The arginine-rich carboxy-terminal domain of the hepatitis B virus core protein mediates attachment of nucleocapsids to cell-surface-expressed heparan sulfate

Peter Vanlandschoot, Freya Van Houtte, Benedikte Serruys and Geert Leroux-Roels

Virus Host Interactions Unit, Center for Vaccinology, Department of Clinical Biology, Microbiology and Immunology, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium

Correspondence
Peter Vanlandschoot
Peter.Vanlandschoot{at}UGent.be


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Binding of hepatitis B virus nucleocapsids to mouse B cells leads to production of nucleocapsid-specific antibodies, class II presentation of peptides and the generation of T helper-1 immunity. This T-cell-independent activation of B cells is thought to result from cross-linking of cell-surface immunoglobulin molecules, if these contain a specific motif in the framework region 1–complementarity determining region 1 junction. In the present study, it was observed that nucleocapsids bound to different B-cell lines, an interaction that was not dependent on cell-surface-expressed immunoglobulins. Furthermore, binding to several non-B-cell lines was observed. Capsids that lacked the carboxy-terminal protamine-like domains did not bind to cells. Treatment of nucleocapsids with ribonucleases enhanced the attachment of nucleocapsids to cells. Various soluble glycosaminoglycans inhibited attachment of nucleocapsids, while treatment of cells with heparinase I also reduced binding. These observations demonstrated that the arginine-rich protamine-like regions of the core proteins are responsible for the attachment of nucleocapsids to glycosaminoglycans expressed on the plasma membranes of cells.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Hepatitis B virus (HBV) is the prototype of the family Hepadnaviridae and is the smallest animal DNA virus known. The genome consists of approximately 3200 nt and is a partially double-stranded, relaxed circular DNA molecule. Four open reading frames – the polymerase gene, the X gene, the pre-core/core gene and the surface gene – have been identified, encoding seven proteins. During infection, capsid proteins, or core antigens (HBcAg), self-assemble to form an icosahedral capsid, a 30–34 nm particle composed of 180 or 240 units of the 21 kDa HBcAg protein. This capsid structure encapsidates pre-genomic RNA together with the P protein. This pre-genomic RNA is reverse transcribed into DNA, an event that occurs while the capsid is still inside the infected cell. These nucleocapsids interact with cytosolically exposed regions of the L protein present in the endoplasmic reticulum-to-Golgi intermediate compartment, into which the virus buds (Seeger & Mason, 2000).

Immunization studies in mice have shown that HBcAg is extremely immunogenic (reviewed by Vanlandschoot et al., 2003). As little as 6 ng HBcAg without adjuvant induces antibody production (Milich et al., 1997b). HBcAg functions as both a T-cell-independent and a T-cell-dependent antigen (Milich & McLachlan, 1986; Milich et al., 1997b; Fehr et al., 1998) and preferentially primes T helper-1 (Th1) cells (Milich et al., 1997b). Interestingly, it was reported that HBcAg-incorporated RNA facilitates the priming of this Th1 immunity (Riedl et al., 2002). B cells that take up HBcAg and present peptides through class II molecules are common in naive mice, and nucleocapsids induce CD80 and CD86 co-stimulatory molecules on naive B cells (Milich et al., 1997a). The existence of naive HBcAg-binding human B cells has been confirmed using a human peripheral blood leukocyte/NOD/Scid mouse model. HBcAg induced the secretion of HBcAg-binding immunoglobulin (Ig) M by naive human cells derived from adult and neonatal (cord blood) donors, when cells were transferred into the spleens of NOD/Scid mice. T cells were not required, confirming that HBcAg behaves as a T-cell-independent antigen for IgM in humans (Cao et al., 2001). It is thought that the unique three-dimensional structure of the HBcAg capsid favours this strong immune response and that activation of naive B cells results from binding and cross-linking of cell-surface Ig molecules by capsids (Milich et al., 1997a). Indeed, a linear motif that binds HBcAg has been identified in the framework region 1–complementarity determining region 1 (FR1–CDR1) junction of HBcAg-specific IgM. This motif was present in heavy chains of the mouse VH1 family and human VH1 and VH7 families (Lazdina et al., 2001). The binding site on the capsids was shown to involve residues 76–80, which lie on the tip of the spikes and form part of the immunodominant epitope (Lazdina et al., 2003).

Although these observations support an interaction of HBcAg with Igs derived from restricted V germ-line gene groups, no molecular evidence for binding of HBcAg to Igs expressed on the surface of B cells has been obtained. The initial purpose of the study presented here was to obtain molecular evidence for such an interaction. However, we found that HBcAg was capable of binding to the surface of different cell types without the need for cell-surface-expressed Igs. Evidence for a possible role of the arginine-rich carboxy-terminal region of the core protein in attachment to glycosaminoglycans (GAGs) on the surface of cells is presented.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
HBcAg.
Full-length HBcAg (subtype ayw) produced in Escherichia coli (HBcAg-c) was obtained from Diasorin. Two full-length HBcAg nucleocapsid preparations (subtype adw), obtained from GlaxoSmithKline, were produced in Saccharomyces cerevisiae (HBcAg-y). Truncated HBcAg (subtype ayw), which lacked aa 145–183, was produced in E. coli and obtained from Biodesign.

Reagents.
Heparin, heparan sulfate, chondroitin sulfate B, hyaluronic acid, dextran sulfate 500 (500 kDa), heparinase I, hyaluronidase, phorbol-12-myristate-13-acetate (PMA) and phosphatidylinositol-specific phospholipase C (PI-PLC) were from Sigma. Human AB serum (HS) was from Bio-Whittaker. Annexin-V–FITC was from Pharmingen.

Antibodies.
Goat anti-human Fcµ IgG, goat anti-human Fab IgG and goat IgG were from Sigma. Mouse anti-human IgM/D/G–FITC and mouse IgG1 and IgG2b were from Pharmingen. Human anti-HBcAg was biotinylated using an ECL protein biotinylation module (RPN 2202; Amersham Pharmacia Biotech).

HBcAg ELISA.
Maxisorb 96-well plates (Nunc) were coated with HBcAg in PBS. Wells were blocked with 0·1 % BSA in PBS, followed by washing three times with 0·05 % Triton X-100. Biotinylated HBcAg-specific mAb (1 µg ml–1) was added and the plates were incubated for 1·5 h at room temperature. mAb was detected with streptavidin labelled with peroxidase. After three washes, 3,3',5,5'-tetramethylbenzidine (Sigma) was added and 30 min later the reaction was stopped with 0·5 M H2SO4.

Cells.
Human peripheral blood mononuclear cells were isolated from buffy coats using Ficoll-Hypaque centrifugation (density=1·077 g ml–1; Nycomed Pharma). Cells were stored in liquid nitrogen. THP-1 cells were grown in cRPMI (RPMI 1640 plus 10 % FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U penicillin ml–1, 50 µg streptomycin ml–1 and 20 µM {beta}-mercaptoethanol). To induce differentiation, 100 nM 1,25-dihydroxyvitamin D3 (1,25-VitD3; Calbiochem) was added for 24 or 48 h. Three Ramos cell lines, Epstein–Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) and Jurkat cells were grown in cRPMI. CHO cells expressing human CD14 and CHO cells transfected with vector only (Jack et al., 1995) were grown in MEM alpha without nucleosides or ribonucleosides (Gibco-BRL) supplemented with 10 % FCS, 2 mM L-glutamine, 50 U penicillin ml–1 and 50 µg streptomycin ml–1. HEK293T and COS-7 cells were grown in Dulbecco's MEM supplemented with 10 % FCS, 2 mM L-glutamine, 50 U penicillin ml–1 and 50 µg streptomycin ml–1. Cultured cells were detached mechanically or using trypsin/EDTA and washed twice with PBS/0·8 % BSA. To study the involvement of GAGs, cells were incubated with 10 ng PMA ml–1, 10 U heparinase I ml–1, 10 U hyaluronidase ml–1 or 1 U PI-PLC ml–1 for 2 h at 37 °C in PBS/0·8 % BSA. CaCl2 (0·5 mM) and MgCl2 (0·5 mM) were added for heparinase I and hyaluronidase treatment of cells. After treatment, cells were washed twice with PBS/0·8 % BSA.

HBcAg cell-binding assay.
Cells were incubated on ice with 5 or 10 µg HBcAg ml–1 in 200 µl PBS/0·8 % BSA for 1 h. In some experiments, after washing, cells were incubated for 30 min in 20 % heat-inactivated HS. HBcAg particles were detected using the biotinylated HBcAg-specific mAb followed by streptavidin labelled with phycoerythrin (SAPE). Cells were analysed on a FACScan flow cytometer (Becton Dickinson). Dead cells that incorporated propidium iodide were gated out of the analysis. At least 5000 cells were counted per analysis. Fluorescence was measured at 530 nm for FITC and 580 nm for PE, and the median fluorescence determined in each case. Signals were acquired in a logarithmic mode for FL1 (FITC) and FL2 (PE). Threshold levels were set according to negative (SAPE only) and isotypic controls.

RNase treatment of HBcAg.
HBcAg (10 µg) was treated with 0·25 µg RNase (a heterogeneous mixture of ribonucleases from bovine pancreas; Roche) in PBS for 60 min at room temperature. HBcAg was separated on a 1 % TAE agarose gel and stained with ethidium bromide to visualize nucleocapsid-associated RNA. Protein was then visualized by staining with Gelcode Blue Stain reagent (Pierce).

SDS-PAGE.
Proteins were analysed by 15 % SDS-PAGE and stained with Gelcode Blue Stain reagent.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Although several observations have suggested that HBcAg binds and activates naive B cells through interaction with a linear motif present in the FR1–CDR1 junction (Lazdina et al., 2001), no molecular evidence for such an interaction with cell-surface-expressed Igs has been presented. Because such evidence is difficult to obtain using primary B cells, we studied the interaction of HBcAg with various B-cell lines to try to confirm this hypothesis.

Interaction of HBcAg with different Ramos-derived cell lines
The first B-cell line used in this study was Ramos 2G6 4C10, a subclone of the well-known Ramos cell line (Klein et al., 1975). This cell line both secretes IgM molecules and expresses them on the surface of the plasma membrane. The IgM molecule belongs to the VH6-encoding gene family (Sale & Neuberger, 1998; Zhang et al., 2001a) and does not contain the HBcAg binding motif. Indeed, when tested in two different diagnostic assays, cell supernatant was negative for the presence of anti-HBc IgM antibodies (data not shown). Nevertheless, as shown in Fig. 1, binding of HBcAg-c to Ramos 2G6 4C10 cells was observed. Because of this unexpected result, binding to the parental Ramos cell line and another subclone, Ramos 2.23, was investigated. Again attachment of HBcAg-c to the IgM-expressing Ramos cells was observed. However, more importantly, HBcAg-c interacted with Ramos 2.23 cells, which do not express cell-surface IgM (Fig. 1).



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Fig. 1. Expression of cell-surface IgM is not required for attachment of HBV nucleocapsids to the Ramos 2.23 cell line. Cells (5x105) were incubated with 5 µg HBcAg-c or anti-IgM–FITC ml–1 for 60 min on ice in PBS/0·8 % BSA. HBcAg particles were detected using the biotinylated HBcAg-specific mAb, followed by SAPE (open curves). Filled curves indicate isotypic controls or controls with no HBcAg added.

 
HBcAg fails to induce apoptotic cell death of Ramos 2G6 4C10 cells
When the structure of the HBcAg capsid was first revealed by cryo-electron microscopy, it was proposed that the orientation of the dimeric spikes distributed over the surface of the capsid shell might be optimal for cross-linking B-cell membrane Ig antigen receptors. Cross-linking of cell-surface-expressed IgM on Ramos cells leads to rapid induction of apoptotic death (Valentine & Licciardi, 1992) and a reduction in the secretion of IgM. If HBcAg interacts with the Ramos-expressed IgM molecules, one might expect a rapid induction of cell death and reduced secretion of IgM. The addition of 2 µg anti-Fcµ antibodies ml–1 resulted in massive apoptotic cell death, as demonstrated by strong staining with annexin V (Fig. 2). The addition of 2 µg anti-Fab antibodies ml–1 also induced apoptosis, but to a lesser extent. Both treatments caused a reduction in IgM secretion (data not shown). The addition of up to 10 µg HBcAg-c ml–1 did not result in the induction of apoptosis (Fig. 2) or in a reduction in IgM secretion (data not shown). These observations, together with the results from the binding assays, demonstrated that attachment of HBcAg-c to Ramos cells did not depend on the presence of cell-surface-expressed Igs.



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Fig. 2. Nucleocapsids fail to induce apoptotic cell death of Ramos 2G6 4C10 cells. Cells (5x105) were incubated in cRPMI for 24 h at 37 °C with 1 or 10 µg HBcAg-c ml–1, 2 µg goat anti-Fcµ ml–1 or 2 µg goat anti-Fab ml–1 (black lines). Cells were stained with annexin V–FITC to detected apoptotic cells. Only cells that did not take up propidium iodide are shown. Shaded curves represent untreated cells.

 
Interaction of HBcAg with EBV-transformed LCLs
Since binding of HBcAg to Ramos cells did not require Igs, we investigated whether attachment to other B-cell-derived cell lines might be independent of cell-surface-expressed antibodies. EBV-transformed LCLs derived from five different donors were tested for binding of HBcAg-c and for the presence of membrane-bound Igs. Attachment of HBcAg-c to all five LCLs was clearly demonstrated (Fig. 3). Two of these LCLs (LCL-GL and LCL-NV) did not express detectable levels of IgM, IgD or IgG molecules. Very low levels of IgM were present on LCL-KH and LCL-AC cells, the latter also expressing some IgD. LCL-JV cells expressed IgG molecules on the cell surface. These observations further demonstrated that no Igs need to be present for binding of HBcAg to B-cell-derived cell lines.



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Fig. 3. Nucleocapsids bind to LCLs that express no or very low levels of cell-surface Igs. LCL cells (5x105) derived from five different donors were incubated with 5 µg HBcAg-c ml–1 or with anti-IgM–, anti-IgD– or anti-IgG–FITC for 60 min on ice in PBS/0·8 % BSA. Cells were incubated in PBS/0·8 % BSA with 20 % HS before HBcAg particles were detected using the biotinylated HBcAg-specific mAb, followed by SAPE. Filled curves represent isotypic controls or controls with no HBcAg added.

 
Interaction of HBcAg with non-B-cell-derived cell lines
Following the observation that attachment of HBcAg to B-cell-derived cell lines appeared to be Ig-independent, we investigated whether binding to other cell types might also be possible. Several different human and non-human cell lines were tested. Remarkably, attachment to all of these cell lines was observed (Fig. 4). Binding occurred to the human pre-monocytic cell line THP-1, as well as to a more mature differentiation stage of these cells obtained by 1,25-vitD3 treatment. HBcAg-c also bound to Jurkat cells, a much-used human T-cell line. In addition, attachment of HBcAg-c was observed to the human HEK293T cell line, various hamster CHO cell lines and to the simian virus 40-transformed African green monkey kidney cell line COS-7.



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Fig. 4. Nucleocapsids bind to cell lines of different species and lineages. THP-1, 1,25-vitD3-treated THP-1, Jurkat, CHO-DHFR and CHO-CD14 cells (5x105) were incubated with 5 µg HBcAg-c ml–1 for 60 min on ice in PBS/0·8 % BSA. HEK293T and COS-7 cells were incubated with 10 µg HBcAg-c ml–1. HBcAg particles were detected using the biotinylated HBcAg-specific mAb, followed by SAPE. Before HBcAg particles on the THP-1 and Jurkat cells were stained, cells were incubated in PBS/0·8 % BSA with 20 % HS. Filled curves indicate isotypic controls or controls with no HBcAg added.

 
All of the above experiments were performed using E. coli-expressed HBcAg. Nucleocapsids produced in yeast were also found to be capable of binding to the cell surface of different cell lines, albeit with lower efficiency (data not shown), demonstrating that binding of nucleocapsids was not a consequence of expression in a specific host cell.

Capsids lacking protamine-like domains do not bind to the surface of HEK293T cells
The binding of HBcAg to cell lines of different species and lineages is difficult to explain. It does, however, suggest that HBcAg probably contains a motif that recognizes a ligand conserved among different cell types and different species. HBcAg contains a highly arginine-rich carboxy-terminal region of 34–36 aa connected to the shell-forming core domain by a flexible linker-peptide sequence (Pumpens & Grens, 1999; Watts et al., 2002). Arginine-rich peptides, called protein transduction domains (PTDs), are known to bind to the surface of different cell types and different species (Futaki, 2002; Green et al., 2003). Based on these observations, we hypothesized that the arginine-rich stretches might be responsible for the binding of nucleocapsids to the cells. Hence, capsids without the protamine-like domain were expected not to bind to the cell surface. To verify this hypothesis, capsids made from core proteins that lacked aa 145–183 were used (Fig. 5a). These truncated capsids did not contain RNA (Fig. 5b, c) (Gallina et al., 1989) and were recognized by the mAb used in the binding assays (Fig. 5d). As hypothesized, truncated capsids were not detected at the surface of LCL-GL and HEK293T cells (Fig. 5e), clearly demonstrating that the arginine-rich protamine-like domains are indeed responsible for the attachment of nucleocapsids to the cell surface.



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Fig. 5. Capsids that lack the protamine-like domain do not bind to the surface of LCL-GL and HEK293T cells. (a)–(c) Truncated and full-length HBcAg (1 µg) were separated by 15 % SDS-PAGE to demonstrate the removal of the protamine-like domain (a). To visualize the absence of encapsidated RNA in truncated capsids, 2·5 µg capsid was analysed on a 1 % agarose gel containing ethidium bromide (b). The same gel was then stained with Gelcode Blue Stain reagent to visualize the nucleocapsids (c). Lanes: 1, truncated HBcAg; 2, full-length HBcAg. (d) The biotinylated anti-HBcAg mAb was shown to bind equally well to truncated ({square}) and full-length ({blacksquare}) capsids. (e) LCL-GL and HEK293T cells (5x105) were incubated with 10 µg truncated (black line) or full-length (grey line) capsids ml–1 for 60 min on ice in PBS/0·8 % BSA. HBcAg particles were detected using the biotinylated HBcAg-specific mAb, followed by SAPE. The dotted line indicates the control with no HBcAg added.

 
RNase treatment of HBcAg enhances binding of nucleocapsids to the surface of HEK293T cells
Encapsidated RNA is known to stabilize the nucleocapsid and to reduce accessibility of the protamine-like domains (Wingfield et al., 1995). Therefore, it was hypothesized that a reduction in RNA content might result in increased availability of the arginine-rich stretches, resulting in increased binding capacity. To reduce the RNA content, particles were treated with RNase, since partial and even complete removal of capsid-associated RNA by RNase treatment has been demonstrated (Wingfield et al., 1995; Riedl et al., 2002; Storni et al., 2004). Indeed, RNase treatment partially destroyed the RNA, as shown on a 1 % agarose gel stained with ethidium bromide (Fig. 6a). Gelcode Blue Stain reagent was used to visualize proteins and demonstrated that RNA co-localized with the capsids (Fig. 6b). It further revealed that RNase treatment did not alter the electrophoretic mobility of the capsids, except for HBcAg-c. This material showed some reduced mobility or aggregation after RNase treatment. RNase treatment did not cause proteolytic breakdown (Fig. 6c) and did not affect the recognition of HBcAg by the mAb used in the binding assays (Fig. 6d). Most importantly, nucleocapsids treated with RNase showed increased binding to the surface of HEK293T cells (Fig. 6e).



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Fig. 6. Enzymic removal of encapsidated RNA enhances binding of nucleocapsids to the surface of HEK293T cells. Ten micrograms of HBcAg-c (lanes 3) and HBcAg-y preparations (lanes 1 and 2) were incubated with or without 0·25 µg RNase in 20 µl PBS for 60 min at room temperature. (a) To visualize breakdown of encapsidated RNA, 5 µl was loaded on a 1 % agarose gel containing ethidium bromide. (b) The same gel was stained with Gelcode Blue Stain reagent to visualize nucleocapsids. (c) To detect possible breakdown of core protein, 5 µl was separated by 15 % SDS-PAGE and stained with Gelcode Blue Stain reagent. (d) The biotinylated anti-HBcAg mAb bound equally well to RNase-treated ({blacksquare}) and mock-treated ({square}) nucleocapsids. (e) HEK293T cells (5x105) were incubated with HBcAg that had been treated with 10 µg RNase ml–1 (black line) or mock treated (shaded curve) for 60 min on ice in PBS/0·8 % BSA. HBcAg particles were detected using the biotinylated HBcAg-specific mAb, followed by SAPE. Filled curves indicate controls with no HBcAg added.

 
Attachment of nucleocapsids to cells involves cell-surface-expressed GAGs
Recently, it has been demonstrated that proteins or larger complexes that contain a PTD can bind to GAGs on the cell surface (Tyagi et al., 2001; Console et al., 2003). If the protamine-like regions are responsible for the attachment of nucleocapsids to the cell, it was considered possible that this binding was to GAGs. Adding heparin, heparan sulfate or chondroitin sulfate B to the binding mixture completely blocked binding of nucleocapsids to HEK293T and LCL-GL cells. The same concentration of dextran sulfate 500 partially inhibited binding, but hyaluronic acid had no effect (Fig. 7). When LCL-GL cells were treated with heparinase I and PMA, attachment of nucleocapsids was reduced strongly and mildly, respectively (Fig. 8). Neither hyaluronidase nor PI-PLC treatment reduced binding to LCL-GL cells (Fig. 8). Binding of nucleocapsids to HEK293T cells was reduced after treatment with heparinase I, but not after treatment with hyaluronidase (data not shown).



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Fig. 7. Binding of nucleocapsids to LCL-GL and HEK293T cells is inhibited by various soluble GAGs. Cells (5x105) were incubated with 10 µg HBcAg-y ml–1 with (shaded curve) or without (black line) 50 µg GAGs ml–1 in PBS for 60 min on ice. HBcAg-y particles were detected using the biotinylated HBcAg-specific mAb, followed by SAPE. The dotted lines indicate controls with no HBcAg added.

 


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Fig. 8. Treatment of LCL-GL cells with PMA and heparinase I reduces attachment of nucleocapsids. Cells (106) were incubated with 10 U heparinase I or hyaluronidase ml–1 for 2 h at 37 °C in PBS/0·8 % BSA with 1 mM CaCl2 and 0·5 mM MgCl2, or were incubated with 10 ng PMA ml–1 or 1 U PI-PLC ml–1 for 2 h at 37 °C in PBS/0·8 % BSA. Treated (shaded curves) and mock-treated (black lines) cells (5x105) were then incubated with 10 µg HBcAg-y ml–1 for 60 min on ice in PBS/0·8 % BSA. HBcAg-y particles were detected using the biotinylated HBcAg-specific mAb, followed by SAPE. The dotted lines indicate controls with no HBcAg added.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Heparan sulfates attached to proteins (proteoglycans) are expressed by most cells. Proteoglycans include the syndecan family of transmembrane proteins, the glypican family attached to the cell membrane by a glycosylphosphatidylinositol (GPI) tail and extracellular matrix proteins such as perlecan. Cell types differ in the proteoglycans they express, and the structure and modifications of the sugar moiety of proteoglycans are cell-type dependent and often change during differentiation or upon activation of cells. In some glycoproteins, like CD44, the carbohydrate side chains are occasionally replaced with heparan sulfate (Van der Voort et al., 2000). There has been increasing awareness that this diversity allows highly selective interactions with different ligands (Iozzo, 2001; Turnbull et al., 2001).

In this study, an unexpected interaction of the nucleocapsid of HBV with the surface of different cell lines was revealed. Binding was inhibited by heparin, heparan sulfate, chondroitin sulfate B and dextran sulfate 500. Reduced binding was observed after enzymic removal of heparin-like molecules from the cell membranes using heparinase I. These data suggested that binding involves heparan sulfates present on cell membranes. Attachment required a certain structure and is not driven solely by charge, since binding was not blocked by hyaluronic acid. This polymer is made up of alternating glucuronic acid and N-acetylglucosamine units. In chondroitin sulfate B, which inhibits binding efficiently, glucuronic acid is replaced by iduronic acid. The binding of HBV nucleocapsids did not show a clear specificity for a certain cell type or species. Binding to a monocytic cell line, B-cell lines, epithelial cell lines and a T-cell line were all demonstrated here. PMA, which causes shedding of transmembrane and GPI-anchored proteins, reduced binding of HBcAg to LCL-GL cells. PI-PLC treatment, which causes shedding of GPI-anchored proteins only, had no effect. This indicated that binding occurred through interaction with a transmembrane proteoglycan on LCL cells. HBcAg binds to Namalwa B cells and a human erythroleukaemia line (K562), which express low levels of heparan sulfates but no glypicans or syndecans (Steinfeld et al., 1996; Zhang et al., 2001b). Treatment of these two cell lines with heparinase I reduced binding of nucleocapsids (data not shown). Taken together, only the presence of heparan sulfates and not the presence of a certain proteoglycan family of proteins seems to be required for binding of nucleocapsids to cell lines. However, such immortalized cell lines are presumably not the best tools for unravelling preferences for particular heparan sulfate modifications and proteoglycans.

Based on sequence similarities between PTD domains (Futaki, 2002; Green et al., 2003) and the protamine-like domain of the core protein, it was hypothesized that this region is responsible for the interaction with cells. Indeed, capsids that lacked the arginine-rich domains did not bind to cells. However, the arginine-rich protamine-like domains (aa 150–183) of the core proteins are assumed to be located inside the capsid structure. In cryo-electron micrographs of E. coli-expressed nucleocapsids assembled from full-length core proteins, an inner shell is evident. This inner shell presumably represents RNA attached to the protamine-like domains (Zlotnick et al., 1997), which are connected to the capsid assembly domains (aa 1–140) by peptides (aa 141–149) that form a mobile array on the interior surface (Watts et al., 2002). Despite the interior localization of the protamine-like domain, trypsin cleaves the recombinant core proteins between residues 150 and 151 (Wingfield et al., 1995; Gallina et al., 1989), while trypsin immobilized to 40 nm gold particles does not (Rabe et al., 2003). This indicates that trypsin probably enters the nucleocapsid through pores of 1·2–1·5 nm diameter (Wynne et al., 1999). Removal of RNA associated with the capsids enhanced trypsin proteolysis (Wingfield et al., 1995). Because the trypsin cleavage site overlaps the RNA-binding motif (aa 150–156; Wingfield et al., 1995), it is reasonable to assume that destruction of RNA results in an increased number of free trypsin cleavage sites. The full-length nucleocapsids used here were expressed in E. coli or S. cerevisiae. These capsids were not serine-phosphorylated and contained RNA, and exposure of the arginine-rich domains therefore seemed unlikely (Rabe et al., 2003). We hypothesize that the unbranched heparan sulfate chains enter the nucleocapsids through the large pores to bind to the protamine-like domains. That these pores allow entry of larger molecules has been suggested by the packaging of ~30 DNA molecules (20–21 nt) per capsid in RNase A-treated nucleocapsids (Storni et al., 2004). Removal of encapsidated RNA by RNase treatment increased binding to the cells, most probably because the interior of the capsids and the arginine-rich sequences became more accessible for the long sugar chains.

In summary, a new and unexpected interaction of HBV nucleocapsids produced in bacteria or yeast with GAGs has been demonstrated. Whether the interaction of nucleocapsids with surface proteoglycans contributes to the unique immunogenicity of recombinant nucleocapsids is now being investigated. Monocytes, macrophages and B cells can express syndecan 1, 2 and 4 (Clasper et al., 1999; Saphire et al., 2001; Manakil et al., 2001). Syndecans contain a conserved cytoplasmic region that interacts indirectly with members of the src/cortactin signalling pathway and tubulin (Zimmermann & David, 1999). Src family kinases are implicated in many cellular events such as cell spreading and cell migration. In B cells, Src protein tyrosine kinases are activated by cross-linking of the B-cell receptor (Geisberger et al., 2003). Multimerization of syndecan 4 induces intracellular recruitment of PIP2 and activates PKC{alpha} (Zimmermann & David, 1999; Simons & Horowitz, 2001). Both syndecan 1 and 4 mediate macropinocytosis of different heparan sulfate-interacting ligands or organisms (Freissler et al., 2000; Fuki et al., 2000; Tkachenko et al., 2004). Syndecan 4, expressed by mouse B-cell lines, transmits a signal for the formation of dendritic processes that might facilitate intercellular communication (Yamashita et al., 1999). These different observations suggest that interactions of nucleocapsids with proteoglycans might contribute to the remarkable immunogenicity of recombinant nucleocapsids. Because nucleocapsids are also highly immunogenic during infection, such interactions might also be significant in vivo.


   ACKNOWLEDGEMENTS
 
The authors wish to thank Dr Michael S. Neuberger (MRC, Cambridge, UK) for the gift of the Ramos and Ramos 2.23 cell lines, Dr Martine Wettendorf (GlaxoSmithKline, Rixensart, Belgium) for providing yeast-expressed HBcAg and Dr Rudy Beyaert (VIB, Ghent, Belgium) for the HEK293T cell line. This research was supported financially by the Concerted Research Initiative of the University of Ghent (GOA Project no. 12050203).


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 3 September 2004; accepted 23 September 2004.