The machinery for endocytosis of epidermal growth factor receptor coordinates the transport of incoming hepatitis B virus to the endosomal network.

Sodium taurocholate cotransporting polypeptide (NTCP) is expressed at the surface of human hepatocytes and functions as an entry receptor of hepatitis B virus (HBV). Recently, we have reported that epidermal growth factor receptor (EGFR) is involved in NTCP-mediated viral internalization during the cell entry process. Here, we analyzed which function of EGFR is essential for mediating HBV internalization. In contrast to the reported crucial function of EGFR-downstream signaling for the entry of hepatitis C virus (HCV), blockade of EGFR-downstream signaling proteins, including mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and signal transducer and activator of transcription (STAT), had no or only minor effects on HBV infection. Instead, deficiency of EGFR endocytosis resulting from either a deleterious mutation in EGFR or genetic knockdown of endocytosis adaptor molecules abrogated internalization of HBV via NTCP and prevented viral infection. EGFR activation triggered a time-dependent relocalization of HBV preS1 to the early and late endosomes and to lysosomes in concert with EGFR transport. Suppression of EGFR ubiquitination by site-directed mutagenesis or by knocking down two EGFR-sorting molecules, signal-transducing adaptor molecule (STAM) and lysosomal protein transmembrane 4ß (LAPTM4B), suggested that EGFR transport to the late endosome is critical for efficient HBV infection. Cumulatively, these results support the idea that the EGFR endocytosis/sorting machinery drives the translocation of NTCP-bound HBV from the cell surface to the endosomal network, which eventually enables productive viral infection.

Use of Indocyanine Green (ICG), a Medical Near Infrared Dye, for Enhanced Fluorescent Imaging-Comparison of Organic Anion Transporting Polypeptide 1B3 (OATP1B3) and Sodium-Taurocholate Cotransporting Polypeptide (NTCP) Reporter Genes.

Molecular and cellular imaging in living organisms have ushered in an era of comprehensive understanding of intracellular and intercellular events. Currently, more efforts have been focused on the infrared fluorescent dyes that facilitate deeper tissue visualization. Both sodium taurocholate cotransporting polypeptide (NTCP) and organic-anion-transporting polypeptide 1B3 (OATP1B3) are capable of carrying indocyanine green (ICG) into the cytoplasm. We compared the feasibility of NTCP and OATP1B3 as reporter genes in combination with ICG. NTCP and OATP1B3 were transduced into HT-29 cells. Genetically modified HT-29 cells were inoculated into nude mice. ICG was administered in vitro and in vivo and the signals were observed under confocal microscopy, flow cytometry, multimode microplate reader, and an in vivo imaging system. Both NTCP- and OATP1B3-expressing cells and xenografts had higher ICG intensities. The OATP1B3-expressing xenograft has a higher ICG uptake than the NTCP-expressing xenograft. NTCP or OATP1B3 combined with ICG could serve as a noninvasive imaging modality for molecular and cellular imaging. OATP1B3 outperforms NTCP in terms of in vivo imaging.

Interaction of human organic anion transporter 2 (OAT2) and sodium taurocholate cotransporting polypeptide (NTCP) with antineoplastic drugs.

The ability of an antineoplastic drug to exert its cytostatic effect depends largely on the balance between its uptake into and extrusion from the cancer cells. ATP driven efflux transporter proteins drive the export of antineoplastic drugs and play a pivotal role in the development of chemoresistance. As regards uptake transporters, comparably less is known on their impact in drug action. In the current study, we characterized the interactions of two uptake transporter proteins, expressed mainly in the liver; the organic anion transporter 2 (OAT2, encoded by the SLC22A7 gene) and the sodium taurocholate cotransporting polypeptide (NTCP, encoded by the SLC10A1 gene), stably transfected in human embryonic kidney cells, with some antineoplastic agents that are routinely being used in cancer chemotherapy. Whereas NTCP did not show any strong interactions with the cytostatics tested, we observed a very strong inhibition of OAT2 mediated [(3)H] cGMP uptake in the presence of bendamustine, irinotecan and paclitaxel. The Ki values of OAT2 for bendamustine, irinotecan and paclitaxel were determined to be 43.3±4.33µM, 26.4±2.34µM and 10.4±0.45µM, respectively. Incubation of bendamustine with OAT2 expressing cells increased the caspase-3 activity, and this increase was inhibited by simultaneous incubation with bendamustine and probenecid, a well-known inhibitor of OATs, suggesting that bendamustine is a substrate of OAT2. A higher accumulation of irinotecan was observed in OAT2 expressing cells compared to control pcDNA cells by HPLC analysis of cell lysates. The accumulation was diminished in the presence of cGMP, the substrate we used to functionally characterize OAT2, suggesting specificity of this uptake and the fact that OAT2 mediates uptake of irinotecan.

Design and synthesis of a novel candidate compound NTI-007 targeting sodium taurocholate cotransporting polypeptide [NTCP]-APOA1-HBx-Beclin1-mediated autophagic pathway in HBV therapy.

Sodium taurocholate cotransporting polypeptide (NTCP) is a multiple transmembrane transporter predominantly expressed in the liver, functioning as a functional receptor for HBV. Through our continuous efforts to identify NTCP as a novel HBV target, we designed and synthesized a series of new compounds based on the structure of our previous compound NT-5. Molecular docking and MD simulation validated that a new compound named NTI-007 can tightly bind to NTCP, whose efficacy was also measured in vitro virological examination and cytotoxicity studies. Furthermore, autophagy was observed in NTI-007 incubated HepG2.2.15 cells, and results of q-PCR and Western blotting revealed that NTI-007 induced autophagy through NTCP-APOA1-HBx-Beclin1-mediated pathway. Taken together, considering crucial role of NTCP in HBV infection, NTCP-mediated autophagic pathway may provide a promising strategy of HBV therapy and given efficacy of NTI-007 triggering autophagy. Our study suggests pre-clinical potential of this compound as a novel anti-HBV drug candidate.

Establishment and characterization of a new cell culture system for hepatitis B virus replication and infection.

Hepatitis B virus (HBV) is a primary cause of chronic liver diseases in humans. HBV infection exhibits strict host and tissue tropism. HBV core promoter (Cp) drives transcription of pregenomic RNA (pgRNA) and plays a key role in the viral life cycle. Hepatocyte nuclear factor 4α (HNF4α) acts as a major transcriptional factor that stimulates Cp. In this work, we reported that BEL7404 â€‹cell line displayed a high efficiency of DNA transfection and high levels of HBV antigen expression after transfection of HBV replicons without prominent viral replication. The introduction of exogenous HNF4α and human sodium taurocholate cotransporting polypeptide (hNTCP) expression into BEL7404 made it permissive for HBV replication and susceptible to HBV infection. BEL7404-derived cell lines with induced HBV permissiveness and susceptibility were constructed by stable co-transfection of hNTCP and Tet-inducible HNF4α followed by limiting dilution cloning. HBV replication in such cells was sensitive to inhibition by nucleotide analog tenofovir, while the infection was inhibited by HBV entry inhibitors. This cell culture system provides a new and additional tool for the study of HBV replication and infection as well as the characterization of antiviral agents.

Binding of Hepatitis B Virus Pre-S1 Domain-Derived Synthetic Myristoylated Peptide to Scavenger Receptor Class B Type 1 with Differential Properties from Sodium Taurocholate Cotransporting Polypeptide.

(1) Background: The myristoylated pre-S1 peptide (Myr47) synthesized to mimic pre-S1 domain (2-48) in large (L) surface protein of hepatitis B virus (HBV) prevents HBV infection to hepatocytes by binding to sodium taurocholate cotransporting polypeptide (NTCP). We previously demonstrated that yeast-derived nanoparticles containing L protein (bio-nanocapsules: BNCs) bind scavenger receptor class B type 1 (SR-B1). In this study, we examined the binding of Mry47 to SR-B1. (2) Methods: The binding and endocytosis of fluorescence-labeled Myr47 to SR-B1 (and its mutants)-green fluorescence protein (GFP) fusion proteins expressed in HEK293T cells were analyzed using flow cytometry and laser scanning microscopy (LSM). Various ligand-binding properties were compared between SR-B1-GFP and NTCP-GFP. Furthermore, the binding of biotinylated Myr47 to SR-B1-GFP expressed on HEK293T cells was analyzed via pull-down assays using a crosslinker and streptavidin-conjugated beads. (3) Conclusions: SR-B1 bound not only Myr47 but also its myristoylated analog and BNCs, but failed to bind a peptide without myristoylation. However, NTCP only bound Myr47 among the ligands tested. Studies using SR-B1 mutants suggested that both BNCs and Myr47 bind to similar sites of SR-B1. Crosslinking studies indicated that Myr47 binds preferentially SR-B1 multimer than monomer in both HEK293T and HepG2 cells.

Clinical features of sodium-taurocholate cotransporting polypeptide deficiency in pediatric patients: case series and literature review.

Sodium-taurocholate cotransporting polypeptide (NTCP) deficiency is a newly reported hereditary bile acid metabolic disease. Here we describe the clinical characteristics of 12 cases of pediatric NTCP deficiency, as well as review 60 previously reported cases in the literature in order to provide better guidance for pediatricians. The clinical records, laboratory and imaging data were collected of 12 cases who were treated at the pediatric infectious disease department of the West China Second University Hospital of Sichuan University, China, from December 2018 to July 2020. PubMed and Wanfang databases were searched and 11 studies including 60 pediatric NTCP deficiency patients from January 2015 to November 2020 were retrieved. In our center, there were 4 girls and 8 boys, with a median age at admission of 9.9 months (range, 2.2 to 70 months). Six patients (50%) had prolonged neonatal jaundice. All of the patients (12/12; 100%) had normal growth and development. The reason for the first visit was prolonged neonatal jaundice (4/12, 33.3%), non-liver related diseases (6/12, 50%) and routine checkup (2/12, 16.7%). Hypercholanemia was documented in 12/12 (100%), elevated aspartate aminotransferase (AST) in 6/12 (50%), and elevated alanine aminotransferase (ALT) in 1/12 (8.3%). All of the patients (12/12; 100%) had homozygous mutations of c.800C>T in SLC10A1. Sixty patients (22 girls and 38 boys) were included in the literature review; 36 (60%) had hyperbilirubinemia after 1 month. The reasons for testing for hypercholanemia were identified in 47/60 cases, and included prolonged neonatal jaundice and neonatal transient cholestasis in 26 (26/47, 55.3%); non-liver related diseases in 14 (14/47, 29.8%); routine medical examination in 3 (3/14, 6.4%); volunteer recruitment in 1 (1/14, 7.1%); dark urine in 1 (1/47, 2.1%). Hypercholanemia was confirmed in 60/60 (100%); 31 (51.7%) had elevated AST, and 10 (16.7%) had elevated ALT. Among 59 Chinese patients, 52 (88.1%) had homozygous mutations of c.800C>T in SLC10A1. The most common symptom of pediatric NTCP deficiency is jaundice. NTCP deficiency can also be detected during routine check-ups. The common biochemical features are hypercholanemia and elevated AST. Screening for c.800C>T mutation in SLC10A1 is useful for primary genetic screening in Chinese infants with persistent hypercholanemia after infectious, structural, and immunological factors are excluded.

HBV Pre-S1-Derived Myristoylated Peptide (Myr47): Identification of the Inhibitory Activity on the Cellular Uptake of Lipid Nanoparticles.

The Myr47 lipopeptide, consisting of hepatitis B virus (HBV) pre-S1 domain (myristoylated 2-48 peptide), is an effective commercialized anti-HBV drug that prevents the interaction of HBV with sodium taurocholate cotransporting polypeptide (NTCP) on human hepatocytes, an activity which requires both N-myristoylation residue and specific amino acid sequences. We recently reported that Myr47 reduces the cellular uptake of HBV surface antigen (HBsAg, subviral particle of HBV) in the absence of NTCP expression. In this study, we analyzed how Myr47 reduces the cellular uptake of lipid nanoparticles (including liposomes (LPs) and HBsAg) without NTCP expression. By using Myr47 mutants lacking the HBV infection inhibitory activity, they could reduce the cellular uptake of LPs in an N-myristoylation-dependent manner and an amino acid sequence-independent manner, not only in human liver-derived cells but also in human non-liver-derived cells. Moreover, Myr47 and its mutants could reduce the interaction of LPs with apolipoprotein E3 (ApoE3) in an N-myristoylation-dependent manner regardless of their amino acid sequences. From these results, lipopeptides are generally anchored by inserting their myristoyl residue into the lipid bilayer and can inhibit the interaction of LPs/HBsAg with apolipoprotein, thereby reducing the cellular uptake of LPs/HBsAg. Similarly, Myr47 would interact with HBV, inhibiting the uptake of HBV into human hepatic cells, while the inhibitory effect of Myr47 may be secondary to its ability to protect against HBV infection.

Role of Sodium Taurocholate Cotransporting Polypeptide as a New Reporter and Drug-Screening Platform: Implications for Preventing Hepatitis B Virus Infections.

PURPOSE: Sodium taurocholate cotransporting polypeptide (NTCP) is a transmembrane protein responsible for delivering indocyanine green (ICG), an ideal infrared fluorescent dye, from extracellular space into the cytoplasm. Additionally, NTCP located in the hepatocyte membrane is the portal for hepatitis B and D virus (HBV/HDV) infections. This study verified the feasibility of NTCP as a reporter and further established a drug-screening platform for HBV/HDV infections. PROCEDURES: NTCP was transduced into HT-29, a colorectal cancer cell line. To examine the use of NTCP as a reporter, NTCP-expressing cells were treated with ICG and examined through flow cytometry, an in vivo imaging system (IVIS), and confocal microscopy. Furthermore, ICG was administrated to NTCP-expressing tumor-bearing nude mice and examined using the IVIS. To study the drug-screening platform, NTCP-expressing cells were treated with cyclosporin A, an NTCP inhibitor, and ICG, and examined using a multimode detection platform. Moreover, nude mice were injected with NTCP inhibitors and ICG, and subsequently, their ICG signal was examined in vivo and in the blood. RESULTS: In the reporter study, the ICG signal was higher in NTCP-expressing cells/tumors than in control cells/tumors after ICG treatment. In the drug-screening platform study, NTCP-expressing cells had decreased ICG intensity after treatment with NTCP inhibitors and ICG. Nude mice that were administered cyclosporin A had lower ICG intensity in the liver and higher intensity in the peripheral tissue and blood. CONCLUSIONS: NTCP and ICG form an ideal reporter system with extensive applications in cancer biology, robust drug-drug interactions, and drug screening in HBV/HDV infections.

Down-regulation of cell membrane localized NTCP expression in proliferating hepatocytes prevents hepatitis B virus infection.

Hepatocyte proliferation could result in the loss of covalently closed circular DNA (cccDNA) and the emergence of cccDNA-cleared nascent hepatocytes, which appear refractory to hepatitis B virus (HBV) reinfection with unknown mechanism(s). Sodium taurocholate cotransporting polypeptide (NTCP) is the functional receptor for HBV entry. In this study, down-regulation of cell membrane localized NTCP expression in proliferating hepatocytes was found to prevent HBV infection in HepG2-NTCP-tet cells and in liver-humanized mice. In patients, lower NTCP protein expression was correlated well with higher levels of hepatocyte proliferation and less HBsAg expression in HBV-related focal nodular hyperplasia (FNH) tissues. Clinically, significantly lower NTCP protein expression was correlated with more active hepatocyte proliferation in CHB patients with severe active necroinflammation and better antiviral treatment outcome. Mechanistically, the activation of cell cycle regulatory genes p53, S-phase kinase-associated protein 2 (SKP2) and cyclin D1 during cell proliferation, as well as proliferative and inflammatory cytokine Interleukin-6 (IL-6) could transcriptionally down-regulate NTCP expression. From these aspects, we conclude that within the milieu of hepatocyte proliferation, down-regulation of cell membrane localized NTCP expression level renders nascent hepatocytes resistant to HBV reinfection. This may accelerate virus clearance during immune-mediated cell death and compensatory proliferation of survival hepatocytes.

Quantitative NTCP pharmacophore and lack of association between DILI and NTCP Inhibition.

The human sodium taurocholate cotransporting polypeptide (NTCP) is a hepatic bile acid transporter. Inhibition of NTCP uptake may potentially also prevent hepatitis B virus (HBV) infection. The first objective was to develop a quantitative pharmacophore for NTCP inhibition. Recent studies showed that hepatotoxic drugs could inhibit bile acid uptake into hepatocytes, without inhibiting canalicular efflux, and cause bile acid elevation in plasma. Hence, a second objective was to examine whether NTCP inhibition is associated with drug induced liver injury (DILI). Twenty-seven drugs from our previous study were used as the training set to develop a quantitative pharmacophore. From secondary screening from a drug database, six retrieved drugs and three drugs not retrieved by the model were tested for NTCP inhibition. Tertiary screening involved drugs known to cause DILI and not cause DILI. Overall, ninety-four drugs were assessed for hepatotoxicity and were assessed relative to NTCP inhibition. The quantitative pharmacophore possessed one hydrogen bond acceptor, one hydrogen bond donor, a hydrophobic feature, and excluded volumes. From 94 drugs, NTCP inhibitors and non-inhibitors were approximately equally distributed across the drugs of most DILI concern, less DILI concern, and no DILI concern, indicating no relationship between NTCP inhibition and DILI risk. Hence, an approach to treat HBV via NTCP inhibition is not expected to be associated with DILI.

NTCP opens the door for hepatitis B virus infection.

A liver bile acids transporter, sodium taurocholate cotransporting polypeptide (NTCP, encoded by SLC10A1) was recently identified as a functional receptor for hepatitis B virus (HBV) and its satellite hepatitis D virus (HDV). NTCP-complemented human hepatoma HepG2 cells (HepG2-NTCP) were shown to support infection of HBV and HDV in vitro, providing a much-needed and convenient cell culture system for the viruses. Identification of NTCP as a functional receptor for HBV has significantly advanced our understanding of the viral life cycle and opened new opportunities for developing anti-HBV interventions. This article forms part of a symposium in Antiviral Research on "An unfinished story: from the discovery of the Australia antigen to the development of new curative therapies for hepatitis B".

microRNA expression in hepatitis B virus infected primary treeshrew hepatocytes and the independence of intracellular miR-122 level for de novo HBV infection in culture.

Infection of Hepatitis B virus (HBV) in hepatocytes has been known to be controlled by multiple cellular factors, while the relationship of the infection and liver microRNAs remains obscure. In this study, a miRNA database, containing 168 unique mature miRNA members from primary hepatocytes of a primate-like animal, northern treeshrew (Tupaia belangeri) that is the only species susceptible for HBV infection other than human and chimpanzee, was established. The relative level of a liver predominant microRNA, miR-122, was markedly increased upon HBV infection of the primary tupaia hepatocyte (PTH). However, introducing neither miR-122 nor its antagonist anti-miR-122 into PTHs, or, HepG2-NTCP that is HepG2 cells with the newly identified receptor sodium taurocholate cotransporting polypeptide (NTCP) did not alter the viral infection on these cells. These data suggest that de novo HBV infection of cultured hepatocytes does not depend on the expression level of intracellular miR-122 of the target cells.