Title: Myristoylated PreS1-domain of the hepatitis B virus L-protein mediates specific binding to differentiated hepatocytes
Abstract: HepatologyVolume 58, Issue 1 p. 31-42 Viral HepatitisFree Access Myristoylated PreS1-domain of the hepatitis B virus L-protein mediates specific binding to differentiated hepatocytes†‡§ Anja Meier, Anja Meier Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, GermanySearch for more papers by this authorStefan Mehrle, Stefan Mehrle Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, GermanySearch for more papers by this authorThomas S. Weiss, Thomas S. Weiss Center for Liver Cell Research, Department of Pediatrics, University Medical Center Regensburg, Regensburg, GermanySearch for more papers by this authorWalter Mier, Walter Mier Department of Nuclear Medicine, University Hospital Heidelberg, Heidelberg, GermanySearch for more papers by this authorStephan Urban, Corresponding Author Stephan Urban [email protected] Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany fax: +49-6221-561946Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany===Search for more papers by this author Anja Meier, Anja Meier Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, GermanySearch for more papers by this authorStefan Mehrle, Stefan Mehrle Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, GermanySearch for more papers by this authorThomas S. Weiss, Thomas S. Weiss Center for Liver Cell Research, Department of Pediatrics, University Medical Center Regensburg, Regensburg, GermanySearch for more papers by this authorWalter Mier, Walter Mier Department of Nuclear Medicine, University Hospital Heidelberg, Heidelberg, GermanySearch for more papers by this authorStephan Urban, Corresponding Author Stephan Urban [email protected] Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany fax: +49-6221-561946Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany===Search for more papers by this author First published: 05 December 2012 https://doi.org/10.1002/hep.26181Citations: 97 † Potential conflict of interest: Nothing to report. ‡ This work received grants from the Bundesministerium für Bildung und Forschung (BMBF), “Innovative Therapieverfahren,” grant number 01GU0702, the Landesstiftung Baden Württemberg and financial support from the Vision 7 GmbH. § The data presented in this study is consistent with the recent publication of Yan et al. describing sodium taurocholate cotransporting polypeptide (NTCP/SLC10A1) as a specific receptor for HBV and HDV infection. Yan H, Zhong G, Xu G, He W, Jing Z, Gao Z, et al. Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. elife 2012;1:e00049. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Abstract Chronic infection with the human hepatitis B virus (HBV) is a global health problem and a main cause of progressive liver diseases. HBV exhibits a narrow host range, replicating primarily in hepatocytes. Both host and hepatocyte specificity presumably involve specific receptor interactions on the target cell; however, direct evidence for this hypothesis is missing. Following the observation that HBV entry is specifically blocked by L-protein-derived preS1-lipopeptides, we visualized specific HBV receptor/ligand complexes on hepatic cells and quantified the turnover kinetics. Using fluorescein isothiocyanate-labeled, myristoylated HBV preS1-peptides we demonstrate (1) the presence of a highly specific HBV receptor on the plasma membrane of HBV-susceptible primary human and tupaia hepatocytes and HepaRG cells but also on hepatocytes from the nonsusceptible species mouse, rat, rabbit and dog; (2) the requirement of a differentiated state of the hepatocyte for specific preS1-binding; (3) the lack of detectable amounts of the receptor on HepG2 and HuH7 cells; (4) a slow receptor turnover at the hepatocyte membrane; and (5) an association of the receptor with actin microfilaments. The presence of the preS1-receptor in primary hepatocytes from some non-HBV-susceptible species indicates that the lack of susceptibility of these cells is owed to a postbinding step. Conclusion: These findings suggest that HBV hepatotropism is mediated by the highly selective expression of a yet unknown receptor* on differentiated hepatocytes, while species specificity of the HBV infection requires selective downstream events, e.g., the presence of host dependency or the absence of host restriction factors. The criteria defined here will allow narrowing down reasonable receptor candidates and provide a binding assay for HBV-receptor expression screens in hepatic cells. (HEPATOLOGY 2013) See Editorial on Page 9 Chronic hepatitis B is a global medical problem caused by the human hepatitis B virus (HBV). About 350 million people are persistently infected and need therapeutic treatment to reduce the risk of developing liver cirrhosis and HCC.1 Since the currently approved medications are mostly noncurative, novel therapeutic strategies are needed.2 HBV, the prototypic member of the hepadnavirus family, is a 42 nm, enveloped, partially double-stranded DNA virus with a restricted host range and an extraordinary tropism to infect the parenchymal liver cells of its host.3 Since HBV properly assembles after transfection with genomic HBV DNA of even nonhepatic cells, the specificity for hepatocytes must be related to an early infection event. One of the proposed restricted steps might be the lack of a hepatocyte-specific receptor. However, this hypothesis needs to be proven. The envelope of HBV consists of proteins termed large (L), middle (M), and small (S) protein. They are encoded in one open reading frame and share the C-terminal S-domain which provides four trans-membrane helices4 and is probably involved in fusion.5 In addition to the S-domain, the M-protein contains an extension of 55 amino acids called preS2. The L-protein has a further N-terminal extension termed preS1. The preS1-domain of L- becomes N-terminally myristoylated and plays a key role in HBV entry into hepatocytes.6 Due to the previous limitation to primary human (PHHs) and primary tupaia belangeri hepatocytes (PTH) and HepaRG cell lines as the only in vitro HBV infection systems, receptor recognition and the mechanism of virus entry and membrane fusion are just about to be understood. Using HepaRG cells7 and primary PTH,8 heparin sulfate proteoglycans (HSPG) were identified as inevitable to initiate HBV infection.9, 10 Since HSPG interaction cannot explain HBV hepatocyte specificity, it is supposed an essential but not very specific step. Using recombinant HBV and hepatitis delta virus (HDV) as a surrogate system to study HBV entry, essential infectivity determinants within the envelope proteins have been identified: (1) N-terminal myristoylation of L is mandatory for infectivity.11, 12 (2) Consecutive removal or insertion of short sequences in the N-terminal 75 amino acids (genotype D) of the preS1-domain abrogates infection.13, 14 Deletions and mutations introduced C-terminally of amino acid 75 including elimination of the M-protein and randomization of the preS2-domain within the L-protein had almost no influence on infectivity.15, 16 (3) Besides preS1, another essential element of HBV/HDV infectivity has been assigned to the antigenic loop (AGL) of the S-domain.17 Replacement of cysteine residues in the AGL rendered HDV and HBV (Yi Ni, unpubl. results) noninfectious. Since some of these cysteines (e.g., Cys-124) participate in intramolecular disulfide bonds, the sensitivity against reducing agents hints at the involvement of disulfide-bridge rearrangements during virus entry.18 Since only L-protein containing SVPs are able to bind hepatocytes from Tupaia belangeri the S-protein/domain is probably not essential for hepatocyte-specific binding.19 Consistent with the results of the mutational analyses, HBV preS1-derived lipopeptides, mimicking the myristoylated N-terminal preS1-infectivity domain, efficiently inhibit HBV and HDV infection of HepaRG cells, PHH, and PTH.7, 20-23 The activity of the peptides requires myristoylation and the integrity of an internal sequence (9-NPLGFFP-15) which is highly conserved between primate hepadnaviruses.21 Since their inhibitory effect remains for several hours after preincubation they probably inactivate a cellular receptor.24 In the present study we used fluorescently labeled, myristoylated HBVpreS1-peptides to analyze the presence and turnover kinetics of this HBVpreS1-specific receptor on hepatocytes from different species. We investigated whether receptor expression coincides with the species specificity of HBV and demonstrate highly specific binding of the preS1-lipopeptide to permissive cells (PHH, PTH, HepaRG). Unexpectedly, we detected specific binding to hepatocytes from non-susceptible species such as mouse, rat, rabbit, and dog, but not pig, cynomolgus, or rhesus monkey. Expression of a functional HBVpreS1-receptor was associated with the differentiation state of the hepatocyte: No binding was observed in undifferentiated HepaRG cells or dedifferentiated PMH and PHH. HepG2 and HuH7 cells were unable to bind the peptide even after dimethyl sulfoxide (DMSO)-induced differentiation. Kinetic studies demonstrated a slow turnover and a constrained lateral movement of the receptor complex at the plasma membrane (PM), possibly due to a cytoskeleton interaction. Abbreviations DIPEA, N,N-Diisopropylethylamine; DMSO, dimethyl sulfoxide; FITC, fluorescein isothiocyanate; ge, genome equivalent; HBTU, O-(benzotriazol-1-yl)-N,N,N ′,N ′-tetramethyluronium hexafluorophosphate; HSPG, heparan sulfate proteoglycan; L-protein, hepatitis B virus large surface protein; PBS, phosphate-buffered saline; PEG, polyethylene glycol; PHH, primary human hepatocytes; PMH, primary mouse hepatocytes; PTH, primary Tupaia belangeri hepatocytes. Materials and Methods Synthesis of Peptides. Peptides were produced by solid phase synthesis employing the Fmoc/tBu strategy with O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate; (HBTU) / N,N-Diisopropylethylamine (DIPEA) activation in an Applied Biosystems 433A peptide synthesizer. Fluorescein isothiocyanate (FITC; Applichem) was coupled to the ϵ-amino group of an introduced D-lysine at position 49. Alternatively, Atto-565-maleimide (Fluka) was linked to a cysteine at the same position. Reversed phase high-performance liquid chromatography (HPLC) was carried out as described (Schieck et al.25). The identity of the peptides was verified by mass spectrometry. Stock solutions (500 μM) of the peptides in 2% DMSO were prepared, diluted with the appropriate medium, and added to the cells at the indicated concentrations. Preparation and Cultivation of Primary Hepatocytes and Cell Lines. PHH were cultivated in serum-free medium as described.26 Tissue samples from liver resections were obtained from patients undergoing partial hepatectomy. Experimental procedures were performed according to the guidelines of the charitable state controlled foundation HTCR (Human Tissue and Cell Research), with the informed patient's consent approved by the local Ethical Committee of the University of Regensburg. PMH were prepared by a two-step standard perfusion protocol using a 2-mM EGTA-containing buffer, followed by a treatment with 3.3 mg/mL collagenase type IV (Sigma-Aldrich).27 Parenchymal cells were enriched through resuspension and centrifugation of the cells in a Percoll solution with a density of 1,063 g/mL. Cryopreserved hepatocytes were bought from Celsis (rabbit, dog, cynomolgus monkey, rhesus monkey, and pig) or BD Gentest (rat and cynomolgus monkey). Cryopreserved PTHs were a kind gift of Maura Dandri (Hamburg). HuH7 and HepG2 cell lines were cultivated in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal calf serum (FCS), L-glutamine (2 mM), penicillin (50 U/mL), and streptomycin (50 μg/mL). HepaRG cells were cultivated as described.7 To induce differentiation, HuH7 and HepG2 cells were treated for 14 days with 0.5% DMSO; dedifferentiation of PMH and PHH was induced by growth in DMSO-free medium for up to 8 days. Binding Assays with Fluorescently Labeled HBVpreS-peptides. Binding experiments were performed in supplemented Williams E medium as described above. For PHH, medium was complemented with 50 μM hydrocortisone and 5 μg/mL insulin. Experiments with primary hepatocytes were carried out either on day 1 after plating (microscopy) or immediately after thawing (flow cytometry). HepaRG cells were tested on day 5 after seeding, or 2 weeks after DMSO-induced differentiation. Cells were incubated at 37°C with the appropriate labeled peptide in medium. Binding competition was carried out by coincubation of HBVpreS/2-48myr-K-FITC with an excess of unlabeled HBVpreS/2-48myr. Infection Inhibition. For infection inhibition, HepaRG cells were preincubated for 30 minutes with peptide and inoculated with a 1:20 dilution of a polyethylene glycol (PEG)-precipitated (50- to 100-fold enrichment) HepG2.2.15/HepAd38-derived virus stock (16 hours at 37°C)7 in medium containing 250 nM peptide and 4% PEG 8000 (Sigma-Aldrich). Cells were repeatedly washed and cultivated for up to 2 weeks. To quantify HBV replication, medium was collected from day 8 to 13 post infection and secreted HBeAg was determined by enzyme-linked immunosorbent assay (ELISA (AxSym, Abbott)). Fluorescence Microscopy. Primary hepatocytes were grown on coverslips, coated with 0.1 mg/mL collagen (Cell Systems). After incubation with peptide at 37°C, cells were washed in phosphate-buffered saline (PBS), fixed (4% paraformaldehyde [PFA]), and mounted in DAPI-containing mounting medium (VectaShield). Microscopy was performed on a Perkin Elmer spinning disk confocal microscope, using a 60× WI (NA 1.2) or 100× oil (NA 1.4) objective, a Hamamatsu C9100-50 camera, and the software Volocity (Perkin Elmer). Quantification of peptide binding was achieved by measurement of the gray values in 60 (Fig. 5A) or 50 (Fig. 5B) circular selections in 3 or 10 representative pictures using ImageJ (NIH, Bethesda, MD). To analyze fluorescence recovery after photobleaching (FRAP), PMH were grown on collagen-coated chambered coverglass (LabTek). Cells were incubated with 400 nM HBVpreS/2-48myr-C-Atto565, diluted in Leiboviz (L-15) phenol red-free medium for 1 hour at 37°C, washed, and supplied with fresh L-15. As a control, cells were stained with 5 μL/mL Vybrant DiI (Invitrogen). Live cell microscopy at 37°C was performed in a heated chamber. Bleaching was achieved using the 568 nm laser, and recovery of fluorescence was monitored for 30 seconds with a frequency of 2 frames per second. Flow Cytometry. The 4 × 105/mL freshly prepared or cryopreserved primary hepatocytes were incubated for 30 minutes at room temperature with 200 nM of the respective peptide. After washing (PBS) flow cytometry was performed using a FACS Calibur and the software Cell Quest Pro (Becton-Dickinson). Competition of binding was performed with a 100-fold excess of unlabeled HBVpreS/2-48myr or control peptides (HBVpreS/2-48myr(D11,13) and HBVpreS/1-48). Cell viability was assessed by propidium iodide. To exclude unspecific binding caused by nonparenchymal cells, hepatocyte preparations were controlled by uptake of acLDL (10 μg/mL for 2 hours at 37°C), a marker for endothelial cells. Results Fluorescently Labeled HBVpreS/2-48myr Specifically Binds to the PM of Hepatic HepaRG Cells. Myristoylated HBV-preS1 lipopeptides inhibit HBV infection of HepaRG cells.21 To investigate specific receptor-interaction, we synthesized a fluorescently labeled variant of HBVpreS/2-48myr (Fig. 1A) and performed binding assays with HepaRG cells. Cosynthetic coupling of one FITC-moiety per molecule HBVpreS/2-48myr-K-FITC was accomplished by introduction of a lysine at position 49. As a control, we synthesized a mutant lipopeptide in which the L-leucine at position 11 and the L-phenylalanine at position 13 were replaced by the respective D-enantiomers (HBVpreS/2-48myr(D11,13)-K-FITC). A second control peptide comprised the wildtype sequence but lacked the N-terminal myristoyl moiety (HBVpreS/1-48-K-FITC). Both peptides are inactive (HBVpreS/2-48myr(D11,13)) or drastically impaired in inhibitory activity (HBVpreS/1-48).21 To ensure that the K-FITC-residue had no effect on the inhibitory activity, we tested HBVpreS/2-48myr-K-FITC in comparison to the nonlabeled HBVpreS/2-48myr in an infection inhibition assay (Fig. 1B). HBVpreS/2-48myr-K-FITC inhibited HBV infection in PHH like HBVpreS/2-48myr, as measured by secreted HBeAg. HBVpreS/2-48myr(D11,13)-K-FITC was inactive, confirming the requirement of the 9-NPLGFFP-15 sequence. HBVpreS/1-48-K-FITC showed only marginal activity. Figure 1Open in figure viewerPowerPoint HBVpreS/2-48myr-K-FITC inhibits HBV infection and specifically binds differentiated HepaRG cells. (A) Schematic representation of the HBV L-protein with its N-terminal myristic acid moiety (myr), the preS1, the preS2, and the S domain (above) in addition to the three fluorescently labeled peptides used in this study (below). HBVpreS/2-48myr-K-FITC (2-48myr-K-FITC) comprises the N-terminal 47 amino acids of the preS1-domain (genotype D) with the highly conserved essential domain 9-NPLGFFP-15 highlighted in blue. The peptide is N-terminally modified with myristic acid at glycine 2 and linked to an FITC-labeled lysine (K-FITC) at the C-terminus. HBVpreS/2-48myr(D11,13)-K-FITC (2-48myr(D11,13)-K-FITC), in which amino acids 11 and 13 were replaced by their respective D-enantiomers (shown by small capitals) and HBVpreS/1-48-K-FITC (1-48-K-FITC) which lacks N-terminal myristoylation were used as controls. (B) HBV infection inhibition assay comparing the three fluorescently labeled peptides with HBVpreS/2-48myr (2-48myr). PHH were infected with an multiplicity of infection (MOI) of 105 GE/cell in the absence (w/o) or the presence of 250 nM unlabeled 2-48myr or labeled 2-48myr-K-FITC, 2-48myr(D11,13)-K-FITC and 1-48-K-FITC. Cell culture supernatants were collected between days 8 and 13 and secreted HBeAg (S/Co) was determined by ELISA. (C) Binding of fluorescently labeled peptides to HepaRG cells. Differentiated HepaRG cells were incubated with 200 nM of the respective peptide, washed, and fixed with 4% PFA. Images were obtained by confocal microscopy. Nuclei were stained with DAPI (blue), bound peptide is shown in green. Note that the cells in the absence of peptide (w/o) show autofluorescence within the cytoplasm but not at the plasma membrane. To examine whether the differences in infection inhibition reflect specific binding properties to susceptible cells, we incubated differentiated HepaRG cells with the three peptides (200 nM) and analyzed cell association by confocal microscopy. As depicted in Fig. 1C, HBVpreS/2-48myr-K-FITC was localized at the PM of HepaRG cells (upper right). Incubation of cells with HBVpreS/2-48myr(D11,13)-K-FITC (lower left) or HBVpreS/1-48-K-FITC (lower right) did not result in significant PM staining, demonstrating the dependency of binding on the sequence integrity and the presence of the myristic acid. The specific peptide signal could clearly be discriminated from the punctuated cellular autofluorescence detected in the absence of peptide (upper left). HBVpreS/2-48myr-K-FITC binding was observed only in the hepatic clusters of HepaRG cells but not in biliary cells (data not shown). HBVpreS/2-48myr-K-FITC Specifically Binds PHH and PTH. Besides HepaRG cells, HBV infects PHH28 and PTH8 and is blocked by acylated HBVpreS-derived lipopeptides.20, 24 We therefore tested PHH and PTH for their ability to bind HBVpreS/2-48myr-K-FITC. We detected a sequence-specific and myristoyl-dependent association of the wildtype but not the control peptide with the PM of PHH (Fig. 2A). Specific binding of HBVpreS/2-48myr-K-FITC to PHH could also be detected in suspended cells by flow cytometry (Fig. 2B). Significant binding, visible by a shift of the cell population towards an approximately 10-fold higher fluorescence signal, was observed only for cells incubated with HBVpreS/2-48myr-K-FITC, but not with HBVpreS/2-48myr(D11,13)-K-FITC (Fig. 2B, dark green line versus orange line). Binding was prevented by an excess of the nonlabeled peptide HBVpreS/2-48myr (blue line) but not with the respective mutant HBVpreS/2-48myr(D11,13) (red line). This substantiates the high specificity of HBVpreS-receptor interaction in PHH. Consistently, PTH bound HBVpreS/2-48myr-K-FITC with comparable efficacy as PHH. Again, binding was prevented by unlabeled HBVpreS/2-48myr but not by the mutant HBVpreS/2-48myr(D11,13) (Fig. 2C). Figure 2Open in figure viewerPowerPoint HBVpreS/2-48myr-K-FITC binds HBV-susceptible PHH and PTH. (A) PHH were incubated for 30 minutes with 200 nM HBVpreS/2-48myr-K-FITC (2-48myr-K-FITC, left) or the mutant HBVpreS/2-48myr(D11,13)-K-FITC (2-48myr(D11,13)-K-FITC, right). After fixation, binding of the peptides was analyzed by confocal microscopy. Specific plasma membrane staining (green) was observed for 2-48myr-K-FITC but not for 2-48myr(D11,13)-K-FITC. Punctured intracellular fluorescence derived by autofluorescence was also detectable in the absence of peptide (data not shown). Nuclei staining (DAPI) is shown in blue. (B) Flow cytometric analysis of peptide binding and binding competition. Cryopreserved PHH were incubated in solution with 200 nM of 2-48myr-K-FITC (dark green), 2-48myr(D11,13)-K-FITC (orange), or the nonmyristoylated peptide HBVpreS/1-48-K-FITC (1-48-K-FITC, light green). Autofluorescence of the cells is shown in pink (w/o peptide). Competition of binding was performed by incubating PHH with 200 nM FITC-labeled 2-48myr-K-FITC in the presence of a 100-fold excess (20 μM) of unlabeled HBVpreS/2-48myr (2-48myr, blue) or HBVpreS/2-48myr(D11,13) (2-48myr(D11,13), red). (C) Quantitative analysis of the experiment shown in (B) compared to a second binding assay performed with cryopreserved PTH. The relative binding capacities of both cell types were normalized by dividing the signal of peptide fluorescence at the cell membrane through the respective signal caused by autofluorescence (w/o peptide). Binding of HBVpreS/2-48myr-K-FITC Is Not Restricted to HBV-Susceptible Hepatocytes. To investigate if HBVpreS1-receptor expression is restricted to hepatocytes from HBV susceptible hosts, we performed binding studies using PMH that are not susceptible to HBV infection.29 Unexpectedly, we observed the same sequence specific and myristoyl-dependent binding of HBVpreS/2-48myr-K-FITC to the PMH surface as for PHH, PTH, and HepaRG cells (Fig. 3A). Specific binding to PMH was confirmed by flow cytometry (data not shown). Binding was also detected in primary rat hepatocytes (PRH) (Fig. 3B). Again, excess of unlabeled HBVpreS/2-48myr reduced the fluorescence to background level (blue line), whereas coincubation with HBVpreS/2-48myr(D11,13) had no effect (Fig. 3B, red line). Accordingly, an HBVpreS1-receptor is present on the hepatocytes of mice and rats. We extended this analysis and tested HBVpreS-peptide binding to primary hepatocytes from rabbits, dogs, cynomolgus monkeys, rhesus monkeys, and pigs. As depicted in Fig. 3C, we found specific binding of HBVpreS/2-48myr-K-FITC to rat, rabbit, and dog hepatocytes but surprisingly not to hepatocytes from cynomolgus and rhesus monkey, despite their closer evolutionary relation to humans. Binding was also not observed on pig hepatocytes. Thus, differentiated hepatocytes from some HBV nonsusceptible species do express the HBVpreS-specific receptor (mouse, rat, dog, and rabbit), while others do not (pig, cynomolgus, and rhesus monkey), indicating that a step downstream specific preS-binding must restrict HBV/HDV-infection in these species. Figure 3Open in figure viewerPowerPoint HBVpreS/2-48myr-K-FITC specifically binds to primary hepatocytes of mouse, rat, rabbit, and dog. (A) Binding of fluorescently labeled peptides to PMH. PMH were incubated with 200 nM of HBVpreS/2-48myr-K-FITC (2-48myr-K-FITC, upper right), HBVpreS/2-48myr(D11,13)-K-FITC (2-48myr(D11,13)-K-FITC, lower left), and HBVpreS/1-48-K-FITC (1-48-K-FITC, lower right). After washing, cells were fixed with 4% PFA and analyzed by confocal microscopy. The autofluorescence of the cells in the absence of peptide (w/o) is depicted in the upper left panel. (B) Freshly isolated PRH were incubated with 200 nM of 2-48myr-K-FITC (dark green), 2-48myr(D11,13)-K-FITC (orange), or 1-48-K-FITC (light green). Binding competition was performed by coincubation of 200 nM 2-48myr-K-FITC in the presence of a 100-fold excess (20 μM) of unlabeled HBVpreS/2-48myr (2-48myr, blue) or the mutant HBVpreS/2-48myr(D11,13) (2-48myr(D11,13), red). Fluorescence was quantified by flow cytometry. (C) Specific binding of 2-48myr-K-FITC to primary hepatocytes from various species. Cryopreserved rat, rabbit, dog, human, pig, cynomolgus, and rhesus monkey hepatocytes were incubated in suspension with 200 nM of the peptide and fluorescence was analyzed by flow cytometry. To exclude possible unspecific binding of the fatty acid residue (myr) we defined the ratio of 2-48myr-K-FITC to the mutant 2-48myr(D11,13)-K-FITC signal as the relative specificity of binding. Shown data represent two (rat, rabbit, dog), three (human, cynomolgus) or one (rhesus, pig) independent experiments. HBVpreS-Receptor Expression Requires Hepatocyte Differentiation. The susceptibility of HepaRG cells to HBV infection depends on a differentiated state of the cells.7, 30 Moreover, PHH lose their susceptibility to HBV when DMSO is withdrawn from the medium.31-34 In order to test if this correlates with the presence of the HBVpreS-receptor we analyzed undifferentiated (5 days after seeding) and differentiated HepaRG cells (28 days after seeding, including a 2-week DMSO treatment) for their ability to bind HBVpreS/2-48myr-K-FITC. As seen in Fig. 4A (upper panel, left), HBVpreS/2-48myr-K-FITC did not stain the PM of undifferentiated cells, whereas binding was seen after differentiation (lower panel, left). This implies a cell state-dependent induction of HBVpreS/2-48myr-receptor expression during HepaRG-differentiation. Accordingly, we investigated whether dedifferentiation of binding competent primary hepatocytes result in an opposite effect. We cultivated PMH in the absence of DMSO and followed their ability to bind HBVpreS/2-48myr-K-FITC over time (Fig. 4A, right panels). While freshly isolated PMH specifically accumulated the peptide at the PM, hepatocytes from the same preparation lost their ability to bind HBVpreS/2-48myr-K-FITC within a few days of cultivation. Loss of binding could be prevented by addition of DMSO (data not shown). This correlates with the fact that PHH lose their susceptibility for HBV during several days of cultivation in the absence of DMSO.28 Thus, expression of an HBVpreS-receptor is linked to pathways controlling the differentiation state of hepatocytes. Figure 4Open in figure viewerPowerPoint Binding of HBVpreS/2-48myr-K-FITC depends on the differentiation state of the cell. (A) Undifferentiated (day 5 post plating, d5) and differentiated HepaRG cells (day 28 post plating and day 14 post-DMSO treatment, d28) were incubated with 200 nM of HBVpreS/2-48myr-K-FITC (left panels). PMH were incubated with the same peptide either in a differentiated state at day 1 post plating (diff. d1, lower right) or in a de-differentiated state after 8 days of cultivation without DMSO (de-diff. d8, upper right). Cells were washed, fixed with 4% PFA, and analyzed by confocal microscopy. Peptide-derived fluorescence is shown in green; nuclei were stained with DAPI (blue). (B) HuH7 and HepG2 cells are refractory to binding of HBVpreS/2-48myr-K-FITC. HuH7 (left panels) and HepG2 (right panels) hepatoma cells were incubated with 200 nM of HBVpreS/2-48myr-K-FITC either after having reached confluency (-DMSO, upper panels) or having been exposed to 2 weeks of DMSO-induced differentiation (+DMSO, lower panels). Cells were washed, fixed with 4% PFA, and analyzed by confocal microscopy. Peptide fluorescence is depicted in green; nuclei were stained with DAPI (blue). Note that even after DMSO-induced differentiation no specific staining at the plasma membrane of HepG2 and HuH7 cells could be observed. To investigate whether HuH7 and HepG2 express detectable amounts of the HBVpreS-receptor following DMSO-induced differentiation, we performed binding experiments with these cells under differentiation conditions. As shown in Fig. 4B (upper panel) dividing cultures of HuH7 and HepG2 cells showed no significant binding of HBVpreS/2-48myr-K-FITC. Cultivation in the presence of 0.5% DMSO for 2 weeks did not render the cells competent for HBVpreS/2-48myr-K-FITC-binding (Fig.4C, lower panels). Nonhepatoma cell lines with (293T, CHO-K1) or without a GAG-matrix (CHO-pgs745 cells) were also refractory for peptide binding (data not shown). This excludes a direct interaction with GAGs, a conclusion that was strengthened by the observation that binding o