Identification of NTCP as an HBV Receptor: The Beginning of the End or the End of the Beginning?

Abstract

See “Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes,” by Ni Y, Lempp FA, Mehrle S, et al, on page 1070.Considering that the hepatitis B virus (HBV) genome was cloned >3 decades ago, the journey toward the identification of its receptor has been extremely slow and tortuous. HBV expresses 3 co-terminal envelope proteins termed large (L), middle (M), and small (S), which contain preS1+preS2+S, preS2+S, and S domain alone, respectively. The preS1 domain is believed to mediate virus attachment to the high-affinity receptor, although anti-preS2 and anti-S antibodies can neutralize infectivity as well. In fact, the S domain mediates initial HBV recruitment to hepatocyte surface via heparan sulfate proteoglycans.1Schulze 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 (301) Google Scholar, 2Leistner 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, 3Sureau C, Salisse J. A conformational heparan sulfate binding site essential to infectivity overlaps with the conserved hepatitis B virus a-determinant. Hepatology;57:985–994.Google Scholar In the last 2 decades, more than a dozen host-binding proteins to the preS1, preS2, or S domain have been identified.4Glebe D. Urban S. Viral and cellular determinants involved in hepadnaviral entry.World J Gastroenterol. 2007; 13: 22-38Crossref PubMed Scopus (223) Google Scholar For a few such proteins, cDNA transfection conferred HBV infection based on the sensitive polymerase chain reaction, but without independent confirmation. Another approach is to determine whether silencing their expression impairs HBV infection in a susceptible cell line. Unfortunately, no such cell line was available until 2002, when Gripon et al5Gripon P. Rumin S. Urban S. et al.Infection of a human hepatoma cell line by hepatitis B virus.Proc Natl Acad Sci U S A. 2002; 99: 15655-15660Crossref PubMed Scopus (930) Google Scholar reported that dimethyl sulfoxide (DMSO) treatment renders a human liver progenitor cell line, HepaRG, susceptible to low-level HBV infection. In this regard, DMSO could prolong the HBV susceptibility of cultured primary human hepatocytes by maintaining cellular differentiation.6Gripon P. Diot C. Theze N. 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 Adding polyethylene glycol during virus incubation also enhances infectivity of cell culture-derived HBV particles, probably by concentrating virions on the cell surface. Such improvements, together with the availability of primary Tupaia hepatocytes (PTH) as a more reliable source of in vitro infection, paved the way for the identification of sodium taurocholate cotransporting polypeptide (NTCP) as the candidate HBV receptor.7Yan H. Zhong G. Xu G. et al.Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.Elife. 2012; 1: e00049Crossref PubMed Scopus (1339) Google Scholar Although this provocative finding from Li’s group attracted lots of attention, the report by Ni et al8Ni Y. Lempp F.A. Mehrle S. et al.Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes.Gastroenterology. 2014; 146: 1070-1083Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar in the current issue of Gastroenterology represents the critical, independent confirmation.In their original eLife report,7Yan H. Zhong G. Xu G. et al.Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.Elife. 2012; 1: e00049Crossref PubMed Scopus (1339) Google Scholar Yan et al employed a novel technique to establish NTCP as a binding partner for the myristoylated peptide 2-48 of the preS1 domain. Lack of NTCP expression in 2 human hepatoma cell lines, HepG2 and Huh7 cells, is consistent with their resistance to HBV infection. Moreover, in vitro culture of PTH led to a rapid loss of NTCP expression, as expected for an HBV receptor. Importantly, silencing NTCP expression by shRNA reduced HBV infectivity in PTH, primary human hepatocyte, and HepaRG cells. It also diminished infectivity of hepatitis delta virus (HDV), which employs envelope proteins of HBV for its transmission. Conversely, transfection with human NTCP (hNTCP) cDNA enabled HBV infection of HepG2 cells, as well as HDV infection of both HepG2 and Huh7 cells. A limitation of that study was that they relied heavily on hepatitis B surface antigen (HBsAg, the secreted S protein) and hepatitis B e antigen (HBeAg, a secreted version of viral core protein) as indicators of successful HBV infection. Measurement of viral mRNA and replicative DNA was achieved mostly by polymerase chain reaction, thus raising issues about the robustness of infection.Consistent with the Yan report, Ni et al from Stephan Urban’s laboratory observed a 3.6-fold induction of hNTCP mRNA in HepaRG cells differentiated by DMSO treatment.8Ni Y. Lempp F.A. Mehrle S. et al.Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes.Gastroenterology. 2014; 146: 1070-1083Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar One of the 2 shRNAs nearly abolished hNTCP expression when delivered by lentivirus vector. Its ability to completely block HBV and HDV infection in HepaRG cells implicates NTCP as the dominant port of HBV/HDV entry in this particular cell line. Transfection of the hNTCP cDNA into HepG2, Huh7, and HepaRG cells conferred binding of myristoylated preS1 peptide. HDV infection was reconstituted more efficiently in hNTCP-transfected Huh7 and HepaRG cells, yet only HepG2 cells showed evidence of core protein expression as well as HBsAg and HBeAg secretion following HBV infection (summarized in Table 1). Therefore, molecules other than hNTCP are required for infectivity of both viruses, and such factors are virus specific and differentially expressed in the 3 cell lines. Treatment of NTCP-reconstituted cells with 2.5% DMSO markedly increased core protein expression, DNA replication, and HBeAg secretion in both HepG2 and Huh7 cell lines. It also enabled HBsAg secretion from Huh7 cells. In the presence of DMSO, NTCP transfected HepG2 cells showed a 100-fold higher level of HBeAg secretion than HepaRG cells. Consequently HBV RNAs, replicative DNA, and even the covalently closed circular DNA, the template for RNA transcription, could be detected by conventional hybridization technique. On the other hand, for unknown reasons, HBsAg secretion remained inefficient in hNTCP-transfected HepG2 cells despite DMSO treatment.Table 1Interplay of Viral, Host, and Differentiation Factors in Sodium Taurocholate Cotransporting Polypeptide (NTCP)-Mediated Hepatitis B Virus (HBV) and Hepatitis Delta Virus (HDV) Infection of Hepatoma Cell LineshNTCPhNTCP + DMSODMSOhNTCPhNTCP + DMSOmNTCPbNot deficient in the binding of myristoylated preS peptide.mNTCPHBVHBVHBVHDVHDVHBVHDVHepG2 (human)+++++++aExcept for hepatitis B surface antigen secretion, which is inefficient.-+/-++++++--cBased on Ref12Huh7 (human)+/-++-++++++-NDHepaRG (human)+/-NDbNot deficient in the binding of myristoylated preS peptide.++++NDNDNDHep1-6 (mouse)-NDND+NDNDNDHep56D (mouse)-NDND+NDNDNDTC5123 (rat)-NDND++NDNDNDDMSO, dimethyl sulfoxide; hNTCP, human NTCP; mNTCP, mouse NTCP; ND, not determineda Except for hepatitis B surface antigen secretion, which is inefficient.b Not deficient in the binding of myristoylated preS peptide.c Based on Ref12Yan H. Peng B. He W. et al.Molecular determinants of hepatitis B and D virus entry restriction in mouse sodium taurocholate cotransporting polypeptide.J Virol. 2013; 87: 7977-7991Crossref PubMed Scopus (139) Google Scholar Open table in a new tab Because the normal function of NTCP is to import bile salts, Ni et al asked whether HBV exploits this property for entry into hepatocytes. Indeed, the myristoylated preS1 peptide inhibited transport of taurocholate into hNTCP transfected HepG2 cells. Conversely, several bile salts inhibited binding of the preS1 peptide and consequently HBV infection of the hNTCP transfected Huh7 cells. Very similar results were obtained by the Li group and by Watashi et al.9Yan H. Peng B. Liu Y. et al.Viral entry of Hepatitis B and D viruses and bile salts transportation share common molecular determinants on sodium taurocholate cotransporting polypeptide.J Virol. 2014 Jan 3; ([Epub ahead of print])Crossref Scopus (180) Google Scholar, 10Watashi K. Sluder A. Daito T. et al.Cyclosporin A and its analogs inhibit hepatitis B virus entry into cultured hepatocytes through targeting a membrane transporter NTCP.Hepatology. 2013 Dec 21; ([Epub ahead of print])Google Scholar Furthermore, Yan et al9Yan H. Peng B. Liu Y. et al.Viral entry of Hepatitis B and D viruses and bile salts transportation share common molecular determinants on sodium taurocholate cotransporting polypeptide.J Virol. 2014 Jan 3; ([Epub ahead of print])Crossref Scopus (180) Google Scholar found residues in the hNTCP molecule critical for bile salt transport were also essential for hNTCP to serve as the HBV receptor. Further studies are needed to determine whether HBV infection interferes with bile salt metabolism.Identification of NTCP as an HBV receptor raises the issue of how this molecule could account for the species specificity of HBV infection, and whether it is possible to establish a small animal model of HBV infection via NTCP reconstitution or humanization. hNTCP is a glycoprotein of 349 residues. The Tupaia homologue could serve as a receptor for HBV and related woolly monkey HBV despite a 30-amino acid insertion.11Zhong G. Yan H. Wang H. et al.Sodium taurocholate cotransporting polypeptide mediates woolly monkey hepatitis B virus infection of Tupaia hepatocytes.J Virol. 2013; 87: 7176-7184Crossref PubMed Scopus (51) Google Scholar In contrast, NTCP from crab-eating monkey lacked affinity for myristoylated preS1 peptide and failed to mediate HBV infection.7Yan H. Zhong G. Xu G. et al.Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.Elife. 2012; 1: e00049Crossref PubMed Scopus (1339) Google Scholar Mouse NTCP failed to confer susceptibility to HBV infection despite its affinity for myristoylated preS1 peptide,8Ni Y. Lempp F.A. Mehrle S. et al.Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes.Gastroenterology. 2014; 146: 1070-1083Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar, 12Yan H. Peng B. He W. et al.Molecular determinants of hepatitis B and D virus entry restriction in mouse sodium taurocholate cotransporting polypeptide.J Virol. 2013; 87: 7977-7991Crossref PubMed Scopus (139) Google Scholar suggesting a role of NTCP beyond high-affinity binding to HBV virions. Unfortunately, hNTCP could not confer HBV infection in the 2 mouse hepatoma cell lines and rat hepatoma cell line tested (Table 1),8Ni Y. Lempp F.A. Mehrle S. et al.Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes.Gastroenterology. 2014; 146: 1070-1083Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar, 12Yan H. Peng B. He W. et al.Molecular determinants of hepatitis B and D virus entry restriction in mouse sodium taurocholate cotransporting polypeptide.J Virol. 2013; 87: 7977-7991Crossref PubMed Scopus (139) Google Scholar thus dashing the hopes for the establishment of a small animal model of HBV infection in the near future.Besides the paper by Ni et al in the current issue of Gastroenterology others have also confirmed the ability of hNTCP to confer susceptibility to HBV infection in HepG2 cells10Watashi K. Sluder A. Daito T. et al.Cyclosporin A and its analogs inhibit hepatitis B virus entry into cultured hepatocytes through targeting a membrane transporter NTCP.Hepatology. 2013 Dec 21; ([Epub ahead of print])Google Scholar (our unpublished observation). Moreover, 2 upcoming papers, one from the Urban laboratory, demonstrated that cyclosporine inhibits HBV infection by targeting NTCP.10Watashi K. Sluder A. Daito T. et al.Cyclosporin A and its analogs inhibit hepatitis B virus entry into cultured hepatocytes through targeting a membrane transporter NTCP.Hepatology. 2013 Dec 21; ([Epub ahead of print])Google Scholar, 13Nkongolo S. Ni Y. Lempp F.A. et al.Cyclosporin A inhibits hepatitis B and hepatitis D virus entry by cyclophilin-independent interference with the NTCP receptor.J Hepatol. 2013 Dec 1; ([Epub ahead of print])PubMed Google Scholar Thus, NTCP serves as an HBV receptor in primary human hepatocytes, PTH, HepaRG, and HepG2 cells. However, one has to bear in mind that the current infection system is based on cell culture-derived HBV particles added at extremely high multiplicity of infection in the presence of polyethylene glycol. In vivo, a few virus particles injected through the bloodstream are sufficient to infect the entire liver of a chimpanzee.14Asabe S. Wieland S.F. Chattopadhyay P.K. et al.The size of the viral inoculum contributes to the outcome of hepatitis B virus infection.J Virol. 2009; 83: 9652-9662Crossref PubMed Scopus (222) Google Scholar Thus, it remains to be seen whether the NTCP system of HBV infection can be improved by the addition of other host factors and whether NTCP can mediate the infection of serum-derived HBV particles. Another question is whether NTCP constitutes the only or major HBV receptor in vivo. In this regard, Li’s group found a naturally occurring polymorphism in the NTCP gene (S267F), which abrogated its ability to support HBV infection in cell culture.9Yan H. Peng B. Liu Y. et al.Viral entry of Hepatitis B and D viruses and bile salts transportation share common molecular determinants on sodium taurocholate cotransporting polypeptide.J Virol. 2014 Jan 3; ([Epub ahead of print])Crossref Scopus (180) Google Scholar About 0.3% of East Asians harbor this mutation in both alleles. HBV infection in any of such individuals will cast doubt on the role of NTCP as the only HBV receptor. Nevertheless, some features of NTCP are compatible with its role as the HBV receptor. Both myristoylated and cholesterol-modified preS1 peptide 2-48, but not the unmodified peptide, could interact with NTCP.9Yan H. Peng B. Liu Y. et al.Viral entry of Hepatitis B and D viruses and bile salts transportation share common molecular determinants on sodium taurocholate cotransporting polypeptide.J Virol. 2014 Jan 3; ([Epub ahead of print])Crossref Scopus (180) Google Scholar In this regard, cholesterol is important for HBV infectivity,15Bremer C.M. Bung C. Kott N. et al.Hepatitis B virus infection is dependent on cholesterol in the viral envelope.Cell Microbiol. 2009; 11: 249-260Crossref PubMed Scopus (70) Google Scholar and L protein is myristoylated at position 2 by a glycine residue, which is dispensable for HBV virion formation but essential for infectivity.16Gripon P. Le Seyec J. Rumin S. et al.Myristylation of the hepatitis B virus large surface protein is essential for viral infectivity.Virology. 1995; 213: 292-299Crossref PubMed Scopus (150) Google ScholarNi et al should be applauded for improving the in vitro HBV infection system, which will prove useful for many different applications, such as the screening of more potent entry inhibitors and testing of the infectivity of viral variants. Identification of NTCP as an HBV receptor should usher in an exciting era of research. An immediate question concerns the nature of co-receptors or co-factors for productive HBV infection, such as those responsible for differential infectivity between NTCP-reconstituted HepG2 and Huh7 cells, as well as those induced by DMSO in these cell lines. As post receptor binding steps for productive HBV infection include membrane fusion (which is most likely mediated by the S domain and may occur at plasma or endosomal membrane), capsid disassembly, nuclear targeting of the relaxed circular DNA genome, and its conversion to the covalently closed circular DNA, the HepG2-enriched and DMSO-induced factors could affect any of these steps. If the example of hepatitis C virus, an RNA virus targeting the liver, is any indication,17Meredith L.W. Wilson G.K. Fletcher N.F. et al.Hepatitis C virus entry: beyond receptors.Rev Med Virol. 2012; 22: 182-193Crossref PubMed Scopus (72) Google Scholar many more host factors for HBV infection are to be discovered. It will be interesting in this regard to reexamine the previously identified binding partners of HBV envelope proteins for their contribution to efficient HBV infection.4Glebe D. Urban S. Viral and cellular determinants involved in hepadnaviral entry.World J Gastroenterol. 2007; 13: 22-38Crossref PubMed Scopus (223) Google Scholar Finally, one may wonder whether the NTCP homologues in woodchucks and ducks mediate infection by the related woodchuck hepatitis virus and duck HBV (DHBV). The previously identified carboxypeptidase D and glycine decarboxylase for DHBV infection18Kuroki K. Eng F. Ishikawa T. et al.gp180, a host cell glycoprotein that binds duck hepatitis B virus particles, is encoded by a member of the carboxypeptidase gene family.J Biol Chem. 1995; 270: 15022-15028Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 19Tong S. Li J. Wands J.R. Carboxypeptidase D is an avian hepatitis B virus receptor.J Virol. 1999; 73: 8696-8702Crossref PubMed Google Scholar, 20Li J. Tong S. Lee H.B. et al.Glycine decarboxylase mediates a postbinding step in duck hepatitis B virus infection.J Virol. 2004; 78: 1873-1881Crossref PubMed Scopus (15) Google Scholar are unrelated to NTCP. They seem to be necessary for DHBV infection, but failed to reconstitute DHBV susceptibility, at least in a chicken hepatoma cell line. See “Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes,” by Ni Y, Lempp FA, Mehrle S, et al, on page 1070. See “Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes,” by Ni Y, Lempp FA, Mehrle S, et al, on page 1070. See “Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes,” by Ni Y, Lempp FA, Mehrle S, et al, on page 1070. Considering that the hepatitis B virus (HBV) genome was cloned >3 decades ago, the journey toward the identification of its receptor has been extremely slow and tortuous. HBV expresses 3 co-terminal envelope proteins termed large (L), middle (M), and small (S), which contain preS1+preS2+S, preS2+S, and S domain alone, respectively. The preS1 domain is believed to mediate virus attachment to the high-affinity receptor, although anti-preS2 and anti-S antibodies can neutralize infectivity as well. In fact, the S domain mediates initial HBV recruitment to hepatocyte surface via heparan sulfate proteoglycans.1Schulze 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 (301) Google Scholar, 2Leistner 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, 3Sureau C, Salisse J. A conformational heparan sulfate binding site essential to infectivity overlaps with the conserved hepatitis B virus a-determinant. Hepatology;57:985–994.Google Scholar In the last 2 decades, more than a dozen host-binding proteins to the preS1, preS2, or S domain have been identified.4Glebe D. Urban S. Viral and cellular determinants involved in hepadnaviral entry.World J Gastroenterol. 2007; 13: 22-38Crossref PubMed Scopus (223) Google Scholar For a few such proteins, cDNA transfection conferred HBV infection based on the sensitive polymerase chain reaction, but without independent confirmation. Another approach is to determine whether silencing their expression impairs HBV infection in a susceptible cell line. Unfortunately, no such cell line was available until 2002, when Gripon et al5Gripon P. Rumin S. Urban S. et al.Infection of a human hepatoma cell line by hepatitis B virus.Proc Natl Acad Sci U S A. 2002; 99: 15655-15660Crossref PubMed Scopus (930) Google Scholar reported that dimethyl sulfoxide (DMSO) treatment renders a human liver progenitor cell line, HepaRG, susceptible to low-level HBV infection. In this regard, DMSO could prolong the HBV susceptibility of cultured primary human hepatocytes by maintaining cellular differentiation.6Gripon P. Diot C. Theze N. 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 Adding polyethylene glycol during virus incubation also enhances infectivity of cell culture-derived HBV particles, probably by concentrating virions on the cell surface. Such improvements, together with the availability of primary Tupaia hepatocytes (PTH) as a more reliable source of in vitro infection, paved the way for the identification of sodium taurocholate cotransporting polypeptide (NTCP) as the candidate HBV receptor.7Yan H. Zhong G. Xu G. et al.Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.Elife. 2012; 1: e00049Crossref PubMed Scopus (1339) Google Scholar Although this provocative finding from Li’s group attracted lots of attention, the report by Ni et al8Ni Y. Lempp F.A. Mehrle S. et al.Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes.Gastroenterology. 2014; 146: 1070-1083Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar in the current issue of Gastroenterology represents the critical, independent confirmation. In their original eLife report,7Yan H. Zhong G. Xu G. et al.Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.Elife. 2012; 1: e00049Crossref PubMed Scopus (1339) Google Scholar Yan et al employed a novel technique to establish NTCP as a binding partner for the myristoylated peptide 2-48 of the preS1 domain. Lack of NTCP expression in 2 human hepatoma cell lines, HepG2 and Huh7 cells, is consistent with their resistance to HBV infection. Moreover, in vitro culture of PTH led to a rapid loss of NTCP expression, as expected for an HBV receptor. Importantly, silencing NTCP expression by shRNA reduced HBV infectivity in PTH, primary human hepatocyte, and HepaRG cells. It also diminished infectivity of hepatitis delta virus (HDV), which employs envelope proteins of HBV for its transmission. Conversely, transfection with human NTCP (hNTCP) cDNA enabled HBV infection of HepG2 cells, as well as HDV infection of both HepG2 and Huh7 cells. A limitation of that study was that they relied heavily on hepatitis B surface antigen (HBsAg, the secreted S protein) and hepatitis B e antigen (HBeAg, a secreted version of viral core protein) as indicators of successful HBV infection. Measurement of viral mRNA and replicative DNA was achieved mostly by polymerase chain reaction, thus raising issues about the robustness of infection. Consistent with the Yan report, Ni et al from Stephan Urban’s laboratory observed a 3.6-fold induction of hNTCP mRNA in HepaRG cells differentiated by DMSO treatment.8Ni Y. Lempp F.A. Mehrle S. et al.Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes.Gastroenterology. 2014; 146: 1070-1083Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar One of the 2 shRNAs nearly abolished hNTCP expression when delivered by lentivirus vector. Its ability to completely block HBV and HDV infection in HepaRG cells implicates NTCP as the dominant port of HBV/HDV entry in this particular cell line. Transfection of the hNTCP cDNA into HepG2, Huh7, and HepaRG cells conferred binding of myristoylated preS1 peptide. HDV infection was reconstituted more efficiently in hNTCP-transfected Huh7 and HepaRG cells, yet only HepG2 cells showed evidence of core protein expression as well as HBsAg and HBeAg secretion following HBV infection (summarized in Table 1). Therefore, molecules other than hNTCP are required for infectivity of both viruses, and such factors are virus specific and differentially expressed in the 3 cell lines. Treatment of NTCP-reconstituted cells with 2.5% DMSO markedly increased core protein expression, DNA replication, and HBeAg secretion in both HepG2 and Huh7 cell lines. It also enabled HBsAg secretion from Huh7 cells. In the presence of DMSO, NTCP transfected HepG2 cells showed a 100-fold higher level of HBeAg secretion than HepaRG cells. Consequently HBV RNAs, replicative DNA, and even the covalently closed circular DNA, the template for RNA transcription, could be detected by conventional hybridization technique. On the other hand, for unknown reasons, HBsAg secretion remained inefficient in hNTCP-transfected HepG2 cells despite DMSO treatment. DMSO, dimethyl sulfoxide; hNTCP, human NTCP; mNTCP, mouse NTCP; ND, not determined Because the normal function of NTCP is to import bile salts, Ni et al asked whether HBV exploits this property for entry into hepatocytes. Indeed, the myristoylated preS1 peptide inhibited transport of taurocholate into hNTCP transfected HepG2 cells. Conversely, several bile salts inhibited binding of the preS1 peptide and consequently HBV infection of the hNTCP transfected Huh7 cells. Very similar results were obtained by the Li group and by Watashi et al.9Yan H. Peng B. Liu Y. et al.Viral entry of Hepatitis B and D viruses and bile salts transportation share common molecular determinants on sodium taurocholate cotransporting polypeptide.J Virol. 2014 Jan 3; ([Epub ahead of print])Crossref Scopus (180) Google Scholar, 10Watashi K. Sluder A. Daito T. et al.Cyclosporin A and its analogs inhibit hepatitis B virus entry into cultured hepatocytes through targeting a membrane transporter NTCP.Hepatology. 2013 Dec 21; ([Epub ahead of print])Google Scholar Furthermore, Yan et al9Yan H. Peng B. Liu Y. et al.Viral entry of Hepatitis B and D viruses and bile salts transportation share common molecular determinants on sodium taurocholate cotransporting polypeptide.J Virol. 2014 Jan 3; ([Epub ahead of print])Crossref Scopus (180) Google Scholar found residues in the hNTCP molecule critical for bile salt transport were also essential for hNTCP to serve as the HBV receptor. Further studies are needed to determine whether HBV infection interferes with bile salt metabolism. Identification of NTCP as an HBV receptor raises the issue of how this molecule could account for the species specificity of HBV infection, and whether it is possible to establish a small animal model of HBV infection via NTCP reconstitution or humanization. hNTCP is a glycoprotein of 349 residues. The Tupaia homologue could serve as a receptor for HBV and related woolly monkey HBV despite a 30-amino acid insertion.11Zhong G. Yan H. Wang H. et al.Sodium taurocholate cotransporting polypeptide mediates woolly monkey hepatitis B virus infection of Tupaia hepatocytes.J Virol. 2013; 87: 7176-7184Crossref PubMed Scopus (51) Google Scholar In contrast, NTCP from crab-eating monkey lacked affinity for myristoylated preS1 peptide and failed to mediate HBV infection.7Yan H. Zhong G. Xu G. et al.Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.Elife. 2012; 1: e00049Crossref PubMed Scopus (1339) Google Scholar Mouse NTCP failed to confer susceptibility to HBV infection despite its affinity for myristoylated preS1 peptide,8Ni Y. Lempp F.A. Mehrle S. et al.Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes.Gastroenterology. 2014; 146: 1070-1083Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar, 12Yan H. Peng B. He W. et al.Molecular determinants of hepatitis B and D virus entry restriction in mouse sodium taurocholate cotransporting polypeptide.J Virol. 2013; 87: 7977-7991Crossref PubMed Scopus (139) Google Scholar suggesting a role of NTCP beyond high-affinity binding to HBV virions. Unfortunately, hNTCP could not confer HBV infection in the 2 mouse hepatoma cell lines and rat hepatoma cell line tested (Table 1),8Ni Y. Lempp F.A. Mehrle S. et al.Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes.Gastroenterology. 2014; 146: 1070-1083Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar, 12Yan H. Peng B. He W. et al.Molecular determinants of hepatitis B and D virus entry restriction in mouse sodium taurocholate cotransporting polypeptide.J Virol. 2013; 87: 7977-7991Crossref PubMed Scopus (139) Google Scholar thus dashing the hopes for the establishment of a small animal model of HBV infection in the near future. Besides the paper by Ni et al in the current issue of Gastroenterology others have also confirmed the ability of hNTCP to confer susceptibility to HBV infection in HepG2 cells10Watashi K. Sluder A. Daito T. et al.Cyclosporin A and its analogs inhibit hepatitis B virus entry into cultured hepatocytes through targeting a membrane transporter NTCP.Hepatology. 2013 Dec 21; ([Epub ahead of print])Google Scholar (our unpublished observation). Moreover, 2 upcoming papers, one from the Urban laboratory, demonstrated that cyclosporine inhibits HBV infection by targeting NTCP.10Watashi K. Sluder A. Daito T. et al.Cyclosporin A and its analogs inhibit hepatitis B virus entry into cultured hepatocytes through targeting a membrane transporter NTCP.Hepatology. 2013 Dec 21; ([Epub ahead of print])Google Scholar, 13Nkongolo S. Ni Y. Lempp F.A. et al.Cyclosporin A inhibits hepatitis B and hepatitis D virus entry by cyclophilin-independent interference with the NTCP receptor.J Hepatol. 2013 Dec 1; ([Epub ahead of print])PubMed Google Scholar Thus, NTCP serves as an HBV receptor in primary human hepatocytes, PTH, HepaRG, and HepG2 cells. However, one has to bear in mind that the current infection system is based on cell culture-derived HBV particles added at extremely high multiplicity of infection in the presence of polyethylene glycol. In vivo, a few virus particles injected through the bloodstream are sufficient to infect the entire liver of a chimpanzee.14Asabe S. Wieland S.F. Chattopadhyay P.K. et al.The size of the viral inoculum contributes to the outcome of hepatitis B virus infection.J Virol. 2009; 83: 9652-9662Crossref PubMed Scopus (222) Google Scholar Thus, it remains to be seen whether the NTCP system of HBV infection can be improved by the addition of other host factors and whether NTCP can mediate the infection of serum-derived HBV particles. Another question is whether NTCP constitutes the only or major HBV receptor in vivo. In this regard, Li’s group found a naturally occurring polymorphism in the NTCP gene (S267F), which abrogated its ability to support HBV infection in cell culture.9Yan H. Peng B. Liu Y. et al.Viral entry of Hepatitis B and D viruses and bile salts transportation share common molecular determinants on sodium taurocholate cotransporting polypeptide.J Virol. 2014 Jan 3; ([Epub ahead of print])Crossref Scopus (180) Google Scholar About 0.3% of East Asians harbor this mutation in both alleles. HBV infection in any of such individuals will cast doubt on the role of NTCP as the only HBV receptor. Nevertheless, some features of NTCP are compatible with its role as the HBV receptor. Both myristoylated and cholesterol-modified preS1 peptide 2-48, but not the unmodified peptide, could interact with NTCP.9Yan H. Peng B. Liu Y. et al.Viral entry of Hepatitis B and D viruses and bile salts transportation share common molecular determinants on sodium taurocholate cotransporting polypeptide.J Virol. 2014 Jan 3; ([Epub ahead of print])Crossref Scopus (180) Google Scholar In this regard, cholesterol is important for HBV infectivity,15Bremer C.M. Bung C. Kott N. et al.Hepatitis B virus infection is dependent on cholesterol in the viral envelope.Cell Microbiol. 2009; 11: 249-260Crossref PubMed Scopus (70) Google Scholar and L protein is myristoylated at position 2 by a glycine residue, which is dispensable for HBV virion formation but essential for infectivity.16Gripon P. Le Seyec J. Rumin S. et al.Myristylation of the hepatitis B virus large surface protein is essential for viral infectivity.Virology. 1995; 213: 292-299Crossref PubMed Scopus (150) Google Scholar Ni et al should be applauded for improving the in vitro HBV infection system, which will prove useful for many different applications, such as the screening of more potent entry inhibitors and testing of the infectivity of viral variants. Identification of NTCP as an HBV receptor should usher in an exciting era of research. An immediate question concerns the nature of co-receptors or co-factors for productive HBV infection, such as those responsible for differential infectivity between NTCP-reconstituted HepG2 and Huh7 cells, as well as those induced by DMSO in these cell lines. As post receptor binding steps for productive HBV infection include membrane fusion (which is most likely mediated by the S domain and may occur at plasma or endosomal membrane), capsid disassembly, nuclear targeting of the relaxed circular DNA genome, and its conversion to the covalently closed circular DNA, the HepG2-enriched and DMSO-induced factors could affect any of these steps. If the example of hepatitis C virus, an RNA virus targeting the liver, is any indication,17Meredith L.W. Wilson G.K. Fletcher N.F. et al.Hepatitis C virus entry: beyond receptors.Rev Med Virol. 2012; 22: 182-193Crossref PubMed Scopus (72) Google Scholar many more host factors for HBV infection are to be discovered. It will be interesting in this regard to reexamine the previously identified binding partners of HBV envelope proteins for their contribution to efficient HBV infection.4Glebe D. Urban S. Viral and cellular determinants involved in hepadnaviral entry.World J Gastroenterol. 2007; 13: 22-38Crossref PubMed Scopus (223) Google Scholar Finally, one may wonder whether the NTCP homologues in woodchucks and ducks mediate infection by the related woodchuck hepatitis virus and duck HBV (DHBV). The previously identified carboxypeptidase D and glycine decarboxylase for DHBV infection18Kuroki K. Eng F. Ishikawa T. et al.gp180, a host cell glycoprotein that binds duck hepatitis B virus particles, is encoded by a member of the carboxypeptidase gene family.J Biol Chem. 1995; 270: 15022-15028Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 19Tong S. Li J. Wands J.R. Carboxypeptidase D is an avian hepatitis B virus receptor.J Virol. 1999; 73: 8696-8702Crossref PubMed Google Scholar, 20Li J. Tong S. Lee H.B. et al.Glycine decarboxylase mediates a postbinding step in duck hepatitis B virus infection.J Virol. 2004; 78: 1873-1881Crossref PubMed Scopus (15) Google Scholar are unrelated to NTCP. They seem to be necessary for DHBV infection, but failed to reconstitute DHBV susceptibility, at least in a chicken hepatoma cell line. Hepatitis B and D Viruses Exploit Sodium Taurocholate Co-transporting Polypeptide for Species-Specific Entry into HepatocytesGastroenterologyVol. 146Issue 4PreviewHepatitis 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. Full-Text PDF