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J. Biol. Chem., Vol. 281, Issue 24, 16563-16569, June 16, 2006

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Clathrin-mediated Endocytosis and Lysosomal Cleavage of Hepatitis B Virus Capsid-like Core Particles^*

From the Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel

Received for publication, February 14, 2006 , and in revised form, April 7, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The hepatitis B virus (HBV) core particle serves as a protective capsid shell for the viral genome and is highly immunogenic. Recombinant capsid-like core particles are used as effective carriers of foreign T and B cell epitopes and as delivery vehicles for oligonucleotides. The core monomer contains an arginine-rich C terminus that directs core particle attachment to cells via membrane heparan sulfate proteoglycans. Here we investigated the mechanism of recombinant core particle uptake and its intracellular fate following heparan sulfate binding. We found that the core particles are internalized in an energy-dependent manner. Core particle uptake is inhibited by chlorpromazine and by cytosol acidification known to block clathrin-mediated endocytosis but not by nystatin, which blocks lipid raft endocytosis. Particle uptake is abolished by expression of dominant negative forms of eps15 and Rab5, adaptors involved in clathrin-mediated endocytosis and early endosome transport, respectively. Endocytosed particles are transported to lysosomes where the core monomer is endoproteolytically cleaved into its distinct domains. Using protease inhibitors, cathepsin B was identified as the enzyme responsible for core monomer cleavage. Finally we found that monomer cleavage promotes particle dissociation within cells. Together, our results show that HBV capsid-like core particles are internalized through clathrin-mediated endocytosis, leading to lysosomal cleavage of the core monomer and particle dissociation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The hepatitis B virus (HBV)² is a small, enveloped DNA virus that infects hepatocytes and replicates through reverse transcription. The viral capsid also termed the core particle is a 30-nm diameter icosahedral structure assembling from 180 or 240 virally encoded core antigen (HBcAg) monomers (1). HBcAg is a 185-amino acid protein that harbors two distinct domains, namely an assembly domain (residues 1–144) that drives particle formation and an arginine-rich domain (residues 150–185) that binds nucleic acids (2). The particle lattice is fenestrated by repetitive 1.5-nm diameter channels that allow partial exposure of the arginine-rich domains of HBcAg on the shell surface (3). The core particle is extremely immunogenic, eliciting strong and long-lasting B and T cell immune responses during infection (4). T cell responses provoked by core particles are essential for keeping HBV replication under control (5).

When expressed in various heterologous systems, HBcAg efficiently self-assembles into capsid-like core particles indistinguishable from native capsid shells (6–8). Particle assembly tolerates fusion of foreign proteins to the HBcAg monomer, making it an attractive carrier of B and T cell epitopes for vaccination purposes (4). Consistently, T cell epitopes carried by capsid-like particles are efficiently presented on class I and class II MHC molecules of antigen-presenting cells (APCs) (9). Also, the capacity of the arginine-rich domain of HBcAg to bind nucleic acids has been utilized by us and by others to incorporate oligodeoxynucleotides (ODN) into the lumen of recombinant core particles (10, 11). The encapsulated ODN are delivered into cultured cells with high efficiency (10). Furthermore, ODN encapsulated in core particles or conjugated to an arginine-rich peptide derived from the C terminus of HBcAg exert enhanced immunostimulatory effects in vivo, and these effects have been suggested to result from their efficient endosomal delivery and activation of toll-like receptor 9 (TLR9) signaling (11–13). These findings led researchers to propose that the core particles are efficiently endocytosed by cells, yet this possibility and the underlying mechanism have not been investigated.

Recently we and others have shown that capsid-like core particles efficiently bind a wide variety of cell types including macrophages and B cells (14, 15). Particle attachment requires the arginine-rich domain of HBcAg and sulfated heparan sulfate proteoglycans on the target cell membrane (14, 15). We further showed that interaction with membrane heparan sulfate facilitates cytokine induction by the core particle in macrophages. Here we investigated core particle cell entry following heparan sulfate binding and the underlying mechanism. We found that the particles are internalized via clathrin-mediated endocytosis involving Eps15 and Rab5. Endocytosed particles are trafficked to lysosomes, where HBcAg is endoproteolytically cleaved into its distinct domains by cathepsin B and disassembles. These findings illuminate the molecular mechanism underlying core particle-mediated delivery of macromolecules for potentially therapeutic purposes and may have implications for HBcAg immunogenicity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Reagents and Plasmids—Sodium azide (NaN₃), 2-deoxy-D-glucose, chlorpromazine, nystatin, chloroquine, aprotinin, pepstatin A, E64d, and Z-Phe-Ala fluoromethylketone (Z-FA-fmk) were from Sigma. Heparin was from Calbiochem. LysoTracker Red was from Molecular Probes. The eGFP-N1 plasmid was from Clontech. The plasmid encoding GFP-tagged Eps15 D95/295 was kindly provided by Prof. Dautry-Varsat (Pasteur Institute, Paris, France) and was previously described (16).

Recombinant HBV Core Particles—The coding regions for the full-length (amino acids 1–185) or the C-terminally deleted versions of HBcAg were PCR-amplified from a HBV genome template (subtype adw) using the appropriate primers and cloned into pRSET B (Invitrogen). DNA constructs were analyzed by DNA sequencing. The particles were prepared in Escherichia coli and purified as previously described (10). For analysis of recombinant capsid-like particles on sucrose gradients, samples from the core particle preparations were layered onto step gradients with six 330-µl steps of 10, 20, 30, 40, 50, and 60% sucrose (w/v) in PBS and centrifuged for 45 min at 4 °C and 55,000 rpm in a TLS-55 rotor (Beckman) using the TL-100 ultracentrifuge as previously described (17). All experiments using recombinant full-length (HBc) core particles were reproducible using bacterially prepared HBV capsids (subtype ayw) generously provided by Prof. Paul Pumpens (Biomedical Research and Study Center, University of Latvia, Riga, Latvia).

View larger version (51K): [in this window] [in a new window]   FIGURE 1. HBc core particles are endocytosed following cell attachment. COS-1 (upper panels) and HepG2 (lower panels) cells were incubated with HBc core particles (1 nM) with no additional drugs (A, C), in the presence of 20 µg/ml heparin (B) or in the presence of 0.1% NaN3 and 50 mM 2 deoxy-D-glucose (D, E) for 4 h. The cells were extensively washed with normal medium (A, B, D) or with medium containing 0.3 mg/ml heparin (C, E) prior to cell fixation. Immunofluorescence was performed using polyclonal anti-HBcAg antibodies (red) and nuclei were stained with Hoechst 33342 (blue) as described under "Experimental Procedures." The cells were visualized by confocal microscopy, and representative optical fields are shown.

Cell Culture—HepG2 and COS-1 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum and antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin). For immunohistochemistry analyses, the cells were seeded on 13 mm coverslips in 24-well plates (Nunc) 1 day before the experiment and incubated with the indicated concentrations of HBc core particles.

Cytosol Acidification—Inhibition of clathrin-coated pit endocytosis by cytosol acidification was performed as previously described (18). Briefly, the cells were pre-treated for 30 min with Dulbecco's modified Eagle's medium containing 10 mM HEPES, pH 7.4 and 30 mM NH₄Cl before addition of particles in Na⁺-free buffer (140 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 1 mM amiloride, and 10 mM HEPES, pH 7.5) for 2 h. The cells were processed for immunofluorescent analysis as described below.

Transfections—For transfection experiments, HepG2 and COS-1 cells were seeded at 60% confluency on 13 mm coverslips in 24-well plates and transfected with 0.5 µg of DNA per well using the calcium phosphate method. 24 h after transfection the cells were incubated with HBc particles for the indicated times.

Immunostaining—Nuclei were stained with Hoechst 33342 (Molecular Probes) by brief incubation prior to cell fixation. The cells were extensively washed with normal medium or medium containing heparin (0.3 mg/ml) and then with PBS prior to fixation with 4% paraformaldehyde for 30 min at room temperature. Fixed cells were permeabilized with 0.5% Triton X-100 in PBS for 25 min at room temperature, washed with PBS containing 0.2% Tween 20 (PBS/T), and blocked with fetal calf serum containing 10% (v/v) skim milk and 0.2% (v/v) Tween 20 for 45 min. Cells were then incubated with polyclonal rabbit anti-HBcAg antibodies (diluted 1:75) in PBS/T containing 10% skim milk for 1 h, washed six times for 5 min with PBS/T and incubated with Rhodamine red X-conjugated anti-rabbit antibodies (Jackson Immunoresearch Laboratories) for 40 min. Finally, the cells were washed six times as above, and the coverslips were mounted in Aqua-polymount (Polysciences). Microscopic images were obtained using a Bio-Rad MRC-1024 confocal system, utilizing an argon-krypton mixed gas laser and mounted on a Zeiss Axiovert microscope. Representative fields of one of three experiments with similar results are shown. In each experiment the presented images were acquired using identical laser intensities.

Immunoblotting—Total cell extracts made in radioimmune precipitation assay lysis buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% Nonidet P-40 (v/v), 0.5% AB-deoxycholate (v/v), 0.1% SDS (v/v), 1 mM dithiothreitol, and a protease inhibitors mixture (Sigma)) were subjected to SDS-PAGE followed by Western blot analysis according to standard protocols. For detection of particles under native conditions, samples from core particle preparations or cell extracts made by three cycles of freezing and thawing in PBS containing protease inhibitors were subjected to electrophoresis through 1% agarose gel in TAE (40 mM Tris acetate, 1 mM EDTA). The proteins were either stained with GelCode (Pierce) or capillary-transferred to a nitrocellulose membrane followed by Western blot analysis according to standard protocols.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
HBc Core Particles Are Internalized via Clathrin-mediated Endocytosis—In this study we used COS-1 and HepG2 cells to study HBV core particle entry into cells and the possible underlying mechanism. Incubation of cells with full-length HBc core particles followed by cell fixation and immunofluorescent microscopy visualization revealed HBcAg-positive staining on the surface and possibly in the cytoplasm of cells but not in the cell nucleus (Fig. 1A). HBcAg-positive staining was observed for all cells examined in at least 10 different optical fields (data not shown), demonstrating efficient particle cell attachment. No signal was detected in mock-treated cells, demonstrating the specificity of the anti-HBcAg antibodies used for the immunofluorescent analysis (data not shown). Particle cell attachment was abolished when soluble heparin was included during the incubation period (Fig. 1B), in accordance with the fact that particle binding to cells is mediated by heparan sulfate (14, 15). Interestingly, when the cells were incubated with particles in the absence of heparin but washed with medium containing heparin to remove externally bound particles at the end of the incubation period, punctate HBcAg-positive staining was observed reminiscent of endosomal or lysosomal vesicles (Fig. 1C). This result suggested that the particles become endocytosed. To test this possibility further, the incubations were preformed in the presence of energy inhibitors (azide and 2-deoxy-D-glucose) that block endocytosis. Depletion of cellular energy had no substantial effect on particle binding to cells but blocked particle uptake (Fig. 1, D and E, respectively). Incubating the cells at 4 °C had a similar effect to energy inhibitors (data not shown). Together these results demonstrate that HBc particles are internalized by endocytosis.

View larger version (53K): [in this window] [in a new window]   FIGURE 2. HBc core particle uptake requires clathrin-mediated endocytosis. A, effect of inhibitors of endocytic pathways on HBc core particle uptake in HepG2 cells. Cells were either untreated or pre-treated with nystatin (25 µg/ml) or chlorpromazine (CPZ; 10 µg/ml) for 30 min prior to incubation with HBc core particles (1 nM) for 2 h in the continuous presence of the drugs. B, core particle uptake is blocked by cytosol acidification. HepG2 cells were either untreated or subjected to cytosol acidification protocol (see "Experimental Procedures" for details) and incubated with HBc core particles (1 nM) for 2 h. HepG2 (C) and COS-1 (D) cells were transiently transfected with a vector expressing either GFP alone (GFP-N1) or GFP fused to Eps1595/295 as described under "Experimental Procedures." 24 h after transfection, the cells were incubated with HBc core particles for 4 h. In all panels, externally bound core particles were removed by washing with medium containing heparin, the cells were fixed and immunostained using polyclonal anti-HBcAg antibodies (red). Nuclei were stained with Hoechst 33342 (blue). Green fields in panels (C) and (D) represent GFP autofluorescence. The cells were visualized by confocal microscopy and representative images are shown. Arrows in the right panels in C mark the location of one HBcAg-positive cell not expressing GFP-Eps1595/295.

It was previously shown that pre-treatment of cells with NH₄Cl followed by incubation of the cells in the presence of amiloride leads to cytosol acidification and blockage of clathrin-coated pit endocytosis (18). HepG2 cells were either untreated or subjected to cytosol acidification prior to incubation with HBc core particles. Immunofluorescent microscopy analysis showed that cytosol acidification resulted in abolishment of the signal representing internalized core (Fig. 2B), consistent with clathrin-mediated endocytosis of the particles into cells.

To further examine the role of clathrin-mediated endocytosis in HBcAg uptake, we used a mutant form of Eps15 (Eps1595/295) that lacks the second and third of the three EH domains of the protein (16). Eps15 binds the AP2 adaptor and overexpression of Eps1595/295 generates a dominant negative effect that blocks the formation of clathrin-coated pits without affecting other endocytic pathways. HepG2 and COS-1 cells were transfected with constructs encoding either Eps1595/295 fused to GFP (GFP-Eps1595/295) or GFP alone. 24 h later the cells were incubated with HBc core particles and assayed for their capacity to internalize the particles by immunofluorescent microscopy. Overexpression of GFP alone had no effect on particle uptake (Fig. 2, C and D). By contrast, in cells overexpressing GFP-Eps1595/295, entry of core particles was diminished. The stronger effect of GFP-Eps1595/295 expression on particle entry into HepG2 cells relative to COS-1 cells likely results from its lower expression level in the latter cell line (compare relative fluorescent intensities of GFP-Eps1595/295 in Fig. 2, C and D). Collectively, these results demonstrate that HBc core particles enter cells via clathrin-mediated endocytosis.

Transport of HBc Core Particles from the Cell Surface Is Modulated by Rab5 GTPase—The small GTPase Rab5 controls vesicular transport of ligands from the plasma membrane to early endosomes and homotypic fusion between endosomes (21). To test the possible involvement of the small GTPase Rab5 in HBc core particle uptake and trafficking, either wild-type (WT) Rab5 or different Rab5 mutants defective in the GTPase site were expressed in COS-1 cells. Rab5 S34N is a dominant negative GDP binding mutant that reduces the endocytosis efficiency of recycling and lysosomally destined ligands (22, 23). Rab5 Q79L is a GTPase-deficient, constitutively active mutant shown to increase ligand endocytosis (22). Expression of WT Rab5 did not affect HBc particle uptake and particles partially co-localized with Rab5 within cells (Fig. 3). In cells expressing the constitutively active Rab5 Q79L, markedly enlarged endosomes were generated in agreement with a previous report (24) and HBcAg strongly accumulated in the Rab5 Q79L-containing endosomes. By contrast, in cells expressing the dominant negative Rab5 S34N, uptake of HBc core particles was strongly diminished. Similar results were obtained in HepG2 cells (data not shown). These results demonstrate that Rab5 regulates the vesicular transport of HBc core particles from the cell surface to early endosomes.

View larger version (114K): [in this window] [in a new window]   FIGURE 3. HBc core particle uptake and transport to early endosomes are regulated by Rab5 GTPase. COS-1 cells were transfected with expression vectors encoding GFP tagged-WT Rab5, Rab5 Q79L or Rab5 S34N as indicated. 24 h after transfection the cells were incubated with HBc core particles (1 nM) for 4 h, washed with medium containing heparin and fixed. The cells were immunostained with anti-HBcAg antibodies (red) and nuclei were stained with Hoechst 33342 (blue). Green fields represent GFP autofluorescence. The cells were visualized by confocal microscopy, and representative images are presented.

To examine the kinetics of core particle internalization and its lysosomal targeting. Cells were incubated with FITC-labeled HBc core particles for various times, and the lysosomes were stained with Lyso-Tracker red. The analysis showed that particles were becoming internalized into cells after 1 h (Fig. 4C). However, at this time point the internalized particles did not co-localize with LysoTracker-red in lysosomes. Limited co-localization of the particles with the lysosomal marker was noted after 2 h whereas the particles extensively co-localized with LysoTracker red after 4 h. These results indicate that core particle routing to lysosomes is relatively slow, resembling the lysosomal trafficking kinetics of HIV-1 Tat in cells (25).

To further investigate the fate of internalized HBcAg, we prepared total extracts from COS-1 cells incubated with HBc particles in the absence or presence of energy inhibitors and washed with normal medium or heparin-containing medium as described in the legend to Fig. 1. The extracts were subjected to Western blot analysis using mac22, a monoclonal antibody that recognizes an epitope between residues 110 and 130 of HBcAg. A 21-kDa reactive band corresponding to HBcAg monomers was detected in the extracts prepared from cells incubated with HBc particles (Fig. 5A, lane 1). Interestingly, an additional band of 16 kDa was observed in the same total extract. No signal was detected in cells incubated with HBc particle in the continuous presence of heparin (Fig. 5A, lane 2), consistent with the immunofluorescent analysis (Fig. 1B). In cells incubated with HBc particles and washed with medium containing heparin prior to cell harvest, the intensity of the 21-kDa band was abolished (Fig. 5A, lane 3). By contrast, the intensity of the 16-kDa band in the same cell extract was unaffected, suggesting it represents endocytosed HBcAg that underwent processing. Consistently, the 16-kDa band was not detected in the extract prepared from energy-deprived cells (Fig. 5A, lane 4). No signal was detected in cells treated with energy inhibitors and washed with heparin (Fig. 5A, lane 5), consistent with the immunofluorescent analysis (Fig. 1E). Similar results were obtained when externally associated particles were removed from the cell surface by trypsin (data not shown). Together, these results indicate that HBcAg is processed following particle entry into cells.

View larger version (65K): [in this window] [in a new window]   FIGURE 4. Endocytosed HBc core particles are targeted to lysosomes. A, COS-1 cells were incubated with FITC-labeled HBc core particles (2 nM) for 6 h, and Lysotracker-Red (200 nM) was added for the last 30 min. The cells were washed with medium containing heparin and nuclei were stained with Hoechst 33342. The cells were fixed and visualized using confocal microscopy. Insets in A are enlarged in B. C, time course analysis of core particle uptake and lysosomal trafficking. Cells were incubated with FITC-labeled HBc core particles (2 nM) for the indicated times, and Lysotracker-Red (200 nM) was added for the last 30 min for each time point. The cells were processed as described in A.

View larger version (42K): [in this window] [in a new window]   FIGURE 5. Endocytosed HBcAg is cleaved at a region linking its assembly and arginine-rich domains. A, COS-1 cells were incubated with HBc core particles (1 nM) with no additional drugs (lanes 1 and 3), in the presence of 20 µg/ml heparin (lane 2) or in the presence of 0.1% NaN3 and 50 mM 2 deoxy-D-glucose (lanes 4 and 5) for 4 h. The cells were extensively washed with normal medium (lanes 1, 2, and 4) or with medium containing 0.3 mg/ml heparin (lanes 3 and 5) before the cells were harvested. Total cell extracts were prepared as described under "Experimental Procedures." Samples from the inputs and from the retrieved extracts were subjected to SDS-PAGE and Western blot analysis using mac22 and anti--tubulin antibodies. B, analysis of the different HBcAg truncation mutants and full-length HBc core particles following SDS-PAGE and Western blotting with mac22 (left panel) or following native agarose gel electrophoresis and protein staining (right panel). HBc-162 and HBc-172 correspond to core particles assembled from HBcAg monomers containing stop codons after positions 162 and 172, respectively. C, analysis of the different HBcAg truncation mutants and full-length HBc core particles by rate zonal ultracentrifugation. Particles were resolved on step gradient of 10 to 60% sucrose as described under "Experimental Procedures." Seven fractions were collected from top and samples from each fraction were analyzed by SDS-PAGE and Western blotting with mac22. Gradient fractions are indicated from top (lane 1) to bottom (lane 7). D, HBc-172 and HBc-162 are endocytosed by cells. COS-1 cells were incubated with HBc, HBc-172 or HBc-162 core particles (1 nM each) for 4 h. The cells were washed with medium containing 0.3 mg/ml heparin and fixed. Immunofluorescence was performed using polyclonal anti-HBcAg antibodies (red), and nuclei were stained with Hoechst 33342 (blue) as described under "Experimental Procedures." The cells were visualized by confocal microscopy and representative optical fields are shown. E, COS-1 cells were incubated with HBc, HBc-172, or HBc-162 core particles (1 nM each) for 4 h and harvested. Total cell extracts were prepared and analyzed by SDS-PAGE and Western blotting. F, same extracts as in A were subjected to electrophoresis through a Tris-Tricine gel and probed with mac22 (left panel) or pac74 (right panel).

View larger version (29K): [in this window] [in a new window]   FIGURE 6. Endoproteolytic cleavage of HBcAg in lysosomes is mediated by cathepsin B and promotes core particle disassembly. A, COS-1 cells were incubated with HBc core particles (1 nM) for 4 h in the absence (lane 1) or presence 100 µM chloroquine (lane 2), 10 µM aprotinin (lane 3), 100 µM pepstatin A (lane 4), 100 µM E64d (lane 5), or 10 µM Z-FA-fmk (lane 6). The cells were washed with medium containing heparin, harvested, and total extracts were subjected to Western blot analysis using mac22. B, analysis of endocytosed HBcAg on a native agarose gel. COS-1 cells were incubated with HBc core particles (1 nM) in the absence (lane 2) or presence (lane 3) of 100 µM chloroquine for 4 h. The cells were washed with medium containing heparin, and extracts were prepared and subjected to electrophoresis on a native agarose gel followed by Western blot analysis using polyclonal anti-HBcAg antibodies as described under "Experimental Procedures." Core particles from the input of lane 2 were analyzed in lane 1.

The lysosome contains various proteases, including serine, aspartic acid, and cysteine proteases responsible for processing of exogenous proteins taken up by endocytosis. The lysosomal localization of the particle suggests that processing of HBcAg is generated by endoproteolytic cleavage in this compartment. To test this possibility, cells were incubated with HBc core particles in the absence or presence of different inhibitors of lysosomal proteases. The cells were washed with medium containing heparin prior to cell harvest and total cell extracts were analyzed by Western blotting. Chloroquine, a general inhibitor of lysosomal proteases prevented cleavage of HBcAg (Fig. 6A, lane 2). Aprotinin and pepstatin A known to inhibit serine and aspartic acid proteases, respectively, did not affect HBcAg processing (Fig. 6A, lanes 3 and 4). By contrast, in the presence of E64d which inhibits cysteine proteases HBcAg cleavage was inhibited (Fig. 6A, lane 5). Furthermore, Z-FA-fmk, a specific inhibitor of the cysteine protease cathepsin B prevented the cleavage (Fig. 6A, lane 6). Thus, we conclude that lysosomal cathepsin B is responsible for the endoproteolytic cleavage of HBcAg.

Finally, we also characterized the mobility of internalized particles on a native agarose gel. Particulate HBV core particles typically migrate as a distinctive band on native agarose gels and their migration is disturbed following particle disassembly (26). Particles taken from the culture media at the end of the incubation period migrated as a distinct band (Fig. 6B, lane 1), indicating the particularity of HBcAg was not disturbed prior to cell attachment and entry. By contrast, the migration of HBcAg present in the lysate of heparin-washed cells was smeary (Fig. 6B, lane 2). The distinct particle migration was largely restored for HBcAg present in the lysate of heparin-washed cells incubated with HBc particles in the presence of chloroquine (Fig. 6B, lane 3). Together these results suggest that HBcAg cleavage promotes particle disassembly following cell entry.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Previous studies by us and by others demonstrated that the arginine-rich domain of HBcAg interacts with sulfated heparan sulfate proteoglycans to mediate core particle attachment to a wide variety of cell types including macrophages and B cells (14, 15). We show here that the attachment step is followed by clathrin-mediated endocytosis of the particles, depending on Eps15 and the small GTPase Rab5. The internalized cores are transported via early endosomes to lysosomes, where HBcAg is cleaved into its distinct domains by cathepsin B and the particle dissociates.

The family of mammalian heparan sulfate proteoglycans includes six GPI-anchored glypicans and four transmembrane syndecans that bind a wide variety of ligands (27). The mechanisms for ligand internalization may vary depending on the nature of the ligand and the proteoglycan that binds it, as well as on additional receptors that may participate in ligand binding. Using chlorpromazine, cytosol acidification and a dominant negative form of Eps15 we were able to show that core particle internalization is specifically attained via clathrin-mediated endocytosis. Clathrin-mediated particle uptake was observed in various cell types, including human THP-1 macrophages (data not shown). Further, in all cell types examined the particles were transported to lysosomes, leading to cathepsin B-dependent cleavage and processing of HBcAg. These findings suggest that a common yet specific pathway exists for particle uptake and trafficking in cells. The specificity may result from the multimeric nature of the particle, clustering heparan sulfate proteoglycans upon binding. The clustering may in turn drive movement of heparan sulfate proteoglycans into clathrin-coated pits. Alternatively, uptake and trafficking of the core particle may follow a constitutive lysosomal turnover pathway of the heparan sulfate proteoglycan serving as its receptor. The latter possibility is in line with the lysosomal route responsible for cellular catabolism of heparan sulfate proteoglycans (28).

Lysosomal targeting of the particle is followed by cathepsin B-mediated HBcAg cleavage. The cleavage site resides in a region linking the assembly and arginine-rich domains of HBcAg. This finding is consistent with a previous in vitro study showing that a protease-sensitive hinge links the assembly and arginine-rich domains of particulate HBcAg (29). The mobility of endocytosed HBcAg through a native agarose gel suggests that the particle becomes destabilized following HBcAg cleavage. This suggestion is consistent with previous studies showing that core particles genetically or proteolytically deleted of the arginine-rich domain are substantially less stable than full-length particles (2, 30, 31). In addition, non-proteolytic changes in HBcAg may be induced by the lysosomal environment (e.g. disulfide bond reduction) to further facilitate particle destabilization.

While attempting to map the cleavage site within HBcAg, we found that particles lacking the 23 C-terminal residues of the monomer (HBc-162) attach and enter cells with similar efficiency to full-length HBc particles. We further found that core particles completely deleted of the arginine-rich domain of HBcAg (residues 150–185) do not bind cells (data not shown), consistent with the role played by this region in capsid cell attachment (14, 15). Thus, residues 150–162 are necessary whereas residues 163–185 are dispensable for core particle cell attachment and entry. Arginine-rich sequences exposed on the shell surface have been suggested to interact with negatively charged heparan sulfate glycosaminoglycans to mediate particle cell attachment (14). Our finding that residues 150–162 of HBcAg are responsible for core particle cell attachment lends further support to this notion because residues 150–159 but not 165–175 of HBcAg were shown to be exposed on the surface of intact core particles (29).

Recombinant HBV core particles have been used as carriers of foreign epitopes and ODN for vaccination and delivery purposes (4, 10–13). Capture of core particles by heparan sulfate proteoglycans followed by their clathrin-mediated uptake and lysosomal trafficking is likely at the basis of these processes. Cathepsin B-mediated cleavage at a position linking the assembly and arginine-rich domains of HBcAg is expected to trigger the release of the encapsulated cargo in the lysosome in a stepwise manner. First, HBcAg cleavage causes detachment of cargo bound by the arginine-rich domain (e.g. ODN) from the particle lattice. Second, because HBcAg cleavage promotes particle dissociation it would eventually allow the physical escape of the encapsulated cargo from the capsid interior. Thus, HBcAg cleavage followed by particle dissociation can facilitate release of the antigenic peptides and encapsulated ODN in lysosomes, allowing peptide loading on class II MHC molecules and TLR9 signaling, respectively. Furthermore, the acidic environment in lysosomes could possibly permit translocation of HBcAg and the delivered cargo to the cytoplasm, as was recently demonstrated for HIV-1 Tat and cationic peptides (25, 32). Cytoplasmic translocation following lysosomal delivery could drive cross-presentation of peptides carried by the particle (9) and could allow delivery of encapsulated macromolecules into the cytosol and the nucleus.

Finally, the possibility that native HBV core particles undergo endocytosis and lysosomal cleavage may have implications for HBcAg immunogenicity during HBV infection. Exogenous HBcAg uptake and processing are important for presentation of HBcAg T cell epitopes on MHC class II molecules (33). Uptake and lysosomal processing by antigen-presenting cells is expected to trigger CD4+ T cell activation, whereas entry and processing by HBV-infected hepatocytes known to express MHC class II but no co-stimulatory molecules is expected to result in immune tolerance (33). Thus, particle uptake and processing during HBV infection is possibly involved in the intricate processes of immune activation and immune evasion by the virus.

	FOOTNOTES

 
^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel. Tel.: 972-8-934-2320; Fax: 972-8-934-4108; E-mail: yosef.shaul{at}weizmann.ac.il.

² The abbreviations used are: HBV, hepatitis B virus; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PBS, phosphate-buffered saline; Z, benzyloxycarbonyl; fmk, fluoromethylketone; GFP, green fluorescent protein; W T, wild type; FITC, fluorescein isothiocyanate; ODN, oligodeoxynucleotides.

³ I. Sominskaya, personal communication.

	ACKNOWLEDGMENTS

 
We thank Prof. Alice Dautry-Varsat for the GFP-Eps15 95/295 construct. We are grateful to Dr. Irina Sominskaya, Dr. Galina Borisova, and Prof. Paul Pumpens for recombinant capsids. We thank Prof. Yosef Yarden, Prof. Sima Lev, Prof. Mike Fainzilber, Dr. Joseph Lotem and Dr. Liora Cahalon for materials. We thank Prof. Nathan Sharon, Dr. Nir Paran, and Dr. Gad Asher for helpful comments and Sylvia Budilovsky for technical assistance.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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