A bile acid transporter as a candidate receptor for hepatitis B and D virus entry

COMMENTARY ON: Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. Huan Yan, Guocai Zhong, Guangwei Xu, Wenhui He, Zhiyi Jing, Zhenchao Gao, Yi Huang, Yonghe Qi, Bo Peng, Haimin Wang, Liran Fu, Mei Song, Pan Chen, Wenqing Gao, Bijie Ren, Yinyan Sun, Tao Cai, Xiaofeng Feng, Jianhua Sui, Wenhui Li in eLife 2012, 1:e00049. doi: 10.7554/eLife.00049. Epub Nov. 13. Copyright © 2012, Yan et al. http://www.ncbi.nlm.nih.gov/pubmed/23150796 Abstract Human hepatitis B virus (HBV) infection and HBV-related diseases remain a major public health problem. Individuals coinfected with its satellite hepatitis D virus (HDV) have more severe disease. Cellular entry of both viruses is mediated by HBV envelope proteins. The pre-S1 domain of the large envelope protein is a key determinant for receptor(s) binding. However, the identity of the receptor(s) is unknown. Here, by using near zero distance photo-cross-linking and tandem affinity purification, we revealed that the receptor-binding region of pre-S1 specifically interacts with sodium taurocholate cotransporting polypeptide (NTCP), a multiple transmembrane transporter predominantly expressed in the liver. Silencing NTCP inhibited HBV and HDV infection, while exogenous NTCP expression rendered nonsusceptible hepatocarcinoma cells susceptible to these viral infections. Moreover, replacing amino acids 157–165 of nonfunctional monkey NTCP with the human counterpart conferred its ability in supporting both viral infections. Our results demonstrate that NTCP is a functional receptor for HBV and HDV. DOI:http://dx.doi.org/10.7554/eLife.00049.001. With 350 million chronically infected individuals worldwide, hepatitis B virus (HBV) is an unsolved global health challenge. Current treatment strategies, based on interferon-alpha or nucleos(t)ide analogues have been shown to control viral infection and reduce liver disease. However, available treatments are far from satisfactory as they largely fail to eradicate HBV or hepatitis delta virus (HDV) [[1]Scaglione S.J. Lok A.S. Effectiveness of hepatitis B treatment in clinical practice.Gastroenterology. 2012; 142: e1361Abstract Full Text Full Text PDF Scopus (118) Google Scholar]. Although the HBV genome replicates in a variety of cell lines, the virus can only infect primary human and Tupaia hepatocytes (PHH and PTH) [2Köck J. Nassal M. MacNelly S. Baumert T.F. Blum H.E. von Weizsacker F. Efficient infection of primary tupaia hepatocytes with purified human and woolly monkey hepatitis B virus.J Virol. 2001; 75: 5084-5089Crossref PubMed Scopus (127) Google Scholar, 3Gripon P. Diot C. Theze N. Fourel I. Loreal O. Brechot C. et al.Hepatitis B virus infection of adult human hepatocytes cultured in the presence of dimethyl sulfoxide.J Virol. 1988; 62: 4136-4143Crossref PubMed Google Scholar] and the bipotent differentiated HepaRG liver progenitor cell line [[4]Gripon P. Rumin S. Urban S. Le Seyec J. Glaise D. Cannie I. et al.Infection of a human hepatoma cell line by hepatitis B virus.Proc Natl Acad Sci USA. 2002; 99: 15655-15660Crossref PubMed Scopus (946) Google Scholar]. Despite tremendous progress in the molecular characterization of HBV replication and assembly, the host determinants mediating the first steps of infection remain poorly defined, limiting the development of robust in vitro models supporting the complete HBV life cycle. Although other hepadnaviruses (e.g., duck hepatitis B virus [DHBV]) share some functional and structural properties with HBV and are therefore used as models for HBV-host interactions, functional data suggest that entry pathways of these viruses differ [[5]Glebe D. Urban S. Viral and cellular determinants involved in hepadnaviral entry.World J Gastroenterol. 2007; 13: 22-38PubMed Google Scholar]. Indeed, the functional relevance of cellular receptors identified for DHBV (such as carboxypeptidase D) could not be confirmed for HBV (for review see [[5]Glebe D. Urban S. Viral and cellular determinants involved in hepadnaviral entry.World J Gastroenterol. 2007; 13: 22-38PubMed Google Scholar]). The pre-S1 domain of the HBV encoded large surface envelope protein plays a role in particle entry. Indeed, a peptide derived from the pre-S1 protein inhibits HBV infection of human hepatocytes [4Gripon P. Rumin S. Urban S. Le Seyec J. Glaise D. Cannie I. et al.Infection of a human hepatoma cell line by hepatitis B virus.Proc Natl Acad Sci USA. 2002; 99: 15655-15660Crossref PubMed Scopus (946) Google Scholar, 6Gripon P. Cannie I. Urban S. Efficient inhibition of hepatitis B virus infection by acylated peptides derived from the large viral surface protein.J Virol. 2005; 79: 1613-1622Crossref PubMed Scopus (272) Google Scholar, 7Meier A, Mehrle S, Weiss TS, Mier W, Urban S. The myristoylated preS1-domain of the hepatitis B virus L-protein mediates specific binding todifferentiated hepatocytes. Hepatology 2012. http://dx.doi.org/10.1002/hep.26181. [Epub ahead of print].Google Scholar] and chimeric uPA/SCID mice [[8]Petersen J. Dandri M. Mier W. Lutgehetmann M. Volz T. von Weizsacker F. et al.Prevention of hepatitis B virus infection in vivo by entry inhibitors derived from the large envelope protein.Nat Biotechnol. 2008; 26: 335-341Crossref PubMed Scopus (329) Google Scholar]. Since HDV utilizes the envelope proteins of HBV, it is assumed to enter hepatocytes via a similar mechanism [[5]Glebe D. Urban S. Viral and cellular determinants involved in hepadnaviral entry.World J Gastroenterol. 2007; 13: 22-38PubMed Google Scholar]. There is accumulating evidence that HBV attaches to cells via heparan sulfate proteoglycans [9Schulze A. Gripon P. Urban S. Hepatitis B virus infection initiates with a large surface protein-dependent binding to heparan sulfate proteoglycans.Hepatology. 2007; 46: 1759-1768Crossref PubMed Scopus (306) Google Scholar, 10Leistner C.M. Gruen-Bernhard S. Glebe D. Role of glycosaminoglycans for binding and infection of hepatitis B virus.Cell Microbiol. 2008; 10: 122-133PubMed Google Scholar, 11Sureau C, Salisse J. A conformational heparan sulfate-binding site essential to infectivity overlaps with the conserved hepatitis B virus a-determinant. Hepatology 2012. http://dx.doi.org/10.1002/hep.26125. [Epub ahead of print].Google Scholar]. Several cell surface proteins have been reported to interact with HBV envelope proteins but none of them have been confirmed to be an essential entry factor [[5]Glebe D. Urban S. Viral and cellular determinants involved in hepadnaviral entry.World J Gastroenterol. 2007; 13: 22-38PubMed Google Scholar]. A recent study by Wenhui Li’s laboratory at the National Institute of Biological Sciences in Beijing, China, identified a novel HBV and HDV receptor candidate [[12]Yan H. Zhong G. Xu G. He W. Jing Z. Gao Z. et al.Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.eLife. 2012; 1: e00049Crossref Scopus (1389) Google Scholar]. Based on the previous mapping studies by Schulze et al. [[13]Schulze A. Schieck A. Ni Y. Mier W. Urban S. Fine mapping of pre-S sequence requirements for hepatitis B virus large envelope protein-mediated receptor interaction.J Virol. 2010; 84: 1989-2000Crossref PubMed Scopus (169) Google Scholar], Wenhui Li’s team established a photo cross-linking assay using a series of synthetic pre-S1 peptides as “bait” to identify interacting proteins expressed in Tupaia hepatocytes to screen for putative HBV entry factors. The cross-linked peptide-protein complexes were purified and analyzed by mass spectrometry (MS). Comparing the MS results of the captured proteins with a Tupaia protein database obtained by deep-sequencing the Tupaia transcriptome, enabled Yan and colleagues to identify sodium taurocholate cotransporting polypeptide (NTCP, also known as SLC10A1) as a hepatocyte surface molecule binding pre-S1. NTCP is a member of the solute carrier family 10 (SLC10), the major bile acid uptake system in human hepatocytes, that localizes to the basolateral hepatocyte membrane (Fig. 1). NTCP is a 349-amino acid integral membrane glycoprotein comprising 7 or 9 transmembrane (TM) domains according to topology studies on a related SLC10 family member, apical sodium-dependent bile acid transporter (ASBT) [14Kullak-Ublick G.A. Stieger B. Meier P.J. Enterohepatic bile salt transporters in normal physiology and liver disease.Gastroenterology. 2004; 126: 322-342Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar, 15Mareninova O. Shin J.M. Vagin O. Turdikulova S. Hallen S. Sachs G. Topography of the membrane domain of the liver Na+-dependent bile acid transporter.Biochemistry. 2005; 44: 13702-13712Crossref PubMed Scopus (41) Google Scholar, 16Hu N.J. Iwata S. Cameron A.D. Drew D. Crystal structure of a bacterial homologue of the bile acid sodium symporter ASBT.Nature. 2011; 478: 408-411Crossref PubMed Scopus (190) Google Scholar]. The ability of NTCP to bind HBV pre-S1 was confirmed using NTCP transfected 293T cells. Silencing NTCP expression in PTHs, HepaRG or PHHs partially reduced HBV or HDV infection. NTCP expression in non-permissive HepG2 or HuH7 hepatoma cells rendered these cells susceptible to low level HBV or HDV infection, respectively. Finally, the authors combined phylogenetic analysis with mutagenesis studies to identify a putative role for NTCP amino acids 157–165 in viral infection. As shown for many other viruses, “cellular receptor proteins” can act in several ways to mediate viral entry, including viral attachment, post-binding transport and viral fusion [[17]Zeisel M.B. Fofana I. Fafi-Kremer S. Baumert T.F. Hepatitis C virus entry into hepatocytes: molecular mechanisms and targets for antiviral therapies.J Hepatol. 2011; 54: 566-576Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar]. Hepatic NTCP expression is regulated by a number of familiar pathways, notably the glucocorticoid receptor, the retinoic acid receptor and hepatic nuclear transcription factors HNF1α, HNF4α, and HNF3β [[18]Geyer J. Wilke T. Petzinger E. The solute carrier family SLC10: more than a family of bile acid transporters regarding function and phylogenetic relationships.Naunyn Schmiedebergs Arch Pharmacol. 2006; 372: 413-431Crossref PubMed Scopus (132) Google Scholar]. NTCP is a member of a family of 7 related solute carrier family transporters and has been shown to interact with a variety of partner proteins. In addition to forming a homodimer, NTCP can dimerise with other members of the SLC10A family, notably SLC10A4 and SLC10A6 (sodium-dependent organic anion transporter, SOAT). NTCP interaction with these partner proteins regulate protein trafficking in vitro and hints at possible mechanisms for viral transport [[19]Bijsmans I.T.G.W. Bouwmeester R.A.M. Geyer J. Faber K.N. van de Graaf S.F.J. Homo- and hetero-dimeric architecture of the human liver Na+-dependent taurocholate co-transporting protein.Biochem J. 2012; 441: 1007-1015Crossref PubMed Scopus (50) Google Scholar]. Resolving these outstanding issues will clarify the role of NTCP in HBV internalization. Furthermore, it will be of interest to explore NTCP or its regulatory factors as antiviral targets. The observations that silencing NTCP did not ablate HBV infection of PHHs and that HepG2-NTCP cells support low level HBV infection suggest that NTCP may not be the sole host factor defining liver permissivity to HBV, highlighting the need for additional studies in this area. In summary, the work of Yan et al. provides an important advance in our understanding of HBV entry and suggests new avenues for the genesis of cell culture and animal model systems that support HBV and HDV infection, enabling the development of new antivirals and immunotherapies. The authors acknowledge support by Inserm, ANRS, University of Strasbourg, the European Union (INTERREG-IV-Rhin Supérieur-FEDER -Hepato-Regio-Net 2009 and 2012), the Laboratoire d’excellence HEPSYS (ANR-10-LAB-28), MRC and NIHR Liver Biomedical Research Unit, University of Birmingham. The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.