Proteoglycans act as cellular hepatitis delta virus attachment receptors - PubMed (original) (raw)
Proteoglycans act as cellular hepatitis delta virus attachment receptors
Oscar Lamas Longarela et al. PLoS One. 2013.
Abstract
The hepatitis delta virus (HDV) is a small, defective RNA virus that requires the presence of the hepatitis B virus (HBV) for its life cycle. Worldwide more than 15 million people are co-infected with HBV and HDV. Although much effort has been made, the early steps of the HBV/HDV entry process, including hepatocyte attachment and receptor interaction are still not fully understood. Numerous possible cellular HBV/HDV binding partners have been described over the last years; however, so far only heparan sulfate proteoglycans have been functionally confirmed as cell-associated HBV attachment factors. Recently, it has been suggested that ionotrophic purinergic receptors (P2XR) participate as receptors in HBV/HDV entry. Using the HBV/HDV susceptible HepaRG cell line and primary human hepatocytes (PHH), we here demonstrate that HDV entry into hepatocytes depends on the interaction with the glycosaminoglycan (GAG) side chains of cellular heparan sulfate proteoglycans. We furthermore provide evidence that P2XR are not involved in HBV/HDV entry and that effects observed with inhibitors for these receptors are a consequence of their negative charge. HDV infection was abrogated by soluble GAGs and other highly sulfated compounds. Enzymatic removal of defined carbohydrate structures from the cell surface using heparinase III or the obstruction of GAG synthesis by sodium chlorate inhibited HDV infection of HepaRG cells. Highly sulfated P2XR antagonists blocked HBV/HDV infection of HepaRG cells and PHH. In contrast, no effect on HBV/HDV infection was found when uncharged P2XR antagonists or agonists were applied. In summary, HDV infection, comparable to HBV infection, requires binding to the carbohydrate side chains of hepatocyte-associated heparan sulfate proteoglycans as attachment receptors, while P2XR are not actively involved.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. Chemical structures, charge and function of the compounds used in the study.
Listed are the structures of the repeating units (disaccharide or lysine) or the individual molecules. Charged groups are depicted in red. Possible acetylations are marked in blue. The number of charged groups per repeating unit or molecule and the function of the substance are listed. Not included are derivatives of the compounds.
Figure 2. Highly-sulfated GAGs interfere with the in vitro HDV infection. A
) HepaRG cells or PHH were infected with HDV for 16 h at 37°C. Uninfected cells or cells treated with 200 nM preS/2-48myr peptide during virus inoculation were used as controls. Overlays of HDV-specific IF (red) and nucleus staining (blue) for the respective infections on HepaRG cells (top) or PHH (bottom) on day 5 p.i. are depicted. B–E) HepaRG cells (B–D) or PHH (E) were pre-incubated for 30 min at 37°C with the indicated substances followed by HDV infection for 16 h at 37°C in presence of the compounds. HDV-specific IF was performed on day 6 p.i. Nuclei were stained with DAPI. The number of HDAg-positive cells and the total cell number were determined. The results are depicted as percentage of infected cells in comparison to the untreated control. F) HepaRG cells were pre-incubated for 30 min at 37°C with 500 nM preS/2-48myr or 100 µg/ml suramin. HDV inoculation was performed for 16 h at 37°C in presence of the substances (pre−/co-inoc.). In parallel HepaRG cells were infected with HDV for 16 h at 37°C without treatment. After inoculation, the cells were washed and 500 nM preS/2-48myr or 100 µg/ml suramin was added to the untreated, HDV infected cells. After 24 h at 37°C all cells were washed and further cultivated. HDV-specific IF was performed on day 6 p.i. Nuclei were stained with DAPI. The number of HDAg-positive cells and the total cell number were determined. The results are depicted as percentage of infected cell in comparison to the untreated control. In total, 735258 cells were counted for the experiments.
Figure 3. The obstruction or removal of negatively charged cellular interaction sites inhibit the HDV infection.
A) HepaRG cells were pre-incubated with increasing concentrations of poly-L-lysine for 30 min at 37°C and subsequently infected in presence of the polycation for 16 h at 37°C. B) HDV was pre-incubated for 1 h at 37°C with heparin (100 µg/ml and 500 µg/ml), suramin (100 µg/ml) and dextran sulfate (100 µg/ml). Unbound polyanions were removed by ultrafiltration. HepaRG cells were incubated with the pre-treated viruses for 8 h at 37°C. C) HepaRG cells were pre-incubated with heparin, suramin, dextran sulfate, pentosan polysulfate or poly-L-lysine for 1 h at 37°C. Cells were washed and incubated with HDV for 8 h at 37°C in absence of the compounds. D) Sulfation of cellular proteoglycans was inhibited by treatment of HepaRG cells with increasing concentrations of sodium chlorate for 48 h prior to HDV infection. E) HepaRG cells were incubated for 1 h at 37°C with the indicated concentrations of heparinase III or 1 µM preS/2-48myr. The cells were washed and inoculated with HDV for 16 h at 37°C in presence of the substances. HDV-specific IF was performed for all experiments on day 6 p.i. Nuclei were stained with DAPI. The number of HDAg-positive cells and the total cell number were determined. The results are depicted as percentage of infected cells in comparison to the untreated control. In total, 822302 cells were counted for the analysis.
Figure 4. Inhibition of HDV and HBV infection by P2XR antagonists and agonists is dependent on their charge. A
) HepaRG cells were infected with serum of an HDV-positive patient (GTA7) in presence of P2XR antagonists (PPADS, suramin and AZ11645373) and agonists (ivermectin) for 16 h at 37°C. PreS/2-48myr peptide (200 nM) was used in parallel as control. HDV-specific IF and nuclei staining was done 5 days p.i. The number of total and HDV-infected cells was quantified using ImageJ software. The results are depicted as percentage of the untreated control. In total, 290368 cells were counted for the analysis. Cell lysates of in parallel infected HepaRG cells were used for an HDAg-specific Western Blot. B) HepaRG cells were infected with HBV in presence of P2XR antagonists (PPADS, suramin and AZ11645373) and agonists (ivermectin) for 16 h at 37°C. Supernatants from days 8–11 p.i. were collected and secreted HBeAg was quantified by ELISA. Experiments were performed in duplicate and repeated at least two times independently. The values presented are mean ± SD. C) HepaRG cells were infected with serum of an HDV-positive patient (GTA7). Ivermectin (1 µM) was added at different time points during or after virus inoculation. HDV-specific IF and nuclei staining was done 6 days p.i. The number of total and HDV-infected cells was quantified using the ImageJ software. The results are depicted as percentage of the untreated control. In total, 128450 cells were counted for the analysis.
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Oscar Lamas was supported by a Sheila Dame Postdoctoral Grant awarded by the European Association for the Study of the Liver (EASL) (www.easl.eu). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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