Duck hepatitis B virus particles bearing the L and S envelope proteins bind a cellular glycoprotein of 180 kDa (gp180) with high affinity and specificity. Binding is mediated by the pre-S region of the L protein and is blocked by neutralizing but not by non-neutralizing monoclonal antibodies to the virus. These and other properties have suggested that gp180 may be a component of the viral entry machinery. Here we report the purification of gp180 from duck liver and the isolation and characterization of cDNA encoding it. DNA sequence analysis of this cDNA indicates that gp180 is a novel member of the basic carboxypeptidase gene family.

Duck hepatitis B virus is a member of the hepadnavirus group, which includes human hepatitis B virus (1). These viruses are small enveloped DNA viruses that infect liver cells, causing acute and chronic hepatitis (1). The cellular receptor for human hepatitis B virus has not been identified. A candidate receptor for the duck hepatitis B virus (DHBV) 1 has recently been described (2, 3). This 180-kDa protein, named gp180, was identified based on its ability to co-precipitate with DHBV particles or recombinant DHBV envelope proteins (2). gp180 was found to bind with high affinity and specificity to the preS region of the DHBV large envelope protein (2). The binding domain within the preS region that is responsible for the gp180 interaction overlaps key areas that have been established as important for viral infectivity (4). The cDNA for gp180 has recently been cloned, revealing that gp180 is a member of the metallocarboxypeptidase gene family (3). Interestingly, gp180 contains an N-terminal signal peptide, three metallocarboxypeptidase-like domains, a putative C-terminal transmembrane domain, and a short cytoplasmic tail. Of the known carboxypeptidases, gp180 has the highest homology to carboxypeptidase E (CPE). Specifically, the first and second domains of gp180 have 39% and 43% amino acid identity with mammalian CPE, and the third domain has 29% amino acid identity.CPE functions in the biosynthesis of numerous peptide hormones and transmitters (5, 6). CPE removes C-terminal Arg and Lys residues remaining after endoprotease cleavage of the prohormone. For many years, CPE was thought to be the only carboxypeptidase involved with neuropeptide biosynthesis. However, recent studies on the Cpe fat /Cpe fat mouse suggest that additional CPE-like carboxypeptidases partially compensate for a defective CPE. These mice have a point mutation within the coding region of the CPE gene that renders the protein inactive (7). Despite the absence of active CPE in the Cpe fat /Cpe fat mice, a reduced but detectable amount of peptide processing occurs, implying that additional carboxypeptidases may perform this step in vivo. A novel carboxypeptidase with CPE-like properties, named metallocarboxypeptidase D (CPD), was identified in bovine pituitary and in other tissues (CPD) (8). In bovine tissues, the major form of CPD is 180 kDa (8). In addition to the 180-kDa form, rat tissues also contain several smaller forms ranging from 100 to 180 kDa (9). In contrast, all other known carboxypeptidases are . The size of the major species of CPD is similar to that of gp180, raising the possibility that these proteins are homologs. In addition, the partial N-terminal amino acid sequence of bovine CPD is similar to that of the duck gp180, and both proteins are

The preSl surface glycoprotein of hepatitis B virus is targeted to the endoplasmic reticulum (ER) and is retained in this organelle when expressed in the absence of other viral gene products. The protein is also acylated at its N terminus with myristic acid. Sequences responsible for its ER retention have been identified through examination of mutants bearing lesions in the preSl coding region. These studies reveal that such sequences map to the N terminus of the molecule, between residues 6 and 19. Molecules in which this region was present remained in the ER; those in which it had been deleted were secreted from the cell. Although all deletions which allowed efficient secretion also impaired acylation of the polypeptide, myristylation alone was not sufficient for ER retention: point mutations which eliminated myristylation did not lead to secretion. These data indicate that an essential element for ER retention resides in a 14-amino-acid sequence that is unrelated to previously described ER retention signals.

Sodium taurocholate cotransporting polypeptide (NTCP) was recently discovered as a hepatitis B virus (HBV) receptor, however, the detailed mechanism of HBV entry is not yet fully understood. We investigated the cellular entry pathway of HBV using recombinant HBV surface antigen L protein particles (bio-nanocapsules, BNCs). After the modification of L protein in BNCs with myristoyl group, myristoylated BNCs (Myr-BNCs) were found to bind to NTCP in vitro, and inhibit in vitro HBV infection competitively, suggesting that Myr-BNCs share NTCP-dependent infection machinery with HBV. Nevertheless, the cellular entry rates of Myr-BNCs and plasma-derived HBV surface antigen (HBsAg) particles were the same as those of BNCs in NTCP-overexpressing HepG2 cells. Moreover, the cellular entry of these particles was mainly driven by heparan sulfate proteoglycan-mediated endocytosis regardless of NTCP expression. Taken together, cell-surface NTCP may not be involved in the cellular uptake of HBV, while presumably intracellular NTCP plays a critical role.

We have identified a 180-kDa cellular glycoprotein (gpl80) that binds with high affinity to duck hepatitis B virus (DHBV) particles. The protein was detected by coprecipitating labeled duck hepatocyte proteins with virions or recombinant DHBV envelope proteins, using nonneutralizing monoclonal antibodies to the virion envelope. Binding of gpl80 requires only the pre-S region of the viral large envelope protein, since recombinant fusion proteins bearing only this region efliciently coprecipitate gpl80. The DHBV-gpl8O interaction is blocked by two independent neutralizing monoclonal antibodies. The protein is found on both internal and surface membranes of the cell, and the species distribution of gpl80 binding activity mirrors the known host range of DHBV infection. Functional gpl80 is expressed in a wide variety of tissues in susceptible ducks. Human hepatitis B virus (HBV) is a small, enveloped DNA virus that causes acute and chronic hepatitis and is strongly associated with the development of hepatocellular carcinoma (5). It is a member of the hepadnavirus group, other members