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Abstract

Hepatitis B virus (HBV) and Hepatitis D virus (HDV) infect human hepatocytes and can cause acute and chronic liver disease, often leading to inflammation, cirrhosis and hepatocellular carcinoma. Despite their global health burden, curative treatment options remain unavailable. In 2023, the entry inhibitor bulevirtide (BLV, formerly Myrcludex B) was approved in Europe for treatment of chronic hepatitis D. BLV is a myristoylated peptide corresponding to the first 2-48 amino acids of the preS1 domain of the HBV large surface protein. BLV blocks viral entry by targeting the HBV/HDV entry receptor sodium taurocholate cotransporting polypeptide (NTCP), which also functions as a major bile salt transporter in the liver. The structure of the NTCP-BLV complex is defined by insertion of the first 21 N-terminal residues of BLV into the NTCP tunnel pore. Previous studies reported prolonged surface retention of BLV, suggesting high stability and slow endocytosis of the receptor-peptide complex. While the molecular binding mode of BLV to NTCP is well characterized, it remains unclear whether BLV modulates the internalization and intracellular trafficking of NTCP, potentially contributing to the antiviral activity of BLV. To elucidate the NTCP-BLV post-binding events, this thesis aimed (i) to characterize the internalization dynamics of preS1-derived peptides using live-cell imaging, (ii) to evaluate strategies for selective labelling of surface-exposed NTCP and, (iii) to study NTCP trafficking in the presence and absence of preS1-derived ligands. Fluorescently labelled BLV and its truncated variant comprising only the first 21 amino acids (HBVpreS/2-21) were synthesized to monitor NTCP-mediated peptide internalization at the single-cell level using 24-hour live-cell imaging. BLV exhibited slow internalization with persistent membrane-associated signals, while HBVpreS/2-21 was rapidly and completely internalized within eight hours and partially degraded over 24 hours. To selectively visualize surface-exposed NTCP, two complementary strategies were employed: genetic code expansion combined with click chemistry and an N-terminal SNAP-tag fusion. These strategies were evaluated for compatibility with the natural bile salt transport and viral receptor functions of NTCP. Site-specific incorporation of non-canonical amino acids (NTCP G144TCO*) preserved bile salt transport and BLV binding but disrupted HDV entry. In contrast, N-terminal SNAP-tagging of NTCP (SNAP-NTCP) maintained HDV susceptibility. Both approaches enabled monitoring NTCP trafficking in the absence and presence of preS1 ligands. Live-cell imaging revealed that SNAP-NTCP was completely internalized within eight hours in the absence of ligands and redistributed throughout the cytoplasm. Although BLV binding did not alter the internalization kinetics of SNAP-NTCP and NTCP-G144TCO*, ligand specific dynamics were observed: colocalization of BLV with SNAP-NTCP persisted intracellularly for at least 12 hours, whereas HBVpreS/2-21 dissociated from NTCP followed by degradation and redistribution of SNAP-NTCP. In conclusion, this study demonstrates distinct NTCP-mediated internalization patterns of preS1-derived peptides. The sustained intracellular stability of the NTCP-BLV complex suggests that the inhibitory potential of BLV extends beyond surface retention and may involve modulation of NTCP trafficking, possibly representing a prerequisite for effective inhibition of HBV and HDV entry. Moreover, SNAP-NTCP provides a versatile tool for future microscopy studies on receptor-mediated viral entry processes.