Original ResearchFull Report: Basic and Translational—LiverHepatitis B and D Viruses Exploit Sodium Taurocholate Co-transporting Polypeptide for Species-Specific Entry into Hepatocytes
Section snippets
Gene Expression Microarray Analysis
Total RNA from HepaRG cells were extracted using the AllPrep DNA/RNA Mini kit (Qiagen, Valencia, CA). RNA was quantified with NanoDrop ND-1000 (Thermo Fisher Scientific, Wilmington, DE). For expression analysis, SentrixH HumanHT-12 v4 bead chips (IlluminaH, San Diego, CA) encompassing 47,231 features were used (Supplementary Material).
Plasmids
Human NTCP complementary DNA (Origene, Rockville, MD) and mNtcp complementary DNA (S. Herzig, Heidelberg, Germany) were subcloned into the puromycin co-expressing
Identification of hNTCP as an HBV PreS-Specific Receptor for HBV and HDV
Receptor candidates that fulfill previously defined criteria for HBV preS binding6, 7, 14 were identified by differential microarray expression screens: HepaRG cells were grown to confluence and receptor expression was induced with 2% DMSO for 14 days. Up-regulated genes (>3.5-fold) were selected by plasma membrane association, basolateral sorting, high expression in the liver, low expression in HepG2/HuH7 cells, and conservation in primates and rodents (Supplementary Figure 1). Serpin C1
Discussion
For decades, identification of specific receptors promoting HBV and HDV entry into hepatocytes remained an unresolved challenge. Heparan sulfate proteoglycans have been assessed as mandatory attachment factors,22, 23 however, they cannot explain hepatotropism and species specificity. Yan et al recently identified hNTCP as an HBV preS-specific receptor3 and showed that hNTCP expression supports infection of HepG2 and HuH7 cells. We applied a bioinformatics-assisted array approach and identified
Acknowledgments
The authors thank Katrin Schöneweis and Christa Kuhn for support in cell culture experiments; Jessika Sonnabend for preparing the Southern blot marker; Walter Mier, Heidelberg, for peptide synthesis and analysis; Anne Nies, Stuttgart, for MRP2 antibodies; and Davide Corti, Bellinzona, for preS and S-specific monoclonal antibodies. The authors are indebted to Ralf Bartenschlager for his continuous intellectual support.
Maria Fälth's current affiliation is Cellzome/GlaxoSmithKline, Heidelberg,
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Conflicts of interest This author discloses the following: Stephan Urban is co-applicant and co-inventor on patents protecting HBV preS-derived lipopeptides (Myrcludex B) for the use of HBV/HDV entry inhibitors. The remaining authors disclose no conflicts.
Funding This work received funding by the Deutsche Forschungsgemeinschaft (DFG) UR72/7-1 and FOR1202/UR72/5-1 to S.U. and the Hartmut Hoffmann Berling International Graduate School HBIGS to S.N.
Author names in bold designate shared co-first authorship