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
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ABSTRACT |
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INTRODUCTION |
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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 1complementarity determining region 1 (FR1CDR1) 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 7680, 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.
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METHODS |
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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-VFITC 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/GFITC 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 ml1) 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 ml1; 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 ml1, 50 µg streptomycin ml1 and 20 µM -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, EpsteinBarr 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 ml1 and 50 µg streptomycin ml1. HEK293T and COS-7 cells were grown in Dulbecco's MEM supplemented with 10 % FCS, 2 mM L-glutamine, 50 U penicillin ml1 and 50 µg streptomycin ml1. 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 ml1, 10 U heparinase I ml1, 10 U hyaluronidase ml1 or 1 U PI-PLC ml1 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 ml1 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.
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RESULTS |
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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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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 3436 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 145183 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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DISCUSSION |
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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 150183) 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 1140) by peptides (aa 141149) 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 1·21·5 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 150156; 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 (2021 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
(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.
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ACKNOWLEDGEMENTS |
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Received 3 September 2004;
accepted 23 September 2004.