Hepatitis B virus (HBV) infections are among the major public health concerns worldwide with more than 250 million of chronically ill individuals. Many of them are additionally infected with the Hepatitis D virus, a satellite virus to HBV. Chronic infection frequently leads to serious liver diseases including cirrhosis and hepatocellular carcinoma, the most common type of liver cancer. Although current antiviral therapies can control HBV replication and slow down disease progress, there is an unmet medical need to identify therapies to cure this chronic infectious disease. Lately, a noteworthy progress in fighting against HBV has been made by identification of the high-affinity hepatic host receptor for HBV and HDV, namely Na⁺/taurocholate cotransporting polypeptide (NTCP, gene symbol SLC10A1). Next to its primary function as hepatic uptake transporter for bile acids, NTCP is essential for the cellular entry of HBV and HDV into hepatocytes. Due to this high-ranking discovery, NTCP has become a valuable target for drug development strategies for HBV/HDV-infected patients. In this review, we will focus on a newly predicted three-dimensional NTCP model that was generated using computational approaches and discuss its value in understanding the NTCP’s membrane topology, substrate and virus binding taking place in plasma membranes. We will review existing data on structural, functional, and biological consequences of amino acid residue changes and mutations that lead to loss of NTCP’s transport and virus receptor functions. Finally, we will discuss new directions for future investigations aiming at development of new NTCP-based HBV entry blockers that inhibit HBV tropism in human hepatocytes.

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.

Na⁺/taurocholate cotransporting polypeptide (NTCP) is a member of the solute carrier (SLC) family 10 transporters (gene symbol SLC10A1) and is responsible for the sodium-dependent uptake of bile salts across the basolateral membrane of hepatocytes. In addition to its primary transporter function, NTCP is the high-affinity hepatic receptor for hepatitis B (HBV) and hepatitis D (HDV) viruses and, therefore, is a prerequisite for HBV/HDV virus entry into hepatocytes. The inhibition of HBV/HDV binding to NTCP and internalization of the virus/NTCP receptor complex has become a major concept in the development of new antiviral drugs called HBV/HDV entry inhibitors. Hence, NTCP has emerged as a promising target for therapeutic interventions against HBV/HDV infections in the last decade. In this review, recent findings on protein–protein interactions (PPIs) between NTCP and cofactors relevant for entry of the virus/NTCP receptor complex are summarized. In addition, strategies aiming to block PPIs with NTCP to dampen virus tropism and HBV/HDV infection rates are discussed. Finally, this article suggests novel directions for future investigations evaluating the functional contribution of NTCP-mediated PPIs in the development and progression of HBV/HDV infection and subsequent chronic liver disorders.

Cellular entry of the hepatitis B and D viruses (HBV/HDV) require binding of the viral surface polypeptide preS1 to the hepatobiliary transporter NTCP. This interaction can be blocked by bulevirtide (BLV, formerly Myrcludex B), a preS1 derivative and approved drug for treating HDV infection. To elucidate the basis of this inhibitory function, we determined a cryo-EM structure of BLV-bound human NTCP. BLV forms two domains, a plug lodged in the bile salt transport tunnel of NTCP and a string that covers the receptor’s extracellular surface. The N-terminally attached myristoyl group of BLV interacts with the lipid-exposed surface of NTCP. Our structure reveals how BLV inhibits bile salt transport, rationalizes NTCP mutations that decrease the risk of HBV/HDV infection, and provides a basis for understanding the host specificity of HBV/HDV. Our results provide opportunities for structure-guided development of inhibitors that target HBV/HDV docking to NTCP.