Evaluation and identification of hepatitis B virus entry inhibitors using HepG2 cells overexpressing a membrane transporter NTCP

Abstract

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.

Association of Genetic Variation of Sodium Taurocholate Cotransporting Polypeptide with Chronic Hepatitis B Virus Infection

“…Recent studies showed that cyclosporin A and its derivatives, neutralizing antibody against HBV surface protein, and compounds known to inhibit the NTCP transporter activity could inhibit HBV entry using NTCP through interfering with the binding between NTCP and the pre-S1 domain of the HBV large envelope protein (Iwamoto et al, 2014;Nkongolo et al, 2013;Watashi et al, 2013), thus, proposing a novel strategy to identify anti-HBV agents by targeting NTCP. Whether NTCP polymorphisms, including the rs2296651 polymorphism, are relevant to the action of anti-HBV agents targeting NTCP may be a focus of future investigation.…”

Characterization of the Anti-HCV Activities of the New Cyclophilin Inhibitor STG-175

“…For HIV-1, the current model is that CypI, by blocking HIV-1 capsid-CypA interactions, prevent the nuclear import of the viral genome and its subsequent integration into the host chromosomes. For HBV, the current model is that CypI exert two independent inhibitory actions: i) an entry block, by binding to NTCP and preventing the viral glycoprotein from interacting with this cell surface receptor [63][64]; and ii) a still unknown post-entry block, likely at a transcription or translation step since CypI such as ALV inhibits HBsAg production upon HBV transfection [65]. Further work is required to fully elucidate the antiviral MoA of CypI for these three viruses.…”

Current and future directions for treating hepatitis B virus infection

“…Iwamoto et al [120] , Watashi et al [121] and Tsukuda et al [122] reported that cyclosporine A and its analogs blocked HBV entry through inhibiting the interaction between NTCP and the HBV large surface protein. HBV entry inhibitors might also be useful for controlling HBV infection in the near future.…”