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Virology 497 (2016) 23–32

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Virology
journal homepage: www.elsevier.com/locate/yviro

Cellular uptake of hepatitis B virus envelope L particles is independent
of sodium taurocholate cotransporting polypeptide, but dependent on
heparan sulfate proteoglycan
Masaharu Somiya a,b,c, Qiushi Liu a,b, Nobuo Yoshimoto a, Masumi Iijima a, Kenji Tatematsu a,
Tadashi Nakai a, Toshihide Okajima a, Kazuyuki Kuroki d, Keiji Ueda e, Shun’ichi Kuroda a,b,n
a

The Institute of Scientific and Industrial Research, Osaka University, Osaka 567-0047, Japan
Graduate School of Bioagricultural Sciences, Nagoya University, Aichi 464-8601, Japan
Japan Society for the Promotion of Science, Tokyo 102-0083, Japan
d
Central Research Resource Branch, Cancer Research Institute, Kanazawa University, Ishikawa 920-1192, Japan
e
Division of Virology, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan
b
c

art ic l e i nf o

a b s t r a c t

Article history:
Received 8 April 2016
Returned to author for revisions
24 June 2016
Accepted 29 June 2016

Sodium taurocholate cotransporting polypeptide (NTCP) was recently discovered as a hepatitis B virus
(HBV) receptor, however, the detailed mechanism of HBV entry is not yet fully understood. We investigated the cellular entry pathway of HBV using recombinant HBV surface antigen L protein particles
(bio-nanocapsules, BNCs). After the modification of L protein in BNCs with myristoyl group, myristoylated BNCs (Myr-BNCs) were found to bind to NTCP in vitro, and inhibit in vitro HBV infection
competitively, suggesting that Myr-BNCs share NTCP-dependent infection machinery with HBV. Nevertheless, the cellular entry rates of Myr-BNCs and plasma-derived HBV surface antigen (HBsAg) particles
were the same as those of BNCs in NTCP-overexpressing HepG2 cells. Moreover, the cellular entry of
these particles was mainly driven by heparan sulfate proteoglycan-mediated endocytosis regardless of
NTCP expression. Taken together, cell-surface NTCP may not be involved in the cellular uptake of HBV,
while presumably intracellular NTCP plays a critical role.
& 2016 Elsevier Inc. All rights reserved.

Keywords:
Bio-nanocapsule
Endocytosis
Hepatitis B virus
Myristoylation
Virus uncoating

1. Introduction
Hepatitis B virus (HBV) infection is one of the major causes of
liver-related diseases, including chronic hepatitis, liver cirrhosis,
and hepatocellular carcinoma (Neuveut et al., 2010). Despite much
effort in the last five decades since the discovery of HBV, the life
cycle of HBV is not yet fully understood at the molecular level.
Generally, viral entry is an important target for antiviral drugs
since entry inhibitors can prevent the binding of virions to cellular
receptors and subsequent viral membrane fusion with the host cell
membrane (Dimitrov, 2004). In regards to the development of
entry inhibitors for HBV, sodium taurocholate cotransporting
polypeptide (NTCP) was recently identified as a functional cellular
receptor for HBV by Yan et al. (2012). Particularly, it was shown
that exogenous expression of NTCP conferred susceptibility to HBV
infection in vitro in otherwise non-susceptible Huh-7 and HepG2
cells (Iwamoto et al., 2014; Yan et al., 2012). However, the detailed
n
Correspondence to: Department of Biomolecular Science and Reaction, The
Institute of Scientific and Industrial Research, Osaka University, Mihogaoka 8-1,
Ibaraki, Osaka 567-0047, Japan.
E-mail address: skuroda@sanken.osaka-u.ac.jp (S. Kuroda).

http://dx.doi.org/10.1016/j.virol.2016.06.024
0042-6822/& 2016 Elsevier Inc. All rights reserved.

mechanism of the NTCP-mediated cellular entry pathway has not
been fully elucidated (Li, 2015; Urban et al., 2014; Watashi et al.,
2014b; Yan et al., 2015). In addition to NTCP, several other cellular
proteins have been proposed as important factors for HBV infectivity. Most importantly, the heparan sulfate proteoglycan
(HSPG) was identified as an initial attachment protein of HBV, and
especially, glypican-5 may be one of the HSPG that is involved in
the cellular entry process (Leistner et al., 2008; Schulze et al.,
2007; Verrier et al., 2016). Additionally, several domains in the
HBV envelope L protein have been shown to play essential roles in
the interactions between HBV and human hepatic cells. For example, Neurath et al. (1986) were the first to find that 10–36
amino acid residues (aa) of the pre-S1 region were essential for the
recognition of human hepatic cells. Using HBV deletion mutants, it
was shown that N-terminal aa of the pre-S1 region were necessary
for HBV infectivity, which was likely due to cellular receptor
binding (Blanchet and Sureau, 2007). Additionally, using pre-S1derived synthetic peptides, 9–15 aa of the pre-S1 region were
shown to be crucial for HBV infection as a synthetic peptide corresponding to those residues suppressed infection in vitro (Schulze
et al., 2010). Recently, it was demonstrated that 9–15 aa of the preS1 region participated in the interaction with NTCP (Yan et al.,

. several hepatic cell lines. myristoylation at the glycine-2 residue was tightly linked to HBV infectivity (Gripon et al. (A) N-terminal amino acid sequence of the L protein of BNCs (Kuroda et al. the cellular entry of HBV-mimicking particles was inhibited by a myristoylated pre-S1 peptide (2–48 aa) regardless of NTCP expression.. Potential lysine residues for myristoylation are highlighted in red. However. 2009). the antigenic loop of the S region was shown to be essential for HBV infection (Abou-Jaoudé and Sureau. 1992).. In the current study. Therefore. we synthesized large amounts of HBV envelope L protein particles in yeast cells (referred to as bio-nanocapsules. and HBsAg particles (referred to as HBV-mimicking particles) were shown to enter HepG2 cells regardless of NTCP expression with similar efficiency. 2014). in lieu of primary human hepatocytes.. Materials BNCs were purified from Saccharomyces cerevisiae AH22Rstrain harboring an HBsAg L protein expression plasmid as described previously (Jung et al. Yamada et al. 1995). 2013) and fusogenic domain (Somiya et al. 2015) are indicated with brackets. / Virology 497 (2016) 23–32 Fig. 2015. Furthermore. 2011.. it is time-consuming and laborious to purify HBV virions from hepatitis B patient plasma or HBV-containing culture medium. 2. Owing to the identification of NTCP as a functional HBV receptor. Essential NTCP-binding domain (Meier et al.1. Salisse and Sureau. these results suggest that the pre-S1 domain is important for HSPG-dependent endocytosis. 1A). we prepared myristoylated BNCs (MyrBNCs) by chemical modification of BNCs with myristoyl groups. Taken together. BNCs. Furthermore.. 2013). have become useful for HBV research because NTCP overexpression confers HBV susceptibility (Iwamoto et al. BNCs can bind and enter into a wide variety of human hepatic cells including those not susceptible to HBV or that lack NTCP (Yamada et al. which are endogenous HBV subviral particles containing myristoylated L proteins. BNCs have been utilized as human hepatic cell-specific drug-delivering nanocarriers both in vitro and in vivo (Somiya and Kuroda.. (B) MALDI-TOF MS data of protease digests of BNCs and Myr-BNCs. Myr-BNCs. followed by the disruption of both membranes. we recently used BNCs to reveal that 9–24 aa of the pre-S1 region exhibit low pH-dependent fusogenic activity (Somiya et al. Amino acids from  5 to  1 correspond to a residual fragment of the chicken lysozyme signal peptide (C-SIG). and intracellular NTCP may exclusively function in the binding of HBV. Myr-BNCs were shown to inhibit HBV infection of NTCP-expressing HepG2 cells through interactions with NTCP in a competitive manner. the rate of cellular entry of BNCs was the same as that of hepatitis B patient plasma-derived HBV surface antigen (HBsAg) particles. 2003). BNCs are hollow nanocapsules displaying L proteins as transmembrane proteins in a manner similar to those in HBV.. These observations demonstrated that BNCs could enter cells through a NTCP-independent pathway and escape from endocytic vesicles following membrane fusion. 2012). it is necessary to obtain substantial amounts of HBV. The glycine residue highlighted in green indicates the N-myristoylation site found in the endogenous L protein of HBV genotype D. 1992). 1A). 1992). Preparation of Myr-BNCs.24 M. 2007. To investigate the intracellular trafficking of HBV with biochemical techniques. Unlike BNCs... Kuroda et al. While the various cellular proteins and viral domains (within the pre-S1 and S regions) described above are indispensable for the cellular attachment and entry of HBV. While BNCs have no myristoyl group at the N-terminus of the L protein. NTCP is not involved in the cellular uptake of HBVmimicking particles. suggesting that myristoylation is necessary for pre-S1 interaction with NTCP (Urban et al. Somiya et al. 2005.. N-succinimidyl myristate and a mouse monoclonal anti-pre-S1 antibody . Meanwhile. 1. 2015) (see Fig. it remains unclear how these factors contribute to these mechanisms at the molecular level. Materials and methods 2. and was recently demonstrated to interact with HSPG (Sureau and Salisse. After encapsulation of drugs. BNCs) (Kuroda et al. 2012) (see Fig.. which was predominantly mediated by HSPG-dependent endocytosis. Jaoude and Sureau. In addition to the aa sequence of the L protein. 2014). Furthermore. which could induce membrane fusion between the envelope membranes of the BNCs and exogenous membranes.

2001). The human hepatocellular carcinoma cell line HepG2. UK) as a secondary antibody. USA) for 30 min.5% (w/v) octyl β-D-glucopyranoside and 0. 10% FBS.8 7 0. Switzerland). fixed with 4% (w/v) paraformaldehyde.000 rpm at 4 °C for 10 min. Samples were solubilized with a mixture of 0. approx.0. and incubated at room temperature for 5 min.47 g software (National Institutes of Health. Japan). 0. Billerica. Louis.. Similar to above. HepG2/NTCP cells were lysed with RIPA buffer (20 mM Tris-HCl pH 7. and one tablet per 25 mL Protease Inhibitor Cocktail) on ice for 10 min. Basel.1 M glycine-HCl buffer (pH 2.25 mg/mL amphotericin B) containing 4% (w/v) polyethylene glycol and 2% (v/v) DMSO were incubated with HBV (subtype. Japan).1857 0. Heparin sodium salt and Hoechst 33342 were purchased from Nacalai Tesque (Kyoto. 1 mM EDTA. The labeling was stopped by mixing with medium containing FBS. and stained with Hoechst 33342.043  10. transferred to blotting membranes. 150 mM NaCl. MA. 5  104 HepG2/NTCP cells/well grown in PMM (Williams E containing 5 mg/mL transferrin. completely dried. GE Healthcare). Sunnyvale. Germany). Beijing. and centrifuged at 15.. data are shown as means 7 SD. cells were washed. 0.0.5) and subjected to western blotting using an anti-pre-S1 antibody and a Trueblot Ultra HRPconjugated anti-mouse IgG antibody (Rockland. 50 mM hydrocortisone.4. and then subjected to MALDI-TOF MS. Briefly. the particles were dissolved in PBS.0  106 HepG2/ NTCP cells were lysed with 1. 1% v/v Triton X-100. and either BNCs or Myr-BNCs (1 mg of protein). Briefly. USA). CA.0  105 HepG2 or HepG2/NTCP cells/well were . ayw. Olympus. Darmstadt.0 mL of lysis buffer (20 mM Tris-HCl pH 8. USA).8  10. USA). 2011). fixed. and then centrifuged at 15. Buckinghamshire. and further incubated with SDS sample buffer at 60 °C for 10 min. 2. USA). washed twice with PBS.0  104 HepG2 or HepG2/NTCP cells/well were seeded in 8-well glass bottomed chamber slides. USA) following published procedures (Kim et al. Chemically synthesized Myr47 peptide (myristoyl groupGTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDQWPEANQVC) was purchased from Scrum (Tokyo. mixed with TA buffer (33% v/v acetonitrile and 0. In vitro HBV infection assay Approx. Absorbance at 450 nm (absorbance at 630 nm was used as background) was measured with the Spectra Maxs 190 (Molecular Devices. and HB patient derived-HBsAg particles (HBV-mimicking particles). 2. N ¼ 3.3.. 2014)) at 200 genome equivalents per cell in the presence of either 20 mg/mL (as protein) of BNCs or Myr-BNCs at 37 °C for 24 h. Buckinghamshire. Bruker. For the immunoprecipitation assay.M. and further digested by Asp-N (Roche). 2 mM L-glutamine.7 cells (Ogura et al. Cells were then incubated with culture medium containing 5 mg/mL (as protein) of each labeled particle at 37 °C for 5. Cytochemical analysis Approx. Preparation of myristoylated BNCs (Myr-BNCs) BNCs were mixed with N-succinimidyl myristate (at a molar ratio of L protein:N-succinimidyl myristate of 1:5) in phosphate buffered saline (PBS) and incubated at room temperature for several hours. BNCs Myr-BNCs Z-average (nm) Polydispersity index (-) Zeta potential (mV) 111.6. UK) in PBS at 25 °C. Myristoylation of BNCs was evaluated by MALDI-TOF MS (Ultraflex III. 150 mM NaCl. anti-NTCP antibody (1:100 dilution).2.067% v/v TFA) saturated with sinapinic acid. 3 mg/mL insulin. 1% v/v Nonidet P-40) containing Protease Inhibitor Cocktail (Roche). MA. and then incubated with a horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (GE Healthcare. the particles were labeled with the lipophilic dye CellVue Claret as described previously (Balogh et al. At the end of the infection. mixed with equal volume of CellVue Claret labeling solution (1/250 dilution in Diluent C). USA). v/v) supplemented with 10% (v/v) fetal bovine serum (FBS). For the detection of cell-surface NTCP.7 7 4. PA. 2.1 0. Tokyo. Flow cytometry analysis Approx. Tokyo. Ltd. The supernatant was mixed with 20 mL of protein G-conjugated Sepharose resin (50% v/v slurry.5 mL centrifugal filter with a 100 kDa cut-off (Merck-Millipore. Worcestershire. USA). Somiya et al. 5 mM dexamethasone. Ten mL of the supernatants were sampled at 0. HepG2 expressing NTCP (HepG2/NTCP). incubated with an anti-NTCP primary antibody.000 rpm at 4 °C for 10 min.5. NTCP was visualized using the ECL Prime Western Blotting Detection Reagent (GE Healthcare.5 h. Diameters and zeta potentials of BNCs and MyrBNCs were measured with Zetasizer Nano ZS (Malvern Instruments. Bethesda. D. 3. Supernatants were mixed with 20 units/mL PNGase-F. China) according to the manufacturer’s instructions. the co-immunoprecipitates were eluted with 0. and incubated with culture medium containing 3% DMSO for 1 day. Ryosuke Suzuki (National Institute of Infectious Diseases. CA.5 117.5% (v/v) trifluoroacetic acid (TFA). Samples were subjected to gel electrophoresis. For the observation of BNCs. Roche. Western blotting and immunoprecipitation assay Western blotting for NTCP was performed as described previously (Okuyama-Dobashi et al. UK) and ChemiDoc 25 system (Bio-rad. Waltham. CA. 100 IU/mL penicillin G.2 71.8 (clone KR127) were purchased from Santa Cruz Biotechnology (Santa Cruz. Hercules. All specimens were observed under a confocal laser scanning microscope (FV-1000. 10 ng/mL epidermal growth factor. 2015). Japan). 5.5% w/v sodium deoxycholate. Japan). 2. and fluorophore-labeled Myr47 (carboxytetramethylrhodamine [TAMRA] was conjugated to the C-terminus) were kindly provided by Dr.1% w/v SDS. the cells were washed with 1 mL of 2% DMSO-PMM three times and then cultured in 1 mL of PMM for 9 days. 2. cells were incubated with 200 nM TAMRAconjugated Myr47 peptide at 37 °C for 1 h. blocked with PBS containing 5% (w/v) skim milk. Briefly. prepared from HepAD38. and 9 days post-infection (dpi).8 7 6. Gilbertsville. Japan). Lipophilic fluorescent dye (CellVue Claret) and a rabbit polyclonal anti-NTCP antibody (HPA042727) were purchased from Sigma-Aldrich (St.. spotted onto target plates. and analyzed with the B3 HBeAg ELISA Kit (Bioneovan Co. 5 ng/mL sodium selenite. 100 mg/mL streptomycin. genotype. Japan). and then stained with Hoechst 33342. and incubated at 4 °C overnight. Myr-BNCs. / Virology 497 (2016) 23–32 Table 1 Physicochemical properties of BNCs and Myr-BNCs. Sodium chlorate was purchased from Wako Pure Chemical (Osaka. and further incubated with 50 nM LysoTracker Red (Thermo Fisher Scientific. Cells were maintained in Ham’s F-12/Dulbecco’s modified Eagle’s medium (50%/ 50%. and 0. incubated at 37 °C for 2 h.1897 0. MO. BNCs and Myr-BNCs were analyzed by dynamic light scattering. Hepatitis B patient-derived HBsAg particles were purchased from Institute of Immunology (Tokyo. MA. The colocalization of particles with LysoTracker Red was analyzed by ImageJ version 1. 2. After the resin was washed five times with lysis buffer. Unreacted N-succinimidyl myristate was removed by ultrafiltration using an Amicon Ultra-0. 2..019 0. BNCs and Myr-BNCs were deglycosylated with peptide N-glycosidase-F (PNGase-F. 6.

respectively. or patient-derived HBsAg particles (5 mg/mL as protein) at 37 °C for 6 h unless otherwise indicated. and analyzed by western blotting with an anti-NTCP antibody. 6.09 and 2865. San Diego. Interaction of Myr-BNCs with NTCP Since human hepatocellular carcinoma-derived cell lines such as HepG2 normally lack NTCP expression (Watashi et al. HepG2/NTCP cells were infected with HBV in the presence of Myr-BNCs or BNCs. 3. to those of the original BNCs (Table 1). USA). 2A). Myr-BNCs. These results showed that Myr-BNCs bind to NTCP and can inhibit HBV entry in a myristoyl group-dependent manner. To evaluate the effect of heparin on HSPG-dependent endocytosis. resulting in the ablation of the L protein N-myristoylation signal (Fig. (B) Interaction of Myr-BNCs with NTCP. Somiya et al. Arrowheads. 1A). Lysates of HepG2/NTCP cells were mixed with BNCs or Myr-BNCs.. heparin sodium salt (100 mg/mL. N ¼3. Results 3. Effect of Myr-BNCs on in vitro HBV infection. Myristoylation of BNCs BNCs were synthesized in yeast cells by expressing the L protein with an N-terminal fusion of the chicken lysozyme signal peptide (C-SIG). 2014a). NTCP. 2013). cells were pre-treated with sodium chlorate (100 mM. . such as Z-average. Fig. and 9 dpi were analyzed by HBeAg ELISA. (A) Expression of NTCP in HepG2 and HepG2/NTCP cells.2. 2C). Since N-myristoylation of the L protein is necessary for HBV infectivity. and patient-derived HBsAg particles) was added to the culture medium. we chemically treated BNCs with a 5-fold molar excess of N-succinimidyl myristate. asterisks. which indicated that N-myristoylation of a pre-S1-derived peptide (2–48 aa) was essential for binding to NTCP (Meier et al. After the incubation with particles.1. protein G contamination. indicating that a portion of the L proteins in BNCs was myristoylated at the N-terminal lysine-(-5). Myr-BNCs. 1B). CA. 3. followed by flow cytometry analysis using the FACSCanto II (BD Biosciences. the N-terminal lysine-(-5) (either at the α-amine or ε-amine) was found to be modified with a myristoyl group by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Fig. and zeta potential. The myristoylated BNCs (Myr-BNCs) were found to have similar physicochemical properties. 3. 2. which facilitates efficient membrane translocation of the L protein across the yeast endoplasmic reticulum membrane and subsequent particle formation (Kuroda et al. 2B). in an in vitro infection assay with cell culture-derived HBV on HepG2/NTCP cells.. final) was added to the culture medium. HepG2 cells overexpressing NTCP (HepG2/NTCP) were used for analyzing the NTCP-mediated cellular entry pathway (Fig. Calculated m/z values of the peptide corresponding to the sequence from  5 to þ 19 aa and its myristoylated derivative (210 Da increase) were 2655. anti-NTCP immunoprecipitates were found to contain only Myr-BNCs (Fig. Among the four lysine residues located in the putative ectodomains of BNCs ( 5 to 164 aa and 258 to 331 aa). This result is consistent with a previous report. Furthermore. After incubating either BNCs or Myr-BNCs with HepG2/NTCP lysates.. Cell lysates were deglycosylated with PNGase-F. were shown to inhibit infection and consequent release of hepatitis B e antigen (HBeAg) (Fig. 1992). (C) Inhibition of HBV infection of HepG2/NTCP cells by Myr-BNCs.26 M. Anti-NTCP immunoprecipitates and no antibody immunoprecipitate controls were analyzed by western blotting with an anti-pre-S1 antibody (upper panel) and an anti-NTCP antibody (lower panel). Cells were incubated with fluorophore-labeled BNCs. / Virology 497 (2016) 23–32 seeded in 6-well plates and incubated in culture medium containing 3% DMSO at 37 °C for 1 d. but not BNCs. final) at 37 °C for 16 h before the assay. For the ablation of sulfate groups on HSPG. the Myr47 peptide (as indicated molar excess of HBsAg proteins in BNCs. Myr-BNCs. polydispersity index. cells were washed twice with PBS and detached with PBS containing 2 mM EDTA. For the evaluation of the effect of the pre-S1 region.45. Culture media collected at 0. data are shown as means 7 SD.

Somiya et al. respectively. NTCP-independent cellular binding and entry of HBV-mimicking particles. (E) Colocalization of NTCP and LysoTracker Red. 20 mm.M. NTCP and endosomes/lysosomes were stained by immunofluorescence and LysoTracker Red. (A) Interaction of Myr47-TAMRA with cell-surface NTCP. (B) Cellular binding and entry of BNCs. Arrowhead indicates colocalization of NTCP and LysoTracker Red. HepG2 and HepG2/NTCP cells were incubated with fluorophore-labeled particles at 37 °C for 6 h. (C) Intracellular localization of HBV-mimicking particles. Colocalization rates were calculated from five different images including that in panel C (data are shown as means 7 SD). respectively. and HBsAg particles. Bars. (D) Colocalization rates of the particles with LysoTracker Red. 3. data are shown as means 7 SD. / Virology 497 (2016) 23–32 27 Fig. HepG2 and HepG2/NTCP cells were incubated with fluorophore-labeled particles at 37 °C for 6 h. HepG2 and HepG2/NTCP cells were treated with Myr47-TAMRA at 37 °C for 60 min. N ¼ 3. Arrows indicate colocalization signal. and then analyzed by flow cytometry. Nuclei and late endosomes were visualized with Hoechst 33342 and LysoTracker Red staining. Myr-BNCs. . and then observed under confocal laser scanning microscopy. Nuclei were visualized with Hoechst 33342 staining.

Thus. data are shown as means 7 SD. This result agreed well with the previous observation that NTCP localizes to the basolateral membrane (Sun et al. similar amounts of intracellular particles were observed in both cell types. and subsequently travel from early endosomes to lysosomes (Macovei et al.28 M. 3B).. the rate of cellular internalization of BNCs into Huh7 cells (NTCP-lacking cells) has been shown to be similar to that of HBV into human hepatocytes (Qiao et al. 2006). we observed that NTCP localizes in endosome/ lysosome in HepG2/NTCP cells as previously reported (Sarkar et al. the entry of HBV-mimicking particles into both cell types was completely blocked at 4 °C. we examined if HSPG was involved in the NTCP-independent endocytosis of HBVmimicking particles. 2012) without permeabilization. 2013). the cellular uptake of particles was also significantly inhibited in both cell types. fluorophore-labeled particles were added to HepG2 and HepG2/NTCP cells at 4 °C and 37 °C. 1994. 3. 3E. 3C and D). 2012). Considering that the sulfate group of HSPG plays a crucial role in the interaction with HBV (Leistner et al. 2008).. / Virology 497 (2016) 23–32 Fig. Somiya et al. 2014a). the endocytosis of Myr-BNCs was less affected by heparin and sodium chlorate. HSPG-dependent endocytosis of BNCs and Myr-BNCs HBV has been shown to enter cells via endocytosis. Therefore. When the particles were incubated with HepG2 and HepG2/NTCP cells for 6 h in the presence of heparin as a competitor for HSPG. these results indicate that nearly all HBV-mimicking particles were internalized within 6 h by endocytosis into HepG2 cells regardless of NTCP expression. and HBsAg particles (namely. N ¼ 3. it is postulated that intracellular NTCPs. Additionally. presumably due to an increase of non-specific interactions of Myr-BNCs with cells by the addition of the hydrophilic myristoyl groups. Schulze et al. Yamada et al. When HepG2 and HepG2/NTCP cells were incubated with HBV-mimicking particles in the presence of Myr47.. the different particles showed similar intracellular localization in both cell types as determined by a cytochemical analysis with LysoTracker Red.. and the amounts of internalized particles were then determined by flow cytometry (Fig. the extracellular particles were removed by trypsinization. the cell-associated and intracellular amounts of each of these particles were the same in both cell types (Fig. As trypsinization removes HBV attached to the cell surface (Watashi et al. Compared with those of BNCs and HBsAg particles. (A) Temperature dependency of cellular binding and entry of HBV-mimicking particles. Myr-BNCs. (B) Cellular entry of HBV-mimicking particles. Furthermore. 4B). As shown in Fig. 2007). However. After trypsinization. a substantial amount of Myr47 peptide was found to bind on the plasma membrane of HepG2/NTCP cells (Fig. 2006). the cellular binding and entry of HBV-mimicking particles were inhibited in both cell types . 1996). trypsinized to remove the extracellular particles. These results indicate that cell-surface NTCP did not contribute to the binding and entry of HBV-mimicking particles. an NTCP-binding peptide. and then analyzed by flow cytometry. Endocytosis of HBV-mimicking particles. After treatment with sodium chlorate... 4. As for the intracellular trafficking. 1998). HSPG is a low-affinity receptor for the initial attachment of HBV (see introduction).. HBV-mimicking particles) were incubated with HepG2 or HepG2/NTCP cells. are involved in the entry process of Myr-BNC and HBV at endosomes/lysosomes. an inhibitor for proteoglycan sulfation (Mislick and Baldeschwieler.3. fluorophore-labeled HBV-mimicking particles were allowed to be internalized by both cell types at 37 °C for 6 h. a fluorescent dye that stains acidic endosomes/lysosomes (Figs.. as shown in Fig. but not the cell-surface NTCPs. although NTCP could interact with Myr-BNCs (presumably as well as with HBsAg particles) in vitro. 2008. and then analyzed by flow cytometry as previously described (Khalil et al. these results indicate that the NTCP-independent endocytosis of HBV-mimicking particles is dependent on HSPG. the cellular entry of particles was equally suppressed in both cell types (Figs.. and thus the cellular binding and entry of BNCs into HepG2 cells were shown to be inhibited in the presence of heparin (Kasuya et al.4. when the fluorophore-labeled forms of BNCs. 4A. and then subjected to flow cytometry.. HepG2 and HepG2/NTCP cells were treated with HBV-mimicking particles at 37 °C for 6 h. NTCP-independent cellular binding and entry of Myr-BNCs When HepG2/NTCP and HepG2 cells were treated with the NTCP-binding fluorophore-labeled Myr47 peptide (corresponding to the 2–48 aa of pre-S1 with an N-terminal myristoyl group (Yan et al. To examine if HBV-mimicking particles also enter cells by endocytosis. The particles were incubated with HepG2 and HepG2/NTCP cells at 37 °C or 4 °C for 3 h.. 3A). 5A and B). Furthermore. 3.

(Fig. 5D).. 5C). Especially. These results suggest that the N-terminal pre-S1 region is involved in the NTCP-independent and HPSG-dependent endocytosis of HBV-mimicking particles. This is likely due to a lower content of the L protein in HBsAg particles. 2001). HSPG-dependent endocytosis of HBV-mimicking particles. Somiya et al. While HSPG was also identified as an . where the L protein accounts for 1–2 mol percent (mol%) of the total envelope protein in HBsAg particles (Heermann et al. / Virology 497 (2016) 23–32 29 Fig.M. (C) Five molar-excess of Myr47 peptide against HBsAg protein in HBV-mimicking particles inhibited the cellular binding and entry of HBV-mimicking particles in both cell types. 1984) compared to 100 mol% in BNCs and Myr-BNCs (Yamada et al. data are shown as means 7SD. 5. the cellular uptake of HBsAg particles was severely affected by Myr47 compared to BNCs and Myr-BNCs. Discussion Following the discovery of NTCP as a potential HBV receptor. (D) Dose-dependent inhibition effect of Myr47 on NTCP-independent cellular binding and entry of HBVmimicking particles in HegG2 cells.. 4. the interaction of NTCP with the myristoylated form of the pre-S1 region has been considered to play a crucial role in the cellular binding and entry of HBV. N ¼ 3. The inhibition effect of Myr47 was dose-dependent in HepG2 cells lacking NTCP (Fig. Heparin and sodium chlorate treatment inhibited the cellular binding and entry of HBV-mimicking particles in HepG2 cells (A) and HepG2/NTCP cells (B).

Although it has not yet been confirmed that HBV virions directly bind to NTCP. 3A). Somiya et al. Meanwhile. HSPG is a membrane-anchored protein. it remained unknown how NTCP and HSPG cooperatively contribute to the cellular binding and entry of HBV at the molecular level.. 3B)...e. HSPG functions as a universal receptor for various types of viruses. In the current study. after a chemical modification of recombinant yeast-derived BNCs with a myristoyl group (Fig. it is highly likely that HBV virions also enter cells by this HSPG-dependent and NTCP-independent endocytic mechanism. 1999. 3 and 4) but rather on HSPG (Fig. through which viruses bind prior to internalization into cells (Bernfield et al. Christianson and Belting. Working hypothesis of the cellular binding and entry of Myr-BNC and HBV in NTCP-deficient (left) and NTCP-expressing (right) human hepatic cells. HSPG-mediated binding of HBV and its subviral particles were observed even in HBV non-susceptible cells such as HeLa and Chinese hamster ovary cells (Leistner et al. 2014).. which expands linear polysaccharide chains in the extracellular domain.30 M. which is indispensable for the establishment of HBV infection. While the Myr47 peptide was shown to bind to cell-surface NTCP (Fig. For example. / Virology 497 (2016) 23–32 Fig.. and inhibit HBV infection of HepG2/NTCP cells (Figs. HBV receptor. 6. 2000). it was demonstrated for the first time herein that myristoylated HBV-mimicking particles (i. 1B). 2007). 2014). the cellular uptake of HBV-mimicking particles was revealed to be dependent not on NTCP (Figs. hepatitis C virus utilizes HSPG as a necessary initial attachment receptor (Baumert et al. and dengue virus uses HSPG as a binding and entry receptor on hepatocytes (Hilgard. the cellular binding and entry of Myr-BNCs were found to be NTCP-independent (Fig. 5). NTCP is a membrane protein containing multiple transmembrane segments. 2B and C). These results indicate . Myr-BNCs) could bind to NTCP in vitro. it can be extremely difficult to obtain substantial amounts of HBV virions for biochemical analyses. 2008. Myr-BNCs were found to interact with NTCP in vitro. On the other hand. Schulze et al. Since the L proteins of HBVmimicking particles and HBV virions exhibit similar membrane topology. These results of the current study provide strong evidence that HBV-mimicking particles are internalized by HSPG-dependent and NTCP-independent endocytosis. These results strongly suggest that Myr-BNCs and HBV share the NTCP-dependent infection machinery.

K.A.... Curr. 2008).. M. T. Enzym. and endocytic vesicles are enriched with NTCP (Fig.2013. Protein Expr. HBV is thought to initially attach to HSPG on the cell surface and then transfer to NTCP (Watashi et al. http://dx. Dimitrov..L.C. Sureau.L. http://dx. Iijima. S. K.1.. matbio. T. 292–299.. In the current study. Evaluation and identification of hepatitis B virus entry inhibitors using HepG2 cells overexpressing a membrane transporter NTCP.0002.A. 2007.2000..12. 2015). E. 2014. as well as the S region. 808–813. 2014. http://dx. 13057–13066. Pap. Blanchet..79. 729–777.. Entry of hepatitis delta virus requires the conserved cysteine residues of the hepatitis B virus envelope protein antigenic loop and is blocked by inhibitors of thiol-disulfide exchange.. Virology 213. Jaoude.. 1994). M..1016/j. Bernfield. Virol. T.H. and S. Watashi.doi.. we demonstrated that cell-surface NTCP is not involved in the cellular binding and uptake of HBV-mimicking particles (Fig.. Res. X.01495-07. 2014). 2. EMBO J.org/10. 2014. Seyffarth. which may prevent the interaction of their myristoylated pre-S1 regions with cell-surface NTCP. This topology change was crucial for the HSPG binding (Seitz et al..S. D...S. 3E and Sarkar et al. Balogh.. 3B). HBV virions and Myr-BNCs may similarly enter cells via HSPG-mediated endocytosis.H. Virol. Yoshimoto. P. but these systems generally require large amounts of HBV virions and long-term cell culture.16.Y.. J. which is indispensable for HBV infection (Schulze et al. G. Hepatology 32.J. but also the HSPG-dependent binding of HBV.. Myristylation of the hepatitis B virus large surface protein is essential for viral infectivity. K. S.H.T. N. N. Baumgarten.N. J. M. Wakita.1128/JVI. Götte.12. 2006). As shown in Figs. 1995.. R. Szeberényi. L.K. T. A simple fluorescent labeling technique to study virus adsorption in Newcastle disease virus infected cells. Schwartz. 2014). 49. Fukasawa. 5841–5849. W. J.doi.doi. 35. hepatitis B virus surface antigen L particles. H..004. in the absence of NTCP.1995. Microb. thus indicating that the pre-S1 region plays a crucial role in HSPG-mediated endocytosis. Aly. Functions of cell surface heparan sulfate proteoglycans. whereby they traffic to late endosomes. 79. M.. Lincecum...doi.2005.1006/viro. 6. M. T. http: .. Suzuki. 2005. C.F.Y. http://dx. Kaneko et al. R..org/10.1016/j.. W. leading to degradation (left panel). Various anti-HBV compounds have been discovered by using in vitro HBV infection systems utilizing NTCP-expressing hepatocellular carcinoma cells (Iwamoto et al. 2011.M.1016/j.. Sureau.. To date. Osaka University Graduate School of Medicine to S. it was demonstrated that pre-S1 region is not exposed after secretion from HBV-infected cells. and K. Fitzgerald.O. Q.doi. Matrix Biol. 2016). I. HSPG may deliver HBV virions from the cell surface to intracellular compartments.. Christianson. Annu. Efficient and rapid purification of drug. Ito.coviro.org/10. Sureau.. The authors thank Dr.1128/JVI. Rumin. Biochem. Nkongolo et al. Aizaki. 443. J. 2014b). Q. 2015. Thomssen. These particles could then bind to intracellular NTCP through the exposed pre-S1 region.org/10. M. Heparan sulfate proteoglycan as a cell-surface endocytosis receptor..1128/JVI. designed experiments.005. was previously shown to interact with HSPG. Tsukuda. Hilgard. performed experiments. which may inhibit not only the NTCPdependent binding of HBV. U. Author contribution M...enzmictec. Reizes. KAKENHI Grant-in-Aid for Scientific Research (S) 16H06314 to S. in the presence of NTCP. M. Park.. 78.. 81. Tatematsu. Nat. 1998).. Meredith. Tokyo. As shown in Fig.K..org/10.1038/nrmicro817. wrote the manuscript. Le Seyec. We previously found that low pH could induce conformational changes in the pre-S1 region in BNCs (Somiya et al.H. Zako. Baumert.729. Large surface proteins of hepatitis B virus containing the pre-s sequence.org/10..68. J. 1984. Virol.2013. Koiwai. J. 2007.. Japan).10460-10466. http://dx.K.K.. Iwamoto...U. http://dx. Gerlich.. A... such as endosomes or lysosomes. K.. M... Koichi Watashi (National Institute of Infectious Diseases) for his helpful comments and discussion regarding this work. 68.doi. Technol. 2000. from Saccharomyces cerevisiae.doi. C.. http://dx. Since Myr-BNCs were shown to inhibit HBV infection in vitro.doi. J. H. Rev.org/ 10. Purif. http://dx. Rev. and then converted to fully-exposed form in dime-dependent manner. Guguen-Guillouzo..and gene-carrying bio-nanocapsules. 52.. Y..org/10.. http://dx. http://dx. G. 4. Urban.. Kuroda. K. Entry of hepatitis B and C viruses – recent progress and future impact.. 13.2011. C.06. S. 1069–1077. Gerlich. K.I. followed by the exposure of the pre-S1 region under acidic conditions. Meanwhile. S. O. 109–122. Ni. L. Lu. H.. McKeating. it is possible that Myr-BNCs could be used as an HBV model to screen entry inhibitors of HBV in high-throughput screening.doi.18713.. 396–402. Biochem.org/10.. Commun. C. Somiya et al. NTCP is mainly found at the basolateral membrane (Sun et al. These observations suggest that the Myr47 peptide.L. M. Niimi. Fujimoto..S. / Virology 497 (2016) 23–32 that the myristoylated pre-S1 region of Myr-BNCs may not bind to cell-surface NTCP.. 10460–10466.org/10.K.-Y.. Microbiol. 2007. J. Felmlee. During the morphogenesis of HBV..10. under development as Myrcludex-B (Petersen et al. P.doi. 2015.. T. M. Sureau and Salisse. Ryosuke Suzuki (National Institute of Infectious Diseases. 255–259.K.052. 1999. Goldmann. References Abou-Jaoudé. 81.S.. KAKENHI Grant-in-Aid for JSPS Fellows 25003835 to M. Virol... 2273–2279.S.. Acknowledgements This work was supported in part by The Naito Foundation grants to S. A.. 2011.00096-07.. Markó. Thus.. M.K. 2014. Recently. 51–55..S...1146/annurev. and S.doi. Csatáry.... discussed data.K. Opin.1053/ jhep. Tanizawa. 2014. J. KAKENHI Grant-in-Aid for Scientific Research (A) 25242043 to S. H. the Myr47 peptide inhibited the cellular binding and entry of HBV-mimicking particles in an NTCP-independent manner.biochem. Heparan sulfate proteoglycans initiate dengue virus infection of hepatocytes.1016/j. and S. Virus entry: molecular mechanisms and biomedical applications. Yan et al. M. Role of the antigenic loop of the hepatitis B virus envelope proteins in infectivity of hepatitis delta virus. L. 31 Conflicts of interest The authors declare no conflicts of interest. 5C and D.. M. These results indicate that the pre-S1 region in HBV as well as in BNCs could undergo conformational changes under acidic condition... Choi.002.2013. and Project MEET. W.. and Myr47-TAMRA were kindly provided by Dr... We thus postulated that the N-terminal pre-S1 region is not fully exposed on the surface of HBV-mimicking particles or HBV virions under neutral conditions. 2013). a Health Labor Sciences Research Grant from the Ministry of Health Labor and Welfare to S.. However. S. HBV virions and Myr-BNCs may enter cells via HSPG-dependent endocytosis and then move to lysosomes. T. and subsequently initiate the uncoating process (endosomal escape) by an unidentified mechanism involving intracellular NTCP and a low pH-dependent fusogenic domain (right panel). H. O. 58–65. Infectivity determinants of the hepatitis B virus pre-S domain are confined to the N-terminal 75 amino acid residues.org/10. inhibitors of HBV cellular binding and entry may be easily screened in cell-based highthroughput assays.. is a pleiotropic anti-HBV drug.. The pre-S1 region. 1994. 2004.. Koloszár... K. 149–155. and M. V. These results showed that Myr47 peptide interferes the binding between HBV-mimicking particles and HSPG. NTCP-overexpressing HepG2 cells. Jeong. Heermann. Kusuhara. Our results herein strongly suggest that HSPG transfers HBV virions to intracellular NTCP for the establishment of HBV infection. Post-translational alterations in transmembrane topology of the hepatitis B virus large envelope protein.. HepG2 cells. Sasaki. Gripon. Bruss.. D. low pH was found to trigger the exposure of the pre-S1 region to the surface of HBV virions (Bruss et al.bbrc.. Okuyama-Dobashi et al. Biophys. K.K. Jung. M. http://dx. By substituting fluorophorelabeled Myr-BNCs for HBV virions.W.. P. Belting. Virol. the N-terminal pre-S1 region is postulated to translocate across the HBV envelope membrane using the hydrophobic myristoyl group as an anchor (Baumert et al.

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