Hepes

The SARS-CoV-2 variant B.1.351 (Beta), 2102-cov-IM-r1-164 was provided by Prof. Michael Schindler (University of Tübingen) and the B.1.617.2 (Delta) variant by Prof. Florian Schmidt (University of Bonn). The BetaCoV/Netherlands/01/NL/2020 (NL-02-2020), B.1.1.7. (Alpha) and hCoV-19/Netherlands/NH-EMC-1720/2021, lineage B.1.1.529 (Omicron) variants were obtained from the European Virus Archive. The hCoV-19/Japan/TY7-503/2021 (Brazil P.1) (Gamma) (#NR-54982) isolate was obtained from BEI resources. SARS-CoV-2 strains were propagated on Vero E6 (NL-02-2020, Delta), VeroE6 overexpressing TMPRSS2 (Alpha), CaCo-2 (Beta) or Calu-3 (Gamma, Omicron) cells. To this end, 70–90% confluent cells in 75 cm² cell culture flasks were inoculated with the SARS-CoV-2 isolate (MOI of 0.03–0.1) in 3.5 ml serum-free medium (MEM, Cat#M4655; Sigma-Aldrich). The cells were incubated for 2 h at 37°C, before adding 20 ml medium containing 15 mM HEPES (Cat#6763.1; Carl Roth). Virus stocks were harvested as soon as a strong cytopathic effect (CPE) became apparent. The virus stocks were centrifuged for 5 min at 1,000g to remove cellular debris, aliquoted, and stored at −80°C until further use.

Rh123, TRQ, Dulbecco's Modified Eagle's Medium (DMEM, high glucose), and fetal bovine serum were purchased from Sigma‐Aldrich. HEPES was obtained from Roth.

HEPES was purchased from Roth. 2ME2 was purchased from Tetrionics (Madison, WI, USA). STX140 was synthesized as described in Leese et al., 2006 (link) (compound 21). Irosustat was synthesized as described in Woo et al., 2000 (link) (compound 17). Unless otherwise stated, chemicals were purchased either from Merck or Sigma. For in vitro experiments, 2ME2, STX140 and Irosustat were dissolved in DMSO at 50 mM and stored in single-use aliquots at -20 °C for up to 6 months. Just before the measurements, this stock solution was diluted in DMSO to obtain a DMSO concentration of 0.2 % (v/v) for all concentrations measured. Therefore, 0.2 % (v/v) DMSO acts as the vehicle control in all in vitro experiments. For in vivo experiments, STX140 was dissolved in a vehicle, namely 40 % 2-hydroxypropyl-β-cyclodextrin (PanReac AppliChem). Vehicle treatment alone served as control. Myelin basic protein (MBP) was extracted from guinea pig brain as described (Eylar et al., 1974 (link), Määttä et al., 1997 (link)).

Functional characterization of the mono- and co-expressed NTCP wild-type and mutant constructs was performed after transient transfection into HEK293 cells as described previously (Müller et al., 2018 (link)). HEK293 cells were cultured as described above and were plated into 24-well plates (Sarstedt, Nümbrecht, Germany) with 3 × 10⁵ cells per well for the transport experiments. Cells were transfected with the indicated constructs with the identical absolute amount of 0.5 μg pDNA per well by Lipofectamine 2000. After 48 h of incubation, cells were washed three times with PBS. Then, cells were pre-incubated with transport buffer (142.9 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO₄ (Roth), 1.2 mM KH₂PO₄, 1.8 mM CaCl₂ (Roth), and 20 mM HEPES (Roth), pH 7.4, 37°C) for 5 min. For uptake experiments, cells were incubated with 300 µl transport buffer containing 10 µM taurocholic acid (TC) spiked with [³H]TC for 10 min at 37°C. Uptake studies were terminated by removing the transport buffer followed by five washing steps in PBS at 4°C. Afterwards, cells were lysed in 1 N NaOH (Roth) with 0.1% SDS and the cell-associated radioactivity of the lysate was determined by liquid scintillation counting. Additionally, protein content per well was determined for data normalization as described (Geyer et al., 2007 (link)).