This research project is supported by Mahidol University and the Thailand Science Research and Innovation (TSRI) separately awarded to A. Wongkajornsilp and K. Sa-ngiamsuntorn. This work was financially supported by the Office of National Higher Education Science Research and Innovation Policy Council through the Program Management Unit for Competitiveness (grant number C10F630093). A. Wongkajornsilp is a recipient of a Chalermprakiat grant of the Faculty of Medicine Siriraj Hospital, Mahidol University. The authors would like to thank Miss Sawinee Seemakhan (Excellent Center for Drug Discovery, Faculty of Science, Mahidol University) for her assistance with the ITC technique.

<ol><li>Levrero, M., Zucman-Rossi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+of+HBV-induced+hepatocellular+carcinoma%5BTitle%5D)+AND+%22Journal+of+Hepatology%22%5BJournal%5D)">Mechanisms of HBV-induced hepatocellular carcinoma.</a> Journal of Hepatology. 64 (1), 84-101 (2016).</li>
<li>Kim, K. -H., Kim, N. D., Seong, B. -L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Discovery+and+development+of+anti-HBV+agents+and+their+resistance%5BTitle%5D)+AND+%22Molecules%22%5BJournal%5D)">Discovery and development of anti-HBV agents and their resistance.</a> Molecules. 15 (9), 5878-5908 (2010).</li>
<li>Shaw, T., Bowden, S., Locarnini, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemotherapy+for+hepatitis+B%3A+New+treatment+options+necessitate+reappraisal+of+traditional+endpoints%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">Chemotherapy for hepatitis B: New treatment options necessitate reappraisal of traditional endpoints.</a> Gastroenterology. 123 (6), 2135-2140 (2002).</li>
<li>Volz, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+entry+inhibitor+Myrcludex-B+efficiently+blocks+intrahepatic+virus+spreading+in+humanized+mice+previously+infected+with+hepatitis+B+virus%5BTitle%5D)+AND+%22Journal+of+Hepatology%22%5BJournal%5D)">The entry inhibitor Myrcludex-B efficiently blocks intrahepatic virus spreading in humanized mice previously infected with hepatitis B virus.</a> Journal of Hepatology. 58 (5), 861-867 (2013).</li>
<li>Mak, L. -Y., Seto, W. -K., Yuen, M. -F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+antivirals+in+clinical+development+for+chronic+hepatitis+B+infection%5BTitle%5D)+AND+%22Viruses%22%5BJournal%5D)">Novel antivirals in clinical development for chronic hepatitis B infection.</a> Viruses. 13 (6), 1169(2021).</li>
<li>Zuccaro, V., Asperges, E., Colaneri, M., Marvulli, L. N., Bruno, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HBV+and+HDV%3A+New+Treatments+on+the+Horizon%5BTitle%5D)+AND+%22Journal+of+Clinical+Medicine%22%5BJournal%5D)">HBV and HDV: New Treatments on the Horizon.</a> Journal of Clinical Medicine. 10 (18), 4054(2021).</li>
<li>Iwamoto, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+and+identification+of+hepatitis+B+virus+entry+inhibitors+using+HepG2+cells+overexpressing+a+membrane+transporter+NTCP%5BTitle%5D)+AND+%22Biochemical+and+Biophysical+Research+Communications%22%5BJournal%5D)">Evaluation and identification of hepatitis B virus entry inhibitors using HepG2 cells overexpressing a membrane transporter NTCP.</a> Biochemical and Biophysical Research Communications. 443 (3), 808-813 (2014).</li>
<li>Tong, S., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+NTCP+as+an+HBV+receptor%3A+the+beginning+of+the+end+or+the+end+of+the+beginning%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">Identification of NTCP as an HBV receptor: the beginning of the end or the end of the beginning.</a> Gastroenterology. 146 (4), 902-905 (2014).</li>
<li>Xuan, J., Chen, S., Ning, B., Tolleson, W. H., Guo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+HepG2-derived+cells+expressing+cytochrome+P450s+for+assessing+metabolism-associated+drug-induced+liver+toxicity%5BTitle%5D)+AND+%22Chemico-Biological+Interactions%22%5BJournal%5D)">Development of HepG2-derived cells expressing cytochrome P450s for assessing metabolism-associated drug-induced liver toxicity.</a> Chemico-Biological Interactions. 255, 63-73 (2016).</li>
<li>Thongsri, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Curcumin+inhibited+hepatitis+B+viral+entry+through+NTCP+binding%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Curcumin inhibited hepatitis B viral entry through NTCP binding.</a> Scientific Reports. 11 (1), 19125(2021).</li>
<li>Sa-Ngiamsuntorn, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+immortalized+hepatocyte-like+cell+line+%28imHC%29+accommodated+complete+viral+lifecycle%2C+viral+persistence+form%2C+cccDNA+and+eventual+spreading+of+a+clinically-isolated+HBV%5BTitle%5D)+AND+%22Viruses%22%5BJournal%5D)">An immortalized hepatocyte-like cell line (imHC) accommodated complete viral lifecycle, viral persistence form, cccDNA and eventual spreading of a clinically-isolated HBV.</a> Viruses. 11 (10), 952(2019).</li>
<li>Watashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cyclosporin+A+and+its+analogs+inhibit+hepatitis+B+virus+entry+into+cultured+hepatocytes+through+targeting+a+membrane+transporter%2C+sodium+taurocholate+cotransporting+polypeptide+%28NTCP%29%5BTitle%5D)+AND+%22Hepatology%22%5BJournal%5D)">Cyclosporin A and its analogs inhibit hepatitis B virus entry into cultured hepatocytes through targeting a membrane transporter, sodium taurocholate cotransporting polypeptide (NTCP).</a> Hepatology. 59 (5), 1726-1737 (2014).</li>
<li>Kaneko, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+tricyclic+polyketide%2C+Vanitaracin+A%2C+specifically+inhibits+the+entry+of+hepatitis+B+and+D+viruses+by+targeting+sodium+taurocholate+cotransporting+polypeptide%5BTitle%5D)+AND+%22Journal+of+Virology%22%5BJournal%5D)">A novel tricyclic polyketide, Vanitaracin A, specifically inhibits the entry of hepatitis B and D viruses by targeting sodium taurocholate cotransporting polypeptide.</a> Journal of Virology. 89 (23), 11945-11953 (2015).</li>
<li>Manta, B., Obal, G., Ricciardi, A., Pritsch, O., Denicola, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tools+to+evaluate+the+conformation+of+protein+products%5BTitle%5D)+AND+%22Biotechnology+Journal%22%5BJournal%5D)">Tools to evaluate the conformation of protein products.</a> Biotechnology Journal. 6 (6), 731-741 (2011).</li>
<li>Martinez Molina, D., Nordlund, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+cellular+thermal+shift+assay%3A+a+novel+biophysical+assay+for+in+situ+drug+target+engagement+and+mechanistic+biomarker+studies%5BTitle%5D)+AND+%22Annual+Review+of+Pharmacology+and+Toxicology%22%5BJournal%5D)">The cellular thermal shift assay: a novel biophysical assay for in situ drug target engagement and mechanistic biomarker studies.</a> Annual Review of Pharmacology and Toxicology. 56, 141-161 (2016).</li>
<li>Appelman, M. D., Chakraborty, A., Protzer, U., McKeating, J. A., van de Graaf, S. F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((N-Glycosylation+of+the+Na%2B-taurocholate+cotransporting+polypeptide+%28NTCP%29+determines+its+trafficking+and+stability+and+is+required+for+hepatitis+B+virus+infection%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">N-Glycosylation of the Na+-taurocholate cotransporting polypeptide (NTCP) determines its trafficking and stability and is required for hepatitis B virus infection.</a> PLoS One. 12 (1), 0170419(2017).</li>
<li>Tsukuda, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+class+of+hepatitis+B+and+D+virus+entry+inhibitors%2C+proanthocyanidin+and+its+analogs%2C+that+directly+act+on+the+viral+large+surface+proteins%5BTitle%5D)+AND+%22Hepatology%22%5BJournal%5D)">A new class of hepatitis B and D virus entry inhibitors, proanthocyanidin and its analogs, that directly act on the viral large surface proteins.</a> Hepatology. 65 (4), 1104-1116 (2017).</li>
<li>Klumpp, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-resolution+crystal+structure+of+a+hepatitis+B+virus+replication+inhibitor+bound+to+the+viral+core+protein%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">High-resolution crystal structure of a hepatitis B virus replication inhibitor bound to the viral core protein.</a> Proceedings of the National Academy of Sciences. 112 (49), 15196-15201 (2015).</li>
<li>Donkers, J. M., Appelman, M. D., van de Graaf, S. F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanistic+insights+into+the+inhibition+of+NTCP+by+myrcludex+B%5BTitle%5D)+AND+%22JHEP+Reports%22%5BJournal%5D)">Mechanistic insights into the inhibition of NTCP by myrcludex B.</a> JHEP Reports. 1 (4), 278-285 (2019).</li>
<li>Saso, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+strategy+to+identify+hepatitis+B+virus+entry+inhibitors+by+AlphaScreen+technology+targeting+the+envelope-receptor+interaction%5BTitle%5D)+AND+%22Biochemical+and+Biophysical+Research+Communications%22%5BJournal%5D)">A new strategy to identify hepatitis B virus entry inhibitors by AlphaScreen technology targeting the envelope-receptor interaction.</a> Biochemical and Biophysical Research Communications. 501 (2), 374-379 (2018).</li>
<li>Baranauskiene, L., Kuo, T. C., Chen, W. Y., Matulis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isothermal+titration+calorimetry+for+characterization+of+recombinant+proteins%5BTitle%5D)+AND+%22Current+Opinion+in+Biotechnology%22%5BJournal%5D)">Isothermal titration calorimetry for characterization of recombinant proteins.</a> Current Opinion in Biotechnology. 55, 9-15 (2019).</li>
<li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure-based+virtual+screening+protocol+for+in+silico+identification+of+potential+thyroid+disrupting+chemicals+targeting+transthyretin%5BTitle%5D)+AND+%22Environmental+Science+%26+Technology%22%5BJournal%5D)">Structure-based virtual screening protocol for in silico identification of potential thyroid disrupting chemicals targeting transthyretin.</a> Environmental Science & Technology. 50 (21), 11984-11993 (2016).</li>
<li>Duff, J. M. R., Grubbs, J., Howell, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isothermal+titration+calorimetry+for+measuring+macromolecule-ligand+affinity%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%3A+JoVE%22%5BJournal%5D)">Isothermal titration calorimetry for measuring macromolecule-ligand affinity.</a> Journal of Visualized Experiments: JoVE. (55), e2796(2011).</li>
<li>Du, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insights+into+protein-ligand+interactions%3A+mechanisms%2C+models%2C+and+methods%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Sciences%22%5BJournal%5D)">Insights into protein-ligand interactions: mechanisms, models, and methods.</a> International Journal of Molecular Sciences. 17 (2), 144(2016).</li>
<li>Sureau, C., Salisse, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+conformational+heparan+sulfate+binding+site+essential+to+infectivity+overlaps+with+the+conserved+hepatitis+B+virus+A-determinant%5BTitle%5D)+AND+%22Hepatology%22%5BJournal%5D)">A conformational heparan sulfate binding site essential to infectivity overlaps with the conserved hepatitis B virus A-determinant.</a> Hepatology. 57 (3), 985-994 (2013).</li>
<li>Herrscher, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hepatitis+B+virus+entry+into+HepG2-NTCP+cells+requires+clathrin-mediated+endocytosis%5BTitle%5D)+AND+%22Cellular+Microbiology%22%5BJournal%5D)">Hepatitis B virus entry into HepG2-NTCP cells requires clathrin-mediated endocytosis.</a> Cellular Microbiology. 22 (8), 13205(2020).</li>
<li>Gripon, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Infection+of+a+human+hepatoma+cell+line+by+hepatitis+B+virus%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">Infection of a human hepatoma cell line by hepatitis B virus.</a> Proceedings of the National Academy of Sciences. 99 (24), 15655-15660 (2002).</li>
<li>Mayati, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+polarization+of+human+hepatoma+HepaRG+cells+in+response+to+forskolin%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Functional polarization of human hepatoma HepaRG cells in response to forskolin.</a> Scientific Reports. 8 (1), 16115(2018).</li>
<li>Sells, M. A., Chen, M. L., Acs, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+of+hepatitis+B+virus+particles+in+Hep+G2+cells+transfected+with+cloned+hepatitis+B+virus+DNA%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA.</a> Proceedings of the National Academy of Sciences of the United States of America. 84 (4), 1005-1009 (1987).</li>
<li>Freyer, M. W., Lewis, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isothermal+titration+calorimetry%3A+experimental+design%2C+data+analysis%2C+and+probing+macromolecule%2Fligand+binding+and+kinetic+interactions%5BTitle%5D)+AND+%22Methods+in+Cell+Biology%22%5BJournal%5D)">Isothermal titration calorimetry: experimental design, data analysis, and probing macromolecule/ligand binding and kinetic interactions.</a> Methods in Cell Biology. 84, 79-113 (2008).</li>
<li>Srivastava, V. K., Yadav, R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSrivastava%2C+VK+%22Data+Processing+Handbook+for+Complex+Biological+Data+Sources%22">Data Processing Handbook for Complex Biological Data Sources</a>. Misra, G. , Academic Press. 125-137 (2019).</li>
<li>Seeger, C., Mason, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sodium-dependent+taurocholic+cotransporting+polypeptide%3A+a+candidate+receptor+for+human+hepatitis+B+virus%5BTitle%5D)+AND+%22Gut%22%5BJournal%5D)">Sodium-dependent taurocholic cotransporting polypeptide: a candidate receptor for human hepatitis B virus.</a> Gut. 62 (8), 1093-1095 (2013).</li>
<li>Seeger, C., Sohn, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+hepatitis+B+virus+with+CRISPR%2FCas9%5BTitle%5D)+AND+%22Molecular+Therapy+-+Nucleic+Acids%22%5BJournal%5D)">Targeting hepatitis B virus with CRISPR/Cas9.</a> Molecular Therapy - Nucleic Acids. 3, 216(2014).</li>
<li>Ni, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hepatitis+B+and+D+viruses+exploit+sodium+taurocholate+co-transporting+polypeptide+for+species-specific+entry+into+hepatocytes%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes.</a> Gastroenterology. 146 (4), 1070-1083 (2014).</li>
<li>Chai, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Properties+of+subviral+particles+of+hepatitis+B+virus%5BTitle%5D)+AND+%22Journal+of+Virology%22%5BJournal%5D)">Properties of subviral particles of hepatitis B virus.</a> Journal of Virology. 82 (16), 7812-7817 (2008).</li>
<li>Moore, A., Chothe, P. P., Tsao, H., Hariparsad, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+the+interplay+between+uptake+transport+and+CYP3A4+induction+in+micropatterned+cocultured+hepatocytes%5BTitle%5D)+AND+%22Drug+Metabolism+and+Disposition%22%5BJournal%5D)">Evaluation of the interplay between uptake transport and CYP3A4 induction in micropatterned cocultured hepatocytes.</a> Drug Metabolism and Disposition. 44 (12), 1910-1919 (2016).</li>
<li>Parvez, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plant-derived+antiviral+drugs+as+novel+hepatitis+B+virus+inhibitors%3A+Cell+culture+and+molecular+docking+study%5BTitle%5D)+AND+%22Saudi+Pharmaceutical+Journal%22%5BJournal%5D)">Plant-derived antiviral drugs as novel hepatitis B virus inhibitors: Cell culture and molecular docking study.</a> Saudi Pharmaceutical Journal. 27 (3), 389-400 (2019).</li>
</ol>

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

HBV infection is initiated via low-affinity binding to heparan sulfate proteoglycans (HSPGs) on hepatocytes25, followed by the binding to NTCP with subsequent internalization through endocytosis26. As NTCP is a crucial receptor for HBV entry, targeting HBV entry can be clinically translated to diminish de novo infection, mother-to-child transmission (MTCT), and recurrence after liver transplantation. Interrupting viral entry would be a feasible alternative cure for chronic HBV infection.
Some critical steps in the above-mentioned protocol are summarized here. While preparing host cells, ensure that hepatocytes reach 100% confluency before adding the maturation medium with 2% DMSO. Culturing subconfluent hepatocytes in 2% DMSO will lead to cell death and detachment. The maturation period can be extended up to 4 weeks to maximize NTCP expression27,28, although a 2 week maturation period is recommended.
Three techniques have been proposed for the quantification of cellular NTCP expression. It is recommended that users select the technique they are most familiar with; here, we preferred flow cytometry to western blot analysis because it is convenient, quick, and easy.
For the production of the cell culture-derived HBV particles (HBVcc), HepG2.2.15 was derived from HepG2 by transient transfection with HBV full-length genome together with the geneticin (G418) resistance gene29. The culture medium should contain 380-500 &#181;g/mL geneticin, otherwise the cells will not produce HBVcc. The low-protein-binding, 0.45 &#181;m&#160;filter should be used to clarify the supernatant to avoid losing HBV yield. For concentrating HBV, polyethylene glycol (PEG) was used with a standard centrifuge. HBV particles can also be concentrated using a sucrose gradient and ultracentrifugation. Multiple freeze and thaw cycles should be avoided since the infectivity will be decreased.
In the anti-HBV entry assay, the hepatocytes must reach 100% confluence before maturation induction. This protocol was developed based on prophylactic treatment. Host cells were exposed to the candidate compounds for 2 h prior to infection with HBV12. After 7 days post infection, HBV infectivity was evaluated using immunofluorescence. All components, concentrations of the blocking solution, primary and secondary antibodies, and washing steps must be optimized to achieve the best result with minimal non-specific staining. Here, we have provided optimized concentrations and techniques.
The TCA uptake assay protocol describes the gold standard for the inhibition of NTCP transport. We modified the protocol to avoid radioactive TCA. The critical steps for the TCA uptake assay are cell washing and harvesting. All these procedures must be performed on ice all the time to prevent further cellular activity-internalization of the compound. After homogenizing the cells and pelleting cell debris, the supernatant must be gently aspirated without disrupting the pellet. If the user&#39;s facility permits the use of the liquid scintillation technique, 3H-taurocholic acid is commonly used in TCA transport and uptake studies. As we validated TCA uptake using ELISA, this alternative technique can substitute the use of the radioactive compound.
ITC is a biophysical method widely used to determine the thermodynamic parameters linked to the interactions between soluble proteins and small molecular weight ligands30,31. This technique can be used to investigate the interaction of various ligands with a small molecule or protein. ITC is a direct and noninvasive method with no additional steps for signal detection, no molecular weight limitations, and no labeling, immobilization, or any other chemical modification. The limitation of ITC is the prerequisite of high concentrations of the interacting materials. Protein and ligand have to be highly purified and sufficiently soluble in water (10-100 &#181;M) at 25 &#176;C for 1 h with stirring. The unstable protein solution can be detected through heat signal change as a function of time.
Human enhanced-NTCP hepatocytes provide an alternative model for the in vitro study of HBV infection but are similar to HepaRG and primary human hepatocytes. These host models are hampered by their limited reliability and susceptibility8,33. The inefficient HBV propagation in HepG2-NTCP or Huh7-NTCP cells was evidenced by low HBV inoculum derived from HBVcc-producing cells. In several studies, HBV was used to infect the target cells at an MOI of 1,00033,34. The subviral particles in HBVcc contain HBV envelope protein (L/M/S) and competitively bind NTCP, resulting in decreased infectivity35. To overcome this limitation, this protocol modified HepaRG or imHC culture with a maturation protocol to maximize NTCP levels. HBVcc derived from HepG2.2.15 was concentrated before being used as inoculum. The HBV infectivity was higher than 80% in differentiated imHC based on the measurement of the HBV core antigen11.
We have described the identification of anti-HBV compounds targeting NTCP to interrupt viral entry and adopted a hepatic maturation procedure to increase NTCP levels. To identify entry inhibitors, hepatocytes were pretreated with candidate compounds for 2 h before the infection with HBV at 4 &#176;C to allow viral binding but not entry. The decrease in HBV infectivity was evaluated on day 7 post infection. The representative data included cyclosporine A, a classical NTCP inhibitor, as a positive control to validate the model.
Since NTCP facilitates both transporter- and receptor-mediated endocytosis functions, HBV entry inhibitors could target at least one of these mechanisms. The inhibition of NTCP transport can be evaluated using a TCA uptake assay based on ELISA, eliminating the radioactive TCA from the classical protocol36. The affinity between NTCP and the entry inhibitor was measured using ITC. This technique provided essential thermodynamic parameters, including the stoichiometry (n), dissociation constant (KD), change in free energy (&#916;G), enthalpy (&#916;H), entropy (&#916;S), and heat capacity of binding (&#916;Cp). Various anti-HBV agents have been identified by molecular docking, such as quercetin, rutin, hesperidin, and luperol, that could specifically bind HBV reverse transcriptase comparable to lamivudine, a nucleoside analog. The compounds were investigated using ITC and exhibited negative Gibb energy (&#8722;&#916;G) ranging from &#8722;9.3 to &#8722;5.2 kcal/mol and KD of 1 &#215; 10-6 to 1 &#215; 10-3 M with HBV polymerase37. Taken together, the data obtained from these aforementioned assays will help screen the antiviral efficacy of candidate compounds that act as HBV entry inhibitors. The established protocol will be useful for discovering novel NTCP antagonists/inhibitors. The suppression of viral entry could be useful in prophylactic and therapeutic treatment.

Hepatitis B virus (HBV) infection is considered a life-threatening disease worldwide. Chronic HBV infection is laden with a risk of liver cirrhosis and hepatocellular carcinoma1. Current anti-HBV treatment focuses mostly on post viral entry using nucleos(t)ide analogs (NAs) and interferon-alpha (IFN-&#945;)2,3. The discovery of an HBV entry inhibitor, Myrcludex B, has identified a novel target for anti-HBV agents4. The combination of entry inhibitors and NAs in chronic HBV has significantly lessened the viral load compared to those targeting viral replication alone5,6. However, the classical hepatocyte model for the screening of HBV entry inhibitors is limited by low viral receptor levels (sodium taurocholate cotransporting polypeptide, NTCP). The overexpression of hNTCP in hepatoma cells (i.e., HepG2 and Huh7) improves HBV infectivity7,8. Nevertheless, these cell lines express low levels of phase I and II drug-metabolizing enzymes and exhibit genetic instability9. Hepatocyte models that can help target distinct mechanisms of candidate anti-HBV compounds such as previral entry, NTCP binding, and viral entry would expedite the identification and development of efficacious combination regimens. The study for anti-HBV activity of curcumin has elucidated the inhibition of viral entry as a new mechanism in addition to post viral entry interruption. This protocol details a host model for the screening of anti-HBV entry molecules10.
The goal of this method is to explore candidate anti-HBV compounds for viral entry inhibition, especially blocking NTCP binding and transport. As NTCP expression is a critical factor for HBV entry and infection, we optimized the hepatocyte maturation protocol to maximize NTCP levels11. In addition, this protocol can differentiate the inhibitory effect on HBV entry as inhibition of HBV attachment versus inhibition of internalization. The taurocholic acid (TCA) uptake assay was also modified using an ELISA-based method instead of a radioisotope to represent NTCP transport12,13. The receptor and ligand interaction was confirmed by their 3D structures14,15. The inhibition of NTCP function can be evaluated by measuring TCA uptake activity16. However, this technique did not provide direct evidence of NTCP binding to the candidate inhibitors. Therefore, the binding can be investigated using various techniques, such as surface plasmon resonance17, ELISA, fluorescence-based thermal shift assay (FTSA)18, FRET19, AlphaScreen, and various other methods20. Among these techniques, ITC is a goal standard in binding analysis because it can observe heat absorption or emission in almost every reaction21. The binding affinity (KD) of NTCP and candidate compounds was directly evaluated using ITC; these affinity values were more precise than those obtained using the in silico prediction model22.
This protocol covers techniques in hepatocyte maturation, HBV infection, and screening for HBV entry inhibitor. Briefly, a hepatocyte model was developed based on imHC and HepaRG cell lines. The cultured cells were differentiated into mature hepatocytes within 2 weeks. The upregulation of NTCP levels was detected using real-time PCR, western blot, and flow cytometry11. Hepatitis B virion (HBVcc) was produced and collected from HepG2.2.15. The differentiated imHC or HepaRG (d-imHC, d-HepaRG) was prophylactically treated with the anti-HBV candidates 2 h prior to the inoculation with HBV virion. The expected outcome of the experiment was the identification of the agents that decrease cellular HBV and infectivity. Anti-NTCP activity was evaluated using the TCA uptake assay. NTCP activity could be suppressed by the agents that specifically bound NTCP. The ITC technique was employed to investigate the feasibility of interactive binding that could predict inhibitors and their target proteins, determining the binding affinity (KD) of the ligand for the receptor via non-covalent interactions of the biomolecular complex23,24. For instance, KD&#160;&#8805; 1 &#215; 103 mM represents weak binding, KD&#160;&#8805; 1 &#215; 106 &#181;M represents moderate binding, and KD&#160;&#8804; 1 &#215; 109 nM represents strong binding. The &#916;G is directly correlated with binding interactions. In particular, a reaction with negative &#916;G is an exergonic reaction, indicating that binding is a spontaneous process. A reaction with a negative &#916;H indicates that the binding processes depend on hydrogen bonding and Van der Waals forces. Both TCA uptake and ITC data could be used to screen for anti-HBV entry agents. The outcomes of these protocols can provide a foundation for not only anti-HBV screening but also the interaction with NTCP as assessed through binding affinity and transport function. This paper describes host cell preparation and characterization, experimental design, and evaluation of the anti-HBV entry together with the NTCP binding affinity.

<table><tbody><tr><td>&lt;strong&gt;Cell lines&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>HepaRG Cells, Cryopreserved</td><td>Thermo Fisher Scientific</td><td>HPRGC10</td><td></td></tr><tr><td>Hep-G2/2.2.15 Human Hepatoblastoma Cell Line</td><td>Merck</td><td>SCC249</td><td></td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>4% Paraformadehyde Phosphate Buffer Solution</td><td>FUJIFLIM Wako chemical</td><td>163-20145</td><td></td></tr><tr><td>BD Perm/Wash buffer</td><td>BD Biosciences</td><td>554723</td><td>Perm/Wash buffer</td></tr><tr><td>Cyclosporin A</td><td>abcam</td><td>59865-13-3</td><td></td></tr><tr><td>EDTA</td><td>Invitrogen</td><td>15575-038</td><td>8 mM</td></tr><tr><td>G 418 disulfate salt</td><td>Merck</td><td>108321-42-2</td><td></td></tr><tr><td>Halt Protease Inhibitor Cocktail&amp;nbsp; EDTA-free (100x)</td><td>Thermo Scientific</td><td>78425</td><td></td></tr><tr><td>HEPES</td><td>Merck</td><td>7365-45-9</td><td></td></tr><tr><td>illustraTM RNAspin Mini RNA isolation kits</td><td>GE Healthcare</td><td>25-0500-71</td><td></td></tr><tr><td>illustra RNAspin Mini RNA Isolation Kit</td><td>GE Healthcare</td><td>25-0500-71</td><td></td></tr><tr><td>ImProm-II Reverse Transcription System</td><td>Promega</td><td>A3800</td><td></td></tr><tr><td>KAPA SYBR FAST qPCR Kit</td><td>Kapa Biosystems</td><td>KK4600</td><td></td></tr><tr><td>Lenti-X Concentrator</td><td>Takara bio</td><td>PT4421-2</td><td>concentrator</td></tr><tr><td>Luminata crescendo Western HRP substrate</td><td>Merck</td><td>WBLUR0100</td><td></td></tr><tr><td>Master Mix (2x) Universal</td><td>Kapa Biosystems</td><td>KK4600</td><td></td></tr><tr><td>Nucleospin DNA extraction kit</td><td>macherey-nagel</td><td>1806/003</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Merck</td><td>P3813</td><td></td></tr><tr><td>Polyethylene glycol 8000</td><td>Merck</td><td>25322-68-3</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant</td><td>Thermo scientific</td><td>P36930</td><td></td></tr><tr><td>Recombinant NTCP</td><td>Cloud-Clone</td><td>RPE421Hu02</td><td></td></tr><tr><td>RIPA Lysis Buffer (10x)</td><td>Merck</td><td>20-188</td><td></td></tr><tr><td>TCA</td><td>Sigma</td><td>345909-26-4</td><td></td></tr><tr><td>TCA Elisa kit</td><td>Mybiosource</td><td>MB2033685</td><td></td></tr><tr><td>Triton X-100</td><td>Merck</td><td>9036-19-5</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td>25200072</td><td>Dilute to 0.125%</td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; Anti-NTCP1 antibody</td><td>Abcam</td><td>ab131084</td><td>1:100 dilution</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; Anti-GAPDH antibody</td><td>Thermo Fisher Scientific</td><td>AM4300</td><td>1:200,000 dilution</td></tr><tr><td>&amp;nbsp;&amp;nbsp; HRP-conjugated goat anti-rabbit antibody</td><td>Abcam</td><td>ab205718</td><td>1:10,000 dilution</td></tr><tr><td>&amp;nbsp;&amp;nbsp; HRP goat anti-mouse secondary antibody</td><td>Abcam</td><td>ab97023</td><td>1:10,000 dilution</td></tr><tr><td>&amp;nbsp;&amp;nbsp; Goat anti-Rabbit IgG Secondary Antibody, Alexa Fluor 488</td><td>Invitrogen</td><td>A-11008</td><td>1:500 dilution</td></tr><tr><td>&lt;strong&gt;Reagent composition&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;1&amp;deg; Antibody dilution buffer&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 1x TBST</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 3% BSA</td><td>Sigma</td><td>A7906-100G</td><td>Working concentration: 3%</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Sodium azide</td><td>Sigma</td><td>199931</td><td>Working concentration: 0.05%</td></tr><tr><td>&lt;strong&gt;Hepatocyte Growth Medium&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; DME/F12</td><td>Gibco</td><td>12400-024</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 10% FBS</td><td>Sigma Aldrich</td><td>F7524</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 1% Pen/Strep</td><td>HyClon</td><td>SV30010</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 1% GlutaMAX</td><td>Gibco</td><td>35050-061</td><td></td></tr><tr><td>&lt;strong&gt;Hepatic maturation medium&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Williams&amp;rsquo; E medium</td><td>Sigma Aldrich</td><td>W4125-1L</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 10% FBS</td><td>Sigma Aldrich</td><td>F7524</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 1% Pen/Strep</td><td>HyClon</td><td>SV30010</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 1% GlutaMAX</td><td>Gibco</td><td>35050-061</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 5 &amp;micro;g/mL&amp;nbsp; Insulin</td><td>Sigma Aldrich</td><td>91077C-100MG</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 50 &amp;micro;M hydrocotisone</td><td>Sigma Aldrich</td><td>H0888-1g</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 2% DMSO</td><td>PanReac AppliChem</td><td>A3672-250ml</td><td></td></tr><tr><td>&lt;strong&gt;IF Blocking solution&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 1x PBS</td><td>Gibco</td><td>21300-058</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 3% BSA</td><td>Sigma</td><td>A7906-100G</td><td>Working concentration: 3%</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 0.2% Triton X-100</td><td>Sigma</td><td>T8787</td><td>Working concentration: 0.2%</td></tr><tr><td>&lt;strong&gt;RIPA Lysis Buffer Solution&lt;/strong&gt;</td><td>Merck</td><td>20-188</td><td>Final concentration: 1X</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Protease Inhibitor Cocktail</td><td>Thermo Scientific</td><td>78425</td><td>Final concentration: 1X</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Na&lt;sub&gt;3&lt;/sub&gt;VO&lt;sub&gt;4&lt;/sub&gt;</td><td></td><td></td><td>Final concentration: 1 mM</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; PMSF</td><td></td><td></td><td>Final concentration: 1 mM</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; NaF</td><td></td><td></td><td>Final concentration: 10 mM</td></tr><tr><td>&lt;strong&gt;Western blot reagent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 10x Tris-buffered saline (TBS)</td><td>Bio-Rad</td><td>170-6435</td><td>Final concentration: 1X</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Tween 20</td><td>Merck</td><td>9005-64-5</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 1x TBST</td><td></td><td></td><td>0.1% Tween 20</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 1x PBS</td><td>Gibco</td><td>21300-058</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Pierce BCA Protein Assay Kit</td><td>Thermo Fisher Scientific</td><td>A53225</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Polyacrylamide gel</td><td>Bio-Rad</td><td>161-0183</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Ammonium Persulfate (APS)</td><td>Bio-Rad</td><td>161-0700</td><td>Final concentration: 0.05%</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; TEMED</td><td>Bio-Rad</td><td>161-0800</td><td>Stacker gel: 0.1%, Resolver gel: 0.05%</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; 2x Laemmli Sample Buffer</td><td>Bio-Rad</td><td>161-0737</td><td>Final concentration: 1X</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; Precision Plus Protein Dual Color Standards</td><td>Bio-Rad</td><td>161-0374</td><td></td></tr><tr><td>&lt;strong&gt;WB Blocking solution/ 2&amp;deg; Antibody dilution buffer&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 1x TBST</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 5% Skim milk (nonfat dry milk)</td><td>Bio-Rad</td><td>170-6404</td><td>Working concentration: 5%</td></tr><tr><td>&lt;strong&gt;1x Running buffer 1 L&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 10x Tris-buffered saline (TBS)</td><td>Bio-Rad</td><td>170-6435</td><td>Final concentration: 1X</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Glycine</td><td>Sigma</td><td>G8898</td><td>14.4 g</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; SDS</td><td>Merck</td><td>7910</td><td>Working concentration: 0.1%</td></tr><tr><td>&lt;strong&gt;Blot transfer buffer 500 mL&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 10x Tris-buffered saline (TBS)</td><td>Bio-Rad</td><td>170-6435</td><td>Final concentration: 1X</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Glycine</td><td>Sigma</td><td>G8898</td><td>7.2 g</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Methanol</td><td>Merck</td><td>106009</td><td>100 mL</td></tr><tr><td>&lt;strong&gt;Mild stripping solution 1 L&lt;/strong&gt;</td><td></td><td></td><td>Adjust pH to 2.2</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; Glycine</td><td>Sigma</td><td>G8898</td><td>15 g</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; SDS</td><td>Merck</td><td>7910</td><td>1 g</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Tween 20</td><td>Merck</td><td>9005-64-5</td><td>10 mL</td></tr><tr><td>&lt;strong&gt;Equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>15 mL centrifuge tube</td><td>Corning</td><td>430052</td><td></td></tr><tr><td>50 mL centrifuge tube</td><td>Corning</td><td>430291</td><td></td></tr><tr><td>Airstream Class II</td><td>Esco</td><td>2010621</td><td>Biological safety cabinet</td></tr><tr><td>CelCulture CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Esco</td><td>2170002</td><td>Humidified tissue culture incubator</td></tr><tr><td>CFX96 Touch Real-Time PCR Detector</td><td>Bio-Rad</td><td>1855196</td><td></td></tr><tr><td>FACSVerse Flow Cytometer</td><td>BD Biosciences</td><td>651154</td><td></td></tr><tr><td>Graduated pipettes (10 mL)</td><td>Jet Biofil</td><td>GSP010010</td><td></td></tr><tr><td>Graduated pipettes (5 mL)</td><td>Jet Biofil</td><td>GSP010005</td><td></td></tr><tr><td>MicroCal PEAQ-ITC</td><td>Malvern</td><td></td><td>Isothermal titration calorimeters</td></tr><tr><td>Mini PROTEAN Tetra Cell</td><td>Bio-Rad</td><td>1658004</td><td>Electrophoresis chamber</td></tr><tr><td>Mini Trans-blot absorbent filter paper</td><td>Bio-Rad</td><td>1703932</td><td></td></tr><tr><td>Omega Lum G Imaging System</td><td>Aplegen</td><td>8418-10-0005</td><td></td></tr><tr><td>Pipette controller</td><td>Eppendorf</td><td>4430000.018</td><td>Easypet 3</td></tr><tr><td>PowerPac HC</td><td>Bio-Rad</td><td>1645052</td><td>Power supply</td></tr><tr><td>PVDF membrane</td><td>Merck</td><td>IPVH00010</td><td></td></tr><tr><td>T-75 cell culture flask</td><td>Corning</td><td>431464U</td><td></td></tr><tr><td>Trans-Blot SD Semi-Dry Transfer Cell</td><td>Bio-Rad</td><td>1703940</td><td>Semi-dry transfer cell</td></tr><tr><td>Ultrasonic processor (Vibra-Cell VCX 130)</td><td>Sonics &amp;amp; Materials</td><td></td><td></td></tr><tr><td>Versati Tabletop Refrigerated Centrifuge</td><td>Esco</td><td>T1000R</td><td>Centrifuge with swinging bucket rotar</td></tr></tbody></table>

a competent hepatocyte model examining hepatitis b virus entry through sodium taurocholate cotransporting polypeptide as a therapeutic target

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load but not result in complete remission. An efficient hepatocyte model for HBV infection would offer a true-to-life viral life cycle that would be crucial for the screening of therapeutic agents. Most available anti-HBV agents target lifecycle stages post viral entry but not before viral entry. This protocol details the generation of a competent hepatocyte model capable of screening for therapeutic agents targeting pre-viral entry and post viral entry lifecycle stages. This includes the targeting of sodium taurocholate cotransporting polypeptide (NTCP) binding, cccDNA formation, transcription, and viral assembly based on imHC or HepaRG as host cells. Here, the HBV entry inhibition assay used curcumin to inhibit HBV binding and transporting functions via NTCP. The inhibitors were evaluated for binding affinity (KD) with NTCP using isothermal titration calorimetry (ITC)-a universal tool for HBV drug screening based on thermodynamic parameters.

Hepatic maturation features were observed, including binucleated cells and polygonal-shaped morphology (Figure 1), especially in the differentiated stage of imHC (Figure 1A). A large increase in NTCP expression was measured in d-HepaRG and d-imHC at 7-fold and 40-fold, respectively (Figure 1B). The highly glycosylated form of NTCP, postulated to confer susceptibility to HBV entry, was detected more in d-imHC than in d-HepaRG (Figure 1C). The differentiated imHC contained 65.9% higher NTCP levels than did the undifferentiated cells (Figure 1D).
Figure 2A shows a summary of the prophylactic treatment. Figure 2B shows HBV immunofluorescence staining performed to evaluate the anti-HBV entry activities on day 7 post infection, while Figure 2C outlines the HBV binding assay protocol. The level of HBV binding on the cell surface receptor was evaluated by real-time PCR (Figure 2D). To determine whether NTCP was the receptor for HBV attachment, TCA uptake was determined for the candidate binding inhibitors (Figure 2E). Based on this model, the putative inhibitory activity of candidate compounds decreased HBV binding through NTCP.
The calorimetric alteration was detected with continuous injections to the sample cell (Figure 3). A standard non-linear least squares regression was plotted based on one binding site fitted well to the data. The solid line indicated the best fit to the experimental values (Figure 3A,B). Table 1 shows thermodynamic parameters correlated with the binding of NTCP with cyclosporine A (CsA) or a candidate compound.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63761/63761fig01.jpg" /> 
Figure 1: Hepatic maturation of HepaRG and imHC with NTCP characterization. Both cell lines were cultured in hepatic maturation medium for 2 weeks. (A) Hepatocyte characteristics such as binucleated cells and polygonal-shaped morphology were exclusively observed in the maturation stage of imHC. Scale bars = 50 &#181;m. (B) The expression of NTCP was upregulated in both HepaRG and imHC. NTCP normalized with GAPDH was higher in imHC than in HepaRG. (C) A highly glycosylated form of NTCP was observed after maturation. (D) Percentage of NTCP elevation in d-imHC was evaluated using flow cytometry. Data are presented as mean &#177; SD. *, **, and *** represent statistical difference with p &#60; 0.05, p &#60; 0.01, and p &#60; 0.001, respectively. Abbreviations: NTCP = sodium taurocholate cotransporting polypeptide; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; NTCP-FITC = fluorescein isothiocyanate-labeled NTCP. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63761/63761fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63761/63761fig02.jpg" /> 
Figure 2: Prophylactic CCM treatment decreased viral entry, intracellular HBV DNA, and TCA uptake activity. (A)&#160;d-imHC&#160;were pretreated with CCM for 2 h prior to the inoculation with HBV. (B) Anti-HBV entry activity was determined by immunofluorescence staining on day 7 post infection. (C) A schematic schedule of HBV binding protocol is presented. (D) The level of bound HBV DNA on hepatocytes was evaluated and compared with 4 &#181;M CsA and the classical HBV entry inhibitor 25 units/mL heparin. (E) The effect of the 10-30 &#181;M CCM on TCA uptake inhibition was mediated through the NTCP receptor. Abbreviations: CCM = curcumin; HBV = hepatitis B virus; TCA = taurocholic acid; d-imHC&#160;= differentiated imHC; CsA = cyclosporine A; HP = heparin. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63761/63761fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63761/63761fig03.jpg" /> 
Figure 3: The affinity of NTCP to individual molecules (CsA or candidate compound) was demonstrated using ITC. The ITC profile for the combination of NTCP with either (A) CsA or (B) curcumin was generated from sequential injection of a compound (150 &#956;M CsA or candidate) into the NTCP solution (15 &#181;M of NTCP, pH 7.0, 25 &#176;C). The calorimetric raw data between differential power (&#181;cal/s) and time (min) were plotted. Data were analyzed based on a one-site binding model where the solid lines indicated the best-fit results. During the injection of ligands (CSA or curcumin) into the NTCP solution, the enthalpy (&#916;H) changed after an increase in the ligand concentration in the sample cell. These data indicate that the binding reaction occurred. Abbreviations: CsA = cyclosporine A; NTCP = sodium taurocholate cotransporting polypeptide; ITC = isothermal titration calorimetry; DP = differential power. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63761/63761fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Protein</td>
			<td rowspan="2">Ligand</td>
			<td>Binding constant (KD)</td>
			<td>Enthalpy change (&#916;H)</td>
			<td>Entropy change (&#916;T&#916;S)</td>
			<td>Gibbs Free Energy change (&#916;G)</td>
			<td rowspan="2">Type of binding</td>
		</tr>
		<tr>
			<td>(M-1)</td>
			<td>(kcal/mol)</td>
			<td>(kcal/mol)</td>
			<td>(kcal/mol)</td>
		</tr>
		<tr>
			<td>Human NTCP (SLA10A1)</td>
			<td>CCM</td>
			<td>1.21 &#215;10-6</td>
			<td>-1</td>
			<td>0.6</td>
			<td>-0.39</td>
			<td>Hydrogen bond</td>
		</tr>
		<tr>
			<td>Human NTCP (SLA10A1)</td>
			<td>TCA</td>
			<td>1 &#215;10-9</td>
			<td>80</td>
			<td>-96</td>
			<td>-16</td>
			<td>Hydrophobic interaction</td>
		</tr>
	</tbody>
</table>
Table 1: Thermodynamic parameters resulting from the interaction between NTCP and individual compounds (CCM and TCA) using ITC. Abbreviations: NTCP = sodium taurocholate cotransporting polypeptide; CCM = curcumin; TCA = taurocholic acid; ITC = isothermal titration calorimetry.

Watch this Scientific Journal Video about A Competent Hepatocyte Model Examining Hepatitis B Virus Entry through Sodium Taurocholate Cotransporting Polypeptide as a Therapeutic Target at JoVE.com

A Competent Hepatocyte Model Examining Hepatitis B Virus Entry through Sodium Taurocholate Cotransporting Polypeptide as a Therapeutic Target

We present a protocol to screen anti-hepatitis B virus (HBV) compounds targeting pre- and post-viral entry lifecycle stages, using isothermal titration calorimetry to measure binding affinity (KD) with host sodium taurocholate cotransporting polypeptide. Antiviral efficacy was determined through the suppression of viral lifecycle markers (cccDNA formation, transcription, and viral assembly).

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load ...

a-competent-hepatocyte-model-examining-hepatitis-b-virus-entry

Nanyang Technological University

Research

JoVE Journal

Biochemistry

2.1K Views.  Mahidol University. Dit protocol is ontworpen om te verifiëren of een kandidaat-anti HBV-verbinding de virale toegang remt, vooral door NTCP-binding te blokkeren. Deze techniek is bedoeld om te onderzoeken of HBV-invoer of een eerste stap van infectie kan worden onderbroken door een kandidaat-verbinding. De procedure wordt gedemonstreerd door Piyanoot Thongsri, een PSP-student uit mijn laboratorium.Om te beginnen met de meting van het natrium taurocholaat co-transporterende polypeptide, of NTCP, incubeer...

Video: A Competent Hepatocyte Model Examining Hepatitis B Virus Entry through Sodium Taurocholate Cotransporting Polypeptide as a Therapeutic Target

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in human non-hepatic and murine cell lines, the efficiency was very low and required long-term selection procedures using HCV replicon constructs expressing dominant antibiotic-selectable markers1-5. HCV in vitro research is therefore limited to human hepatoma cell lines permissive for virus entry and completion of the viral life cycle. Due to HCVs narrow species tropism, there is no immunocompetent small animal model available that sustains the complete HCV replication cycle 6-8. Inefficient replication of HCV in non-human cells e.g. of mouse origin is likely due to lack of genetic incompatibility of essential host dependency factors and/or expression of restriction factors.
We investigated whether HCV propagation is suppressed by dominant restriction factors in either human cell lines derived from non-hepatic tissues or in mouse liver cell lines. To this end, we developed two independent conditional trans-complementation methods relying on somatic cell fusion. In both cases, completion of the viral replication cycle is only possible in the heterokaryons. Consequently, successful trans-complementation, which is determined by measuring de novo production of infectious viral progeny, indicates absence of dominant restrictions.
Specifically, subgenomic HCV replicons carrying a luciferase transgene were transfected into highly permissive human hepatoma cells (Huh-7.5 cells). Subsequently, these cells were co-cultured and fused to various human and murine cells expressing HCV structural proteins core, envelope 1 and 2 (E1, E2) and accessory proteins p7 and NS2. Provided that cell fusion was initiated by treatment with polyethylene-glycol (PEG), the culture released infectious viral particles which infected na&iuml;ve cells in a receptor-dependent fashion.
To assess the influence of dominant restrictions on the complete viral life cycle including cell entry, RNA translation, replication and virus assembly, we took advantage of a human liver cell line (Huh-7 Lunet N cells 9) which lacks endogenous expression of CD81, an essential entry factor of HCV. In the absence of ectopically expressed CD81, these cells are essentially refractory to HCV infection 10 . Importantly, when co-cultured and fused with cells that express human CD81 but lack at least another crucial cell entry factor (i.e. SR-BI, CLDN1, OCLN), only the resulting heterokaryons display the complete set of HCV entry factors requisite for infection. Therefore, to analyze if dominant restriction factors suppress completion of the HCV replication cycle, we fused Lunet N cells with various cells from human and mouse origin which fulfill the above mentioned criteria. When co-cultured cells were transfected with a highly fusogenic viral envelope protein mutant of the prototype foamy virus (PFV11) and subsequently challenged with infectious HCV particles (HCVcc), de novo production of infectious virus was observed. This indicates that HCV successfully completed its replication cycle in heterokaryons thus ruling out expression of dominant restriction factors in these cell lines. These novel conditional trans-complementation methods will be useful to screen a large panel of cell lines and primary cells for expression of HCV-specific dominant restriction factors.

We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in ...

Immunology and Infection

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

"Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally infected chimpanzees, most in vitro models require several hundreds to thousands of viral genomes per cell in order to initiate a transient infection. Additionally, static 2D cultures of primary human hepatocytes (PHH) allow only short-term studies due to their rapid dedifferentiation. Here, we describe 3D liver-on-a-chip cultures of PHH, either in monocultures or in cocultures with other nonparenchymal liver-resident cells. These offer a significant improvement to studying long-term HBV infections with physiological host cell responses. In addition to facilitating drug efficacy studies, toxicological analysis, and investigations into pathogenesis, these microfluidic culture systems enable the evaluation of curative therapies for HBV infection aimed at eliminating covalently closed, circular (ccc)DNA. This presented method describes the set-up of PHH monocultures and PHH/Kupffer cell co-cultures, their infection with purified HBV, and the analysis of host responses. This method is particularly applicable to the evaluation of long-term effects of HBV infection, treatment combinations, and pathogenesis.

The goal of this protocol is to provide a step-by-step guide to perform 3-D &#34;liver-on-a-chip&#34; infection experiments with the hepatitis B virus.

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally ...

Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist pMHC, through a process termed co-agonism. This protocol describes an experimental system to investigate co-agonism during human CD8+ T cell activation by expressing human MHC class I molecules presenting pre-determined peptides as single polypeptides (single chain MHC) in a xenogeneic cell line. We expressed single chain MHCs under conditions where low levels of agonist single chain p-MHC complexes and high levels of non-stimulatory single chain p-MHC complexes were expressed. Use of this experimental system allowed us to compare CD8+ T cell responses to agonist pMHC in the presence or absence of non-stimulatory pMHC. The protocol describes cell line transfection with single chain MHC constructs, generation of stable cell lines, culture of hepatitis B virus-specific human CD8+ T cells and T cell activation experiments simultaneously quantifying cytokine production and degranulation. The presented methods can be used for research on different aspects of CD8+ T cell activation in human T cell systems with known peptide MHC specificity.

This protocol describes the use of single chain MHC class I complexes to investigate molecular interactions in human CD8+ T cell activation: generation of engineered antigen presenting cells expressing single chain constructs, culture of human CD8+ T cell clone and T cell activation experiments.

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist ...

Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of liver cancer. This is a major concern, especially in developing countries. Failure of the immune system to mount an effective response against HBV leads to chronic infection. Although HBV vaccine is present and novel antiviral medicines are being created, eradication of virus-reservoir cells remains a major health topic. Described here is a method for the generation of viral antigen (Ag) -specific CD8+ cytotoxic T lymphocytes (CTLs) derived from induced pluripotent stem cells (iPSCs) (i.e., iPSC-CTLs), which have the ability to suppress HBV replication. HBV replication is efficiently induced in mice through hydrodynamic injection of an HBV expression plasmid, pAAV/HBV1.2, into the liver. Then, HBV surface Ag-specific mouse iPSC-CTLs are adoptively transferred, which greatly suppresses HBV replication in the liver and blood as well as prevents HBV surface Ag expression in hepatocytes. This method demonstrates HBV replication in mice after hydrodynamic injection and that stem cell-derived viral Ag-specific CTLs can suppress HBV replication. This protocol provides a useful method for HBV immunotherapy.

Presented here is a protocol for the effective suppression of hepatitis B virus (HBV) replication in mice by utilizing adoptive cell transfer (ACT) of stem cell-derived viral antigen (Ag)-specific T lymphocytes. This procedure may be adapted for potential ACT-based immunotherapy of HBV infection.

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of ...

A Cell Culture Model for Producing High Titer Hepatitis E Virus Stocks

Hepatitis E virus is the leading cause of liver cirrhosis and liver failure with increasing prevalence worldwide. The single-stranded RNA virus is predominantly transmitted by blood transfusions, inadequate sanitary conditions and contaminated food products. To date the off-label drug ribavirin (RBV) is the treatment of choice for many patients. Nonetheless, a specific HEV treatment remains to be identified. So far, the knowledge about the HEV life cycle and pathogenesis has been severely hampered because of the lack of an efficient HEV cell culture system. A robust cell culture system is essential for the study of the viral life cycle which also includes the viral pathogenesis. With the method described here one can produce viral titers of up to 3 x 106 focus forming unit/mL (FFU/mL) of non-enveloped HEV and up to 5 x 104 FFU/mL of enveloped HEV. Using these particles, it is possible to infect a variety of cells of diverse origins including primary cells and human, as well as animal cell lines. The production of infectious HEV particles from plasmids poses an infinite source, which makes this protocol exceedingly efficient.

Described here is an effective method on how to produce high viral titer stocks of hepatitis E virus (HEV) to efficiently infect hepatoma cells. With the presented method, both non-enveloped, as well as enveloped viral particles can be harvested and used for inoculating various cell lines.

Hepatitis E virus is the leading cause of liver cirrhosis and liver failure with increasing prevalence worldwide. The single-stranded RNA virus is ...

Modeling Hepatitis B Virus Infection in Non-Hepatic 293T-NE-3NRs Cells

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV models are limited to hepatoma cell lines (such as HepG2 and Huh7) overexpressing a functional HBV receptor, sodium taurocholate co-transporting polypeptide (NTCP). Here, we used 293T-NE-3NRs (293T&#160;overexpressing human NTCP, HNF4&#945;, RXR&#945; and PPAR&#945;) and HepG2-NE (HepG2 overexpressing NTCP) as model cell lines.&#160;HBV infection in these cell lines was performed either by using concentrated HBV virus particles from HepG2.2.15 or co-culturing HepG2.2.15 with the target cell lines. HBcAg immunofluorescence for HBcAg was performed to confirm HBV infection. The two methods presented here will help us study HBV infection in non-hepatic cell lines.&#160;&#160;

This manuscript describes a detailed protocol for Hepatitis B virus (HBV) infection in novel engineered 293T cells (293T-NE-3NRs, expressing human NTCP, HNF4&#945;, RXR&#945; and PPAR&#945;) and traditional hepatic cells (HepG2-NE, expressing human NTCP).

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV ...

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the treatment of many hematopoietic diseases in humans, as their use offers stable transgene expression due to the vector&#39;s ability to integrate into the host genome. This method demonstrates the application of ex vivo gene therapy of hepatocytes to a large animal model of hereditary tyrosinemia type I. This process consists of 1) isolation of primary hepatocytes from the autologous donor/recipient animal, 2) ex vivo gene delivery via hepatocyte transduction with a lentiviral vector, and 3) autologous transplant of corrected hepatocytes via portal vein injection. Success of the method generally relies upon efficient and sterile removal of the liver resection, careful handling of the excised specimen for isolation of viable hepatocytes sufficient for re-engrafting, high-percentage transduction of the isolated cells, and aseptic surgical procedures throughout to prevent infection. Technical failure at any of these steps will result in low yield of viable transduced hepatocytes for autologous transplant or infection of the donor/recipient animal. The pig model of human type 1 hereditary tyrosinemia (HT-1) chosen for this approach is uniquely amenable to such a method, as even a small percentage of engraftment of corrected cells will lead to repopulation of the liver with healthy cells based on a powerful selective advantage over native-diseased hepatocytes. Although this growth selection will not be true for all indications, this approach is a foundation for expansion into other indications and allows for manipulation of this environment to address additional diseases, both within the liver and beyond, while controlling for exposure to viral vector and opportunity for off-target toxicity and tumorigenicity.

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

This protocol is intended to describe porcine hepatocyte isolation and ex vivo gene delivery to cure models of metabolic diseases via autologous cell transplantation. Although this particular model enjoys unique advantages that favor successful therapy, the application is a relevant foundation to address additional diseases and indications.

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the ...

Medicine

Identifying Inhibitors of the HBx-DDB1 Interaction Using a Split Luciferase Assay System

There is an urgent need for novel therapeutic agents for hepatitis B virus (HBV) infection. Although currently available nucleos(t)ide analogs potently inhibit viral replication, they have no direct effect on the expression of viral proteins transcribed from a viral covalently closed circular DNA (cccDNA). As high viral antigen load may play a role in this chronic and HBV-related carcinogenesis, the goal of HBV treatment is to eradicate viral proteins. HBV regulatory protein X (HBx) binds to the host DNA damage-binding protein 1 (DDB1) protein to degrade structural maintenance of chromosomes 5/6 (Smc5/6), resulting in activation of viral transcription from cccDNA. Here, using a split luciferase complementation assay system, we present a comprehensive compound screening system to identify inhibitors of the HBx-DDB1 interaction. Our protocol enables easy detection of interaction dynamics in real time within living cells. This technique may become a key assay to discover novel therapeutic agents for treatment of HBV infection.

Here, we present a method for screening anti-hepatitis B viral agents that inhibit the HBx-DDB1 interaction using a split luciferase assay system. This system allows easy detection of protein-protein interactions and is suitable for identifying inhibitors of such interactions.

There is an urgent need for novel therapeutic agents for hepatitis B virus (HBV) infection. Although currently available nucleos(t)ide analogs potently ...

A Competent Hepatocyte Model Examining Hepatitis B Virus Entry through Sodium Taurocholate Cotransporting Polypeptide as a Therapeutic Target

Samenvatting

Meer video's verkennen