Hepatitis B and D viral receptors
Authors: Jisu Li, Jack Wands
Journal: Hepatology (2015)

Potential conflict of interest: Nothing to report. See Article on Page 35. The entry process of hepatitis B virus (HBV) into hepatocytes has been a long‐standing puzzle. HBV is an enveloped virus, with its nucleocapsid shielding a partially double‐stranded DNA genome. Cloned HBV genome could initiate DNA replication if forced into HepG2 or Huh7 human hepatoma cell lines by transient transfection. This opened the door for powerful reverse genetic approaches to characterize, in great detail, the late events of the HBV life cycle, including RNA transcription, protein translation, DNA replication, and virion release from the cell. However, neither HepG2 nor Huh7 cells are susceptible to HBV infection. Initially, primary human hepatocytes were the only model for in vitro infection with HBV, which has now been supplemented with primary Tupaia hepatocytes and differentiated HepaRG cells, a human liver progenitor cell line. HBV infectivity in vitro was found to be extremely low, which could be partially overcome by adding 4% polyethylene glycol (PEG) during virus incubation.1 One way to facilitate the identification of HBV receptor(s) is to better define the viral components involved. HBV expresses three coterminal envelope proteins named large (L), middle (M), and small (S) through alternative translation initiation. Consequently, the L protein contains an extra pre‐S1 domain compared to the M protein, which, in turn, has an extra pre‐S2 domain than the S protein. Mutational analysis identified the amino terminus of the pre‐S1 domain, including the myristoylation signal, as well as the “a” determinant in the S domain, as essential for HBV infectivity. Yan and colleagues identified sodium taurocholate cotransoporting polypeptide (NTCP) as a binding partner for myristoylated pre‐S1 peptide 2‐48, as shown in Fig. 1. Silencing NTCP expression diminished HBV infectivity; conversely, ectopic NTCP expression rendered HepG2 cells susceptible to HBV infection.2 These provocative findings have since been independently confirmed.3Figure 1: Cell‐surface receptors mediating HBV entry into hepatocytes. The envelope of HBV virions is composed of large (L), middle (M), and small (S) envelope proteins, with L protein having an extra pre‐S1 domain than M, and M having an extra pre‐S2 domain than S. The internal nucleocapsid shields partially double‐stranded viral DNA. The virions initially bind with low affinity to HSPG, at least partly through the S domain. Through an unknown mechanism, HBV subsequently switches its binding to NTCP through the pre‐S1 domain and with high affinity. Verrier et al. identified GPC5 as the core protein of HSPG mediating initial HBV contact, thus making GPC5 antibodies and siRNAs potential therapeutics to block HBV/HDV infection.7 Other agents that inhibit viral entry through the receptors are shown in the dotted squares. Abbreviation: abs, antibodies.Heparin columns have been used to purify HBV particles. Employing primary Tupaia hepatocytes and HepaRG cells, respectively, two groups found that heparin could inhibit HBV infection.4 Preincubating HBV with heparin produced a similar effect, confirming heparin targets HBV rather than the host cells. Considering that heparin is a soluble form of highly sulfated (negatively charged) glycosaminoglycans (GAGs), the GAG side chains of heparin sulfate proteoglycans (HSPGs) probably serve as a low‐affinity HBV receptor. Indeed, pretreatment of cells with an inhibitor of HSPG sulfation diminished HBV infectivity. Likewise, treatment of cells with heprinase I or III to remove the cell‐surface glycans also compromised HBV infectivity. It should be emphasized that HBV infectivity is usually measured by hepatitis B e antigen (HBeAg) released from infected cells, which requires many postentry steps including nucleocapsid disassembly, release of the HBV genome, its conversion to covalently closed circular DNA, and HBV‐RNA transcription. Thus, there is the formal possibility that NTCP or HSPG work at a postentry step. In this regard, hepatitis delta virus (HDV) requires HBV envelope proteins for virion formation and infectivity, but its negative‐stranded RNA genome of HDV replicates through antigenomic RNA, and its nucleocapsid is formed by hepatitis delta antigen (HDAg), which are different from the HBV life cycle. Comparative analysis of HBV and HDV infectivity will help to establish whether the host factor affects the shared steps of virus binding, endocytosis, and uncoating. In this regard, NTCP reconstitution could render Huh7 cells susceptible to HDV infection,2 whereas HSPG was essential for HDV infection of differentiated HepaRG cells as well.6 Therefore, NTCP serves as the high‐affinity receptor for HBV and HDV, whereas HSPG appears to be a low‐affinity receptor for both viruses. The HSPG gene family contains agrin, perlecan, syndecan 1‐4, and glypican 1‐6. Which member mediates HBV/HDV infectivity was the issue addressed by Verrier et al. in this issue of Hepatology.7 They established single clones of Huh7 cells stably transfected with human NTCP. One of one hundred twenty clones was highly susceptible to HDV infection, even in the absence of PEG, into which expression of the 12 HSPG genes was silenced using small interfering RNA (siRNA) pools. In this context, siRNAs directed against glypican 5 (GPC5) reduced HDAg‐positive cells by 44%, in contrast to 25% reduction achieved by the next most effective siRNAs (syndecan 2). GPC5 siRNAs also diminished HDV infectivity in primary human hepatocytes, as well as HBV infectivity in NTCP‐HepG2 cells. Moreover, a rabbit GPC5 antibody dose dependently inhibited HDV RNA in NTCP‐Huh7 cells to a level of 70%. Finally, evidence was presented supporting GPC5 as an HBV docking receptor, as depicted in Fig. 1. Thus, siRNAs against GPC5 diminished HBV binding to NTCP‐HepG2 cells, whereas GPC5 antibody diminished both HBV binding to NTCP‐HepG2 cells and HDV binding to NTCP‐Huh7 cells. Moreover, preincubation of HBV with increasing concentrations of soluble GPC5 dose dependently diminished HBV binding to NTCP‐HepG2 cells. Owing to the inability of siRNAs to completely “knock out” GPC5 expression, it is unclear whether GPC5 is the only HSPG serving as an HBV/HDV receptor, or whether the HSPG‐type of low‐affinity receptor is absolutely required for HBV/HDV infectivity. This can be established by the new gene knockout technology if GPC5 is dispensable for cell survival.8 The renewed identity of the low‐affinity HBV receptor as a gene product, rather than a sugar molecule, makes all the tools of molecular biology available to further study the HBV/HDV entry process. For example, is GPC5 expression dependent on hepatocyte differentiation and can be induced by dimethyl sulfoxide in HepaRG cells (this is the case for NTCP)? Will GPC5 overexpression increase HBV or HDV infectivity in HepG2 or Huh7 cells overexpressing NTCP? Will GPC5 complementary DNA transfection render NTCP‐Huh7 cells susceptible to HBV infection, considering that GPC5 is expressed at a lower level in Huh7 than HepG2 cells?7 Given that HSPG and NTCP mediate separate steps of virus binding, will a combination of NTCP and GPC5 siRNAs inhibit HBV/HDV infectivity to a greater extent than either siRNA alone? There is currently controversy as to whether HSPG interacts with the pre‐S domain or the S domain.4 Coimmunoprecipitation experiments might help solve this issue. A previous study suggested that PEG increases HBV infectivity by promoting virus attachment to the cell surface through HSPG.4 This phenomenon can be now studied by comparing the infection‐promoting effect of PEG in cells overexpressing GPC5 versus those depleted of GPC5. Finally, the ability of GPC5 antibody to block viral infection is attractive for postexposure prophylaxis, probably together with myristoylated pre‐S1 peptide 2‐48 and merits further investigation.