Abstract
A 120-kilodalton protein (p120) was identified in the duck liver that binds to several truncated versions of duck hepatitis B virus (DHBV) pre-S envelope protein, suggesting p120 may serve as a DHBV co-receptor. The amino acid sequences of tryptic peptides from purified p120 were found to be the duck p protein of the glycine decarboxylase complex (DGD). DGD cDNA cloning revealed extensive protein conservation with the chicken homologue except for several insertions in the N-terminal leader sequence. The DGD cDNA contained no in-frame AUG codon at the predicted initiation site of the open reading frame, and site-directed mutagenesis experiments established an AUU codon as the translational initiator. The DGD protein expressed in rabbit reticulocyte lysates bound truncated DHBV pre-S protein identical to that of p120 derived from duck liver confirming DGD as p120. Moreover, transfection studies in liver- and kidney-derived cells revealed both cell surface and cytoplasmic expression of the protein. Cloning of the glycine decarboxylase cDNA will permit a direct test of whether it functions as a cell surface co-receptor or as a co-factor in the DHBV replication cycles.
Highlights
The human hepatitis B virus and related animal viruses form hepatotropic DNA viruses or hepadnaviruses (1); because the early events of hepatocyte infection are unclear, studies were initiated via the duck hepatitis B virus (DHBV)[1] model in a multifold approach to identify candidate cell surface receptor proteins
Interactive proteins for the pre-S domain of DHBV large envelope protein were identified and cDNAs cloned to verify their potential role as DHBV receptor/co-receptor in nonsusceptible cell lines
Transfection of duck carboxypeptidase D cDNA into liver- and kidney-derived cell lines, conferred efficient DHBV binding and entry[2], indicating p170 pre-S-binding protein served as a primary DHBV receptor
Summary
Purification and Microsequencing of the p120 Protein—Frozen duck liver (40 gm) was homogenized in 300 ml of lysis buffer (2). Deletion construct DGD27/2.3.1 was made in a manner similar to that of DGD24a/2.3.1, except that the sense primer used was DGD27 rather than DGD24a In this construct, the 5Ј end of the DGD cDNA lies at position 335 and AUU346 would be the 5Ј-most potential initiation codon. Construct tr1/2 was derived from clone 2.3.1 by attaching an artificial in-frame AUG codon into the 5Ј end It expresses a DGD protein composed of residues 52–1024 but lacks 12 N-terminal residues of the mature protein. This construct was obtained by PCR amplification of clone 2.3.1 using primers tr[1] and tr[2] followed by cloning into the KpnI - XhoI sites of pBluescript vector. As a positive control for DGD expression, primary duck hepatocytes were grown on coverslips and stained as described above
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