The gp180/p170 protein was originally identified as a binding partner for the pre-S region of DHBV large envelope protein (15
). Since the pre-S region is believed to be the ligand for the cell surface receptor protein (11
), p170/gp180 represents a potential candidate molecule in this regard. The role of p170/gp180 as a DHBV receptor has been strengthened by the following observations. First, the p170/gp180–pre-S interaction is species specific since the chicken homologue binds the pre-S protein poorly if at all (16
). Furthermore, DHBV infection of hepatocytes is also species specific. Second, we have previously found that the interaction between GST–pre-S fusion protein and p170 was competitively inhibited by incubation with DHBV particles, suggesting that the binding site was present on native large envelope protein present on intact viral particles (25
). Third, many DHBV mutants with linker substitutions in the pre-S region are no longer infectious when inoculated into PDH cultures (17
). Indeed, these pre-S mutant proteins have also been shown to lose binding activity to gp180 (10
). Fourth, the critical binding site for p170 has been mapped to pre-S residues 87 to 102, and a pre-S peptide composed of residues 80 to 104 has been found capable of binding p170 molecule (though quite weakly [25
]). Of the four types of murine anti-pre-S MAbs that neutralize viral infectivity in cell culture, three antibodies have been shown to bind to this region of the pre-S protein (83 to 90, 91 to 99, and 100 to 107; [4
]). The clustering of several neutralizing epitopes in this pre-S domain is consistent with the concept of the location of a receptor contact site. Further support is provided by the observation that a synthetic peptide covering pre-S residues 80 to 104 inhibited DHBV infection of PDH (18
This study was designed to directly assess the ability of DCPD to mediate virus binding to reconstituted intact cells. By performing comparative studies with PDH, we attempted to verify whether DCPD was the major DHBV receptor in vivo. A significant amount of the DCPD protein expressed in transiently transfected Bosc cells was available on the cell surface, as revealed by IF staining (Fig. a) and by Western blot analysis (data not shown). Binding of DHBV particles to DCPD-transfected Bosc cells was independently demonstrated by three techniques: (i) IF staining of the large envelope protein (Fig. ), (ii) Western blot analysis of the large envelope protein (Fig. A), and (iii) Southern blot analysis of viral DNA (Fig. B). The specificity of virus binding was established by trace background binding found in nontransfected or mock-transfected Bosc cells and in cells transfected with vector backbone, plasmids encoding nonrelevant cell surface proteins, or DCPD deletion mutants that have lost affinity for the pre-S protein.
Reconstitution of DHBV binding is not restricted to Bosc cells but may also take place in other liver- or kidney-derived cell lines, including 293, COS, and LMH (Fig. A). The quantitative difference in virus binding (Bosc > 293 > LMH) may be explained, in part, by a higher level of DCPD expression in Bosc cells due to the presence of simian virus 40 large T antigen as well as by higher transfection efficiency (about 50%) in Bosc and 293 cells than in LMH cells (<10%). In fact, transiently transfected Bosc and 293 cells have higher virus binding capacities than native PDH (25a
). However, binding of DHBV particles does not require overexpression of the DCPD receptor protein in reconstituted cells, since a 293-4 cell line that stably expresses low levels of the DCPD protein binds DHBV particles quite well (Fig. B).
While our previous study (25
) revealed an overlap between the critical p170 binding site on the pre-S domain (amino acids 87 to 102) and the binding sites of three classes of neutralizing MAbs (amino acids 83 to 107), the present study demonstrates directly that such neutralizing monoclonal antibodies strikingly inhibit DHBV binding to DCPD reconstituted cells (Fig. A). Furthermore, pre-S peptide 80-104, which covers the clustered neutralizing antibody binding sites, was found to significantly inhibit DHBV infection of PDH (18
) as well as block DHBV binding to DCPD reconstituted cells (Fig. C). These results together with the ability of energy-depleting reagents to inhibit DHBV entry into both PDH (14
) and DCPD-reconstituted cells (Fig. C) strongly suggest that DCPD is the major viral receptor in duck hepatocytes.
Trypsin pretreatment of reconstituted cells revealed the presence of internalized DHBV DNA (Fig. A). We confirmed that the trypsin-resistant signal represents internalized intracellular DHBV DNA by the following experiments. First, no trypsin-resistant signal was detectable in DCPD-transfected LMH cells preincubated with virus inoculum at a nonpermissive temperature of 4°C, which prevents capping and internalization of viral particles (Fig. B and data not shown). Second, the trypsin-resistant signals were greatly reduced or eliminated by performing the viral infection experiments in the presence of energy-depleting agents, as shown by the studies presented in Fig. C.
In addition to the observations reported here, several lines of indirect evidence also suggest that DCPD may serve as a DHBV receptor. For example, Sunyach et al. (23
) found that pre-S residues 88 to 90, an essential region of the DCPD binding site, were critical for viral binding to PDH and also for viral infectivity. In addition, Bruns and colleagues (3
) reported that at a low multiplicity of infection, the pre-S domain enhances DHBV infection. Although the mechanism(s) of this phenomenon remains to be elucidated, the region responsible for such enhancement corresponds to the binding site of p170. Moreover, other investigators have directly tested role of DCPD as the primary DHBV receptor (2
). Based on the ability of truncation mutants of the pre-S protein to compete for the interaction between DCPD and immobilized pre-S protein, Breiner et al. (2
) proposed that DCPD binding site is composed of a main binding region located at residues 85 to 115 and an auxiliary binding in the N terminus. This finding is in accord with our observation that a small pre-S peptide composed of residues 80 to 104 is capable of binding to p170, although much less efficiently than full-length pre-S protein (25
). Finally, it has been determined that soluble DCPD inhibits DHBV infection of PDH in a dose-dependent manner (2
). It remains to be determined if pretreatment of PDH with antibodies against DCPD will block subsequent DHBV infection.
Breiner et al. found that DCPD is expressed only in the Golgi apparatus of Huh7 cells transfected with DCPD cDNA (2
). They also reported that transfection of DCPD cDNA into Huh7 human hepatocellular carcinoma cells mediated binding of a fusion construct of pre-S–green fluorescent protein as well as fluorescence-labeled viral particles (2
). In this regard, DHBV may bind nonspecifically to human hepatocellular carcinoma cell lines such as HepG2 (12
) and Huh7 (25a
). In our studies, DCPD transfection failed to increase DHBV binding to Huh7 cells (25a
). In the Bosc cells, we found that the DCPD molecule is available on the cell surface (Fig. a), and this was confirmed by confocal microscopy (data not shown). Cell surface expression was still detectable if cells were fixed by nonpermeabilizing agents such as formaldehyde. By Western blot analysis, it was found that trypsin treatment could remove approximately one-half of the DCPD signal (25a
), which also suggests cell surface expression. Discrepancies related to cellular location may be due in part on the high level of expression of DCPD achieved in transfected Bosc cells.
In conclusion, cumulative evidence from different groups, including the direct transfection experiments, strongly suggest that DCPD serves as an avian hepatitis B virus receptor. It is of interest that another protease, aminopeptidase N, has been found to serve as a coronavirus receptor (7
). Whether the enzymatic function of the DCPD protein is required for its biologic function as a viral receptor remains to be established. With the recent cloning of the human homologue of DCPD (24
), it will be important to determine whether this molecule can serve as a hepadnavirus receptor for HBV as well. Identification of DCPD as a DHBV receptor has several implications. For example, the availability of transfected cell lines stably expressing the DCPD molecule will provide a useful system for study of the early events of the viral life cycle as well as permit study of antiviral agents that may block binding of virus to its receptor.
Although DCPD-reconstituted cells were capable of binding and internalizing viral particles, no viral replication was observed even in LMH cells, which support DHBV DNA replication when transfected with cloned DHBV DNA (5
). Thus, additional factors are required for productive viral replication in established cell lines. Such factors, for example, may be responsible for fusion event(s), nuclear translocation of viral genome, or the repair of viral genome prior to formation of covalently closed circular DNA. In this regard, we have previously identified p120 as a binding partner for truncated pre-S peptide (18
). Molecular cloning has revealed p120 to be duck glycine decarboxylase (18a
). It will be interesting to test if this molecule can serve as a cofactor for DHBV infection in combination with DCPD.