Hepatitis B and D Viruses Exploit Sodium Taurocholate Co-transporting Polypeptide for Species-Specific Entry into Hepatocytes

doi:10.1053/j.gastro.2013.12.024

Gastroenterology

Volume 146, Issue 4, April 2014, Pages 1070-1083.e6

https://doi.org/10.1053/j.gastro.2013.12.024 Get rights and content

Background & Aims

Hepatitis B and D viruses (HBV and HDV) are human pathogens with restricted host ranges and high selectivity for hepatocytes; the HBV L-envelope protein interacts specifically with a receptor on these cells. We aimed to identify this receptor and analyze whether it is the recently described sodium-taurocholate co-transporter polypeptide (NTCP), encoded by the SLC10A1 gene.

Methods

To identify receptor candidates, we compared gene expression patterns between differentiated HepaRG cells, which express the receptor, and naïve cells, which do not. Receptor candidates were evaluated by small hairpin RNA silencing in HepaRG cells; the ability of receptor expression to confer binding and infection were tested in transduced hepatoma cell lines. We used interspecies domain swapping to identify motifs for receptor-mediated host discrimination of HBV and HDV binding and infection.

Results

Bioinformatic analyses of comparative expression arrays confirmed that NTCP, which was previously identified through a biochemical approach is a bona fide receptor for HBV and HDV. NTCPs from rat, mouse, and human bound Myrcludex B, a peptide ligand derived from the HBV L-protein. Myrcludex B blocked NTCP transport of bile salts; small hairpin RNA-mediated knockdown of NTCP in HepaRG cells prevented their infection by HBV or HDV. Expression of human but not mouse NTCP in HepG2 and HuH7 cells conferred a limited cell-type–related and virus-dependent susceptibility to infection; these limitations were overcome when cells were cultured with dimethyl sulfoxide. We identified 2 short-sequence motifs in human NTCP that were required for species-specific binding and infection by HBV and HDV.

Conclusions

Human NTCP is a specific receptor for HBV and HDV. NTCP-expressing cell lines can be efficiently infected with these viruses, and might be used in basic research and high-throughput screening studies. Mapping of motifs in NTCPs have increased our understanding of the species specificities of HBV and HDV, and could lead to small animal models for studies of viral infection and replication.

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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We found the infectivity of splicing-deficient HBV was decreased 100-1,000-fold compared with that of wild-type HBV in hu-FRG mice. Another mutant, A487C, which loses the most abundant spliced RNA (SP1), also exhibits severely impaired infectivity. SP1 hypothetically encodes a novel protein HBc^SP1 (HBc^-Cys) that lacks the C-terminal cysteine from full-length HBc. In the SHifter assay, HBc^SP1 was detected in wild-type viral particles at a ratio of about 20-100% vs. conventional HBc, as well as in the serum of patients with chronic hepatitis B, but not in A487C particles. When infection was conducted with a shorter incubation time of 4-8 h at lower PEG concentrations in HepG2-NTCP cells, the entry of the A487C mutant was significantly slower. SP1 cDNA complementation of the A487C mutant succeeded in rescuing its infectivity in hu-FRG mice and HepG2-NTCP cells. Moreover, cryo-electron microscopy revealed a disulfide bond between HBc cysteine 183 and 48 in the HBc intradimer of the A487C capsid, leading to a locked conformation that disfavored viral entry in contrast to the wild-type capsid.
Prior studies unveiled the potential integration of the HBc^-Cys protein into the HBV capsid. We confirmed the proposal and validated its identity and function during infection.
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This study describes the potent activity of BLV against multiple laboratory strains spanning all HBV/HDV A–H/1–8 genotype combinations and the most diverse collection of HDV clinical samples tested to date, including HBV/HDV genotype combinations less frequently observed in the clinic. Overall, all isolates and laboratory strains displayed similar in vitro nanomolar sensitivity to BLV. This broad-spectrum antiviral activity of BLV has direct implications on potential simplified treatment for any patient infected with HDV, regardless of genotype, and supports the new 2023 EASL Clinical Practice Guidelines on HDV that recommend antiviral treatment for all patients with CHD.
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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

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Original ResearchFull Report: Basic and Translational—LiverHepatitis B and D Viruses Exploit Sodium Taurocholate Co-transporting Polypeptide for Species-Specific Entry into Hepatocytes

Background & Aims

Methods

Results

Conclusions

Section snippets

Gene Expression Microarray Analysis

Plasmids

Identification of hNTCP as an HBV PreS-Specific Receptor for HBV and HDV

Discussion

Acknowledgments

Cell

Virology

Virology

Curr Top Membr

J Biol Chem

Hepatology

Viral and cellular determinants involved in hepadnaviral entry

World J Gastroenterol

Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus

elife

Hepatitis B virus biology

Microbiol Mol Biol Rev

Hepatitis B virus hepatotropism is mediated by specific receptor recognition in the liver and not restricted to susceptible hosts

Hepatology

The myristoylated preS1-domain of the hepatitis B virus L-protein mediates specific binding todifferentiated hepatocytes

Hepatology

Infection of a human hepatoma cell line by hepatitis B virus

Proc Natl Acad Sci U S A

Original Research
Full Report: Basic and Translational—Liver
Hepatitis B and D Viruses Exploit Sodium Taurocholate Co-transporting Polypeptide for Species-Specific Entry into Hepatocytes