the presence of suramin, dextran sulfate, dextran, andhyaluronic acid. HBsAg secreted between day 7 to 11 postinfection wasdetermined. (C) Quantification of HBV binding (4 hours at 37°C) toHepaRG cells in presence of heparin (63 �g/mL and 630 �g/mL) orsuramin (100 �g/mL). Cell-associated genome equivalents were quan-tified by real-time PCR.


cells, we quantified cell association of virions in the pres-ence of suramin in a concentration known to block infec-tion (100 �g/mL). As a comparison, we analyzed thecellular binding of HBV in presence of 2 different heparinconcentrations. For suramin, a 2.2-fold reduction inbinding could be observed. At the highest concentration(630 �g/mL), heparin showed a 7.7-fold decreased bind-ing in contrast to the control (Fig. 2C).

To provide additional evidence for the requirement ofsulfated cell surface molecules for HBV infection, we in-hibited the cellular adenosine triphosphate–sulfurylase byaddition of sodium chlorate to the culture medium.25 Asdepicted in Fig. 3A chlorate concentrations greater than50 mM reduced HBV infection of HepaRG cells to back-ground level, indicating the requirement of sulfated cellsurface structures. To test whether chlorate-mediated in-hibition of HBV infection correlates with the reduction invirus binding, we quantified the ability of HepaRG cellsto bind HBV after preincubation with increasing concen-trations of sodium chlorate. As depicted in Fig. 3B, Hep-aRG cells devoid of sulfated GAGs were significantlyimpaired to bind HBV in comparison with the control(36% at 80 mM chlorate). To rule out side effects, Hep-aRG cells were cultured for 2 days in the presence ofsodium chlorate (80 mM) and subsequently infected inabsence or presence of the reagent. In both cases, HBsAgsecretion was reduced to background levels. However,addition of the inhibitor 24-48 hours after inoculationwith the virus had no effect (Supplementary Fig. 3).

Neutralization of Negative Charges on HepaRGCells by Polycations Abrogates HBV Infection. Tosubstantiate the importance of cellular polyanionic bind-ing sites for HBV, we preincubated HepaRG cells withthe heparin antagonists poly-L-lysine and protamine aswell as polybrene and infected the cells in presence of thesubstances. As depicted in Fig. 4A, poly-L-lysine and po-lybrene strongly decreased HBV infection when appliedat 2 or 1.5 �g/mL, respectively; protamine displayed areduction to 19% at 200 �g/mL.

Preincubation of HepaRG cells with heparin, suramin,and dextran sulfate at concentrations that inhibit HBVentry when present during virus inoculation did not im-pair infection, whereas preincubation with poly-L-lysine,

Fig. 3. Sulfation of cellular GAGs is required for the interaction of HBVwith hepatocytes. The sulfation of cellular proteoglycans was inhibited bythe pretreatment of HepaRG cells with increasing concentrations ofsodium chlorate for 48 hours before infection (A) or binding (4 hours at4°C). (B) Infection efficacy was quantified by measurement of HBsAgsecreted between days 7 and 11 postinfection. HBV binding to HepaRGcells was determined by real-time PCR and presented as cell-associatedgenome equivalents per 106 cells.

Fig. 4. The obstruction of negatively charged cellular interaction sitesinhibits HBV infection. (A) HepaRG cells were incubated for 1 hour at37°C with increasing concentrations of poly-L-lysine, polybrene, or pro-tamine. Subsequently infection was performed in the presence of thesubstances. (B) HepaRG cells were preincubated with either polyanionic(heparin, suramin, and dextran sulfate) or polycationic (poly-L-lysine,polybrene, and protamine) compounds for 1 hour at 37°C beforeinfection. Cells were washed and infected in absence of the substances.Secreted HBsAg of days 7 through 11 postinfection is presented aspercentage of the control.


protamine, and polybrene did (Fig. 4B). Unexpectedly,the sulfated polymers heparin and dextran sulfate led toan approximately 1.6-fold and 2.3-fold increased infec-tion. This might be explained by the creation of artificialbinding sites through accumulation of soluble carbohy-drates on the cell surface.

Enzymatic Removal of GAGs by Heparinases Re-duces HBV Binding to and Infection of HepaRG Cells.To specify the particular type of GAG responsible forHBV binding to hepatocytes, we took advantage of thedifferent specificities of heparinase I and III. Althoughheparinase I cleaves heparin and heparan sulfate, hepari-nase III exclusively addresses the latter. As shown in Fig.5A, heparinase I–treated HepaRG cells showed reduced

HBV binding when compared with the control (24.5% at71 mIU/mL and 12.6% at 143 mIU/mL). Similarly, he-parinase III digestion decreased viral attachment by 68%at 38 mIU/mL, indicating that hepatocyte cell surface–exposed heparan sulfate is the key binding element forHBV.

To prove the relevance of the heparinase I/III-medi-ated reduction of HBV binding for infection, HepaRGcells were preincubated with 4, 42, and 167 mIU/mL(heparinase I) or 21 mIU/mL (heparinase III), followedby inoculation with HBV for 16 hours in absence of theenzymes (Fig. 5B). Heparinase I decreased infection in adose-dependent manner, with maximal reduction to 40%of the control. Heparinase III reduced infection approxi-mately 2-fold. A more pronounced effect of the enzymescould be observed when the heparinases were present dur-ing virus inoculation (Supplementary Fig. 4). The slowkinetics of cellular HBV binding and uptake requires aprolonged incubation time for efficient infections. Theinhibitory effect of heparinase I/III may therefore be par-tially counteracted by the re-emergence of newly synthe-sized heparan sulfate proteoglycans (HSPGs) on thesurface of HepaRG cells.

Polyethylene Glycol (PEG) Enhances the Binding ofHBV to Cell-Surface GAGs. PEG has been shown toenhance HBV infection in PHH26 and HepaRG cells.4

To investigate whether the effect of PEG on HBV bind-ing is mediated by cell surface-exposed GAGs, we used theCHO-K1 cell line and its GAG-deficient derivativeCHO-pgsA745. CHO-pgsA745 cells lack xylosyltrans-ferase and do not express heparan and chondroitin sul-fates.23 In comparison, we quantified binding of HBV todifferentiated and nondifferentiated HepaRG cells in thepresence or absence of 4% PEG.

Quantification of HepaRG cell-associated HB virionsin the presence of PEG showed that binding is indepen-dent of the cell differentiation state. In fact, nondifferen-tiated HepaRG cells bound HBV with slightly higher(10.4 ge/cell in comparison with 5.1 ge/cell) efficacy (Fig.6A, right). Taken into account that 116 HBV ge/cell wereapplied but only 4.4% (differentiated cells) or 9.0% (non-differentiated cells) were cell associated after 4 hours, weconcluded that only a limited number of viral bindingsites are available on the cell surface. Consistent with ear-lier studies, binding was reduced (13.7-fold in differenti-ated versus 16.2-fold in nondifferentiated cells) in theabsence of PEG (Fig. 6A, left). A similar degree of HBVcell association including a PEG-mediated enhancementof binding (14.7-fold) was also observed for the nonsus-ceptible CHO-K1 cell line (Fig. 6B). Surprisingly, HBVbinding to CHO-pgsA745 cells was not enhanced byPEG. The observation that HBV binding can only be

Fig. 5. Enzymatic removal of cell-surface GAGs reduces HBV bindingand infection. HepaRG cells were incubated for 1 hour at 37°C with theindicated concentrations of the GAG lyases heparinase I and III. The cellswere washed, and binding (A) or infection (B) was performed in absenceof the enzymes. The amount of cell-associated HBV was quantified viareal-time PCR; infection was analyzed by secreted HBsAg from day 7 to11 postinfection.


reduced by heparin in the presence of PEG (Fig. 6B right)indicates that PEG promotes GAG-mediated virus–cellcontacts.

L-Protein Rich HBV Particles Selectively Bind toHeparin. To biochemically characterize the interactionof HBV particles to GAGs, we applied HepG2.2.15 cell-derived viral and subviral HBV particles to a heparin af-finity chromatography column and determined theirelution behavior. Most DNA-containing particles boundto heparin-sepharose under physiological salt conditions

(140 mM NaCl) and eluted at NaCl concentrations of atleast 240 mM (Fig. 7A left). To prove that these fractionscontain virions, we (1) performed a western blot detectingL-protein (Fig. 7A, middle) which is mostly absent in 22nm spherical subviral particles but enriched in virions andfilaments5 and (2) simultaneously used this fraction incomparison with the input to infect HepaRG cells (Fig.7A, right). The presence of L-protein and HBV-DNA inthe 340 mM NaCl fraction and the unabated infectivityindicates binding of infectious virions to heparin. To in-vestigate the contribution of the preS-part of the L-pro-tein to heparin binding, we took advantage of previousobservations demonstrating the feasibility to enzymati-cally remove preS1 and preS2 from L-protein by tryp-sin27,28 without destroying the S-protein. To that aim, the340 mM elution fraction of a heparin-affinity chromatog-raphy [Fig. 7B, top (underlined)] was trypsinized (5 hoursat 37°C). As a control, the same fraction was incubatedwith phosphate-buffered saline. Although re-applicationof the untreated particles did not alter the ability to againbind to heparin and elute at 340 mM NaCl, HBV-DNA(Fig. 7B, bottom) and S-protein (Fig 7B, middle) wereexclusively found in the flow-through fraction after tryp-sin treatment. The unchanged immunological traceabil-ity of S-protein in the flow-through fraction indicates thattrypsin did not destroy the protein. However, and in ac-cordance with the described sensitivity of the preS-do-main against trypsin, only residual amounts of L-proteinwere detectable after protease treatment when using apreS-specific polyclonal antiserum (Fig. 7B, middle).This suggests a contributory role of the preS-domain ofthe HBV L-protein for GAG binding.

DiscussionGlycoconjugates are cell-surface structures with im-

portant physiologic functions, such as cell– cell and cell–matrix interactions, adhesion, signaling, differentiation,and development.29 It is therefore not surprising thatpathogens, including viruses selected these molecules asattachment receptors. We provide evidence that HBVinfection requires the initial interaction with hepatocytesurface HSPGs and thus, for the first time, identify cellu-lar molecules that serve as a primary attachment receptorfor HBV.

Evidence for this conclusion is based on our observa-tions that HBV binding to and infection of HepaRG cellsis sensitive to (1) inhibition with distinct soluble GAGs,(2) the prevention of GAG sulfation by chlorate, and (3)the enzymatic removal of GAGs from the cell surface.Additionally, although not susceptible, cells with a defectin the biosynthesis of heparan and chondroitin sulfatesexhibited a reduced ability to bind HBV. The fact that

Fig. 6. GAG-mediated cell-association of HBV is enhanced by PEG.(A) Binding of HBV to nondifferentiated and differentiated HepaRG cellswas performed either in absence (left) or presence (right) of 4% PEG.Binding was quantified by a real-time PCR and is presented as cell-associated genome equivalents per well. (B) Binding of HBV (4 hours at37°C) to CHO-K1 (black bars) and the GAG-deficient CHO-pgsA745(gray bars) cells in the absence (left) and presence of PEG (middle andright) and heparin (right). HBV genome equivalents were quantified byreal-time PCR and are given as absolute values and as ratios in the table.


heparin, as a soluble GAG variant, and heparinase III, as aheparan sulfate–specific lyase, interfere with both HBVattachment and productive infection suggests thatHSPGs act as initial HBV binding sites on the hepatocytesurface. This conclusion is also supported by a recentstudy by Leistner et al., using primary Tupaia hepato-cytes.30 Consistent with our results, Ying et al.31 previ-ously demonstrated an almost complete abrogation ofHBV-binding to PHH and the nonsusceptible HepG2cell line in the presence of heparin and dextran sulfate.However, they did not find a change in binding of HBVto HepG2 cells after heparinase treatment. This differ-

ence might be explained by the absence of PEG as a pro-moter of GAG-binding.

Our data indicate that heparin and other highly sul-fated polyanions bind to positively charged structures onthe surface of virions, resulting in inhibition of cellularbinding and abrogation of infection. Although infectionis reduced more than 100-fold, the decrease in virus bind-ing is less pronounced (up to 8-fold). However, consider-ing the low number of available cellular binding sites(5-30 ge/cell) this might be attributable to superimposedunspecific binding events that limit the detection range ofour binding assay.

HSPGs are expressed by many cells. However, theydiffer in the types and the extent of proteoglycans theyexpress and show variations in the heparan sulfate finestructure, such as modifications in chain length, the de-gree of sulfation, and the positions of the sulfate groups.The composition of cell-surface proteoglycans can changeduring differentiation or on activation of cells. Remark-ably, liver heparan sulfates display some unique features,such as a low sulfation density in close proximity to thecore protein and enriched N-sulfated glucosamine resi-dues and 2-O-sulphated uronic acids at their distalends.20,21 This is consistent with our findings that highlysulfated GAGs (heparin, heparan sulfate) and GAG vari-ants (dextran sulfate) were the most effective substances to

4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™Fig. 7. Selective interaction of infectious, L-protein–enriched HBV

particles with heparin-sepharose. (A) A 100-fold concentrate of aHepG2.2.15 cell supernatant, containing virions and subviral particles,but no naked cores, was applied to a heparin-sepharose affinity column.The input, flow through, and elution fractions (240, 340, 540, 1,000,and 2,000 mM NaCl) were analyzed for their HBV DNA content bydot-blot (left). The 340 mM elution fraction was analyzed for its L-proteincontent by a preS1-specific western blot using the monoclonal antibodyMa18/7 (middle). The infectivity of the 340 mM NaCl elution fraction onHepaRG cells in comparison with the respective input fraction wasmeasured (right). The ratio of virions evident from the input and the 340mM NaCl elution fraction of the heparin-affinity chromatography (left) isthe same as the ratio between the control and the 340 mM NaCl elutionfraction used to inoculate the cells for the infection of HepaRG cells(right). The infection was analyzed by quantification of HBsAg andhepatitis B e antigen secretion between days 7 and 11 postinfection. (B)A heparin-affinity chromatography was performed as described in (A).The fractions were analyzed for their L-protein content by an immune dotblot using the preS-specific polyclonal antiserum H863 (top). Trypsindigestion of the 340 mM elution fraction (underlined) was performed for5 hours at 37°C, and the products were reapplied to the heparin column.The fractions were analyzed by immune dot blots detecting either the L-(preS) or the S-protein (middle). As a control, an undigested sample ofthe 340 mM fraction was reapplied to the column. Note that trypsindigestion results in an almost complete disappearance of preS-specificsignals but does not diminish the S-signal, indicating that the integrity ofS-protein remains unchanged. The fractions of the trypsin-treated samplewere analyzed for their DNA content by a quantitative real-time PCR andare presented as genome equivalents/milliliter. The amount of the DNAin the fractions is additionally given as percentage of the input.


inhibit binding and infection whereas decreased sulfationled to a progressive loss of inhibition. Thus, the degree ofsulfation plays the key role in that interaction processwhereas the nature of the polysaccharide backbone is lessimportant.

In accordance with the presence of proteoglycans inmany cell types, we and others3 found that binding ofHBV is not restricted to susceptible HepaRG cells orPHH. Thus, at the level of attachment, the observed hostrange and the species specificity of HBV can only be ex-plained within the limits of the foresaid preference ofHBV for highly sulfated liver HSPGs. Productive infec-tion, however, must be triggered by additional, more spe-cific processes. One of these steps might be related to theinhibitory activity of acylated peptides comprising theN-terminal 47 amino acids of the preS1 domain.10,12 In-terestingly, these lipopeptides neither bind heparin norinterfere with binding of HBV to HepaRG cells at con-centrations known to block infection (unpublished data).This indicates that they function in a postattachment stepand emphasizes the assumption that HBV enters hepato-cytes by a multistep process.

Previous reports4,26 indicated that addition of PEG toPHH or HepaRG cells enhances HBV infection withoutinducing artificial fusion of the viral and the cellularmembranes. This enhancement was related to an in-creased cell association of HBV DNA. However, the na-ture of the molecules involved in the interaction wasunclear. We provide evidence that PEG promotes virusbinding by increasing the GAG-dependent attachment ofHBV to HepaRG cells by approximately the same factoras it increases infection. This conclusion is based on ourobservation that enhancement of binding in the presenceof PEG was detected in all cells, with the exception of theGAG-deficient CHO-pgsA745. This result also excludesa possible unspecific PEG-mediated precipitation of viri-ons on the cell surface and opens the question of whetherPEG can also enhance infection of other pathogens thatuse GAGs as attachment receptors.

Initial reports on the role of suramin suggested that itinhibits the DHBV DNA polymerase activity.32 How-ever, subsequent reports showed that DHBV replicationwas only inhibited when suramin was present during virusinoculation and not at later times.33 Suramin also inter-feres with hepatitis delta virus infection, an RNA virusoidthat replicates independently of the reverse transcrip-tase.24 Using a semiquantitative PCR-based binding as-say, Funk et al.34 demonstrated that suramin reduced thebinding of DNA-containing DHBV particles to embry-onic duck hepatocytes. We here extend this finding toHBV and propose a mechanism of action of suramin,

namely, mimicking highly sulfated cellular structures thatare required for attachment.

Although we and others35 present biochemical evi-dence that heparin-sepharose binds HB virions, we cur-rently do not know the exact binding site. Our datastrongly suggest that it is located in the preS domain of theL-protein because (1) L-protein–enriched virions displayselectivity toward heparin. (2) Removal of the preS do-main abrogates the virus–heparin interaction. Because ofthe functional mapping of sequence requirements forHBV and hepatitis delta virus infectivity,8,36-38 the first 75amino acids of the preS1 domain likely play a crucial rolein this process.

Taken together, we propose a multistep entry pathwayof HBV. It initializes through an L-protein–dependentbinding to cell-associated HSPGs and is followed by ahighly specific step involving a myristoylated N-terminaldomain of preS1.

Acknowledgment: We thank Stephanie Held for tech-nical assistance, Isabel Vogler, Silke Schmidt, and KerryMills for help with the establishment of the HBV bindingassay, Caroline Gahler for the preparation of virus stocks,and Stefan Seitz for many helpful discussions. We alsothank Jeffrey D. Esko for providing the CHO-pgsA745cell line. We are indebted to Ralf Bartenschlager, whocontinuously supports our work.

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