Fig. 2: Binding of preS1 and patient-derived SVPs as model systems to study HBV-NTCP interaction.
From: Structure of antiviral drug bulevirtide bound to hepatitis B and D virus receptor protein NTCP
a Fluorescence microscopy and (b) fluorescence detection of HEK-NTCP cells and non-NTCP expressing HEK293 cells, incubated with or without 200 nM fluorescent preS1-AX568 peptide (representing genotype D, GtD) in the presence and absence of sodium. Expression levels of NTCP in HEK-NTCP cells were confirmed with qPCR. NBD-TC was used to demonstrate NTCP-mediated bile salt transport, whereas 250 μM TC was used as an inhibitor of preS1-peptide binding. Data in (b) present the means ± SD of quadruplicate measurements. *Significantly higher preS1-AX568 fluorescence compared to all other columns, §significantly different preS1 binding and #significant preS1 binding inhibition, all p < 0.0001. c Schematic of HBV subviral particles. The hepatitis B virus surface proteins LHBs, MHBs, and SHBs differ in the N-terminal additions (preS1, preS2) and N-glycosylation pattern within the S-domain. The antigenic loop (AGL) within the S-domain and the N-terminal preS1, together with the myristic acid covalently attached to Gly2, are determinants for HBV infectivity. d Silver-stained polyacrylamide gels of highly purified SVP preparations from two different patients: K826 HBV-GtA and ID1 HBV-GtD. Both show clear bands of non- and N-glycosylated SHBs (p24/gp27 kDa), single and double N-glycosylated MHBs (gp33/ggp36 kDa), and non- and N-glycosylated preS1-containing LHBs (p39/gp42 kDa). Non-reduced SHBs-dimers (48 kDa or 54 kDa, depending on glycosylation status) and human serum albumin (67 kDa) are also indicated. e, f Binding of nanodisc-reconstituted wild-type or G158R mutant of NTCP-eYFP to SVPs from (e) patient K826 (HBV GtA) and (f) patient ID1 (HBV GtD). NTCP-eYFP fluorescence was detected using a fluorescence microtiter plate reader at 485 nm excitation and 535 nm emission. Data present the means ± SD of triplicate measurements of background-subtracted relative fluorescence units (RFU). *Significantly higher fluorescence intensity between SVP-coated wells incubated with and without nanodisc-reconstituted NTCP-eYFP (p = 0.0013, and p = 0.0492 for panels (e) and (f), respectively, according to one-way ANOVA with Dunnett’s multiple comparison test).
