Hepatitis B virus (HBV) entry has been analyzed using infection-susceptible cells, including primary human hepatocytes, primary tupaia hepatocytes, and HepaRG cells. Recently, the sodium taurocholate cotransporting polypeptide (NTCP) membrane transporter was reported as an HBV entry receptor. In this study, we established a strain of HepG2 cells engineered to overexpress the human NTCP gene (HepG2-hNTCP-C4 cells). HepG2-hNTCP-C4 cells were shown to be susceptible to infection by blood-borne and cell culture-derived HBV. HBV infection was facilitated by pretreating cells with 3% dimethyl sulfoxide permitting nearly 50% of the cells to be infected with HBV. Knockdown analysis suggested that HBV infection of HepG2-hNTCP-C4 cells was mediated by NTCP. HBV infection was blocked by an anti-HBV surface protein neutralizing antibody, by compounds known to inhibit NTCP transporter activity, and by cyclosporin A and its derivatives. The infection assay suggested that cyclosporin B was a more potent inhibitor of HBV entry than was cyclosporin A. Further chemical screening identified oxysterols, oxidized derivatives of cholesterol, as inhibitors of HBV infection. Thus, the HepG2-hNTCP-C4 cell line established in this study is a useful tool for the identification of inhibitors of HBV infection as well as for the analysis of the molecular mechanisms of HBV infection.

Chronic hepatitis B virus (HBV) infection is a major public health problem worldwide. Although nucleos(t)ide analogs inhibiting viral reverse transcriptase are clinically available as anti-HBV agents, emergence of drug-resistant viruses highlights the need for new anti-HBV agents interfering with other targets. Here we report that cyclosporin A (CsA) can inhibit HBV entry into cultured hepatocytes. The anti-HBV effect of CsA was independent of binding to cyclophilin and calcineurin. Rather, blockade of HBV infection correlated with the ability to inhibit the transporter activity of sodium taurocholate cotransporting polypeptide (NTCP). We also found that HBV infection-susceptible cells, differentiated HepaRG cells and primary human hepatocytes expressed NTCP, while nonsusceptible cell lines did not. A series of compounds targeting NTCP could inhibit HBV infection. CsA inhibited the binding between NTCP and large envelope protein in vitro. Evaluation of CsA analogs identified a compound with higher anti-HBV potency, having a median inhibitory concentration <0.2 μM. Conclusion: This study provides a proof of concept for the novel strategy to identify anti-HBV agents by targeting the candidate HBV receptor, NTCP, using CsA as a structural platform. (Hepatology 2014;59:1726–1737)

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.