Human Hepatitis B Virus Receptor Research Articles

Quantification of Hepatitis B Virus Covalently Closed Circular DNA in Infected Cell Culture Models by Quantitative PCR.

Persistence of the human hepatitis B virus (HBV) requires the maintenance of covalently closed circular (ccc)DNA, the episomal genome reservoir in nuclei of infected hepatocytes. cccDNA elimination is a major aim in future curative therapies currently under development. In cell culture based in vitro studies, both hybridization- and amplification-based assays are currently used for cccDNA quantification. Southern blot, the current gold standard, is time-consuming and not practical for a large number of samples. PCR-based methods show limited specificity when excessive HBV replicative intermediates are present. We have recently developed a real-time quantitative PCR protocol, in which total cellular DNA plus all forms of viral DNA are extracted by silica column. Subsequent incubation with T5 exonuclease efficiently removes cellular DNA and all non-cccDNA forms of viral DNA while cccDNA remains intact and can reliably be quantified by PCR. This method has been used for measuring kinetics of cccDNA accumulation in several in vitro infection models and the effect of antivirals on cccDNA. It allowed detection of cccDNA in non-human cells (primary macaque and swine hepatocytes, etc.) reconstituted with the HBV receptor, human sodium taurocholate cotransporting polypeptide (NTCP). Here we present a detailed protocol of this method, including a work flowchart, schematic diagram and illustrations on how to calculate "cccDNA copies per (infected) cell".

Sodium taurocholate cotransporting polypeptide is the limiting host factor of hepatitis B virus infection in macaque and pig hepatocytes

Ntcp is the key host factor limiting HBV infection in cynomolgus and rhesus macaques and in pigs. In rodents (mouse, rat) and dogs, transfer of hNTCP supports viral entry but additional host factors are required for the establishment of HBV infection. This finding paves the way for the development of macaques and pigs as immunocompetent animal models to study HBV infection in vivo, immunological responses against the virus and viral pathogenesis. (Hepatology 2017;66:703-716).

Site‐Specific Engineering of Chemical Functionalities on the Surface of Live Hepatitis D Virus

The genetic code expansion strategy, the recently emerged pyrrolysine (Pyl)-based system in particular, has become a generally applicable method for site-specific incorporation of unnatural amino acids (UAAs) into a protein of interest in bacteria, yeast, mammalian cells, and even in animals. However, this technique has yet to be applied to intact and live viruses, which is largely due to the fragile nature as well as the complicated assembly process of many human viruses. To address this challenge, we here coupled the genetic-code expansion strategy with an engineered virus assembly process in human hepatocytes to site-specifically introduce unnatural chemical groups onto virus surface proteins by using hepatitis D virus (HDV) as a model system. HDV has infectedmore than 15 million people worldwide, and currently there are no drugs clinically available against this virus. HDV is a satellite virus of human hepatitis B virus (HBV), which has infected two billion people and among them about 240 million are currently chronically infected. Both HBV and HDV share the same envelope proteins for infection of hepatocytes. Study of HBV and HDV infection has long been hampered by the lack of efficient and easily accessible in vitro infection system. Recently, a bile acid transporter predominantly expressed in liver, sodium taurocholate cotransporting polypeptide (NTCP) was identified as a functional receptor for HDV and HBV. The NTCP complemented human hepatoma cell line HepG2 provided a feasible in vitro infection system for studying HBV and HDV infection. However, the lack of methods to selectively label, monitor, and/or manipulate an intact virus under living conditions still restricts investigations into molecular details of the infection. Many problems are due to the distinct topological features of the critical viral proteins, as well as complex virus assembly processes. For example, HDV has developed a tightly regulated assembly process to produce infectious viral particles in human hepatocytes: the HDV RNAs were first encapsulated with delta antigens and then packaged with three HBV envelope proteins, namely large (L), middle (M), and small (S) proteins, to produce the intact viral particle before being secreted to the extracellular space (Supporting Information, Figure S1). The resulting HDV, with a diameter of 36 nm, is one of the smallest animal viruses known to date. It is therefore exceedingly difficult to chemically label this tiny virus with delicate structures under living conditions. Furthermore, the virus surface envelope proteins contain many chemically active amino acids (for example, cysteine and lysine) that are essential for virus entry in host cells. Conjugation or modification of these natural residues will severely compromise viral infectivity. A noninvasive strategy for manipulation of living viral particles without impairment of their viability and infectivity is thus highly desired. Bioorthogonal reactions have revolutionized our ability to label and manipulate various biomolecules and even whole cells and organisms under living conditions. As a critical step for applying such chemistry for virus labeling, several approaches have been reported for installation of bioorthogonal handles, typically in the form of UAAs into proteins from sub-viral-like particle (SVP) or intact virus. For instance, site-specific or residue-specific incorporation of UAAs bearing an azide or an alkyne moiety into SVP has been demonstrated in bacterial cells by several laboratories. These methods allow the conjugation of SVPs with various fluorescent dyes or therapeutic reagents for biomedical or biomaterial applications. However, SVPs are non-infectious and not suitable for investigating virus infection mechanisms. Indeed, SVPs produced from prokaryotic cells lack posttranslational modifications, particularly on their surface envelope proteins, and therefore differ from the native SVPs. Although attempts have been made to extend some of these methods for virus production in mammalian cells, such strategies typically require the metabolic replacement of a specific type of amino acid permissive only to simple groups (for example, azide and ketone) from the entire virus proteome, which may disrupt the virion assembly process or permute the vulnerable virion structure, resulting in compromised viral infectivity. Taken together, a general approach for precise labeling and manipulation of intact [*] S.-X. Lin, M.-Y. Yang, Prof. Dr. P. R. Chen Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center and Peking-Tsinghua Center for Life Sciences Peking University, Beijing 100871 (China) E-mail: pengchen@pku.edu.cn H. Yan Graduate program in School of Life Sciences Peking University, Beijing (China) H. Yan, L. Li, B. Peng, S. Chen, Prof. Dr. W.-H. Li National Institute of Biological Sciences, Beijing (China) E-mail: liwenhui@nibs.ac.cn [] These authors contributed equally to this work.

Persistent human hepatitis B virus infection in cynomolgus monkeys: A novel animal model in the search for a cure?

Persistent human hepatitis B virus infection in cynomolgus monkeys: A novel animal model in the search for a cure?

Myristoylated PreS1-domain of the hepatitis B virus L-protein mediates specific binding to differentiated hepatocytes

Chronic infection with the human hepatitis B virus (HBV) is a global health problem and a main cause of progressive liver diseases. HBV exhibits a narrow host range, replicating primarily in hepatocytes. Both host and hepatocyte specificity presumably involve specific receptor interactions on the target cell; however, direct evidence for this hypothesis is missing. Following the observation that HBV entry is specifically blocked by L-protein-derived preS1-lipopeptides, we visualized specific HBV receptor/ligand complexes on hepatic cells and quantified the turnover kinetics. Using fluorescein isothiocyanate-labeled, myristoylated HBV preS1-peptides we demonstrate (1) the presence of a highly specific HBV receptor on the plasma membrane of HBV-susceptible primary human and tupaia hepatocytes and HepaRG cells but also on hepatocytes from the nonsusceptible species mouse, rat, rabbit and dog; (2) the requirement of a differentiated state of the hepatocyte for specific preS1-binding; (3) the lack of detectable amounts of the receptor on HepG2 and HuH7 cells; (4) a slow receptor turnover at the hepatocyte membrane; and (5) an association of the receptor with actin microfilaments. The presence of the preS1-receptor in primary hepatocytes from some non-HBV-susceptible species indicates that the lack of susceptibility of these cells is owed to a postbinding step. These findings suggest that HBV hepatotropism is mediated by the highly selective expression of a yet unknown receptor* on differentiated hepatocytes, while species specificity of the HBV infection requires selective downstream events, e.g., the presence of host dependency or the absence of host restriction factors. The criteria defined here will allow narrowing down reasonable receptor candidates and provide a binding assay for HBV-receptor expression screens in hepatic cells.

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

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.

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

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.

Hepatocyte Polarization Is Essential for the Productive Entry of the Hepatitis B Virus

Human hepatitis B virus (HBV) is characterized by a high species specificity and a distinct liver tropism. Within the liver, HBV replication occurs in differentiated and polarized hepatocytes. Accordingly, the in vitro HBV infection of primary human hepatocytes (PHHs) and the human hepatoma cell line, HepaRG, is restricted to differentiated, hepatocyte-like cells. Though preparations of PHH contain up to 100% hepatic cells, cultures of differentiated HepaRG cells are a mixture of hepatocyte-like and biliary-like epithelial cells. We used PHH and HepaRG cells and compared the influence of virus inoculation dose, cell differentiation, and polarization on productive HBV infection. At multiplicities of genome equivalents (mge) >8,000, almost 100% of PHHs could be infected. In contrast, only a subset of HepaRG cells stained positive for HBcAg at comparable or even higher mge. Infection predominantly occurred at the edges of islands of hepatocyte-like HepaRG cells. This indicates a limited accessibility of the HBV receptor, possibly as a result of its polar sorting. Multidrug resistance protein 2 (MRP2), a marker selectively transported to the apical (i.e., canalicular) cell membrane, revealed two polarization phenotypes of HepaRG cells. HBV infection within the islands of hepatocyte-like HepaRG cells preferentially occurred in cells that resemble PHH, exhibiting canalicular structures. However, disruption of cell-cell junctions allowed the additional infection of cells that do not display a PHH-like polarization. HBV enters hepatocytes via the basolateral membrane. This model, at least partially, explains the difference of PHH and HepaRG cells in infection efficacy, provides insights into natural HBV infection, and establishes a basis for optimization of the HepaRG infection system.

Viral and cellular determinants involved in hepadnaviral entry

Hepadnaviridae is a family of hepatotropic DNA viruses that is divided into the genera orthohepadnavirus of mammals and avihepadnavirus of birds. All members of this family can cause acute and chronic hepatic infection, which in the case of human hepatitis B virus (HBV) constitutes a major global health problem. Although our knowledge about the molecular biology of these highly liver-specific viruses has profoundly increased in the last two decades, the mechanisms of attachment and productive entrance into the differentiated host hepatocytes are still enigmatic. The difficulties in studying hepadnaviral entry were primarily caused by the lack of easily accessible in vitro infection systems. Thus, for more than twenty years, differentiated primary hepatocytes from the respective species were the only in vitro models for both orthohepadnaviruses (e.g. HBV) and avihepadnaviruses (e.g. duck hepatitis B virus [DHBV]). Two important discoveries have been made recently regarding HBV: (1) primary hepatocytes from tree-shrews; i.e., Tupaia belangeri, can be substituted for primary human hepatocytes, and (2) a human hepatoma cell line (HepaRG) was established that gains susceptibility for HBV infection upon induction of differentiation in vitro. A number of potential HBV receptor candidates have been described in the past, but none of them have been confirmed to function as a receptor. For DHBV and probably all other avian hepadnaviruses, carboxypeptidase D (CPD) has been shown to be indispensable for infection, although the exact role of this molecule is still under debate. While still restricted to the use of primary duck hepatocytes (PDH), investigations performed with DHBV provided important general concepts on the first steps of hepadnaviral infection. However, with emerging data obtained from the new HBV infection systems, the hope that DHBV utilizes the same mechanism as HBV only partially held true. Nevertheless, both HBV and DHBV in vitro infection systems will help to: (1) functionally dissect the hepadnaviral entry pathways, (2) perform reverse genetics (e.g. test the fitness of escape mutants), (3) titrate and map neutralizing antibodies, (4) improve current vaccines to combat acute and chronic infections of hepatitis B, and (5) develop entry inhibitors for future clinical applications.

N-terminal myristylation of HBV preS1 domain enhances receptor recognition.

The N-terminal portion of the large envelope protein of the human hepatitis B virus (HBV), the preS1 domain, plays a fundamental role in cell attachment and infectivity. Recent investigations have suggested that myristylation of preS1 Gly2 residue is essential for viral infectivity, but the importance of this post-translational modification on HBV-receptor interaction has not been elucidated completely. In this study we produced, using stepwise solid-phase chemical synthesis, the entire preS1[1-119] domain (adw2 subtype), and compared its receptor binding activity with the myristylated form, myristyl-preS1[2-119] in order to define the importance of fatty acid modification. Both synthetic proteins were fully characterized in terms of structural identity using TOF-MALDI mass spectrometry and analysis of tryptic fragments. Circular dichroism measurements indicated a low content of ordered structure in the preS1 protein, while the propensity of the myristylated derivative to assume a conformationally defined structure was more evident. HBV-receptor binding assays performed with plasma membranes preparations from the hepatocyte carcinoma cell line HepG2 clearly showed that the preS1[1-119] domain recognizes the HBV receptor, and confirmed that binding is occurring through the 21-47 region. The myristylated derivative recognized HBV receptor preparations with higher affinity than the preS1 domain, suggesting that the conformational transitions induced in the preS1 moiety by fatty acid post-translational modification are important for efficient attachment of viral particles to HBV receptors.