Role of the Asialoglycoprotein Receptor in Binding and Entry of Hepatitis C Virus Structural Proteins in Cultured Human Hepatocytes

Abstract

We used a baculovirus-based system to prepare structural proteins of hepatitis C virus (HCV) genotype 1a. Binding of this preparation to cultured human hepatic cells was both dose dependent and saturable. This binding was decreased by calcium depletion and was partially prevented by ligands of the asialoglycoprotein receptor (ASGP-R), thyroglobulin, asialothyroglobulin, and antibody against a peptide in the carbohydrate recognition domain of ASGP-R but not preimmune antibody. Uptake by hepatocytes was observed with both radiolabeled and dye-labeled HCV structural proteins. With hepatocytes expressing the hH1 subunit of the ASGP-R fused to green fluorescent protein, we could show by confocal microscopy that dye stain cointernalized with the fusion protein in an area surrounding the nucleus. Internalization was more efficient with a preparation containing p7 than with one that did not. The two preparations bound to transfected 3T3-L1 cells expressing either both (hH1 and hH2) subunits of the ASGP-R (3T3-22Z cells) or both hH1 and a functionally defective variant of hH2 (3T3-24X cells) but not to parental cells. Additionally, uptake of dye-labeled preparation containing p7 was observed with 3T3-22Z cells but not with 3T3-L1 or 3T3-24X cells or with the preparation lacking p7, suggesting that p7 regulates the internalization properties of HCV structural proteins. Our observations suggest that HCV structural proteins bind to and cointernalize with the ASGP-R in cultured human hepatocytes.Hepatitis C virus (HCV) infection has become a major health problem affecting an estimated 170 million people worldwide. Persistent infection occurs in more than 70% of people infected with HCV, which may be complicated by cirrhosis and/or hepatocellular carcinoma (24, 33). Despite highly competitive and extensive research in this field, a highly effective treatment is not yet available. The mainstay of anti-HCV therapy, alpha interferon (IFN-␣) or pegylated IFN-␣, together with ribavirin leads, at best, to viral clearance in ca. 41 to 54% of patients infected with HCV genotype 1a (34,38). Since mechanisms of HCV infection remain unclear, characterization of these mechanisms is now a major issue for the development of new strategies for anti-HCV treatment and prevention.

DC-SIGN and L-SIGN Are High Affinity Binding Receptors for Hepatitis C Virus Glycoprotein E2

“…Furthermore E2 binds to the hepatoblastoma cell line HepG2, which does not express CD81 (25). More recently two novel E2 binding receptors have been identified on HepG2 cells, the scavenger receptor type B class I (SR-BI) (25) and the galactose binding C-type lectin asialoglycoprotein receptor (26). The lack of an efficient cell culture model has precluded functional confirmation of these receptor candidates at the level of virus entry.…”

“…In the case of HCV, it is tempting to speculate that subsequent to interaction with L-SIGN on endothelial cells, the virus could be transmitted to hepatocytes where it uses a specific receptor for entry. Candidates include CD81 (23), the scavenger receptor SR-BI (25), the LDL receptor (21,22), or the asialoglycoprotein receptor (26), which is expressed on liver hepatocytes. Like DC-SIGN and L-SIGN, the asialoglycoprotein receptor attaches HCV through binding of glycan residues (galactose or N-acetylgalactosamine) on the viral glycoprotein E2.…”

Viroporins: structure, function and potential as antiviral targets

“…A controversial role for p7 channel activity during virus entry has been proposed, based upon enhanced hepatocyte uptake of HCV-LP containing p7 (Saunier et al, 2003), as well as inhibitory effects of p7 inhibitors added during the infection process (Griffin et al, 2008). However, despite immuno-gold detection of E2-p7 complexes in HCV-like particles (Isherwood & Patel, 2005), recent studies have failed to demonstrate the presence of HA-tagged p7 within infectious virions .…”

Cellular Binding of Hepatitis C Virus Envelope Glycoprotein E2 Requires Cell Surface Heparan Sulfate

“…E2 is thought to initiate viral attachment (6,8,9), whereas E1 may be involved in virus-cell membrane fusion (6,9). Several cell surface proteins, the tetraspanin CD81 (10,11), the LDL receptor (12), the asialoglycoprotein receptor (13) and scavenger receptor B1 (14), have been proposed to play a role in mediating E2 binding and/or HCV internalization. However, it is still unclear whether any of these molecules can act as a functional cellular receptor mediating viral entry and initiation of infection.…”