Structure of antiviral drug bulevirtide bound to hepatitis B and D virus receptor protein NTCP

Abstract

Cellular entry of the hepatitis B and D viruses (HBV/HDV) requires binding of the viral surface polypeptide preS1 to the hepatobiliary transporter Na⁺-taurocholate co-transporting polypeptide (NTCP). This interaction can be blocked by bulevirtide (BLV, formerly Myrcludex B), a preS1 derivative and approved drug for treating HDV infection. Here, 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.

Introduction

With approximately 1.5 million individuals infected annually, the hepatitis B virus (HBV) continues to pose a substantial global health challenge, despite the availability of an effective vaccine¹. HBV, a small, enveloped DNA virus, causes acute and chronic infection of the liver and the chronic form, in particular, significantly contributes to the overall burden of liver-related diseases, such as cirrhosis and hepatocellular carcinoma (HCC)^2,3. Additionally, co-infection of HBV-infected patients with the hepatitis D virus (HDV), an enveloped RNA satellite virus that uses HBV surface proteins in HBV/HDV co-infected cells for envelopment and infection, enhances the severity of liver disease^4,5,6. Since HBV and HDV share the HBV surface proteins to target hepatocytes for infection, their entry mechanisms have been studied extensively^7,8. Viral entry involves initial low-affinity binding to cellular heparan sulfate proteoglycans (HSPGs)^9,10,11,12 followed by high-affinity binding to the human Na⁺-taurocholate co-transporting polypeptide (NTCP), an interaction crucial for HBV/HDV infection (Fig. 1)^8,13,14. NTCP (SLC10A1), a member of the solute carrier transporter family, is one of four transporters involved in the enterohepatic circulation of bile salts^15,16,17. Located in the basolateral membrane of hepatocytes, NTCP plays a vital role in transporting approximately 80% of bile salts, particularly conjugated bile salts, from the bloodstream into hepatocytes^18,19. Its function is linked to maintaining bile salt homeostasis, essential for the digestion and absorption of dietary fats¹⁸. In 2012, NTCP was identified as a receptor for HBV/HDV^8,13,14. In 2022, four independent studies reported structures of NTCP, revealing nine transmembrane helices (TMs) arranged in two domains, termed panel and core^20,21,22,23. Two of these helices create a crossing motif (X-motif), near which two sodium ion binding sites are located²³. The highest-resolution structure revealed the binding sites for two bile salt molecules in a continuous tunnel that links the extracellular and cytoplasmic side of the basolateral membrane²³.

The viral envelopes of HBV and HDV contain three surface proteins: large (LHBs), middle (MHBs), and small (SHBs), generated from a single open reading frame through alternative start codons^24,25,26. They contain the same C-terminus but differ by N-terminal additions and N-glycosylation status. The SHBs contain four transmembrane helices that are embedded in the viral membrane and are linked by intra- and intermolecular disulfide bridges. The MHBs only contain a preS2-domain, whereas the LHBs contain a preS2- and a preS1-domain located N-terminal to the SHBs²⁷. During protein synthesis, the LHBs undergo a posttranslational modification via myristoylation at a conserved glycine residue (Gly2) in the preS1-domain^24,28,29. Previous studies have shown that the first 75 residues of preS1 are important for viral infectivity of HBV/HDV^30,31. Residues ⁹NPLGFFP¹⁵ are essential for infectivity and binding and are highly conserved within the ten known genotypes of HBV (GtA-GtJ)³². Previous studies suggest that residues Gly2-Asp48 bind to NTCP³². Specific regions of NTCP, including extracellular loop 1 (ECL1) that connects TM2 and TM3 (residues Arg84-Asn87), and the extracellular surface of TM5 (residues Lys157-Leu165), have been shown to interact with the preS1 peptide^8,14,33. The exact mechanism of NTCP-mediated virus entry remains elusive; however, it is thought to occur via endocytosis, which is known to recruit various host factors for entry initiation^34,35. During active HBV infections, two distinct species of viral particles are produced: infectious virus particles and a far larger excess of non-infectious subviral particles (SVPs)^36,37,38. Whilst both species contain all three viral envelope proteins, allowing for recognition and binding to NTCP as a receptor, SVPs lack genetic material and are therefore not infectious^38,39. Thus, SVPs provide an alternative model system for the study of HBV/HDV receptor interaction.

It was recognized that strategies aimed at inhibiting or disrupting binding of preS1 to NTCP hold promise in preventing HBV/HDV infection. In 2023, the commercially available drug Hepcludex® (also known as bulevirtide (BLV), formerly Myrcludex B), received market approval for the treatment of chronic HDV infection in Europe^{32,40,41,42,43}. The sequence of BLV is derived from that of preS1 from HBV genotype C (residues Gly2-Gly48) with a shortening of the 11 additional N-terminal amino acids and one amino acid substitution, Gln46Lys³². BLV exhibits a remarkably high inhibitory constant (IC₅₀ = 140 pM) against HBV and HDV in primary human hepatocytes and HepaRG cells^32,44. BLV has also been demonstrated to inhibit the NTCP-mediated uptake of bile salts, but at an IC₅₀ in the nanomolar range⁴⁵.

We here report a cryo-electron microscopy (cryo-EM) structure of BLV-bound human NTCP, simultaneously providing insight into the mechanism of BLV inhibition and into the interaction between NTCP and the viral preS1 peptide. Coupled with functional analysis, our study advances the molecular understanding of how BLV blocks HBV/HDV infection. These findings hold promise for developing additional therapeutic interventions against HBV/HDV, preventing viral entry into hepatocytes, and thus reducing HBV/HDV-related liver damage. Furthermore, our data may help rationalize why HBV infectivity is affected by variations in NTCP among different species.

Results and discussion

Interaction of bile salts, preS1, and patient-derived SVPs with NTCP

NTCP was shown to transport bile salts in a strictly sodium-dependent manner⁴⁶ and we also observed that a fluorescently labeled taurocholate derivative (4-nitrobenzo-2-oxa-1,3-diazole-taurocholic acid, NBD-TC) is taken up into NTCP-expressing HEK293 cells in a sodium-dependent manner (Fig. 2a). Transport of NBD-TC can only be detected by using a transport buffer containing 143 mM NaCl, while NBD-TC fluorescence is undetectable in sodium-free buffer (equimolar substitution of NaCl with choline chloride). In contrast, the fluorescently labeled preS1 derivative preS1-AX568 binds to NTCP-expressing HEK293 cells under both sodium-containing and sodium-free conditions, indicating that preS1-binding to NTCP does not require sodium (Fig. 2b). Binding of preS1-AX568 to NTCP can be blocked by preincubation with 250 µM taurocholic acid (TC) (Fig. 2a, b), confirming previous data in NTCP-expressing HepG2 hepatoma cells¹³. We then analyzed if preparations of HBV SVPs obtained from patients chronically infected with HBV are capable of binding recombinantly expressed NTCP protein, as used for structure determination in the present study. HBV SVPs contain all surface proteins, namely SHBs, MHBs, and the preS1-bearing LHBs that are also enveloping the HBV/HDV virus particles (Fig. 2c). The SVP preparations represent the HBV genotypes D (GtD, patient ID1) and A (GtA, patient K826) (Fig. 2d). In both preparations the preS1-containing LHBs can be clearly detected by silver-stained polyacrylamide gel electrophoresis, although patient K826 showed a more prominent signal than patient ID1 (Fig. 2d). Plates were then coated with these patient-derived SVPs and binding of nanodisc-reconstituted, eYFP-labeled NTCP²³ was observed by fluorescence measurement. We found a concentration-dependent binding of NTCP (25–100 nM) to GtA and GtD SVPs, demonstrating binding of NTCP to the SVPs. As a control, we used an NTCP variant carrying the mutation G158R, which abolished binding to the SVPs (Fig. 2e, f).

Functional analysis and BLV on TC transport

Bile salt transport in NTCP-expressing HEK293 cells was also analyzed with the radiolabeled bile acid ³H-TC that showed robust uptake into the cells in the presence of media containing sodium (Fig. 3a). TC transport was inhibited at increasing concentrations of BLV with an inhibition constant value (IC₅₀) of 195 nM (95% CI, 174–218 nM) (Fig. 3a). At high concentrations, BLV fully abolished transport. This is consistent with the previously shown result for myristoylated preS1 inhibition of TC uptake into NTCP-expressing HEK293 cells (IC₅₀ of 190 nM)^13,47, indicating that our NTCP construct is functional and suitable for further studies on interaction with BLV….