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. 2019 Apr 23;116(17):8487-8492.
doi: 10.1073/pnas.1811064116. Epub 2019 Apr 5.

Epidermal Growth Factor Receptor Is a Host-Entry Cofactor Triggering Hepatitis B Virus Internalization

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Free PMC article

Epidermal Growth Factor Receptor Is a Host-Entry Cofactor Triggering Hepatitis B Virus Internalization

Masashi Iwamoto et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Sodium taurocholate cotransporting polypeptide (NTCP) is a host cell receptor required for hepatitis B virus (HBV) entry. However, the susceptibility of NTCP-expressing cells to HBV is diverse depending on the culture condition. Stimulation with epidermal growth factor (EGF) was found to potentiate cell susceptibility to HBV infection. Here, we show that EGF receptor (EGFR) plays a critical role in HBV virion internalization. In EGFR-knockdown cells, HBV or its preS1-specific fluorescence peptide attached to the cell surface, but its internalization was attenuated. PreS1 internalization and HBV infection could be rescued by complementation with functional EGFR. Interestingly, the HBV/preS1-NTCP complex at the cell surface was internalized concomitant with the endocytotic relocalization of EGFR. Molecular interaction between NTCP and EGFR was documented by immunoprecipitation assay. Upon dissociation from functional EGFR, NTCP no longer functioned to support viral infection, as demonstrated by either (i) the introduction of NTCP point mutation that disrupted its interaction with EGFR, (ii) the detrimental effect of decoy peptide interrupting the NTCP-EGFR interaction, or (iii) the pharmacological inactivation of EGFR. Together, these data support the crucial role of EGFR in mediating HBV-NTCP internalization into susceptible cells. EGFR thus provides a yet unidentified missing link from the cell-surface HBV-NTCP attachment to the viral invasion beyond the host cell membrane.

Keywords: EGFR; HBV; NTCP; entry; transporter.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Critical role of EGFR in supporting the infection by HBV and HDV. (A, ac) HepG2-NTCP cells (a), dHepaRG cells (b), and PHHs (c) transfected with siRNA against NTCP (si-NTCP), siRNA against EGFR (si-EGFR), or nonrelevant siRNA (si-control) were used for HBV infection assay and examined by detecting extracellular HBs (Upper Left graphs in ac), intracellular HBc (Center images in a and b), cccDNA (Upper Right graph in a), and HBV DNAs (Lower Right in a). Protein production of EGFR, NTCP, and actin are also shown (Lower Left in ac). (B) HepG2-NTCP cells that were transduced with a lentiviral vector expressing siRNA-resistant EGFR (EGFRre) or GFP as a control and then transfected with si-control or si-EGFR were evaluated for HBV infection by detecting extracellular HBs (Upper Left graph) and intracellular HBc (Right images). EGFR and actin proteins 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) were subjected to the infection assay for HDV (a) or HEV (b). An anti-HEVLP antibody was used for a positive control for HEV entry inhibition. Statistical significance was determined using Student’s t test, **P < 0.01. N.S., not significant.
Fig. 2.
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 secretion of 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 replication was 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 a positive 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 to HBV 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-attached cells (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 with siRNAs (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 cells were 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 were observed 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 the internalized preS1 speckles (bd). Percentages of cells exhibiting preS1 internalization are indicated in the graph (Lower) (see Materials and Methods).
Fig. 3.
Fig. 3.
EGFR relocalization triggers the preS1–NTCP internalization. (A) HepG2-NTCP or HepG2 cells attached with preS1-probe at 4 °C were stimulated with EGF or left unstimulated (no stimulation) at 37 °C for 30 min and observed by confocal microscopy (preS1-probe, red; NTCP, green; EGFR, purple; nucleus, blue). White arrows indicate the colocalization of preS1, NTCP, and EGFR (Left, d and h). The percentages of cells showing the internalization for preS1, 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 with or without NTCP were harvested to immunoprecipitate with anti-EGFR antibody (IP: EGFR) or normal mouse IgG as a negative control (IP: IgG) and to detect NTCP or EGFR in the precipitates. NTCP, EGFR, and actin in the total cell lysate were also detected (input). (B, Center) HepG2, dHepaRG, and Huh7 cells were subjected to coimmunoprecipitation assay for endogenous protein interaction. (B, Right) Recombinant NTCP (rNTCP) was incubated with or without recombinant EGFR (rEGRF) in vitro, which was subjected to coimmunoprecipitation analysis as shown above.
Fig. 4.
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 preS1 attachment (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 min of 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, ad) Proposed model for HBV–NTCP–EGFR internalization. HBV attaches to NTCP on the cell surface and is recruited to the NTCP–EGFR complex, which dynamically translocates from the cell surface to the intramembrane vesicles for viral internalization (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 entry process after HBV–NTCP attachment. **P < 0.01; *P < 0.05. N.S., not significant.

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