Long-term trans -inhibition of the hepatitis B and D virus receptor NTCP by taurolithocholic acid

Human hepatic bile acid transporter Na þ /taurocholate cotransporting polypeptide (NTCP) represents the liver-speci ﬁ c entry receptor for the hepatitis B and D viruses (HBV/HDV). Chronic hepatitis B and D affect several million people worldwide, but treatment options are limited. Recently, HBV/HDV entry inhibitors targeting NTCP have emerged as promising novel drug candidates. Nevertheless, the exact molecular mechanism that NTCP uses to mediate virus binding and entry into hepatocytes is still not completely understood. It is already known that human NTCP mRNA expression is downregulated under cholestasis. Furthermore, incubation of rat hepatocytes with the secondary bile acid taurolithocholic acid (TLC) triggers internalization of the rat Ntcp protein from the plasma membrane. In the present study, the long-term inhibitory effect of TLC on transport function, HBV/HDV receptor function, and membrane expression of human NTCP were analyzed in HepG2 and human embryonic kidney (HEK293) cells stably overexpressing NTCP. Even after short-pulse preincubation, TLC had a signi ﬁ cant long-lasting inhibitory effect on the transport function of NTCP, but the NTCP protein was still present at the plasma membrane. Furthermore, binding of the HBV/HDV myr-preS1 peptide and susceptibility for in vitro HDV infection were signi ﬁ cantly reduced by TLC preincubation. We hypothesize that TLC rapidly accumulates in hepatocytes and mediates long-lasting trans -inhibition of the transport and receptor function of NTCP via a particular TLC-binding site at an intracellularly accessible domain of NTCP. Physiologically, this trans -inhibition might protect hepatocytes from toxic overload of bile acids. Pharmacologically, it provides an interesting novel NTCP target site for potential long-acting HBV/HDV entry inhibitors. NEW & NOTEWORTHY The hepatic bile acid transporter NTCP is a high-af ﬁ nity receptor for hepatitis B and D viruses. This study shows that TLC rapidly accumulates in NTCP-expressing hepatoma cells and mediates long-lasting trans -inhibition of NTCP ’ s transporter and receptor function via an intracellularly accessible domain, without substantially affecting its membrane expression. This domain is a promising novel NTCP target site for pharmacological long-acting HBV/HDV entry inhibitors.


INTRODUCTION
Although an effective vaccine is available, hepatitis B virus (HBV) and hepatitis D virus (HDV) infections remain a major global health problem. More than 250 million people worldwide are chronically infected with HBV, and thousands of them suffer from hepatocellular carcinoma (HCC) or develop terminal liver cirrhosis. About 5% of chronic HBV carriers are coinfected with HDV, a situation often associated with disease progression and increased mortality rates (1). The hepatitis D virus is an HBV satellite virus, and both share identical envelope proteins (2). Current standard therapies include nucleoside reverse transcriptase inhibitors and interferon. However, both of these are not curative in the majority of cases (3)(4)(5).
In 2012, Yan et al. (6) identified the liver bile acid transporter Na þ /taurocholate cotransporting polypeptide (NTCP, gene symbol SLC10A1) as the high-affinity hepatic entry receptor for HBV and HDV. NTCP belongs phylogenetically to the solute carrier family SLC10 and is typically expressed at the basolateral membrane of hepatocytes (7,8), explaining the clear hepatotropism of HBV and HDV. NTCP, together with other bile acid carriers (including ASBT, OSTa/b, and BSEP), is essential for the maintenance of the enterohepatic circulation of bile acids between liver and gut (9). In detail, NTCP mediates a sodium-dependent reuptake of mostly conjugated bile acids from portal blood back into hepatocytes (7).
It is already known that HBV and HDV attach to NTCP via their myristoylated preS1 domain comprising the NH 2 -terminal amino acids 2-48 of the large HBV surface protein, briefly called myr-preS1 lipopeptide (10,11). To date, it is generally accepted that myr-preS1-mediated HBV/HDV binding to NTCP represents the mandatory high-affinity attachment step of viral entry into hepatocytes (12,13). Previously, we showed that HBV/HDV myr-preS1-mediated viral binding to NTCP directly interferes with the physiological bile acid transport function of NTCP (14). Briefly, the myr-preS1 peptide blocked the bile acid transport in primary human hepatocytes and NTCP-overexpressing HepG2 hepatoma cells in a concentration-dependent manner. Vice versa, bile acids significantly inhibited myr-preS1 binding to NTCP as well as in vitro HBV infection (14). This effect most likely occurs via cis-inhibition at overlapping binding sites for bile acids and the myr-preS1 peptide at respective extracellular accessible domains of NTCP. In support of this assumption, we have recently shown that mutation of amino acid G158 at the second extracellular loop of human NTCP completely abolished myr-preS1 peptide binding to NTCP without affecting the bile acid transport function of NTCP (15). Other amino acids of NTCP, such as S267, which is located within transmembrane domain 8 (TMD8) in proximity to the extracellular loop connecting TMD8 and TMD9, proved to be essential for the binding of both bile acids and myr-preS1 peptide (16,17). Interestingly, the naturally occurring NTCP variant S267F showed reduced transport activity for taurocholic acid (TC) but maintained transport of other substrates such as rosuvastatin and estrone-3-sulfate (16,18). Patients bearing this S267F variant were less susceptible to chronic HBV infection and showed decreased risk for HCC and liver cirrhosis under chronic HBV infection (19)(20)(21)(22)(23). Overall, these data indicate that the extracellular NTCP-binding sites for its transport substrates and for the myr-preS1 peptide overlap, but both can be separated by mutation (14,15,17).
In contrast to this cis-inhibitory effect, pulse preincubation with the secondary bile acid taurolithocholic acid (TLC) followed by intensive washout had a long-lasting inhibitory effect on the transport function of NTCP, which cannot be explained by cis-inhibition (14,24). Interestingly, this effect was not observed after preincubation with other bile acids such as TC, glycocholic acid (GC), or tauroursodeoxycholic acid (TUDC) (14). It was shown that NTCP mRNA expression is downregulated in patients with cholestasis, probably due to signals induced by bile acid overload (25). While longterm incubation with TLC in rat hepatocytes significantly decreased the plasma membrane expression of rat Ntcp, plasma membrane expression of human NTCP remained unaffected under comparable conditions (24). However, it is currently unclear how TLC mediates this long-term inhibitory effect on human NTCP without affecting substantially its membrane expression. Furthermore, it is of interest if and how this effect influences the susceptibility of hepatocytes for HBV/HDV infection. To address these questions, we analyzed the effect of pulse preincubation of TLC on the transport function, membrane expression, myr-preS1 peptide binding, and in vitro HDV infection in NTCP-overexpressing HepG2 and HEK293 cells. We found a long-lasting inhibitory effect of TLC on the NTCP transporter and receptor function as well as on in vitro HDV infection that might be explained by trans-inhibition of NTCP via a particular TLC-binding site at an intracellularly accessible domain of NTCP.

Cell Lines and Induction
Human embryonic kidney (HEK293; Thermo Fisher Scientific, Waltham, MA) cells, stably expressing the human NTCP protein, NH 2 -terminally tagged with the HA-epitope and COOH-terminally with FLAG-tag were maintained at 37 C, 5% CO 2 , and 95% humidity in Dulbecco's modified Eagle medium (DMEM)/Ham's F12 medium (Thermo Fisher Scientific) supplemented with 10% fetal calf serum (Sigma-Aldrich, Taufkirchen, Germany), 4 mM L-glutamine (PAA, C€ olbe, Germany), and penicillin/streptomycin (PAA). Human hepatoma HepG2 cells, stably transfected with NTCP-FLAG [see more detailed description in (14)], and HepG2-tet-on cells (BD Clontech, Heidelberg, Germany) were cultured under the same conditions in DMEM with all supplements listed above, except for L-glutamine. For induction of the transgene, the medium was supplemented with 1 mg/mL tetracycline (Roth, Karlsruhe, Germany) for HA-NTCP-FLAG-HEK293 cells (further referred to as NTCP-HEK293 cells) or 2 mg/mL doxycycline (Sigma-Aldrich) for NTCP-FLAG-HepG2 cells (further referred to as NTCP-HepG2 cells). NTCP-HepG2 cells were used for all HDV infection experiments as reported before (14). HepG2-tet-on cells were transiently transfected using X-tremeGENE 9 (Roche Diagnostics, Basel, Germany) with a pcDNA5-FRT-TO plasmid (Thermo Fisher Scientific) containing HA-NTCP-FLAG for plasma membrane expression studies. All cell lines were tested negatively for mycoplasma contamination according to the protocol of Uphoff and Drexler (26).  (27), which represents an adapted Norman's method (28). Briefly, 1 mg (2.7 mM) LC (Tokyo Chemical Industry, Tokyo, Japan) was dissolved in 80 mL of anhydrous dioxane. To this solution, 10 mL of triethylamine (Sigma-Aldrich) was added, followed by incubation for 10 min at 23 C. Then, 10 mL of 1% ethyl chloroformate (Thermo Fisher Scientific) solved in dioxane was added and incubated for additional 30

Determination of the Inhibitory Concentrations for [ 3 H]TC Uptake
Bile acid transport measurements were performed in NTCP-HEK293 cells with [ 3 H]TC as reported before (30). Briefly, cells were seeded onto polylysine-coated 96-well plates, induced with 1 mg/mL tetracycline, and grown to 100% confluence over 72 h at 37 C. After preincubation with TLC, cells were washed three times with tempered DMEM. Then, medium was replaced by 80 mL DMEM containing the respective inhibitor or solvent alone (positive control), and cells were further incubated for 5 min at 37 C. Bile acid transport experiments were started by adding 20 mL DMEM containing 25 mM [ 3 H]TC (final concentration: 5 mM). Experiments were stopped after 10 min by washing twice with ice-cold phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH 2 PO 4 , 7.3 mM Na 2 HPO 4 , pH 7.4). For negative control, the NTCP-HEK293 cells were not induced with tetracycline (-tet). Cell-associated radioactivity of [ 3 H]TC was quantified by liquid scintillation counting in a Packard Microplate Scintillation Counter TopCount NXT (Packard Instrument Company, Meriden, CT). Transport rates were determined in disintegrations per minute (dpm). The mean of the negative control was subtracted, and the net transport was expressed as % of positive control. Irbesartan was purchased from Sigma-Aldrich. Inhibitory concentrations (IC 50 ) were calculated using GraphPad Prism 6 (GraphPad, San Diego, CA).

Nonpermeabilized Immunofluorescence Assay
After bile acid incubation, NTCP-HEK293 as well as HepG2-tet-on cells, transiently expressing the HA-NTCP-FLAG protein, were fixed with 3% paraformaldehyde (Roth) and blocked with 5% BSA (Sigma-Aldrich) in PBS for 30 min at RT. Subsequently, monoclonal mouse anti-HA antibody (1:400 dilution for HEK293 and 1:800 for HepG2 cells, Thermo Fisher Scientific, Cat. No. 32-6700, Lot No. QL217972) was incubated for 1 h at 37 C, followed by goat anti-Mouse IgG Alexa Fluor 488 (1:800 dilution, Thermo Fisher Scientific, Cat. No. A-11001, Lot No. 1907294) and nuclear staining with Hoechst 33342 (1 mg/mL). NTCP-HEK293 cells were qualitatively analyzed on Leica DMI6000 B inverted fluorescent microscope or quantitatively measured using multimode microplate reader Spark 10 M (Tecan, M€ annedorf, Switzerland) as reported before (33), with the following settings: fluorescence mode with bottom detection, 488 ± 20 nm bandwidth monochromator for excitation, optimal gain, 4 Â 4 reads per well, and 535 ± 25 nm bandwidth emission filter. For quantification of relative fluorescence of HepG2-tet-on transiently expressing the HA-NTCP-FLAG protein, samples were assessed on Leica DMI6000 B fluorescent microscope and analyzed using LAS-X imaging software (Leica). Cell-based fluorescence was determined by defined regions of interest (ROI), and data are presented as the mean background-subtracted fluorescence intensity of nontransfected HepG2 cells.

Lactate Dehydrogenase Release Cytotoxicity Assay
Pierce lactate dehydrogenase release (LDH) cytotoxicity assay (Thermo Fisher Scientific, Cat. No. 88954) was performed according to the manufacturer's protocol. Briefly, NTCP-HEK293 and NTCP-HepG2 cells were incubated with bile acids over 2 h or 6 h at 37 C. After 2 h, 50 mL of each sample and control medium was transferred to a new well plate, 50 mL of reaction mixture were added, and incubated for 30 min at RT. Finally, the reaction was stopped by adding 50 mM of stop solution and measured by ELISA reader (PHOmo, anthos Mikrosysteme GmbH, Krefeld, Germany). Lysis control (maximal LDH-control) was incubated 45 min with lysis buffer at 37 C.

MTS Cytotoxicity Assay
The CellTiter 96 AQueous MTS colorimetric cell viability assay (Promega, Cat. No. G3581) was performed according to the manufacturer's protocol. NTCP-HEK293 and NTCP-HepG2 cells were incubated 2 h with 100 mL DMEM without phenolred (Thermo Fisher Scientific) containing bile acids at 37 C and 5% CO 2 atmosphere. Thereafter, 20 mL of CellTiter 96 AQueous One Solution Reagent were added and incubated 1 h at 37 C and 5% CO 2 . Using fluorescence reader GloMax (Promega) absorbance was recorded at 450 nm.

Surface Biotinylation and Western Blotting
NTCP-HepG2 cells were seeded onto six-well plates, were induced (NTCP-FLAG expression) or not induced (paternal cells without NTCP-FLAG expression), and were cultured for 3 days to reach confluence. For biotinylation, cells were rinsed three times with PBS. Then, cells were incubated at 4 C on a rocking shaker at 30 rpm for 60 min with 0.5 mg EZ-Link NHS-PEG 12 -Biotin solved in 1,000 mL PBS per well. Biotinylation was stopped by rinsing three times with culture medium and then two times with PBS. Cells of the respective wells were harvested in 1 mL lysis buffer (PBS containing 1.2% Triton X100, Sigma-Aldrich) and 10 mL of protease inhibitor cocktail (Thermo Fisher Scientific, Cat. No. 87785). The lysate was centrifuged at 10,000 g for 2 min to remove debris and DNA. Protein concentration of the supernatant was determined by bicinchoninic acid (BCA) assay following the manufacturer's protocol (Novagen, Darmstadt, Germany, Cat. No. 71285-3) and was measured with NanoDrop One (Thermo Fisher Scientific) photometer in BCA assay application. Cell lysates were adjusted to 400 ng/ mL by dilution with lysis buffer. For each cell lysate, 50 mL streptavidin magnetic beads (Pierce, Cat. No. 88817) were equilibrated with lysis buffer. For pull down, 50 mL equilibrated beads were incubated at 4 C overnight with 900 mL of lysate within 1.5 mL Eppendorf tubes on rotating stand. On the next day, beads were adsorbed to the tube wall by a magnetic stand, surplus buffer was withdrawn, 900 mL fresh buffer was added, and beads were resuspended and rotated for 10 min at 4 C. This washing process was repeated three times. To prepare for Western blotting, beads were adsorbed by a magnetic stand and the surplus was removed. Then, the beads were resuspended in 65 mL Laemmli buffer and incubated at 80 C and 1,200 rpm for 10 min. After centrifugation, 20 mL of the obtained supernatant per well was separated on 10% SDS gel followed by Western blotting. The nitrocellulose membrane was blocked for 1 h at RT in 5% milk powder (Sigma-Aldrich) in tris-buffered saline-Tween 20 [TBS-T; 137 mM NaCl, 10 mM Tris (Roth), pH 8.0, 0.05% Tween-20 (Roth)] and then stained with primary anti-FLAG antibody (anti-FLAG rabbit antibody, 1:2,000 dilution, Sigma-Aldrich, Cat. No. F7425, Lot No. 0000099715) overnight in 5% milk powder in TBS-T at 4 C. The membrane was washed three times in TBS-T and incubated at RT with secondary anti-rabbit-HRP antibody (anti-rabbit-HRP goat, 1:4,000 dilution, Thermo Fisher Scientific, Cat. No. 31460, Lot No. UK293475). Both antibodies were widely used before (34,35). After final washing in TBS-T, the membrane was rinsed with Roti-Lumin substrate (Roth, Cat. No. P078.1), transferred to an imager, and exposed for 20 min (Intas Science Imaging Instruments GmbH, G€ ottingen, Germany). Image analysis was done with Image Lab 6.1 (Bio-Rad Laboratories, CA).

Statistics
Data are shown as means ± SD. Prism software (GraphPad Prism 6.0) was used for data presentation and statistical analysis. Statistical analysis was performed by one-way or two-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison post hoc test as indicated in the figure legends. For IC 50 calculations, statistical analysis was done by two-way ANOVA and Sidak's multiple comparisons post hoc test.

Graphical Abstract
Chemical structures of bile acids and DHEAS were generated using PubChem (National Center for Biotechnology Information, Bethesda, MD).

TLC-Induced Inhibition of NTCP's Transporter and Receptor Function
To analyze the effect of bile acid preincubation on the transporter and virus receptor function of NTCP, NTCP-HEK293 cells were used for transport experiments with the fluorescent bile acid NBD-TC and for binding experiments with the red fluorescent myr-preS1-AX568 peptide. In addition, anti-HA immunofluorescence was performed to detect the HA-NTCP-FLAG protein. Because the HA-coupled NH 2terminus of NTCP is outward oriented and these experiments were performed under nonpermeabilized conditions, fluorescence signals indicate plasma membrane expression of NTCP under these experimental conditions. As expected, positive control cells without bile acid preincubation showed strong accumulation of NBD-TC and significant binding of the myr-preS1-AX568 peptide to the surface of the cells (Fig.  1A). In addition, clear fluorescence staining of NTCP was detected at the plasma membrane of nearly all cells. This pattern was not much different, when NTCP-HEK293 cells were preincubated with 25 mM TC or TUDC for 30 min followed by thorough washout before the respective experiments. As both bile acids demonstrated a potential for NTCP cis-inhibition of bile acid transport and myr-preS1 peptide binding in our previous study (14), this indicates that the washout of the preincubated bile acids was sufficient to avoid such a cis-inhibition. In the case of taurochenodeoxycholic acid (TCDC), NBD-TC and myr-preS1-AX568 peptide showed a trend for lower relative fluorescence, but NTCP expression was still clearly detected at the plasma membrane. In contrast, preincubation of 25 mM TLC nearly abolished NBD-TC transport and myr-preS1-AX568 peptide binding, but plasma membrane expression of NTCP was apparently not changed (Fig. 1A). The latter indicates that the drop in NTCP transport and receptor function was not caused by internalization or degradation of NTCP in these experiments. These qualitative data were verified by quantification of the relative fluorescence units for NBD-TC transport, myr-preS1-AX568 peptide binding, and anti-HA immunofluorescence (Fig. 1B). Most bile acids had no significant effect on the NBD-TC transport and myr-preS1 binding functions of NTCP. TCDC again showed a trend for lower NDB-TC transport and significantly reduced myr-preS1-AX568 peptide binding compared with the solventtreated NTCP-HEK293 cells. Even more pronounced, 25 mM TLC and also lithocholic acid (LC) significantly reduced the NBD-TC transport via NTCP and the myr-preS1-AX568 peptide binding to levels of $50% compared with the positive control. Of note, none of these bile acids induced any cytotoxic effect in the LDH cytotoxicity assay after preincubation even with 50 mM, indicating full cell viability in this experimental setup. NTCP cell surface expression detected by anti-HA immunofluorescence was also not significantly altered by preincubation with any of the bile acids used.

TLC Preincubation Does Not Abolish Transport of [ 3 H]TLC via NTCP
During these experiments, the question arose whether the inhibitory effect of TLC on the transporter and receptor function of NTCP is limited only to the substrate NBD-TC. Because this is not a physiological bile acid, the effect of bile acid preincubation on the transporter and receptor function of NTCP was also investigated with the radiolabeled bile acid  (Fig. 2, A and D 2C). Of note, the inhibitory effect of TLC preincubation was only effective when the preincubation was performed in sodium-containing buffer, meaning that the sodium-dependent TLC uptake via NTCP during the preincubation phase is a prerequisite for this effect. Finally, the transport of [ 3 H]TLC via NTCP was analyzed after 2-h preincubation with 25 mM TLC itself and with 25 mM GC (Fig. 2B). Remarkably, TLC preincubation did not alter its own ([ 3 H]TLC) transport and basically behaved like GC preincubation only in the case of [ 3 H]TLC as transport substrate. These data indicate that TLC preincubation retains not only plasma membrane expression of NTCP but also its principal transport function, at least for TLC. Therefore, it is unlikely that NTCP is just inactivated, for example, by phosphorylation, or dislocated from the plasma membrane by TLC preincubation. To verify that these effects are not just caused by cytotoxic side effects of the preincubated bile acids, additional MTS cytotoxicity assay was performed with TLC and GC under comparable experimental conditions (Fig. 2E). We found no cytotoxic effect for bile acid preincubation that could explain the specific long-term inhibitory effect of TLC.

Inhibitory Effect of TLC Depends on Its Intracellular Concentration
Next, speed and duration of the inhibitory effect of TLC preincubation on the transport function of NTCP were analyzed. For these experiments, NTCP-HEK293 cells were preincubated with 25 mM TLC over time periods ranging from 10 s up to 6 h (Fig. 3, A and C). In these experiments, the TC transport activity of NTCP started to decrease significantly after 2 min of TLC preincubation and reached the nadir already at 20 min (Fig. 3C). Longer preincubation times of up to 6 h did not further decrease the transport activity (Fig.  3A). Again, this effect was specific for [ 3 H]TC as the tested transport substrate of NTCP, whereas TLC preincubation had no significant effect on its own ([ 3 H]TLC) transport activity via NTCP even over longer periods of time (Fig. 3C, right). Furthermore, it was analyzed how long this inhibitory effect of TLC may last and if NTCP's transport activity for TC would recover at any time. To address this question, NTCP-HEK293 cells were preincubated with 25 mM TLC over 2 h. After intensive washing, cells were further incubated in bile acid-free DMEM over time periods of 2 h up to 8 h at 37 C. As clearly   Fig. 3B, the inhibitory effect of TLC preincubation on the transport activity of NTCP remained relatively constant at its low level, even in the absence of TLC over 8 h post incubation. This indicates that TLC preincubation induced a long-lasting inhibitory effect on the TC transport activity of NTCP. Interestingly, the NTCP surface expression remained constant, even for very long periods of preincubation (up to 6 h) and postincubation (up to 8 h) (Fig. 3, A

TLC-Induced Inhibition of HDV Infection
Next, it was analyzed if the inhibitory effect of TLC also affects the susceptibility of NTCP-HepG2 cells for in vitro HDV infection. Because HDV and HBV share identical surface proteins, HDV infection can be regarded as a surrogate of HBV infection. As in vitro infection experiments require incubation of the NTCP-HepG2 cells with HDV virus particles over several hours, it must be noted that only longterm effects can be monitored under these experimental conditions. NTCP-HepG2 cells were incubated with increasing concentrations of TLC or TC over 2 h, followed by intensive washing and incubation with bile acid-free HDV infection medium for 6 h (Fig. 4E). TLC significantly inhibited HDV infection in a concentration-dependent manner. With 25 mM TLC preincubation, the number of HDV-infected cells per well was reduced to $50%. These data go in line with a strong inhibition of NBD-TC uptake that was also significantly inhibited over 6 h postincubation with 25 mM TLC (Fig. 3A). After 2-h preincubation with 250 mM TLC, the HDV infection rate even dropped to nearly zero infected cells per well. In contrast, 2-h preincubation with TC at 2.5 mM, 25 mM, and 250 mM concentrations reduced the HDV infection rate only slightly, but significantly, compared with the control (without bile acid preincubation). At the highest concentration of 250 mM TC, the infection rate still remained at 50% of the control (Fig. 4E). Of note, NTCP surface expression was mostly maintained at all bile acid preincubation conditions, with TC and TLC at least above 60% of the positive control in nonpermeabilized HepG2 cells expressing the HA-NTCP-FLAG protein (Fig. 4B, top). Additionally, cell surface biotinylated NTCP-HepG2 cells did not show any significant decrease in plasma membrane expression of NTCP after preincubation with TLC or TC (Fig. 4B, bottom). Therefore, the decrease in HDV infection rates cannot be explained by a quantitative loss of NTCP plasma membrane expression.
For comparison, 2.5 mM, 10 mM, 25 mM, and 250 mM TLC or TC were used for cis-inhibition of HDV infection (Fig. 5C). In these experiments, the medium contained HDV virus particles and TLC/TC during the 6-h in vitro infection period. As expected, both bile acids had comparable potent inhibitory effects on the HDV in vitro infection rates, with only 8% infected cells per well in the presence of 250 mM TC, and hardly any infected cells per well in the presence of 250 mM TLC, compared with the control (without bile acid coincubation). Both bile acids fully maintained cell viability (measured by LDH cytotoxicity assay) after 6 h of bile acid coincubation and at 4 days postincubation even at the highest concentrations, so that toxic effects on the cells can be excluded (Fig. 5, A and B). This was basically confirmed by additional MTS cytotoxicity tests on NTCP-HepG2 cells that showed full cell viability after 2-h incubation with up to 250 mM TLC (Fig. 4C). These data clearly indicate that TC only had strong inhibitory effects on HDV infection during coincubation (cis-inhibition), whereas TLC showed comparable inhibitory effects after preincubation (trans-inhibition) and during coincubation (cis-inhibition).
To analyze if parts of the trans-inhibitory effect may be induced by bile acid reflux from the cells during the HDV infection, bile acid concentrations were additionally determined in the supernatant at the end of the HDV infection period. Only trace amounts of bile acids refluxed from the cells after preloading with 25 mM and 250 mM of TC or TLC in the preincubation medium (Fig. 4D vs. Fig. 4A). These were determined to 7 mM for TLC and 4 mM for TC after preincubation with 250 mM of the respective bile acid. Of note, both concentrations were not sufficient to cis-inhibit in vitro HDV infection (see Fig. 5C) and, therefore, bile acid reflux from the cells is not a relevant factor for interpretation of the in vitro HDV infection experiments after bile acid preincubation.    = 120). B, bottom: NTCP-HepG2 cells were incubated for 2 h with 25 mM and 250 mM of TC or TLC, or with solvent alone (positive control) and were subjected to surface biotinylation. The used biotinylation agent EZ-Link NHS-PEG 12 -Biotin is highly hydrophilic due to its polyethyleneglycol 12 spacer arm and allows no membrane penetration. Data represent means ± SD of combined data from four independent experiments. ÃSignificantly lower values compared with positive control with P < 0.001 (one-way ANOVA with Dunnett's multiple comparison post hoc test). In addition, a representative Western blot is depicted. C: cell viability was assessed under comparable experimental conditions with 25 mM and 250 mM of TC or TLC, or with solvent alone (positive control). Data represent means ± SD of three independent experiments each with triplicate determinations (n = 9). E: NTCP-HepG2 cells were preincubated for 2 h with the indicated concentrations of TC or TLC at 37 C, followed by intensive washing. Then, cells were incubated with bile acid-free HDV infection medium, containing 700 HDV genome equivalents/cell for 6 h at 37 C. Medium was changed every 3-4 days. At days 9-11 post infection, cells were fixed and immunostained against the expression of HDV antigen (HDAg), as a marker of HDV infection. The numbers of infected cells per well were counted using fluorescence microscopy. Data represent means ± SD of three independent experiments each with triplicate determinations (n = 9). ÃSignificantly lower infection rates compared to positive control with P < 0.001 (two-way ANOVA with Dunnett's multiple comparison post hoc test). DMEM, Dulbecco's modified Eagle's medium; HDV, hepatitis D virus; HepG2, HepG2 hepatoma cells; NTCP, Na þ /taurocholate cotransporting polypeptide; ROI, region of interest; TC, taurocholic acid; TLC, taurolithocholic acid.
with and without TLC preincubation in the presence of increasing concentrations of different cis-acting ligands. Ursodeoxycholic acid (UDC) was used as a prototypic substrate of NTCP, irbesartan as a potent inhibitor of NTCP, and myr-preS1 as a surrogate parameter for HBV/HDV binding. As expected, TLC preincubation decreased the absolute [ 3 H]TC transport rate dramatically by 79% (Fig.  6A). UDC, irbesartan, and myr-preS1, as cis-acting inhibitors, then reduced the transport rate beyond this value to nearly zero at the respective highest inhibitor concentrations (Fig. 6B). Interestingly, IC 50 values for [ 3 H]TC transport inhibition did not differ substantially between the two experimental conditions, indicating that TLC transinhibition affects the overall transport rate of NTCP, but not its cis-binding kinetics for substrates, inhibitors, and myr-preS1.

DISCUSSION
Under physiological conditions, bile acids undergo an efficient enterohepatic circulation. This process consists of their continuous hepatocellular canalicular efflux, intestinal reabsorption, and hepatic reuptake. The latter process is mediated by NTCP and members of the organic anion transporting polypeptide (OATP, gene symbol SLCO) family (7). During cholestasis, this circulation is disrupted and bile acids start to accumulate in the liver. Via the farnesoid X receptor (FXR, gene symbol NR1H4) and some other nuclear receptors, this intrahepatic bile acid increase leads to an adaptive transcriptional regulation of the hepatic bile acid transporters to prevent hepatocytes from bile acid-induced cellular toxicity (36). Consequently, NTCP is downregulated under cholestasis to reduce the hepatic bile acid uptake, and alternative basolateral efflux transporters such as OSTa/b, MRP3 (gene symbol ABCC3), or MRP4 (gene symbol ABCC4) are induced to facilitate the transport of bile acids from cholestatic hepatocytes back into the blood (7,25,37).
In addition, bile acid uptake via NTCP undergoes dynamic regulation at the protein level (38). In rat hepatocytes as well as in Huh7 hepatoma cells stably expressing rat Ntcp or human NTCP, the plasma membrane expression of the Ntcp/NTCP protein as well as its bile acid transport rates are stimulated by cAMP (24,(39)(40)(41)(42)(43). In contrast, bile acids such as TLC induced retrieval of rat Ntcp from the plasma membrane of rat hepatocytes and rat Ntcp-expressing HepG2 cells and thereby limit the bile acid uptake rate (24,44,45). As TLC additionally induces retrieval of Bsep and Mrp2 from the canalicular membrane of hepatocytes, this bile acid, overall, produces acute cholestasis by preventing biliary organic anion and bile acid secretion (46,47). For rat Ntcp, this retrieval process, initiated by cholestatic bile acids, is quite well understood (38) and involves clathrin-dependent endocytosis via the dileucine motif L222/L223 (48). In contrast, short-term regulation of human NTCP has only been sparsely analyzed so far. Interestingly, human NTCP transport rates can also be inhibited by preincubation with TLC. However, TLC did not decrease the plasma membrane expression of human NTCP in HuH cells as in the rat (24). A previous study by Schonhoff et al. (24) analyzed more closely whether this TLC-induced transport inhibition of human NTCP may involve phosphoinositide-3-kinase (PI3K)-dependent activation of protein kinase PKCɛ. However, as they could not find a role of the PI3K/PKB pathway, the cellular mechanism that TLC uses to inhibit the transport function of human NTCP is still unclear.
Based on the data of the present study, we suppose that TLC-induced transport inhibition of NTCP involves TLC binding to NTCP from the cytoplasmic side and, thus, can be classified as a trans-inhibition mode that was already discussed in a previous publication by Schonhoff et al. (24). In addition to the bile acid transport function of NTCP, its HBV/HDV virus receptor function was analyzed as well, to figure out whether trans-inhibition of NTCP could contribute to a better understanding of NTCP-mediated HBV/HDV entry and its pharmacological inhibition. We confirmed that preincubation with particular bile acids significantly inhibited the transport function of NTCP. TLC and LC were equally potent in this regard, whereas TCDC showed only partial inhibition (Fig. 1). In contrast, other bile acids such as TC, GC, or TUDC showed no effect on the transport function of NTCP. This clearly demonstrates that this is a compound-specific effect and does not generally occur in the presence of bile acids. Based on the experimental setup used in the present study, it can be excluded that this inhibitory effect only occurs by competitive cis-inhibition at the outwardopen bile acid-binding site of NTCP. Remarkably, apart from the transport function, the HBV/HDV receptor binding function of NTCP (measured by myr-preS1 peptide binding) was also significantly inhibited by TLC, LC, or TCDC preincubation. Of note, under these experimental conditions and in agreement with the previous study by Schonhoff et al. (24), NTCP was still detected at the plasma membrane ( Fig. 1 and  Fig. 4B). It, therefore, can be excluded that a retrieval of NTCP from the plasma membrane, which was shown for rat Ntcp under comparable experimental conditions (see earlier text in DISCUSSION), is responsible for this drop of NTCP's transporter and receptor function. An alternative explanation for the long-lasting inhibitory effect of TLC could be that the intracellular accumulation of this bile acid would induce cell stress and so cells would require longer time to fully recover their transport function. However, as the MTS cytotoxicity assays showed full cell viability after 2-h preincubation with TLC (Fig. 2E) and NTCP-HEK293 cells still showed full transport activity at least for TLC (Fig.  2B), this possibility seems unlikely.
The inhibitory effect of TLC on the NTCP transporter function occurred within seconds and reached its maximum at 20 min. This maximum could not be further increased Figure 6. Cis-binding kinetics of NTCP at TLC trans-inhibition. NTCP-HEK293 cells were incubated with tetracycline to induce NTCP expression and were used for bile acid transport experiments with 5 mM [ 3 H]TC over 10 min at 37 C. Cells without tetracycline treatment were used as negative control. Measurements were performed with solvent alone (positive control) and with increasing concentrations of ursodeoxycholic acid (UDC), irbesartan, and myr-preS1 peptide as cis-acting inhibitors of TC uptake in two experimental setups. For the first setup, cells were preincubated with 25 mM TLC over 1 h at 37 C in DMEM, then washed three times with DMEM before the [ 3 H]TC uptake experiments were started (results shown in gray). For the second setup, cells were preincubated with pure DMEM over 1 h at 37 C before the same inhibition experiment was performed (results shown in black). The mean of the negative control was subtracted to calculate net [ 3 H]TC transport rates expressed as dpm (A) or as % of control (B) at the y-axis. Half maximal inhibitory concentrations (IC 50 ) were calculated from the data expressed as % of control by nonlinear regression analysis using the equation log(inhibitor) vs. response (GraphPad Prism 6). Absolute transport rates are expressed as dpm. Data represent means ± SD of two independent experiments, each with quadruplicate determinations (n = 8). ÃSignificantly lower transport rates with P < 0.01 (two-way ANOVA with Sidak's multiple comparisons post hoc test). DMEM, Dulbecco's modified Eagle's medium; HEK293, human embryonic kidney cells; NTCP, Na þ /taurocholate cotransporting polypeptide; TC, taurocholic acid; TLC, taurolithocholic acid.
after prolonged preincubation up to 6 h (Fig. 3, A and C). Interestingly, TLC preincubation did not inhibit all functions of NTCP. Although the myr-preS1 binding competence as well as the transport activity for the NTCP substrates TC and DHEAS were significantly reduced, this was not the case for TLC itself (Fig. 2B). This indicates that the NTCP protein is not entirely inactivated, for example, by covalent protein modifications. NTCP is instead selectively inhibited but still active in transporting TLC as a substrate. We hypothesized that TLC does not share the typical binding site and transport route of the bile acid and steroid substrates of NTCP. Thus, TLC transport is still maintained even when TLC preincubation inhibits transport of TC and DHEAS. Another interesting observation was that the inhibitory effect of TLC on the transporter and receptor function of NTCP was longlasting (Fig. 3B). Even under very long incubation of the NTCP-HEK293 cells in the absence of TLC for up to 8 h, the same inhibitory effect was observed as directly after preincubation and washing. Therefore, this long-lasting effect might indicate that TLC trans-binding to NTCP occurs with high affinity.
Another question was if reflux of TLC after the preincubation phase may be responsible for the inhibitory effect and so would only mimic a cis-inhibition. However, in respective control experiments, only trace amounts of TCL refluxed from the cells after washout that would not be sufficient to explain the measured effects by cis-inhibition alone (Fig. 4,  A and D). This was most pronounced in the HDV infection experiments, where TLC concentrations below 10 mM had only minimal effects in cis-inhibition experiments (Fig. 5C). These concentrations would be achieved at most in the extracellular medium after preincubation with 250 mM TLC. In contrast, preincubation with 250 mM TLC completely abolished HDV infection in the trans-inhibition approach (Fig.  4E). These findings go in line with the observation that TLC only gains its trans-inhibitory potential when the preincubation is performed in sodium-containing buffer, which is the prerequisite for NTCP-mediated TLC uptake and accumulation in the preincubation phase (Fig. 2). In contrast, under sodium-free preincubation conditions, TLC largely lost its inhibitory potential, because its uptake via NTCP is abolished in the absence of sodium.
Finally, the question was addressed if the trans-inhibition of NTCP after TLC preincubation only affects the transport rates of the extracellularly provided substrate or if, in addition, the binding kinetics of an extracellular ligand were altered. Bile acids typically show competitive inhibition at the same extracellular binding site after coincubation, that is, when both bile acids are provided from the extracellular site of the transporter (7,24,45). However, in the experimental setup of the present study, TLC was first accumulated in the cells and washed out from the extracellular compartment, so that TLC is supposed to bind to an intracellularly accessible domain of NTCP (trans), whereas the transporter substrates and the myr-preS1 peptide were provided from the extracellular site (cis). In addition to the extracellularly provided transporter substrate [ 3 H]TC, different compounds were coincubated together with [ 3 H]TC to verify if these still interfere competitively with the transporter function of NTCP, even under trans-inhibition with TLC. As shown in Fig. 6, UDC, irbesartan, and myr-preS1 all inhibited the [ 3 H] TC transport rate with comparable inhibition kinetics, irrespective of TLC preincubation. Nevertheless, the absolute transport rate of [ 3 H]TC was reduced significantly by TLC preincubation. These data clearly indicate that TLC represents a long-lasting trans-acting noncompetitive inhibitor of the transporter and HBV/HDV receptor function of NTCP. Furthermore, these data demonstrate that NTCP is still functionally active, so inactivation of NTCP by posttranslational modifications or abrogation from the plasma membrane can be excluded.
Trans-inhibition of membrane transporters by their substrates has previously been reported for other carriers, including ion transporters (49), ATP-binding cassette (ABC) transporters like human TAPL (50), ModBC from Methanosarcina acetivorans (51), TmrAB from Thermus thermophilus (52), and the ABC importer MetNI from Escherichia coli (53). Furthermore, trans-inhibition has been described for various amino acid transporters (54)(55)(56)(57). Also, bile acid transporters from the OATP family have been reported to be trans-inhibited, for example, by cyclosporine A (CsA) (58). Similar to the present study, CsA acted as cis-inhibitor during coincubation with the substrate as well as trans-inhibitor after preincubation and washing before the transport experiments. In the case of OATP1B1 and OATP1B3, CsA preincubation induced a concentration-dependent and long-lasting inhibitory effect on the transport function over at least 16 h (58, 59). From the group of ABC transporters, Stieger et al. showed transinhibition of rat Bsep by the cholestatic estrogen metabolite estradiol-17b-glucuronide (E 2 17G) (60,61). In addition, Akita et al. (62) observed that sulfated bile acids show transinhibition of rat Bsep. Most trans-inhibitors described in these publications showed similar inhibition patterns, including time-and dose dependency of the inhibitory effect, rapid and long-lasting inhibition of the respective transporter, as well as high-affinity binding of the inhibitor to the trans-inhibition site of the transporter. In the present study, all these characteristics were also observed for the NTCP trans-inhibitor TLC, which showed a time-and dose-dependent inhibition of the bile acid transport. Furthermore, trans-inhibition was maintained over at least 8 h after removing of TLC from the incubation medium and, therefore, was long-lasting (Fig. 3B).
Blocking of HBV/HDV infection of hepatocytes by preventing virus binding to NTCP or blocking of endocytosis of the virus/NTCP receptor complex is a promising novel treatment strategy for acute and chronic HBV/HDV infections. This might be achieved by downregulation of NTCP expression in the hepatocytes, for example, by tumor necrosis factor-a or interleukin-6 (63, 64). In addition, retrieval of NTCP from the plasma membrane by induction of clathrin-or caveolin-dependent endocytosis of NTCP might be a option (65,66). However, most approaches focus on the cis-inhibition of the virus binding to NTCP, which can be achieved by peptide-based drugs such as Myrcludex B, mimicking the myr-preS1 peptide, or by small molecules that address the extracellular myr-preS1-and/or bile acids-binding site of NTCP (64,67). Myrcludex B provided proof-of-concept that NTCPdirected inhibition can effectively suppress HBV and HDV infection and was very recently approved as the first drug treatment of HDV-infected patients in Europe and Russia (68). Based on the data of the present study, we suggest a novel potential target site of trans-acting NTCP inhibitors that can block virus binding to NTCP and, thus, may prevent HBV/HDV infection. Although TLC as a cholestatic bile acid is not an appropriate candidate, other compounds could mimic this long-lasting, trans-inhibitory effect of TLC and could be even more effective then short-acting cis-inhibitors of NTCP. To define this potential trans-located binding site of NTCP more closely, 3-D structural information of the human NTCP protein would be needed and would provide the possibility to address this target site by docking strategies.
In conclusion, we found that even after short pulse preincubation, TLC had a potent and long-lasting inhibitory effect on the transporter function of NTCP, while NTCP was still present at the plasma membrane. Furthermore, binding of the HBV/HDV myr-preS1 peptide and susceptibility for in vitro HDV infection were significantly reduced. We hypothesize that TLC rapidly accumulates in hepatocytes and mediates long-lasting trans-inhibition of the transporter and receptor function of NTCP via a particular TLC-binding site at an intracellularly accessible domain of NTCP. Physiologically, this trans-inhibition might protect hepatocytes from toxic overload of bile acids. Pharmacologically, it provides a promising novel NTCP target site for potential longacting HBV/HDV entry inhibitors.