More than 350 million people worldwide are chronically infected with hepatitis B virus (HBV), facing the prospects of disease progression to cirrhosis, liver failure and—in many instances—cancer. More than 50% of liver cancers are due to HBV infection; however, research into viral infection is limited. Disease is more severe when patients are co-infected with hepatitis D virus (HDV). Described as a satellite RNA virus with similar envelope protein structures, HDV can only cause disease in association with HBV and is thought to infect cells in a similar manner of entry. Although the pre-S1 domain of the large envelope L- protein is necessary for HBV and HDV to bind to cells and infect them,1 the identity of the receptor involved has remained unknown. Without a receptor, researchers are severely hampered in making progress with this global public health problem.
Using a combined –omics approach, however, Yan et al. have identified sodium taurocholate cotransporting polypeptide (NTCP) as the receptor involved in pre-S1 binding, demonstrating that it is essential to HBV and also HDV infectivity.2 In order to uncover the cell surface binding site of the two viruses, Yan and co-authors used tree shrew hepatic cells (PTHs) in culture for binding studies. Tree shrews, Tupaia belangeri, are one of only two non-human species sensitive to HBV infection. Using various competitive binding assays involving mutated non-binding variants and inhibitors of receptor interaction, the researchers confirmed that their synthetic pre-S1 peptide behaved similarly to HBV and HDV.
Once confirmed, the researchers added the synthetic pre-S1 peptide to the PTHs in culture, inducing photo-crosslinking with the putative receptor by UV irradiation. Cross-linked products were affinity purified, then separated by SDS-PAGE before in-gel trypsin digestion. The researchers identified the digestion fragments by mass spectrometry (MS) using an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific). They examined the tandem MS data against a tree shrew hepatic protein database previously constructed from transcriptome deep sequencing, identifying the cross-linked fragment products by matching them to a homologue of human (h) NTCP.
Yan et al. confirmed their choice of NTCP as a presumed HBV and HDV receptor by cloning and expressing it, then challenging the expression products in binding studies with their synthetic pre-S1 peptide.
The researchers then investigated whether NTCP was required for HBV and HDV viral infectivity, transfecting PTHs with agents to interfere specifically with the NTCP gene. Gene silencing dramatically reduced HDV and HBV infectivity in PTHs and in the human cell line HepaRG. HepaRG cells are sensitive to HBV and HDV infection only once differentiated. Researchers also found that NTCP mRNA levels increased with differentiation. Furthermore, inducing NTCP expression by transfection of two human cell lines normally immune to HBV/HDV infection, HepG2 and Huh-7, rendered the hepatic cultures susceptible to viral infection.
As a final step, Yan and co-workers compared the hNTCP protein sequence with that of the crab-eating monkey’s (Macaca fascicularis) NTCP (mkNTCP). NTCP is widely conserved among mammals, but specific sequence differences occur naturally, possibly conferring resistance to HBV infection and to the low number of species susceptible in the wild. Although the two share 96.3% sequence homology, mkNTCP cannot bind HDV or the pre-S1 synthetic peptide. The researchers made a series of alterations in the hNTCP and mkNTCP proteins between amino acid (aa) residues 157–165, either abolishing or restoring HDV or pre-S1 synthetic peptide binding, depending on the aa motif substituted. Inserting mkNTCP aa157–165 into hNTCP abolished binding, whereas inserting the hNTCP sequence into mkNTCP restored infectivity.
Although other factors might play roles in facilitating viral entry, the discovery of NTCP as a cell surface receptor is an important step in determining pathways of viral pathogenesis and elucidating new treatment options.
1. Le Seyec, J., et al. (1999) “Infection process of the hepatitis B virus depends on the presence of a defined sequence in the pre-S1 domain,” Journal of Virology, 73 (pp. 2052–7).
2. Yan, H., et al. (2012) “Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus,” eLife, 1, doi: 10.7554/eLife.00049.
Post Author: Amanda Maxwell. Mixed media artist; blogger and social media communicator; clinical scientist and writer.
A digital space explorer, engaging readers by translating complex theories and subjects creatively into everyday language.