The specific core fucose-binding lectin Pholiota squarrosa lectin (PhoSL) inhibits hepatitis B virus infection in vitro

Glycosylation of proteins and lipids in viruses and their host cells is important for viral infection and is a target for antiviral therapy. Hepatitis B virus (HBV) is a major pathogen that causes acute and chronic hepatitis; it cannot be cured because of the persistence of its covalently closed circular DNA (cccDNA) in hepatocytes. Here we found that Pholiota squarrosa lectin (PhoSL), a lectin that specifically binds core fucose, bound to HBV particles and inhibited HBV infection of a modified human HepG2 cell line, HepG2-hNTCP-C4, that expresses an HBV receptor, sodium taurocholate cotransporting polypeptide. Knockout of fucosyltransferase 8, the enzyme responsible for core fucosylation and that aids receptor endocytosis, in HepG2-hNTCP-C4 cells reduced HBV infectivity, and PhoSL facilitated that reduction. PhoSL also blocked the activity of epidermal growth factor receptor, which usually enhances HBV infection. HBV particles bound to fluorescently labeled PhoSL internalized into HepG2-hNTCP-C4 cells, suggesting that PhoSL might inhibit HBV infection after internalization. As PhoSL reduced the formation of HBV cccDNA, a marker of chronic HBV infection, we suggest that PhoSL could impair processes from internalization to cccDNA formation. Our finding could lead to the development of new anti-HBV agents.


PhoSL blocks EGFR activation, which inhibits HBV infection. To determine how PhoSL inhibits
HBV infection, we considered two possibilities: (1) PhoSL affects the protein dynamics of the host cells, or (2)  PhoSL binds to HBV.First, we tested whether PhoSL inhibits HBV infection by affecting protein activity in host cells.EGFR activation enhances HBV entry 24 .EGFR is a heavily N-glycosylated protein, and core fucosylation affects the high-affinity binding of epidermal growth factor (EGF) to EGFR 25 .EGFR is activated by EGF binding, receptor dimerization, autophosphorylation of its intracellular domain, and oligomerization.Thus, we hypothesized that PhoSL inhibits HBV infection by blocking EGFR activation (Fig. 2A).We measured EGFR phosphorylation as a measure of its activation in the presence or absence of PhoSL.After cell seeding and culture for 1 day, HepG2-hNTCP-C4 cells were starved in serum-free medium for 1 day.Then, the cells were pretreated with PhoSL or gefitinib (an inhibitor of EGFR activation) and stimulated with EGF (100 ng/mL).PhoSL blocked EGFR phosphorylation as measured by immunoblot (Fig. 2B,C).Next, we investigated whether PhoSL could block EGFR activation by inhibiting the binding of EGF to EGFR.We treated HepG2-hNTCP-C4 cells with EGF-tetramethylrhodamine conjugate (EGF-TAMRA).Unfortunately, despite EGF-TAMRA-mediated activation of EGFR detected by immunoblot, we did not observe EGF-TAMRA bound to the cells by confocal microscopy (Supplementary Fig. S1 online).Because HepG2 cells express low levels of EGFR 26 , we tested EGF-TAMRA binding to a human pancreatic cancer cell line, PANC-1, which expresses high levels of EGFR.PANC-1 cells are core fucosylated like HepG2-hNTCP-C4 cells are, and PANC-1 cells expressed higher levels of EGFR than HepG2-hNTCP-C4 cells (Supplementary Fig. S2 online).Using confocal microscopy, we observed EGF-TAMRA bound to PANC-1 cells, and PhoSL blocked EGF-TAMRA binding (Supplementary Fig. S2 online).These data indicate that PhoSL inhibits EGFR activation by blocking EGF binding to EGFR.Finally, to compare the effect of PhoSL with that of gefitinib on HBV infection, HepG2-hNTCP-C4 cells were inoculated with HBV particles in the presence of PhoSL or gefitinib (Fig. 2D).Interestingly, although the inhibition of EGFR phosphorylation by PhoSL was weaker than by gefitinib, the level of secreted HBeAg after treatment with PhoSL was lower than that of gefitinib (Fig. 2E).These results suggest that PhoSL can inhibit HBV infection by blocking EGFR activation.
PhoSL inhibits HBV infection in HepG2-hNTCP-C4 WT and FUT8 KO cells.Next, we examined whether PhoSL inhibits HBV infection by binding to HBV .We compared HBV infection of HepG2-hNTCP-C4 wild-type (WT) cells with PhoSL pretreatment (at 4 °C for 1 h) or by simultaneous treatment with PhoSL and HBV particles.After culturing the treated and inoculated cells for 9 days, we found that HBeAg secretion levels from cells simultaneously treated with HBV and PhoSL were lower than from cells pretreated with PhoSL before HBV inoculation (Fig. 3A).In addition, we investigated whether PhoSL inhibited HBV infection of core fucosylation-deficient HepG2-hNTCP-C4 cells.FUT8 is responsible for core fucosylation of N-glycans 13 , and core fucosylation disappears after FUT8 knockout (KO) 27 .We confirmed the absence of core fucose on HepG2-hNTCP-C4 FUT8 KO cells by confocal microscopy and flow cytometry with PhoSL-FITC (Fig. 3B, C).The level of HBeAg secretion from HepG2-hNTCP-C4 FUT8 KO cells was lower than that from WT cells after HBV inoculation (Fig. 3D).This difference could be explained by a decrease in HBV particle endocytosis mediated by the heparan sulfate proteoglycan (HSPG) pathway 17 or the NTCP-EGFR pathway 24 .Core fucosylation increases endocytosis of bio-nano capsules 12 , which can bind to HSPG but not to NTCP, and increases the efficiency of EGFR activation 25 .We inoculated HepG2-hNTCP-C4 WT and FUT8 KO cells with HBV in the presence or absence of PhoSL for 1 day.After removal of free HBV and PhoSL, the cells were cultured for 9 days.PhoSL decreased the levels of HBeAg secretion from WT and FUT8 KO cells (Fig. 3D).The level of HBeAg secretion from FUT8 KO cells treated with PhoSL decreased to approximately 40% of those cells that were not treated with PhoSL.These results indicate that PhoSL can inhibit HBV infection by not only binding to host cells but also by binding to HBV itself, as PhoSL did not bind to HepG2-hNTCP-C4 FUT8 KO cells.
HBV bound by PhoSL is internalized into HepG2-hNTCP-C4 WT cells.Finally, we tested the second possibility of how PhoSL inhibits HBV infection: whether PhoSL binds HBV particles.HBV consists of a nucleocapsid, and an envelope that is formed from lipids and three proteins: the large, medium, and small hepatitis B surface antigens (L-HBsAg, M-HBsAg, and S-HBsAg, respectively).These transmembrane proteins have a common carboxy-terminal sequence corresponding to that of the S protein.The S domain is often N-glycosylated 28 .First, to check whether HBsAg in conditioned medium containing HBV particles from HepAD38.7, a cell line that produces HBV particles 29 , was core fucosylated, we performed immunoprecipitation of HBsAg and lectin blot with PhoSL.Lectin blot with PhoSL revealed that HBsAg from HepAD38.7 cells was core fucosylated (Fig. 4A).Next, to examine whether PhoSL binds to HBV particles, we prepared agarose cross-linked to PhoSL (PhoSL-agarose) and mixed it with conditioned medium containing HBV particles from HepAD38.7 The increasing gradient of blue color represents PhoSL concentrations of 0.5, 1, 2.5, 5, and 10 μg/mL, respectively.Gray indicates 0 μg/mL PhoSL.Live cells were measured with the CellTiter-Glo Luminescent Cell Viability Assay kit.Dunnett's test was performed.There were no statistically significant differences between 0.5, 1, 2.5, 5, and 10 μg/mL PhoSL vs. 0 μg/mL PhoSL.Error bars show SD; n = 3. cells.PhoSL-agarose captured thirteen times more HBV particles than control agarose, as measured by quantitative polymerase chain reaction (qPCR) (Fig. 4B).Finally, to reveal the significance of PhoSL binding to HBV on HBV infection inhibition, we labeled PhoSL with Cy3 and observed the dynamics of PhoSL-Cy3 on HepG2-hNTCP-C4 cells with or without HBV inoculation.Cy3 labeling did not affect the inhibitory efficiency of HBV infection by PhoSL (Fig. 4C).Because PhoSL inhibited cccDNA formation (Fig. 1C), we investigated whether PhoSL affected HBV dynamics by binding to HBV particles.PhoSL-Cy3 was internalized into HepG2-hNTCP-C4 cells with and without HBV inoculation for up to 30 min at 37 °C (Fig. 4D).Then, HepG2-hNTCP-C4 cells were treated with PhoSL-Cy3 in the presence or absence of HBV particles and were trypsinized, which detaches bound HBV from the cell surface 17 .The mean fluorescence intensities of PhoSL-Cy3 with HBV inoculation were higher than those without HBV inoculation at 37 °C for 15 min and 24 h by flow cytometry (Fig. 4E).These data suggest that HBV particles bound to PhoSL are internalized into HepG2-hNCP-C4 cells, and PhoSL may inhibit HBV infection after the internalization.We used these data to develop a schematic that presents how PhoSL can inhibit HBV infection by binding to host cells and HBV particles (Fig. 5).

Discussion
In this study, we showed that PhoSL, a lectin that specifically and strongly binds to core fucose, inhibited HBV infection in a dose-dependent manner without cytotoxicity (Fig. 1).Moreover, PhoSL inhibited HBV infection by binding to HepG2-hNTCP-C4 cells and HBV particles (Figs. 2, 3, 4).Glycans on host cells and viruses are important factors for virus infection 9 .Viruses are internalized using the endocytosis machinery of host cells, and changes in glycans can affect that machinery.EGFR, a heavily N-glycosylated receptor tyrosine kinase, is a coreceptor for HBV infection, and its activation enhances HBV endocytosis via NTCP 24 , an essential receptor for HBV.EGFR activation by autophosphorylation occurs by the binding of its ligand, EGF, which is followed by receptor dimerization 30 .EGFR is N-glycosylated around the EGF binding site 31 .EGFR binds EGF with two distinct affinities: high affinity (Kd ≈ 300 pM) and low affinity (Kd ≈ 2 nM) 32 .Core fucosylation affects high- affinity binding of EGF to EGFR 25 , indicating that N-glycans with core fucose are important regions of EGFR that affect EGF binding.In Supplementary Fig. S2 online, PhoSL (10 μg/mL) blocked the binding of EGF-TAMRA (100 ng/mL or ≈17 nM) to EGFR.Therefore, PhoSL seems to prevent EGF binding to EGFR by steric hindrance.
We also found that PhoSL binds to HBV particles (Fig. 4B).HBV is an enveloped virus, and the envelope consists of lipids and proteins, including HBsAg.HBsAg in conditioned medium containing HBV particles from HepAD38.7 cells was core fucosylated (Fig. 4A).Therefore, PhoSL seems to bind to HBV by recognizing core fucose of HBsAg.Cyanovirin-N, a lectin produced by the cyanobacterium Nostoc ellipsosporum, binds to gp120, a protein on the HIV envelope, and inhibits HIV infection by HIV membrane fusion 33,34 .Recently, it was reported that PhoSL inhibits SARS-CoV-2 infection by binding to S protein, a protein on the SARS-CoV-2 envelope 35 .We found that PhoSL inhibited HBV infection of HepG2-hNTCP-C4 FUT8 KO cells, which cannot bind PhoSL (Fig. 3D).Thus, PhoSL may inhibit HBV infection by binding to HBV particles.The early phase of HBV infection consists of HBV attachment, endocytosis, membrane fusion, and cccDNA formation 36 .To reveal the significance of PhoSL binding to HBV particles on the inhibition of HBV infection, we investigated PhoSL-Cy3 dynamics with HBV inoculation.Flow cytometry analysis showed that the signals from PhoSL-Cy3 increased in the presence of HBV particles (Fig. 4E).Because trypsinization detaches HBV particles from cell surfaces 17 , the increase in PhoSL-Cy3 seems to indicate that PhoSL-bound HBV particles can be internalized .These data suggest that PhoSL-bound HBV particles might inhibit processes after the endocytosis.Possible mechanisms responsible for PhoSL-mediated inhibition of HBV infection were described in Fig. 5. PhoSL treatment could contribute to the development of novel anti-HBV therapies.
The HepG2-hNTCP-C4 FUT8 KO cell line was established by a CRISPR-Cas9 system.HepG2-hNTCP-C4 WT cells were transfected with the PX462 plasmid (Addgene) containing hFUT8 single guide RNA (sgRNA) using Lipofectamine 2000 (Invitrogen) and selected with puromycin (2 μg/mL) for 24 h.After medium replacement, live cells were transferred from six-well plates to 150-mm dishes and cultured for a further two weeks.KO clones were established by isolating colonies with a cloning cylinder.KO was confirmed by genome sequencing.The sgRNAs used are listed in Table 1.
HepAD38.7 cells, a tetracycline-regulated HBV-producing cell line that was established using HepG2 cells 29 , were kindly provided by Dr. Christoph Seeger at Fox Chase Cancer Center (Philadelphia, PA, USA) and were Table 1.sgRNAs for human FUT8 KO in HepG2-NTCP-C4 cells.

Preparation of HBV particles.
To obtain HBV particles for HBV infection experiments, HepAD38.7 cells were induced for HBV replication by omitting tetracycline from the culture medium.The culture medium of confluent HepAD38.7 cells grown without tetracycline was collected each week for 2 weeks, and HBV particles were precipitated from the medium with PEG 8000 (final concentration 6%) at 4 °C overnight.The precipitate was pelleted by centrifugation, and the precipitated HBV particles were resuspended in phosphate-buffered saline (PBS), concentrated, and filtered through a 0.45-µm filter (Millipore).The HBV DNA was quantified by qPCR.
HBeAg ELISA.HBeAg in supernatants from HepG2-hNTCP-C4 cells with or without HBV infection was measured using a commercially available HBeAg ELISA kit.The absorbances were measured at 450 nm and 630 nm on a SpectraMax 190 microplate reader (Molecular Devices) or an SH-1200Lab microplate reader (Corona Electric).
For cccDNA preparation, cell pellets were lysed by resuspension in a hypotonic buffer of 1 × TE buffer containing 1% sodium dodecyl sulfate.Then, NaCl (0.5 M) was added, and the lysate was incubated overnight at 4 °C.After centrifugation, the supernatant was treated with proteinase K (0.2 mg/mL).DNA was extracted from the supernatant with phenol-chloroform-isoamyl alcohol and purified by ethanol precipitation.To isolate the cccDNA, purified DNA was digested with Plasmid-Safe ATP-Dependent DNase (Lucigen) and purified by phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation.
HBV RNA was isolated with TRIzol (Invitrogen), and cDNA was synthesized by reverse transcription.qPCR was performed using a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems).The primers used are listed in Table 2.
PhoSL inhibition assay to measure EGFR activation.HepG2-hNTCP-C4 WT and PANC-1 cells 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 and 5% CO 2 .The cells were starved for 1 day by changing to a serum-free medium.Then, gefitinib (10 μM) as a positive control or PhoSL (10 μg/mL) was added to the medium 3 h or 10 min before stimulation with EGF (100 ng/mL) or EGF-TAMRA (100 ng/ml).The cells were incubated for 10 min at 37 °C under humidified conditions and 5% CO 2 , washed with PBS, and harvested with cell scrapers in ice-cold TNE buffer (10 mM Tris-HCl pH 7.8, 1% NP-40, 1 mM EDTA, and 0.5 M NaCl) containing 1 × protease inhibitor cocktail and 1 × phosphatase inhibitor cocktail 2. After 15 min incubation on ice, the lysates were Table 2. Primers for qPCR of HBV DNA, cccDNA, and RNA.Ref. 38,40 .
PhoSL-mediated precipitation of HBV particles.PhoSL was cross-linked to Pierce NHS-Activated Agarose Dry Resin (Thermo Fisher Scientific) in accordance with the manufacturer's protocol.Next, the PhoSLagarose was rotated with the conditioned medium containing HBV particles isolated from HepAD38.7 cells for 2 h at 4 °C.After washing with Tris-buffered saline, DNA was extracted from the PhoSL-agarose, and qPCR was performed using a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems).

Statistical analysis.
All statistical analyses were performed with EZR software 40 (https:// www.jichi.ac.jp/ saita ma-sct/ Saita maHP.files/ statm edEN.html), and data were presented as mean ± standard deviation (SD) where applicable.Dunnett's test and Student's t test with Bonferroni correction were used.

Figure 3 .
Figure 3. PhoSL inhibited HBV infection of HepG2-hNTCP-C4 WT and FUT8 KO cells.(A) HBeAg secretion from HepG2-hNTCP-C4 cells treated with PhoSL and infected with HBV particles was measured by ELISA.The cells were pretreated with PhoSL at 4 °C for 1 h before inoculation with HBV particles, simultaneous treatment with PhoSL and HBV particles, or inoculation with HBV particles in the absence of PhoSL.The values were normalized to the mean of HBeAg secretion levels in the absence of PhoSL.Student's t test, *P < 0.05.Error bars show SD, n = 3. (B) HepG2-hNTCP-C4 WT and FUT8 KO cells were stained with PhoSL-FITC and observed using confocal microscopy (left).Green: PhoSL-FITC; blue: nucleus (Hoechst 33,342 stain).Bright-field images are shown on the right.Scale bars show 100 μm.(C) Flow cytometry of HepG2-hNTCP-C4 WT and FUT8 KO cells with or without PhoSL-FITC.Black: WT; red: FUT8 KO.Solid lines without fill show the cells stained with PhoSL-FITC.Filled areas show unstained cells.(D) HBeAg secretion from HepG2-hNTCP-C4 WT and FUT8 KO cells infected with HBV particles in the absence or presence of PhoSL (10 μg/ml) was measured by ELISA.The values were normalized to the mean of WT cells in the absence of PhoSL treatment.Black: WT; orange: FUT8 KO.The darker colors show the HBeAg secretion levels from cells treated with PhoSL.The lighter colors show the HBeAg secretion levels from cells not treated with PhoSL.Student's t test with Bonferroni correction, **P < 0.01, vs. no PhoSL.Error bars show SD, n = 3.

Figure 5 .
Figure 5. PhoSL inhibits HBV infection by binding to host cells and HBV particles.(Left) PhoSL inhibits EGFR activation by blocking the binding of EGF to EGFR.(Right) PhoSL binds to HBV particles.HBV particles bound by PhoSL are internalized into host cells.PhoSL seems to inhibit HBV infection after internalization.

Table 3 .
Primary antibodies and dilution ratios for immunoblots.