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.