The Na⁺/taurocholate co-transporting polypeptide (NTCP, gene symbol SLC10A1) is both a physiological bile acid transporter and the high-affinity hepatic receptor for the hepatitis B and D viruses (HBV/HDV). Virus entry via endocytosis of the virus/NTCP complex involves co-factors, but this process is not fully understood. As part of the innate immunity, interferon-induced transmembrane proteins (IFITM) 1–3 have been characterized as virus entry-restricting factors for many viruses. The present study identified IFITM3 as a novel protein–protein interaction (PPI) partner of NTCP based on membrane yeast-two hybrid and co-immunoprecipitation experiments. Surprisingly, IFITM3 knockdown significantly reduced in vitro HBV infection rates of NTCP-expressing HuH7 cells and primary human hepatocytes (PHHs). In addition, HuH7-NTCP cells showed significantly lower HDV infection rates, whereas infection with influenza A virus was increased. HBV-derived myr-preS1 peptide binding to HuH7-NTCP cells was intact even under IFITM3 knockdown, suggesting that IFITM3-mediated HBV/HDV infection enhancement occurs in a step subsequent to the viral attachment to NTCP. In conclusion, IFITM3 was identified as a novel NTCP co-factor that significantly affects in vitro infection with HBV and HDV in NTCP-expressing hepatoma cells and PHHs. While there is clear evidence for a direct PPI between IFITM3 and NTCP, the specific mechanism by which this PPI facilitates the infection process remains to be identified in future studies.

Na⁺/taurocholate cotransporting polypeptide (NTCP, gene symbol SLC10A1) is a hepatic bile acid uptake carrier participating in the enterohepatic circulation of bile acids. Apart from its transporter function, NTCP acts as the high-affinity liver-specific receptor for the hepatitis B virus (HBV), which attaches via its preS1-peptide domain of the large surface protein to NTCP, subsequently leading to endocytosis of the virus/NTCP-receptor complex. Although the process of NTCP-dependent HBV infection of hepatocytes has received much attention over the last decade, the precise molecular sites of the virus/NTCP interaction have not been fully identified. Inspection of the primary protein sequence of human NTCP revealed 139YIYSRGIY146 as a highly conserved tyrosine-rich motif. To study the role of Y139, Y141 and Y146 amino acids in NTCP biology, the aforementioned residues were substituted with alanine, phenylalanine or glutamate (mimicking phosphorylation) using site-directed mutagenesis. Similar to wt NTCP, the Y139A, Y141A, Y146A, Y141F, Y146F, and Y146E mutants were expressed at the plasma membrane of HEK293 cells and exhibited intact bile acid transport function. Y146A, Y146E, and Y146F demonstrated transport kinetics comparable to wild-type NTCP with K_m values of 57.3–112.4 µM and V_max values of 6683–7579 pmol/mg protein/min. Only Y141E was transport deficient, most likely due to an intracellular accumulation of the mutant protein. Most importantly, Y146A and Y146E mutation completely abrogated binding of the viral preS1-peptide to NTCP, while the Y146F mutant of NTCP showed some residual binding competence for preS1. Consequently, the NTCP mutants Y146A and Y146E, when expressed in HepG2 hepatoma cells, showed complete loss of susceptibility for in vitro HBV infection. In conclusion, tyrosine 146, and to some extent tyrosine 141, both belonging to the tyrosine-rich motif 139YIYSRGIY146 of human NTCP, are newly identified amino acid residues that play an essential role in the interaction of HBV with its receptor NTCP and, thus, in the process of virus entry into hepatocytes.

The hepatic bile acid transporter Na⁺/taurocholate co-transporting polypeptide (NTCP) was identified in 2012 as the high-affinity hepatic receptor for the hepatitis B and D viruses (HBV/HDV). Since then, this carrier has emerged as promising drug target for HBV/HDV virus entry inhibitors, but the synthetic peptide Hepcludex^® of high molecular weight is the only approved HDV entry inhibitor so far. The present study aimed to identify small molecules as novel NTCP inhibitors with anti-viral activity. A ligand-based bioinformatic approach was used to generate and validate appropriate pharmacophore and QSAR (quantitative structure–activity relationship) models. Half-maximal inhibitory concentrations (IC₅₀) for binding inhibition of the HBV/HDV-derived preS1 peptide (as surrogate parameter for virus binding to NTCP) were determined in NTCP-expressing HEK293 cells for 150 compounds of different chemical classes. IC₅₀ values ranged from 2 µM up to >1000 µM. The generated pharmacophore and QSAR models were used for virtual screening of drug-like chemicals from the ZINC¹⁵ database (~11 million compounds). The 20 best-performing compounds were then experimentally tested for preS1-peptide binding inhibition in NTCP-HEK293 cells. Among them, four compounds were active and revealed experimental IC₅₀ values for preS1-peptide binding inhibition of 9, 19, 20, and 35 µM, which were comparable to the QSAR-based predictions. All these compounds also significantly inhibited in vitro HDV infection of NTCP-HepG2 cells, without showing any cytotoxicity. The best-performing compound in all assays was ZINC000253533654. In conclusion, the present study demonstrates that virtual compound screening based on NTCP-specific pharmacophore and QSAR models can predict novel active hit compounds for the development of HBV/HDV entry inhibitors.

Chronic hepatitis B virus (HBV) infection is a global health threat. Mutations in the surface antigen of HBV (HBsAg) may alter its antigenicity, infectivity, and transmissibility. A patient positive for HBV DNA and detectable but low-level HBsAg in parallel with anti-HBs suggested the presence of immune and/or diagnostic escape variants. To support this hypothesis, serum-derived HBs gene sequences were amplified and cloned for sequencing, which revealed infection with exclusively non-wildtype HBV subgenotype (sgt) D3. Three distinct mutations in the antigenic loop of HBsAg that caused additional N-glycosylation were found in the variant sequences, including a previously undescribed six-nucleotide insertion. Cellular and secreted HBsAg was analyzed for N-glycosylation in Western blot after expression in human hepatoma cells. Secreted HBsAg was also subjected to four widely used, state-of-the-art diagnostic assays, which all failed to detect the hyperglycosylated insertion variant. Additionally, the recognition of mutant HBsAg by vaccine- and natural infection-induced anti-HBs antibodies was severely impaired. Taken together, these data suggest that the novel six-nucleotide insertion as well as two other previously described mutations causing hyperglycosylation in combination with immune escape mutations have a critical impact on in vitro diagnostics and likely increase the risk of breakthrough infection by evasion of vaccine-induced immunity.

Identification of Na⁺/taurocholate co-transporting polypeptide (NTCP) as high-affinity hepatic entry receptor for the Hepatitis B and D viruses (HBV/HDV) opened the field for target-based development of cell-entry inhibitors. However, most of the HBV/HDV entry inhibitors identified so far also interfere with the physiological bile acid transporter function of NTCP. The present study aimed to identify more virus-selective inhibitors of NTCP by screening of 87 propanolamine derivatives from the former development of intestinal bile acid reabsorption inhibitors (BARIs), which interact with the NTCP-homologous intestinal apical sodium-dependent bile acid transporter (ASBT). In NTCP-HEK293 cells, the ability of these compounds to block the HBV/HDV-derived preS1-peptide binding to NTCP (virus receptor function) as well as the taurocholic acid transport via NTCP (bile acid transporter function) were analyzed in parallel. Hits were subsequently validated by performing in vitro HDV infection experiments in NTCP-HepG2 cells. The most potent compounds S985852, A000295231, and S973509 showed in vitro anti-HDV activities with IC₅₀ values of 15, 40, and 70 µM, respectively, while the taurocholic acid uptake inhibition occurred at much higher IC₅₀ values of 24, 780, and 490 µM, respectively. In conclusion, repurposing of compounds from the BARI class as novel HBV/HDV entry inhibitors seems possible and even enables certain virus selectivity based on structure-activity relationships.

Mammalian hepadnaviruses have likely been evolving alongside their hosts for millions of years. Domestic cat HBV (DCHBV) has been detected in cats from several countries, but its genealogy, epidemiology, and host range remain unclear. Besides DCHBV, the only hepadnavirus identified among carnivores is the ringtail HBV (RtHBV). Because there is a gap in the felid fossil record of approximately 5–7 million years between the late Oligocene and the early Miocene, carnivore-derived viruses might help to shed light on Felidae evolution. Here, we screened 2260 sera and 154 paraffin-embedded liver samples from cats and 2123 sera from dogs sampled in Europe and South and Central America between 2018 and 2020 by PCR for DCHBV. We identified DCHBV genotype A (GtA) in 0.6% (7/1,195; 95% CI, 0.2–1.2) of cats sampled in Germany, France, Croatia, and Bulgaria and a genetically divergent DCHBV genotype B (GtB; 10.8% genomic sequence distance) in 0.2% of cats (2/1,065; 95% CI, 0.0–0.7) from Brazil. The detection rates of the two genotypes did not differ significantly (Fisher, P = .19). Viral loads ranged from 4 × 101–6 × 106 for DCHBV GtA to 5–7 × 103 for DCHBV GtB DNA copies per milliliter of serum. None of the cat livers or dog sera tested positive by PCR. Immunoglobulin G against the DCHBV core antigen (anti-DCHBc) was detected in 8/504 cat sera (1.6%; 95% CI, 0.7–3.1), without significant variation between countries (χ2, P = .17), and in none of 180 dog sera by indirect immunofluorescence assay (IFA). Neither IFA (Fisher, P = .11; n = 311) nor PCR (Fisher, P = .63; n = 699) positivity was significantly associated with increased liver enzymes in cats, respectively. Coevolutionary reconciliations of virus and host phylogenies and Bayesian hypothesis testing suggested evolutionary origins of DCHBV during the Miocene, ∼8–17 million years ago (mya) from ancestral carnivores, consistent with long-term evolution. The long-term association of DCHBV with felines aids in elucidating orthohepadnaviral infection patterns and felid genealogy.