Pholiota squarrosa lectin (PhoSL), a lectin binding to core-fucose specifically, inhibits HBV infection

doi:10.21203/rs.3.rs-2112428/v1

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Pholiota squarrosa lectin (PhoSL), a lectin binding to core-fucose specifically, inhibits HBV infection

https://doi.org/10.21203/rs.3.rs-2112428/v1

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published 15 Apr, 2023

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Glycosylation in host cells and viruses is an important factor in viral infection and a target for anti-viral therapy. Hepatitis B virus (HBV) is a major pathogen causing acute /chronic hepatitis. To achieve the cure, new anti-HBV agents are needed. Here we found that Pholiota squarrosa lectin (PhoSL), a lectin that specifically binds core-fucose, inhibited HBV infection to a human NTCP-expressing HepG2 cell called C4, a cell line susceptible to HBV infection. PhoSL bound to HBV particles. Fut8KO-C4 cells markedly lost HBV infectivity, and addition of PhoSL facilitated the loss of infectivity. Furthermore, we found that PhoSL blocked the activation of epidermal growth factor receptor (EGFR), a process enhancing HBV infection. Observation of the dynamics of fluorescent labeled PhoSL on C4 cells on the infection HBV showed that PhoSL-bound HBV was incorporated into host cells, suggesting that PhoSL could inhibit HBV infection after internalization. Since PhoSL reduced cccDNA formation, the process from internalization to cccDNA formation should be impaired by PhoSL. We believe that this finding should lead to development of new anti-HBV agents.

In viral infection, attachment and entry to host cells are the first and essential steps. Viruses bind to their specific receptors on the cell surface followed by endocytosis. Many cell surface proteins including virus receptors as well as viral envelope proteins are glycoproteins. There are many reports that change in the glycosylation status affects a variety of virus infection and its endocytosis¹. For example, the linkage of sialic acids on host cells is involved in infection of influenza virus² and the inhibition of sialidase is a target for influenza therapy³. Sialylation of host cells are also associated with the infection of SARS-CoV-2⁴.

Hepatitis B virus (HBV) is a major pathogen that causes acute /chronic hepatitis as well as hepatitis C virus and approximately 300 million individuals are infected chronically worldwide in 2019⁵. They will be suffering from developing liver cirrhosis and hepatocellular carcinoma⁶. Previous study showed that HBV bound to heparan sulfate on hepatocytes as the first step of infection. In 2012, sodium taurocholate cotransporting polypeptide (NTCP) was identified as an essential receptor for HBV and hepatitis delta virus (HDV)⁷. NTCP was originally known to be an integral membrane glycoprotein that functions in the enterohepatic circulation of bile acids. It is reported that glycosylation of NTCP is involved in its biological function⁸.

Our previous study demonstrated that core fucosylation was the key glycosylation in pseudo-HBV particles (Bionano capsule, BNC) entry into hepatoma cells, using a variety of oligosaccharide-remodeling cells⁹. Core fucose is synthesized by a reaction of fucosyltransferase 8 (Fut8)¹⁰. Since core fucose is involved in the endocytosis of receptors^11–13, knockdown/up-regulation of Fut8 changed the level of BNC incorporation into hepatoma cells. Association of NTCP with a scaffold protein, myosin-9, was observed in increase in core fucose levels in hepatoma cells. However, the later study showed infection process was different between HBV and BNC. While BNC binds to heparan sulfate alone on cell surface¹⁴, HBV binds to both heparan sulfate and NTCP.

We previously found Pholiota squarrosa lectin (PhoSL) as a lectin binding to core-fucose and PhoSL is stable under high temperature and acidic conditions¹⁵. Many lectins, proteins binding to glycan specifically, are reported as antiviral agents¹⁶. Griffithsin (GRFT), Cyanovirin-N (CV-N) and Scytovirin (SVN) are lectins binding to mannose-rich oligosaccharides and inhibit HIV-1 infection in the range without cytotoxicity^17–19. Furthermore, Cyanovirin-N (CV-N) has been studied in the pre-clinical phase²⁰.

In this study, we investigated effects of PhoSL on HBV infection to human NTCP expressing HepG2 cells, C4. In addition, molecular mechanisms underlying by which PhoSL inhibited HBV infection were analyzed. As results, we found that core-fucosylation of cellular proteins were important for HBV infection and PhoSL inhibited HBV entry process from internalization to cccDNA formation by binding to HBV itself and blocking EGFR activation.

Reagents

Williams E medium (Gibco or Sigma), Fetal bovine serum (Sigma or Nichirei Bioscienses), Penicillin-Streptomycin Mixed Solution (nacalai tesque), GlutaMAX I (Gibco), L-glutamine (nacalai tesque), Insulin-Transferrin-Selenium (Gibco), Holo-transferrin (FUJIFILM Wako), Insulin (Sigma), Hydrocortisone (Sigma or nacalai tesque), Dexamethasone (Sigma), Epidermal growth factor (Thermo Fisher or PeproTech), G418 (FUJIFILM Wako), EGF-TAMRA (Thermo Fisher), Hoechst33342 (nacalai tesque), Dimethyl sulfoxide (FUJIFILM Wako), Polyethylene Glycol - MW 8000 (MP Biomedicals), HBeAg ELISA Kit (Bioneovan or rsbio)

Antibodies and lectins

Anti EGFR mAb (#ab52894, Abcam), Anti Phospho-EGFR (Tyr1068) mAb (#2234, Cell Signaling Technology), Anti β-actin mAb-HRP (#5125, Cell Signaling Technology), PhoSL, PhoSL-FITC and PhoSL-biotin were provided by Dr. Kobayashi of J-Chemical, streptavidin-FITC (#SA-5001, Vector Laboratories)

Cells

HepG2-hNTCP-C4 WT was obtained from the National Institute of Infectious Diseases (Tokyo, Japan)²¹ and maintained in the primary hepatocyte maintenance medium (PMM), which consisted of Williams E medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% Penicillin-Streptomycin Mixed Solution(Stabilized) or (100 units/mL penicillin, 100 µg/mL streptomycin, 0.025 µg/ml amphotericin B), 2 mM GlutaMAX I or 2 mM L-glutamine, 1 x Insulin-Transferrin-Selenium or (5 µg/mL transferrin, 3 µg/mL human insulin, 5 ng/mL sodium selenite), 0.25 µg/mL amphotericin, 50 µM hydrocortisone, 5 µM dexamethasone, 10 ng/mL epidermal growth factor (EGF), and 0.5 mg/mL G418. In PhoSL inhibition assay to EGFR activation, HepG2-hNTCP-C4 WT was maintained in DMEM/F-12 supplemented with 10% heat-inactivated FBS, 1% Penicillin-Streptomycin Mixed Solution (Stabilized), 2 mM GlutaMAX I and 10 mM HEPES.

HepG2-hNTCP-C4 Fut8KO was established by Crispr-Cas9 system. HepG2-hNTCP-C4 WT was transfected with PX462 (Addgne) containing hFut8 sgRNAs using Lipofectamine 2000 (Invitrogen) and selected by puromycin at 2 µg/ml for 24 h. After the medium replacement, living cells were re-inoculated from 6 well plate to 150 mm dish and cultured for further two weeks. KO clones were established by isolating the formed colony with a cloning cylinder. KO was confirmed by genome sequencing. sgRNAs were listed in Table.1

(Table.1) sgRNAs for knock out of human Fut8 in HepG2-NTCP-C4.

hFut8 sgRNA sense 1	5’-caccGAATGAGCATAATCCAACGCC-3’
hFut8 sgRNA antisense 1	5’-aaacGGCGTTGGATTATGCTCATTC − 3’
hFut8 sgRNA sense 2	5’-caccGACCTTGCTGTTTTATATAGG-3’
hFut8 sgRNA antisense 2	5’-aaacCCTATATAAAACAGCAAGGTC-3’

HepAD38.7 cell, a tetracycline (tet) regulated HBV producing cell line, which was established using HepG2 cell²², was kindly provided by Dr. Christoph Seeger at Fox Chase Cancer Center（Philadelphia, U.S.）and maintained in a DMEM/F12 supplemented with 10% heat-inactivated FBS, 1% antibiotic-antimycotic solution, 5 µg/mL insulin, 0.5 mg/mL G418 and 400 ng/mL tetracycline (tet).

PANC-1 was obtained from RIKEN BioResource Research Center and maintained in a RPMI1640 supplemented with 10% heat-inactivated FBS, 1% Penicillin-Streptomycin Mixed Solution　(Stabilized) and 2 mM GlutaMAX I.

Preparation of HBV

To obtain HBV for HBV infection experiments, HepAD38.7 cells were induced for HBV replication by omitting tetracycline in the culture medium. The culture medium of confluent HepAD38 cells without tet was accumulated every week for 2 weeks and HBV was precipitated with 6% PEG8000 (final concentration) at 4˚C overnight. The precipitate was pelleted by centrifugation and the precipitated HBV was suspended in PBS, concentrated, and filtered through a 0.45 µm filter (Millipore). The HBV DNA was quantified by qPCR.

HBV infection to HepG2-hNTCP-C4

HepG2-hNTCP-C4 was seeded on a type I collagen-coated 24-well plate (IWAKI) at 0.5 or 1 × 10⁵ cells/well, and cultured for 1 day at 37°C under humidified conditions of 5% CO₂. Cells were then infected with HBV at 1000 genome equivalent of infection (GEI) in the presence or absence of 0.5, 1, 2.5, 5, 10 µg/ml PhoSL or 10 µM Gefitinib in 0.5 mL of PMM containing 2% dimethyl sulfoxide (DMSO) and 4% PEG 8000. 24 h after infection, cells were washed twice with PMM containing 2% DMSO and cultured in 0.5 mL of PMM containing 2% DMSO for 9 days. Finally, supernatant and cells were harvested for analysis.

HBeAg ELISA

HBeAg in culture supernatant was measured using a commercially available HBeAg ELISA kit. The absorbances was measured at 450 nm and 630 nm on SpectraMax 190 (Molecular Devices) or Grating Microplate Reader SH-1200 Lab (CORONA ELECTRIC).

qPCR

HBV DNA was extracted as follows. Cells were lysed with hypotonic Buffer (20mM Tris-HCl [pH7.5-7.8], 50mMNaCl, 5mM MgCl₂, 0.1% 2-ME) and the supernatant was treated with 1% SDS and 0.2 mg/ml proteinase K overnight at 56℃. After incubation, the DNA was extracted with phenol-chloroform-isoamyl alcohol (PCI extraction) and purified by ethanol precipitation.

For cccDNA preparation, the cells were lysed with hypotonic buffer. The pellet was suspended in TE-1% SDS and lysed. Then, NaCl was added to the lysate at a concentration of 0.5 M and stood still overnight at 4℃. After centrifugation, the supernatant was treated with 0.2 mg/mL proteinase K. DNA was extracted with phenol-chloroform-isoamyl alcohol and purified by ethanol precipitation. To obtain cccDNA, purified DNA was digested with Plasmid-Safe ATP-Dependent DNase and purified by PCI extraction and ethanol precipitation.

HBV RNA was prepared with TRIzol (Invitrogen) and cDNA was synthesized by reverse transcription.

qPCR was performed using QuantStudi 6 Flex Real-Time PCR System (Life Technologies). Primers were listed in Table.2.

(Table.2) Primers for qPCR of HBV DNA, cccDNA, RNA²³

HBV DNA forward primer	5′-CTTCATCCTGCTGCTATGCCT-3′
HBV DNA reverse primer	5′-AAAGCCCAGGATGATGGGAT-3′
cccDNA forward primer	5’-GTCTGTGCCTTCTCATCTGC-3’
cccDNA reverse primer	5’-GCACAGCTTGGAGGCTTGAA-3’
HBV RNA forward primer	5’-ATCATCTCATGTTCATGTCCTAC-3’
HBV RNA reverse primer	5’-GGAGATCTCGAATAGAAGGAAAG-3’

Cytotoxicity assay

HepG2-hNTCP-C4 WT was seeded on a type I collagen coated 96-well plate (IWAKI) at 3000 cells/well and cultured for 1 day at 37°C under humidified conditions of 5% CO₂. After changing to fresh medium, cells were cultured with 0, 0.25, 0.5, 1, 2.5, 5, 10 µg/ml of PhoSL for 1 day. Viability was measured using CellTiter-Glo Luminescent Cell Viability Assay (Promega) and chemiluminescence intensity was measured with Multi-grating Microplate Reader SH-9000 (CORONA ELECTRIC).

PhoSL inhibition to EGFR activation

HepG2-hNTCP-C4 WT and PANC-1 were seeded on type I collagen-coated 60 mm dishes or type I collagen-coated 6-well plates (IWAKI) and cultured at 37°C under humidified conditions of 5% CO₂. Cells were starved for 1 day by changing to serum-free medium. 3 h or 10 min before EGF stimulation, gefitinib as a positive control or PhoSL was added at 10 µM or 10 µg/ml, respectively. EGF stimulation was performed at 100 ng/ml. After adding EGF or EGF-TAMRA and incubating for 10 min at 37°C, 5% CO₂ in a humidified condition, cells were washed with PBS and harvested in ice-cold TNE buffer (10 mM Tris-HCl [pH 7.8], 1% NP40, 1 mM EDTA, and 0.5 M NaCl) containing 1 × Protease Inhibitor Cocktail and 1 × Phosphatase Inhibitor Cocktail 2 by cell scrapers. After 15 min incubation on ice, the lysates were sonicated by Bioruptor UCD-200T (Cosmo Bio) and centrifugated at 20000 × g for 20 min at 4°C. The supernatants were collected as lysates.

Immuno blot

Lysates were subjected to 10% SDS-PAGE under reducing conditions, and subsequently transferred to PVDF membrane (Millipore, Billerica). After blocking with TBST containing 5% skim milk for 1 h at room temperature, the membrane was incubated with primary antibody in TBST containing 5% skim milk over night at 4℃. Primary antibody incubations for the phosphor-EGFR were in TBST containing 5% BSA. After three washes with TBST (10 min, room temperature), the membrane was incubated with anti rabbit IgG pAb – HRP [1 : 10000 dilution] in TBST containing 5% skim milk for 1 h at room temperature. After washing with TBST three times (10 min, room temperature), the signal was detected with Chemi-Lumi One Super (nacalai tesque) and imaged with Fusion Solo S (Vilber Lourmat). Band intensities were quantified with Fiji (https://imagej.net/software/fiji/). Primary antibody dilution ratios were described in Table.3.

(Table.3) Primary antibodies and dilution ratios for immune blot

Anti EGFR mAb	1 : 3000
Anti Phospho-EGFR (Tyr1068) mAb	1 : 1000
Anti β-actin mAb – HRP	1 : 10000

Preparation of PhoSL-Cy3

PhoSL was labeled by Amersham Cy3 Mono-Reactive Dye (GE Healthcare). Free Cy3 was removed by Zeba Spin Desalting Columns (Thermo Fisher Scientific).

Fluorescence staining

HepG2-hNTCP-C4 WT and Fut8 KO were seeded at 6 × 10⁵ cells on type I collagen-coated glass base dishes (IWAKI) and cultured for 1 day at 37°C under humidified conditions of 5% CO₂. After washing with PBS, cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. After three washes with PBS (5 min, room temperature), cells were stained with PhoSL-FITC [1 : 10000 dilution] and Hoechst33342 [1 : 1000 dilution] in PBS containing 2% BSA for 1 h at 4℃. After washing with PBS three times (5 min, room temperature), cells were observed and imaged with confocal laser-scanning microscope FLUOVIEW FV10i (OLYMPUS).

For EGF-TAMRA binding assay, cells were starved for 1 day by changing to the serum-free mediim. PhoSL was added at 10 µg/ml 10 min before EGF stimulation. EGF-TAMRA was mixed at 100 ng/ml. After 10 min incubation at 37°C in humidified conditions, cells were washed with PBS and fixed with 4% paraformaldehyde.

For PhoSL-Cy3 observation, HepG2-hNTCP-C4 were seeded at 1 × 10⁴ cells/ well on collagen-coated 8-well chamber slides (Matsunami Glass) and incubated at 37°C under humidified conditions with 5% CO₂ for 3 days. Cells were then infected with HBV at 2000 G.E.I in 0.5 mL of PMM containing 2% dimethyl sulfoxide (DMSO), 4% PEG 8000, and 10 µg/ml of PhoSL-Cy3. After washing with PBS, cells were fixed with 4% paraformaldehyde, and slides were mounted with Fluoro-KEEPER Antifade Reagent, Non-Hardening Type with DAPI (nacalai tesque). Cells were observed and imaged with a TCS-SP8 confocal microscope (Leica).

Flow cytometry

To confirm core-fucose depletion, HepG2-hNTCP-C4 WT and Fut8 KO were detached with Accutase (nacalai tesque) and centrifuged at 200 × g for 5 min at 4°C. After washing with PBS, cells were stained with PhoSL-FITC [1 : 10000 dilution] in PBS containing 1% BSA for 1 h at 4℃. After washing twice with PBS, flow cytometric analysis was performed with an Attune NxT Flow Cytometer (Thermo Fisher). In supplementary Fig. 2, HepG2-hNTCP-C4 WT and PANC-1 were stained with PhoSL-biotin [1 : 100 dilution] and streptavidin-FITC [1 : 100 dilution].

For quantification of PhoSL-Cy3, HepG2-hNTCP-C4 WT was seeded at 6 × 10⁵ cells/well on type I collagen-coated 6-well plate (IWAKI) and cultured for 1 day at 37°C under humidified conditions of 5% CO₂. Then, cells were infected with HBV at 2000 G.E.I in PMM containing 2% dimethyl sulfoxide (DMSO) and 4% PEG 8000 in the presence of 5 µg/ml PhoSL-Cy3. After 1 h incubation at 4℃, cells were placed at 37℃ and trypsinized after 0, 15 min and 24 h. Flow cytometric analysis was performed using a BD FACSCanto II Flow Cytometer (BD Biosciences). Data were analyzed with FlowJo (https://www.flowjo.com/solutions/flowjo/downloads).

Statistical analysis

All statistical analyses were performed with EZR (https://www.jichi.ac.jp/saitama-sct/SaitamaHP.files/statmedEN.html), and data were presented as mean ± SD where applicable. Dunnett’s test and Student’s t-test were used.

PhoSL inhibits HBV infection to HepG2-hNTCP-C4

Viruses bind to their specific receptors followed by the endocytosis machinery of host cells. Core-fucose is a key modulator of signaling receptor’s dynamics^11,24,25 and enhances the endocytosis of pseudo HBV (Bionano capsule, BNC)⁹. Antibodies are an effective therapy for cancers and autoimmune diseases, which bind to each specific antigens (receptors or ligands) and block these signal transductions via steric hindrance²⁶. These findings prompted us to hypothesized Pholiota squarrosa lectin (PhoSL), a lectin binding to core-fucose specifically and strongly¹⁵, could inhibit HBV infection.

First, we examined whether PhoSL inhiibits HBV infection to HepG2-hNTCP-C4. HepG2-hNTCP-C4 is a clone established from a human hepatoma cell line HepG2 overexpressing hNTCP, a receptor of HBV, and susceptible to HBV infection²⁷. HepG2-hNTCP-C4 were inoculated with HBV in the presence of PhoSL for 1 day. After washing out of free HBV and PhoSL, the cells were cultured for 9 days, as shown in Fig. 1A. Treatment with PhoSL dramatically decreased the level of HBeAg (Fig. 1B), cccDNA (Fig. 1C), HBV DNA (Fig. 1D) and HBV RNA (Fig. 1E) in a dose-dependent manner. Then, we checked whether PhoSL has cytotoxicity to HepG2-hNTCP-C4 using CellTiter-Glo Luminescent Cell Viability Assay, a homogeneous method to determine the number of viable cells in culture based on quantitation of the ATP present. Some lectins have cytotoxicity¹⁶. However, PhoSL didn’t almost affect cell viability and/or growth in the range of 0.5–10 µg/ml (Fig. 1F). These data suggested PhoSL has the effect to HBV infection not caused by cytotoxicity.

PhoSL blocks EGFR activation, but this inhibitory effect is a part of the inhibitory pathway of HBV infection by PhoSL

As the inhibitory pathways, we considered two pathwasy: 1) PhoSL affects protein’s dynamics of the host cells. 2) PhoSL binds to HBV. First, we tested PhoSL inhibits HBV infection by affecting protein’s activity on the host cells. Epidermal growth factor receptor (EGFR) activation enhances HBV entry²⁸. EGFR is a heavily N-glycosylated protein and core-fucose is important of EGFR activation²⁵. EGFR activation is caused through EGF binding, dimerization and autophosphorylation. We hypothesized PhoSL inhibits HBV infection by blocking EGFR activation (Fig. 2A). We checked EGFR phosphorylation with or without PhoSL. After cell seeding and culture for 1 day, HepG2-hNTCP-C4 cells were starved in Serum free medium for 1 day. Next, the cells were pretreated with PhoSL or Gefitinib (EGFR activation inhibitor) and stimulated by EGF at 100 ng/ml. PhoSL blocked EGFR phosphorylation by EGF stimulation (Fig. 2B, C). Next, we demonstrated how PhoSL blocks EGFR activation. EGFR activation is occurred after binding EGF, dimerization and oligomerization. To examine whether PhoSL inhibits EGF binding to EGFR, we observed HepG2-hNTCP-C4 treated with EGF-TAMRA using confocal microscopy. Unfortunately, despite EGF-TAMRA activated EGFR, we couldn’t observe the signal of bound EGF-TAMRA (Supplementary Fig. 1A, B). Because HepG2 expresses low level of EGFR²⁹, we checked EGF-TAMRA binding to PANC-1, a cell line expressing high level of EGFR. PANC-1 was core-fucosylated as well as HepG2-hNTCP-C4 and expressed higher level of EGFR (Supplementary Fig. 2A, B). After addition of EGF-TAMRA, we observed bound EGF-TAMRA to PANC-1 and PhoSL blocked EGF-TAMRA binding (Supplementary Fig. 2D). These data indicate that PhoSL inhibits EGFR activation by blocking EGF binding. Finally, to compare the effect of PhoSL with that of Gefitinib to HBV infection, HepG2-hNTCP-C4 cells were inoculated with HBV in the presence of PhoSL or Geftinib (Fig. 2D). Interestingly, although the inhibition efficiency of PhoSL to EGFR phosphorylation was weaker than that of Gefitinib, the level of secreted HBeAg of treatment with PhoSL was lower than that of Gefitinib (Fig. 2E). These results suggest that PhoSL can block EGFR activation, but it is a part of the inhibitory pathway of HBV infection by PhoSL.

PhoSL inhibits HBV infection not only HepG2-hNTCP-C4 WT but also Fut8KO

Next, we examined whether PhoSL inhibits HBV infection not only by blocking protein’s activation on host cells but also by affecting HBV. We compared the inhibitory efficiency of pretreatment with that of simultaneous treatment to HepG2-hNTCP-C4 WT. HepG2-hNTCP-C4 WT was pretreated with PhoSL at 4℃ for 1 h. After washing out of free PhoSL, the cells were inoculated with HBV and cultured for 9 days. The level of HBeAg secretion from simultaneous treatment was lower than that of pretreatment (Fig. 4A). In addition, we investigated whether PhoSL inhibits HBV infection to core-fucosylation deficient HepG2-hNTCP-C4. Alpha-(1,6)-fucosyltransferase (Fut8) is the unique fucosyltransferase responsible for core-fucosylation of N-glycans¹⁰ (Fig. 3A) and core-fucose disappears by Fut8 knock out²⁴. We checked core-fucose deletion on HepG2-hNTCP-C4 Fut8 KO by lectin florescent stain and flow cytometry analysis with PhoSL-FITC (Fig. 3B, C). Both experiments showed core-fucose on HepG2-hNTCP-C4 was deleted completely by Fut8 knock out. To examine PhoSL inhibits HBV infection by affecting HBV, HepG2-hNTCP-C4 WT and Fut8KO inoculated with HBV in the presence of PhoSL for 1 day. After washing out of free HBV and PhoSL, the cells were cultured for 9 days. PhoSL decreased the levels of HBeAg secretion from not only WT but also Fut8KO (Fig. 3D). These results indicate PhoSL inhibits HBV infection through affecting hosts cells and HBV.

PhoSL binds to HBV and HBV bound by PhoSL is incorporated into HepG2-hNTCP-C4 WT

Finally, we demonstrated that PhoSL can bind to HBV. HBV consists of the nucleocapsid and the envelope formed by lipids and three proteins, L-HBsAg, M-HBsAg and S-HBsAg. These transmembrane proteins have a common carboxy-terminal sequence corresponding to that of the S protein. The S domain is often N-glycosylated (Fig. 4A)³⁰. To examine whether PhoSL binds to HBV, we prepared PhoSL cross-linked agarose and mixed with the conditioned medium containing HBV from HepAD38, a cell line producing HBV. PhoSL cross-linked agarose captured thirteen times more HBV particles than control agarose (Fig. 4B). HBV infection cycle consists of viral attachment, internalization, membrane fusion and replication³¹. To reveal the significance of PhoSL binding to HBV in HBV infection inhibition, we observed PhoSL-Cy3 on HepG2-hNTCP-C4 with or without HBV inoculation. PhoSL-Cy3 inhibited HBV infection as well as PhoSL (Fig. 4C). Although MFIs of PhoSL-Cy3 were almost not different between with and without HBV inoculation at 4℃ for 30 min, MFIs with HBV inoculation were higher than those without HBV inoculation at 37℃ for 15 min and 24 h (Fig. 4D, E). These data suggest that HBV bound by PhoSL is incorporated into HepG2-hNTCP-C4 and the possibility that PhoSL inhibits HBV infection after internalization.

Hepatitis B is a worldwide problem occurred by Hepatitis B virus (HBV) infection. HBV chronical infection is a risk factor of developing liver cirrhosis and hepatocellular carcinoma⁶. Current anti-HBV therapies include nucleoside analogs and interferons. Nucleos(t)ide analogs inhibit HBV replication competitively^32,33. Pegylated interferons (IFNs) modulate the host immune response to HBV infection and induce the degradation of cccDNA in hepatocytes. However, long-term treatment with nucleoside analogs can select drug-resistant viruses and IFNs can present severe adverse effects with low tolerability.

Glycosylation is a target of anti-viral therapy^3,20. In this study, we showed PhoSL, a lectin binding to core-fucose specifically and strongly, inhibited HBV infection in a dose-dependent manner without cytotoxicity (Fig. 1). Moreover, we revealed that PhoSL inhibits HBV infection by binding to HepG2-hNTCP-C4 and HBV particles (Fig. 2,3,4). Glycans on both host cells and viruses are important factors in virus infection¹. Viruses entry using the endocytosis machinery of host cells and glycans affect that machinery. Epidermal growth factor receptor (EGFR), a heavily N-glycosylated receptor tyrosine kinase, is a coreceptor of HBV infection and its activation enhances HBV endocytosis via NTCP²⁸, an essential receptor for HBV. We discovered that PhoSL can blocked EGFR activation (Fig. 2). Although, we couldn’t demonstrate how PhoSL block EGFR activation, PhoSL probably prevents EGF binding to EGFR because core-fucosylation is involved in EGF binding efficiency²⁵.

Despite the inhibitory effect to EGFR activation by PhoSL is lower than gefitinib, PhoSL suppressed HBV infection more strongly (Fig. 2E). These data indicates that blocking EGFR activation is a part of the inhibitory pathway of HBV infection by PhoSL. As another possibility of PhoSL inhibitory pathway, we discovered PhoSL binds to HBV particles (Fig. 4B). In Fig. 3D, PhoSL inhibits HBV infection to HepG2-hNTCP-C4 Fut8KO. HBV is an enveloped virus and the envelope consists of lipids and HBsAg. HBsAg is often N-glycosylated³⁰. Cyanovirin-N, a lectin produced by the cyanobacterium Nostoc ellipsosporum, binds to gp120, a protein on HIV envelope, and inhibits HIV infection by HIV membrane fusion^34,35. To reveal the significance of PhoSL binding to HBV in HBV infection inhibition, we observed PhoSL-Cy3 dynamics in HBV inoculation. MFIs of PhoSL-Cy3 in the presence of HBV was higher than in the absence and PhoSL-Cy3 were incorporated into HepG2-hNTCP-C4. The fact of PhoSL binding to HBV and these data suggest that PhoSL binding to HBV blocks a process after endocytosis. We uncovered a part of the mechanism how PhoSL inhibits HBV infection (Fig. 5). We believe that this discovering contributes to new anti-HBV therapies.

PhoSL, Pholiota squarrosa lectin;

HBV, Hepatitis B virus;

NTCP, sodium taurocholate cotransporting polypeptide;

C4, a human NTCP expressing HepG2 cell;

Fut8, Alpha-(1,6)-fucosyltransferase;

EGFR, Epidermal Growth Factor Receptor;

Acknowledgements

This work was supported by the Research Program on Hepatitis from the Japan Agency for Medical Research and Development, AMED.

Author contributions

T.O., K.U., and E.M. designed experiments; T.O., H.M., Y.A., M.M, and R.M. performed experiments; T.O., H.M., Y.A., M.M., S.T., J.K., Y.K., K.U., and E.M. discussed data; and T.O. and E.M. wrote the manuscript. M.U., K. U. and E.M supervised this study.

Data availability statement

The data presented in this study are available from the corresponding author on reasonable request. The original blot images were available in Supplementary figures.

Financial support

Supported by Japan Agency for Medical Research and Development (grant numbers 22fk0310505h0001

Conflict of Interest

We have no conflicts interest to disclose in this study.

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No competing interests reported.

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Pholiota squarrosa lectin (PhoSL), a lectin binding to core-fucose specifically, inhibits HBV infection

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