We sought to determine whether HCV structural proteins (HCV-SP) produced in insect cells could bind and enter into cultured human hepatocytes. Our results provide several lines of evidence indicating that HCV-SP binds to the ASGP-R in these cells and that ASGP-R may in part be responsible for their internalization into cultured hepatocytes.
The arguments for the involvement of the ASGP-R in HCV-SP binding are as follows. First, the binding of HCV-SP was inhibited by anti-ASGP-R CRD peptide antibody but not by preimmune antibody. The CRD is the extracellular part of the ASGP-R responsible for binding terminal nonreducing galactose residues and N
-acetylgalactosamine residues of desialated N-linked tri- or tetra-antennary glycans, conferring part of its specificity to this receptor (41
). The CRD of hH1 requires calcium for proper binding conformation and sugar binding (41
). Consistent with that, calcium chelation by EGTA inhibited the binding of HCV-SP to cells. Second, the binding of HCV-SP to cells was partially inhibited by ASGP-R ligands such as asialo-orosomucoid (63
) and Tg. Tg is the major component of colloid substance in the thyroid and is a large glycoprotein involved in thyroid hormone synthesis. Tg has been reported to bind an ASGP-R-related receptor in the thyroid (11
). Finally, the transfection of a nonpermissive cell line in order to express both subunits of the human liver ASGP-R conferred HCV-SP binding to these cells (3T3-22Z cells). Altogether, these results suggest that HCV-SP may also bind to ASGP-R in hepatocytes.
Several mechanisms may be relevant to explain the binding of HCV-SP to ASGP-R. First, recombinant glycoproteins produced in insect cells generally lack sialic acid residue at the extremity of their mature carbohydrate domain (27
). It is thus likely that HCV-SP lack sialic acid, as already reported for recombinant HCV envelope proteins produced in mammalian cells (61
). Moreover, asialo-Tg was more potent than Tg in inhibiting the binding of HCV-SP to human hepatocytes. Evidence that the penultimate galactose and N
-acetylglucosamine residues of the complex asialated carbohydrate of Tg bind to ASGP-R in thyrocytes has been previously established (11
). Therefore, the simplest explanation for Tg-induced inhibition of HCV-SP binding is the presence of asialocarbohydrates on the Tg molecule. Interestingly, uptake of circulating desialated Tg by hepatocytes has previously been described (45
). It is indeed well known that a small proportion of Tg is released in the bloodstream (49
), albeit the role is not yet understood.
Although largely mediated by their sugar moiety, the binding of various ligands to ASGP-R occurs within a broad range of affinity and with a certain degree of specificity (62
). Thus, asialo-orosomucoid and asialo-Tg have very different binding affinities in liver and thyroid (11
). In this respect, modifications in either number or position of antennas in the carbohydrate moiety of asialoglycoproteins are monitored by drastic changes in their affinity of binding to ASGP-R (62
). This has raised the possibility that determinants other than asialation of the carbohydrate termini are involved in binding to ASGP-R. In fact, several reports suggest that maturation of the carbohydrate structure of asialoglycoproteins expressed in insect cells differ from that observed in mammals (39
). It is therefore highly probable that the carbohydrate structures of HCV-SP and asialo-Tg are different. Moreover, the idea that the carbohydrate domain is the sole determinant of binding to the ASGP-R is no longer operative (17
). Thus, point mutations in the protein frame of ASGP-R ligands, even if still asialated, lead to a dramatic decrease of their binding affinity. This may perhaps be due to modifications in maturation of their carbohydrate domain or change of conformation of that domain but may also relate to the importance of other determinants on the protein core (17
). In the thyroid, the Tg content in iodotyrosine residues plays a critical role in Tg binding (59
). We therefore think that HCV-SP binding to ASGP-R may not be solely determined by its carbohydrate structure and may have some pathophysiological relevance.
Additional arguments suggest that the binding to ASGP-R is followed by other events and that the ASGP-R is involved in the internalization of HCV-SP as well. First, transfection of nonpermissive cells expressing both subunits of the human liver ASGP-R (3T3-22Z cells) conferred HCV-SP/p7(+) binding and entry into these cells. As expected, entry was not observed with a dual-transfectant cell line expressing a function defective variant of the hH2 subunit of ASGP-R (3T3-24X cells), suggesting that full ASGP-R functionality was required. The reason why 3T3-22Z cells did not uptake HCV-SP/p7(−) remains unknown. However, in a similar manner, HCV-SP/p7(−) uptake appeared not to be as effective as for HCV-SP/p7(+) in HepG2 cells. It has been hypothesized that the lack of p7 may lead to conformational changes of envelope proteins (36
), as reported for closely related virus (21
), that are perhaps important for cell binding and entry of the HCV virion. In fact, the discrepancy between the two types of HCV-SP preparations observed in HepG2 cells could in part be due to their differential affinity for ASGP-R and/or a conformational issue important to trigger internalization. This discrepancy could be even more obvious with 3T3-22Z cells that, in fact, express less ASGP-R than do HepG2 cells (Fig. ). This view would also be consistent with an involvement of noncarbohydrate determinants in the mechanism of entry of HCV-SP after binding to ASGP-R.
In GFP-hH1-transfected HepG2 cells, both HCV-SP and GFP-ASGP-R fusion protein cointernalized and then colocalized in a region surrounding the nucleus. It has been previously reported that, after internalization, asialo-orosomucoid is targeted to the lysosomial compartment in an acidification-dependent manner in HepG2 cells (63
). Additionally, a recycling of ASGP-R to the cell surface after ligand delivery has been suggested (for a review, see reference 62
). In contrast to these studies, we observed here that both types of HCV-SP were targeted with ASGP-R to an area surrounding the nucleus, likely within the rough endoplasmic reticulum and/or nuclear envelope compartments. Consistent with this hypothesis we detected, after its uptake by nontransfected HepG2 cells, radiolabeled HCV-SP mainly in an endoplasmic reticulum membrane-enriched compartment. It is well known that newly synthesized misfolded proteins in the endoplasmic reticulum are targeted toward the proteasome system for protein degradation. Variants of the hH2 and hH1 subunits of ASGP-R, hH2a and hH1i5, respectively, have also been suggested to undergo such a processing (29
). However, if this were the case for GFP-hH1 as well, this subunit would also be targeted toward the endoplasmic reticulum compartment in basal conditions, a hypothesis that, in fact, our observations do not support. We, therefore, think that targeting of the GFP-ASGP-R fusion protein toward the endoplasmic reticulum compartment triggered upon HCV-SP binding may be specific.
ASGP-R binding and pathway of entry may perhaps constitute an alternative pathway to induce an immune response against HCV. Several arguments suggest an involvement of ASGP-R in immune responses. First, ASGP-R has been previously suggested as being one potential target in chronic hepatic autoimmune disease in humans (40
) and, experimentally, antibodies against ASGP-R are produced in animals challenged with woodchuck hepatitis virus (15
). Second, HCV is known to favor the occurrence of autoantibodies, such as those observed in autoimmune thyroid diseases (55
). The existence of a negative correlation between the occurrence of various autoantibodies and anti-ASGP-R autoantibodies has been reported in chronically HCV infected patients (25
). Thus, a balance is observed between hepatic autoimmune response and other autoimmune disorders upon HCV infection. Third, recognition of complex N-linked glycans by lectins is probably important for the development of an appropriate immune response. Thus, altered protein glycosylation is involved in triggering autoimmune diseases in animal models, although the mechanism remains poorly understood as yet (37
). This suggests that ASGP-R plays some role in regulating the immune system.
ASGP-R expression has also been described in dendritic cells (69
). These cells play a pivotal role in the immune response against infectious pathogens (23
), including the induction of appropriate CTL response against dengue virus, another member of the flavivirus group (50
). Several arguments suggest that dendritic cells could be also involved in the clearance of HCV infection. There is growing evidence that dendritic cells of chronically HCV infected patients present with an impaired antigen processing and presentation, leading to incomplete activation of HCV-specific T cells (4
). This phenomenon has also been linked to the observation that HCV could directly infect dendritic cells (46
). In addition, several type II C-type (calcium-dependent) lectins are expressed at the surface of dendritic cells, which have been implicated in antigen uptake and targeting toward proteasome or endoplasmic reticulum compartments. Antigen processing and association with molecules of the major histocompatibility complex class I occur in the endosplasmic reticulum before presentation to the immune system at the cell surface (23
). It is thought that the CTL response is usually triggered via such a mechanism. Although no direct evidence is available so far, our present data are consistent with the fact that HCV-SP/GFP-hH1 complexes could undergo processing in the endoplasmic reticulum compartment in hepatocytes. It is therefore tempting to speculate that HCV-SP entry through the ASGP-R pathway may trigger the processing of HCV-SP-derived peptides through the major histocompatibility complex class I processing pathway and lead to antigen presentation to the immune system by hepatocytes or dendritic cells.
Certainly, other factors or receptors may also be involved in HCV-SP binding and entry into HepG2 cells. CD81 was recently reported to bind recombinant HCV E2 envelope protein (52
). However, no expression of CD81 was detected by reverse transcription-PCR in the HepG2 cells that we used, whereas a clear expression was detected in Molt-4 cells (not shown). This, obviously, precluded CD81 from taking any part in HCV-SP binding or entry in HepG2 cells. The low-density lipoprotein receptor (LDL-R) has been implicated in the binding and entry of HCV into cells (1
) and also requires calcium for ligand binding (3
). The HCV virion has been reported to complex with low-density lipoprotein and very-low-density lipoprotein in the serum (54
). This binding of HCV to lipoproteins in the plasma has even been proposed as an explanation for the lack of detection of viral envelope proteins with various antibodies (53
). We are currently not sure whether LDL-R is a direct or indirect target for HCV to enter into cells and whether HCV-SP directly interacts with this receptor or not. We also do not know whether, in addition to ASGP-R, another receptor is involved in HCV-SP/p7(−) internalization that would not be required in that of HCV-SP/p7(+). Nevertheless, the sum of our observations suggest that ASGP-R is involved in the binding and uptake of both HCV-SP preparations by human hepatocytes and that this ASGP-R should be considered a potential component of this process.