Epidermal growth factor receptor is a host-entrycofactor triggering hepatitis B virus internalizationMasashi Iwamotoa,b, Wakana Sasoa,c, Ryuichi Sugiyamaa, Koji Ishiid, Mio Ohkie, Shushi Nagamorif, Ryosuke Suzukia,Hideki Aizakia, Akihide Ryog, Ji-Hye Yunh, Sam-Yong Parke, Naoko Ohtanii, Masamichi Muramatsua, Shingo Iwamib,j,k,Yasuhito Tanakal, Camille Sureaum, Takaji Wakitaa, and Koichi Watashia,j,k,n,1

aDepartment of Virology II, National Institute of Infectious Diseases, 162-8640 Tokyo, Japan; bMathematical Biology Laboratory, Department of Biology,Faculty of Sciences, Kyushu University, 812-8581 Fukuoka, Japan; cThe Institute of Medical Science, The University of Tokyo, 108-8639 Tokyo, Japan;dDepartment of Quality Assurance and Radiological Protection, National Institute of Infectious Diseases, 162-8640 Tokyo, Japan; eDrug Design Laboratory,Yokohama City University Graduate School of Medical Life Science, 230-0045 Yokohama, Japan; fDepartment of Collaborative Research, Nara MedicalUniversity, 634-8521 Kashihara, Japan; gDepartment of Microbiology, Yokohama City University School of Medicine, 236-0004 Yokohama, Japan;hDepartment of Biochemistry, College of Life Science & Biotechnology, Yonsei University, 03722 Seoul, South Korea; iGraduate School of Medicine, OsakaCity University, 545-8585 Osaka, Japan; jCore Research for Evolutional Science and Technology (CORE), Japan Science and Technology Agency (JST), 332-0012Saitama, Japan; kMIRAI, Japan Science and Technology, 332-0012 Saitama, Japan; lDepartment of Virology and Liver Unit, Nagoya City University GraduateSchool of Medicinal Sciences, 467-8601 Nagoya, Japan; mLaboratoire de Virologie Moléculaire, Institut National de la Transfusion Sanguine, 75739 Paris,France; and nDepartment of Applied Biological Science, Tokyo University of Science, 278-8510 Noda, Japan

Edited by Francis V. Chisari, The Scripps Research Institute, La Jolla, CA, and approved March 12, 2019 (received for review June 27, 2018)

Sodium taurocholate cotransporting polypeptide (NTCP) is a host cellreceptor required for hepatitis B virus (HBV) entry. However, thesusceptibility of NTCP-expressing cells to HBV is diverse depending onthe culture condition. Stimulation with epidermal growth factor (EGF)was found to potentiate cell susceptibility to HBV infection. Here, weshow that EGF receptor (EGFR) plays a critical role in HBV virioninternalization. In EGFR-knockdown cells, HBV or its preS1-specificfluorescence peptide attached to the cell surface, but its internaliza-tion was attenuated. PreS1 internalization and HBV infection could berescued by complementation with functional EGFR. Interestingly, theHBV/preS1–NTCP complex at the cell surfacewas internalized concom-itant with the endocytotic relocalization of EGFR. Molecular interactionbetween NTCP and EGFR was documented by immunoprecipitationassay. Upon dissociation from functional EGFR, NTCP no longer func-tioned to support viral infection, as demonstrated by either (i) theintroduction of NTCP point mutation that disrupted its interactionwithEGFR, (ii) the detrimental effect of decoy peptide interrupting theNTCP–EGFR interaction, or (iii) the pharmacological inactivation ofEGFR. Together, these data support the crucial role of EGFR in mediat-ing HBV–NTCP internalization into susceptible cells. EGFR thus providesa yet unidentified missing link from the cell-surface HBV–NTCP attach-ment to the viral invasion beyond the host cell membrane.

HBV | NTCP | EGFR | entry | transporter

Hepatitis B virus (HBV), an enveloped virus carrying threeenvelope proteins [small, middle, and large surface proteins

(LHBs)], infects humans, chimpanzees, and tupaias with a se-lective tropism to the liver (1). HBV infection is initiated byinteraction between the viral envelope proteins and host factorson a cell surface through heparan sulfate proteoglycan, followedby specific virus attachment to its host receptor, sodium taur-ocholate cotransporting polypeptide (NTCP). Subsequent viralinternalization into cells and membrane fusion proceed throughan endocytosis-dependent manner. However, it has been largelyunknown how virus–cell attachment triggers internalization andwhich host factor (or factors) mediates this process. Clarificationof this entry process is essential for a better understanding of theHBV life cycle, its host tropism, and the eventual formation ofcovalently closed circular DNA (cccDNA) in the nucleus, whichserves as a template for HBV replication (2–4).NTCP was identified as a host receptor essential for HBV entry

(5). Expression of NTCP confers HBV susceptibility to humanhepatic cell lines such as HepG2, Huh7, or undifferentiated Hep-aRG cells, which are originally nonsusceptible to infection (6, 7).Nonetheless, some human-derived hepatic or nonhepatic cell linesremain resistant to HBV entry, even though NTCP overexpressionenabled viral attachment to the cell surface (8–10). Furthermore,

NTCP-complemented HepG2 cells were diversely susceptible toHBV infection, depending on the culture condition, despite sus-tained levels of NTCP expression (SI Appendix, Fig. S1B). Theseobservations likely indicate that NTCP expression is not sufficientfor supporting robust HBV infection. In this study, we identify thatepidermal growth factor (EGF) receptor (EGFR) is a host cofactorthat is required for NTCP’s viral receptor function and determinesthe host cell’s ability to support viral internalization.

ResultsEGFR Is Critically Involved in HBV/HDV Infection. From a screening ofbioactive ligands (Materials and Methods), we found that EGF clearlyaugmented the susceptibility of NTCP-complemented cells (HepG2-NTCP cells) to HBV infection, although the expression of NTCP wasnot affected (SI Appendix, Fig. S1B). We then examined the role ofEGF-related host factor(s) in HBV infection. Contribution of EGFRon HBV infection was evaluated by using siRNA knockdown. Asshown in Fig. 1A, siRNA-mediated knockdown of endogenousEGFR drastically reduced HBV infection to HepG2-NTCP cells(Fig. 1 A, a). A similar observation was obtained in differentiatedHepaRG (dHepaRG) cells and primary human hepatocytes (PHHs)

Significance

For achieving efficient entry into host cells, viruses utilizemultiple host factors for mediating the stepwise entry process.Sole expression of sodium taurocholate cotransporting poly-peptide (NTCP), a cellular receptor for hepatitis B virus (HBV), isnot sufficient for the efficient viral internalization into hepa-tocytes. Here, we identify epidermal growth factor receptor(EGFR) as a host factor that interacts with NTCP and mediatesHBV internalization. Dissociation of the NTCP–EGFR interactionor functional depletion of EGFR attenuated the viral in-ternalization to NTCP-expressing cells and infection. Our datasuggest that HBV enters human hepatocytes using an intrinsicNTCP–EGFR complex as a driving force. EGFR is thus an entrycofactor required for HBV infection of the human liver.

Author contributions: M.I. and K.W. designed research; M.I., W.S., R. Sugiyama, and K.W.performed research; M.O., A.R., J.-H.Y., S.-Y.P., C.S., and K.W. contributed new reagents/analytic tools; M.I., W.S., R. Sugiyama, K.I., S.N., R. Suzuki, H.A., N.O., M.M., S.I., Y.T., T.W.,and K.W. analyzed data; and M.I. and K.W. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1To whom correspondence should be addressed. Email: kwatashi@nih.go.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1811064116/-/DCSupplemental.

Published online April 5, 2019.

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(Fig. 1 A, b and c). Cell viability, NTCP transporter activity, andcell-surface NTCP expression were not reduced by EGFRknockdown (SI Appendix, Fig. S2). To exclude the possibility of ansiRNA off-target effect, we transduced EGFR cDNA resistant tosi-EGFR (EGFRre) by lentivirus vector and found that the HBVsusceptibility of si-EGFR–treated HepG2-NTCP cells was signif-icantly rescued by complementation with EGFRre (Fig. 1B, lane 2vs. lane 4), which reached no significant difference in HBV in-fection of the original cells (Fig. 1B, lane 1 vs. lane 4). These re-sults suggest that EGFR is critically involved in the process ofHBV infection. To address the specific role of the EGFR in viralinfection, we examined the contribution of EGFR to the infectionby other two viruses, hepatitis D and E viruses (HDV and HEV,respectively). As shown in Fig. 1C, knockdown of EGFR signifi-cantly reduced the infection of HDV, but not HEV (Fig. 1C).

EGFR Knockdown Attenuates Virus Internalization. We next in-vestigated which step in the HBV life cycle was affected by de-pletion of EGFR. HBV attaches to hepatocytes and subsequentlyinternalizes into cells through endocytosis, which eventually leads tothe formation of cccDNA in the nucleus; cccDNA produces HBVRNAs and drives HBV replication to produce HBV particles (Fig.2A) (4). During the life cycle, the activity of HBV replication wasnot affected by depletion of EGFR (Fig. 2B), as demonstrated usingHepG2.2.15.7 cells (which autonomously replicate HBV but do notsupport HBV attachment and internalization) (11, 12). While viralattachment was not decreased in EGFR-knockdown cells (Fig. 2C),the level of HBV DNA internalized into cells was significantly re-duced by knockdown of EGFR (Fig. 2D). These data are consistentwith the results that EGFR depletion blocked the infection by bothHBV and HDV (Fig. 1C), two viruses that share the same mech-anisms during the viral internalization process. The amino-terminalpreS1 region of the LHBs, especially amino acids 2 to 48, is essentialfor HBV entry through NTCP attachment, and a peptideconsisting of this region labeled with fluorescence is fre-quently used for the analysis of HBV/HDV entry (5, 13, 14).This tetramethylrhodamine-labeled myristoylated preS1 (aminoacids 2 to 48) peptide (preS1-probe) still bound to the cells under

depletion of EGFR, in contrast to its complete loss of binding tothe NTCP-knockdown cells (Fig. 2E), in agreement with the resultof the attachment assay (Fig. 2C). To examine the process afterattachment, we observed incorporation of preS1-probe using high-magnification confocal microscopy, as previously reported (15).After preS1-probe attachment to the cell surface at 4 °C andwashing out free probe (indicated as 0 h in Fig. 2F), the cells weretransferred to 37 °C to allow preS1-probe incorporation up to12 h. In HepG2-NTCP cells transfected with nonrelevant siRNA(si-control), preS1-probe was incorporated inside the cells, withthe formation of speckled patterns increasing with incubation time(Fig. 2 F, Upper, a–d) as reported (8, 15). In contrast, EGFR-knockdown cells showed a much lower frequency of preS1 in-corporation, with retaining the plasma membrane localization atleast up to 12 h (Fig. 2 F, Upper, e–h). The quantified number ofcells showing preS1 incorporation was significantly decreasedupon EGFR depletion (Fig. 2 F, Lower). These results clearlysuggest that EGFR plays a significant role in HBV internalization.

EGFR Relocalization Triggers the preS1–NTCP Internalization. EGFRis a receptor tyrosine kinase functionally regulated by its phos-phorylation that is induced by ligands such as EGF (16). EGFstimulation induces EGFR activation through its dimerizationand autophosphorylation, triggering downstream signaling suchas the mitogen-activated protein kinase and phosphatidylinositol3-kinase (PI3K) pathways and inducing the endocytosis of EGFRitself (17). In preS1-attached cells, EGF also triggered EGFRrelocalization with speckled patterns within as early as 30 minafter stimulation (Fig. 3 A, Left, c vs. g). Interestingly, EGFstimulation also induced a rapid relocalization of preS1-probeand NTCP to the speckled distribution in parallel with EGFRendocytosis such that preS1-probe, NTCP, and EGFR werecolocalized (Fig. 3 A, Left, h, white arrows and Right). A coimmu-noprecipitation assay showed that NTCP overproduced in 293T cellswas copurified with EGFR (Fig. 3 B, Left, a). Coprecipitation ofendogenous NTCP with EGFR was also observed in HepaRGcells (Fig. 3 B, Center, a). Recombinant NTCP (18) also copre-cipitated with recombinant EGFR in vitro (Fig. 3 B, Right, a).

Fig. 1. Critical role of EGFR in supporting the in-fection by HBV and HDV. (A, a–c) HepG2-NTCP cells(a), dHepaRG cells (b), and PHHs (c) transfected withsiRNA against NTCP (si-NTCP), siRNA against EGFR (si-EGFR), or nonrelevant siRNA (si-control) were usedfor HBV infection assay and examined by detectingextracellular HBs (Upper Left graphs in a–c), in-tracellular HBc (Center images in a and b), cccDNA(Upper Right graph in a), and HBV DNAs (LowerRight in a). Protein production of EGFR, NTCP, andactin are also shown (Lower Left in a–c). (B) HepG2-NTCP cells that were transduced with a lentiviralvector expressing siRNA-resistant EGFR (EGFRre) orGFP as a control and then transfected with si-controlor si-EGFR were evaluated for HBV infection bydetecting extracellular HBs (Upper Left graph) andintracellular HBc (Right images). EGFR and actinproteins are also shown (Lower Left). (C, a and b)HepG2-NTCP cells (prepared as in A, a) (a) or PLC/PRF/5 cells treated with the indicated agents (b) weresubjected to the infection assay for HDV (a) or HEV(b). An anti-HEVLP antibody was used for a positivecontrol for HEV entry inhibition. Statistical signifi-cance was determined using Student’s t test, **P <0.01. N.S., not significant.

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Wang et al. (19) reported that NTCP colocalized and comigratedwith EGFR during EGF-induced trafficking in the absence ofHBV, consistent with our data. These results raise the possibilitythat HBV utilizes the intrinsic EGFR endocytosis pathway forviral internalization, which should be further analyzed in the

future. The above data suggest that EGFR relocalization triggersthe preS1–NTCP internalization.

Functional Interaction with EGFR Is Required for NTCP Receptor Function.Based on the above results, we hypothesized that EGFR mediates

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Fig. 2. EGFR is involved in HBV internalization. (A) HBV life cycle. HBV attaches to the cell surface through NTCP (attachment), followed by internalization inside cells(internalization) and translocation into the nucleus to form cccDNA. cccDNA drives HBV replication by transcription of HBV RNAs, nucleocapsid formation, and secretionof HBV particles (replication). Evaluation of the activity of each step is shown in C and E (attachment), D and F (internalization), and B (replication). (B) HBV replicationwas evaluated by quantifying extracellular HBV DNA from HepG2.2.15.7 cells transfected with the indicated siRNA at 9 d posttransfection. Entecavir was used as apositive control to suppress HBV replication. (C) For evaluating viral attachment, HepG2-NTCP cells transfected with siRNAs (as indicated in Fig. 1 A, a) were exposed toHBV at 4 °C to allow viral attachment to the cell surface. After washing, cell-surface HBV DNA was quantified. (D) For quantifying the internalized HBV, HBV-attachedcells (prepared as in C) were transferred to 37 °C to allow viral internalization into the cells and to quantify intracellular HBV DNA. (E) HepG2-NTCP cells transfected withsiRNAs (as in Fig. 1 A, a) were exposed to preS1-probe (Right images). The fluorescence intensities are shown in the graph (Left). (F) siRNA-transfected HepG2-NTCP cellswere attached to preS1-probe at 4 °C for 1 h, washed of free preS1-probe, and then transferred to 37 °C to allow incorporation of preS1-probe up to 12 h. Cells wereobserved using high-magnification confocal microscopy for preS1-probe internalization (red) after 0, 4, 8, and 12 h of incubation at 37 °C (Upper). White arrows show theinternalized preS1 speckles (b–d). Percentages of cells exhibiting preS1 internalization are indicated in the graph (Lower) (see Materials and Methods).

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HBV–NTCP internalization after HBV attachment to the cellsurface. To address whether EGFR is essential for HBV inter-nalization, we performed loss-of-function analyses using threedifferent approaches: mutation, competition, and pharmacologicalinhibition.First, we determined the region of NTCP responsible for the

interaction with EGFR (Materials and Methods and SI Appendix,Fig. S3A). By screening the NTCP fragment peptides consistingof 20 aa, we found that the amino acid 131 to 150 region [NTCP(131–150)] showed the highest binding signal to EGFR (SI Ap-pendix, Fig. S3 A and B). Further mutagenesis analysis revealedthat upon replacement of Gly with Ala at amino acids 144 and 148of NTCP (NTCPG144,148A), NTCP lost the ability to bind to EGFR(Fig. 4 A, a). Notably, expression of NTCPG144,148A rendered thelevel of preS1 binding (Fig. 4B) and bile acid transport to cells (SIAppendix, Fig. S4) equivalent to that of NTCPWT. However, preS1internalization into NTCPG144,148A-expressing cells was less ob-served than that in NTCPWT-producing cells (Fig. 4 C, b–d vs. f–h).In both of these cells, EGF stimulation induced the endocytosis ofEGFR at 30 min after stimulation (Fig. 4 D, a and e), but theinternalization of preS1 and NTCP was significantly attenuated inNTCPG144,148A-expressing cells (Fig. 4 D, f and g), in contrast tothe colocalization of EGFR, NTCP, and preS1 in speckles inNTCPWT-expressing cells (Fig. 4 D, d, white arrows). This loss ofEGFR–NTCP interaction, which dissociated preS1–NTCPlocalization from EGFR, resulted in the reduction in the in-fection by both HBV and HDV (Fig. 4 E). This result wassupported by a competition analysis using NTCP (131–150).As shown in SI Appendix, Fig. S3C, excess amounts of NTCP(131–150) peptide interfered with the NTCP–EGFR copre-cipitation (SI Appendix, Fig. S3 C, a). Introduction of this NTCP(131–150) as a decoy peptide similarly attenuated the EGF-induced

internalization of preS1–NTCP (SI Appendix, Fig. S5 B, j and k),without affecting the preS1–cell binding itself (SI Appendix, Fig. S5A).This decoy peptide thereby remarkably reduced the HBV infection(Fig. 4F). Moreover, pharmacological inactivation of EGFR bytreatment with a known inhibitor, gefitinib (20), also attenuated thepreS1 internalization (SI Appendix, Fig. S6 A, g) and thus reducedHBV infection in both dHepaRG cells and PHHs (Fig. 4G). Basedon the above data, we concluded that the functional interaction withEGFR is required for NTCP’s ability to support viral infection.

DiscussionEGFR shuttles between the cell-surface plasma membrane and theendosomes through membrane trafficking machinery (17). In thisstudy, the HBV preS1 ligand that was bound to the HBV receptorNTCP at the cell surface was shown to be internalized along withEGFR. We therefore propose a model for internalization of HBVas shown in Fig. 4 H, a, in which HBV interacts with NTCP at thecell surface to be recruited to NTCP–EGFR complex that dynam-ically translocates between the plasma membrane and intracellularvesicles. By taking advantage of the interaction with EGFR, HBV isinternalized into intracellular vesicles, thereby acquiring the op-portunity for subsequent acidification-dependent membrane fusionleading to the release of the nucleocapsid into the cytoplasm (Fig. 4H, a). When NTCP is prevented from interacting with EGFR, ei-ther by introducing mutations in NTCP (Fig. 4 H, b), bymasking the binding interface with a decoy peptide (Fig. 4 H, c),or by inactivation of EGFR (Fig. 4 H, d), cells no longer sup-port HBV entry. Thus, NTCP’s viral receptor function appearsdependent on a functional interaction with EGFR. Activated EGFRtriggers the downstream signaling, including Ras and PI3K, andinduces the endocytosis of EGFR itself (17). It is of particularimportance to understand whether the downstream signaling or

Fig. 3. EGFR relocalization triggers the preS1–NTCPinternalization. (A) HepG2-NTCP or HepG2 cells at-tached with preS1-probe at 4 °C were stimulatedwith EGF or left unstimulated (no stimulation) at37 °C for 30 min and observed by confocal micros-copy (preS1-probe, red; NTCP, green; EGFR, purple;nucleus, blue). White arrows indicate the colocali-zation of preS1, NTCP, and EGFR (Left, d and h). Thepercentages of cells showing the internalization forpreS1, NTCP, and EGFR are indicated in the graph(Right). **P < 0.01; *P < 0.05. N.S., not significant. (B,Left) 293T cells overexpressing EGFR together withor without NTCP were harvested to immunoprecipitatewith anti-EGFR antibody (IP: EGFR) or normal mouseIgG as a negative control (IP: IgG) and to detect NTCPor EGFR in the precipitates. NTCP, EGFR, and actin inthe total cell lysate were also detected (input). (B,Center) HepG2, dHepaRG, and Huh7 cells were sub-jected to coimmunoprecipitation assay for endoge-nous protein interaction. (B, Right) RecombinantNTCP (rNTCP) was incubated with or without recom-binant EGFR (rEGRF) in vitro, which was subjected tocoimmunoprecipitation analysis as shown above.

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Fig. 4. Functional interaction with EGFR is required for NTCP’s ability to mediate viral infection. (A) 293T cells overproducing EGFR and either NTCPWT,NTCPG97,101A, or NTCPG144,148A were lysed and immunoprecipitated with an anti-EGFR antibody (IP: EGFR) or were recovered without immunoprecipitation(input). NTCP, EGFR, and actin were detected. (B and C) Huh7 (B) or HepG2 (C) cells overexpressing either NTCPWT or NTCPG144,148A were examined for preS1attachment (B) as well as preS1 internalization at the indicated time points (C) as in Fig. 2 E and F. The white arrows show internalized preS1 vesicles in C. (D)EGFR (purple), NTCP (green), preS1-probe (red), and the nucleus (blue) were detected in HepG2-NTCPWT, HepG2-NTCPG144,148A, and HepG2 cells after 30 minof EGF stimulation. White arrows indicate the colocalization of preS1, NTCP, and EGFR (Left, d). The percentages of cells showing the internalization of preS1,NTCP, and EGFR are indicated on the graph (Right). (E, a and b) HepG2-NTCPWT and HepG2-NTCPG144,148A cells (a) as well as Huh7-NTCPWT and Huh7-NTCPG144,148A cells (b) were used for the infection assays with HBV and HDV, respectively, as in Fig. 1. (F and G) HBV infection assay using HepG2-NTCP (F)and dHepaRG cells and PHH (G) upon treatment with or without the indicated compounds [NTCP (11–30) or NTCP (131–150) in F; gefitinib in G]. HBs levels(F and G) and cccDNA levels (F) were detected to evaluate HBV infection. (H, a–d) Proposed model for HBV–NTCP–EGFR internalization. HBV attaches to NTCPon the cell surface and is recruited to the NTCP–EGFR complex, which dynamically translocates from the cell surface to the intramembrane vesicles for viralinternalization (a). Dissociation of the NTCP–EGFR interaction either by introducing point mutations in NTCP (b), by binding competition with a decoy peptide(c), or by functional inactivation of EGFR (d), deprives NTCP of the ability to support HBV infection. Thus, EGFR plays a critical role in mediating the entryprocess after HBV–NTCP attachment. **P < 0.01; *P < 0.05. N.S., not significant.

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the EGFR endocytosis machinery is essential for mediating HBVinternalization.The NTCP–EGFR interaction likely occurs irrespective of the

presence of HBV (Fig. 3B) (19). What is the original biologicalsignificance of the NTCP–EGFR interaction for uninfectedcells? Transporter activity and cell-surface expression of NTCPwere not affected by the knockdown of EGFR in the experimentsin SI Appendix, Fig. S2 B and C. NTCP plays an important rolefor constituting the enterohepatic circulation of bile acidsthrough transporting bile acids into hepatocytes (21), a phe-nomenon that should be tightly and rapidly regulated dependingon the concentration of bile acids. It is possible that EGFRcontributes to the rapid regulation of cell-surface NTCP levels byresponding to the change in serum bile acid levels. It has beenreported that the exposure of bile acids at high concentrationinduced the phosphorylation of EGFR and its relocalizationfrom the cell surface (22, 23). Given that the phosphorylation ofEGFR induces its endocytosis as rapidly as within 5 min (24), theNTCP–EGFR association may help rapid down-regulation ofcell-surface NTCP by sensing high concentrations of bile acidsand preventing excess bile acid transport to cells and subsequentcytotoxicity. Further analysis should shed light on a novel regu-lation system for bile acid homeostasis that involves EGFR.In this study, we showed that a decoy peptide [NTCP (131–150)]

and gefitinib clearly inhibited HBV infection (Fig. 4 F and G). It issuggested that the functional interaction of EGFR and NTCP

represents a target for the development of anti-HBV agents (SIAppendix, Fig. S7). Precise analysis of the mode of the NTCP–EGFR interaction should be further analyzed in the future. Be-cause EGFR has been the target of anticancer agents (25), it wouldbe of interest to analyze the effect of such drugs on HBV/HDVinfection. Thus, our findings have a significant impact not only forunderstanding the molecular basis for HBV/HDV infection, butalso for the development of antiviral drugs.

Materials and MethodsHBV used in this study was derived from Hep38.7-Tet cells (genotype D) (6,11). PHHs, dHepaRG, and HepG2-NTCP were infected with HBV as previouslydescribed (6). HBV infection was evaluated by detecting HBV surface protein(HBs), HBV core protein (HBc), cccDNA, and/or HBV DNA (6, 11).

Additional experimental procedures are described in SI Appendix, Mate-rials and Methods.

ACKNOWLEDGMENTS. Plasmids for the production of lentivirus were kindlyprovided by Dr. Hiroyuki Miyoshi, RIKEN. This study was supported by theJapan Society for the Promotion of Science KAKENHI (Grants JP17H04085,JP66KT0111, and JP16K19145); the JST CREST program; JST MIRAI pro-gram; the Japan Agency for Medical Research and Development, AMED(Grants JP18fk0310114j0002, JP18fk0310101j1002, JP18fk0310103j0202,JP18fm0208019j0002, JP18fk0210036j0001, JP17fk0310103j0001, andJP18fm0208019h0202); Takeda Science Foundation; PharmacologicalResearch Foundation, Tokyo; and The Japan Food Chemical ResearchFoundation.

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