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. 2004 Feb;78(4):1873-81.
doi: 10.1128/jvi.78.4.1873-1881.2004.

Glycine decarboxylase mediates a postbinding step in duck hepatitis B virus infection

Jisu Li  1 , Shuping Tong, Hong Bock Lee, Ana Luisa Perdigoto, Hans Christian Spangenberg, Jack R Wands
Affiliations

Glycine decarboxylase mediates a postbinding step in duck hepatitis B virus infection

Jisu Li et al. J Virol. 2004 Feb.

Abstract

Envelope protein precursors of many viruses are processed by a basic endopeptidase to generate two molecules, one for receptor binding and the other for membrane fusion. Such a cleavage event has not been demonstrated for the hepatitis B virus family. Two binding partners for duck hepatitis B virus (DHBV) pre-S envelope protein have been identified. Duck carboxypeptidase D (DCPD) interacts with the full-length pre-S protein and is the DHBV docking receptor, while duck glycine decarboxylase (DGD) has the potential to bind several deletion constructs of the pre-S protein in vitro. Interestingly, DGD but not DCPD expression was diminished following prolonged culture of primary duck hepatocytes (PDH), which impaired productive DHBV infection. Introduction of exogenous DGD promoted formation of protein-free viral genome, suggesting restoration of several early events in viral life cycle. Conversely, blocking DGD expression in fresh PDH by antisense RNA abolished DHBV infection. Moreover, addition of DGD antibodies soon after virus binding reduced endogenous DGD protein levels and impaired production of covalently closed circular DNA, the template for DHBV gene expression and genome replication. Our findings implicate this second pre-S binding protein as a critical cellular factor for productive DHBV infection. We hypothesize that DCPD, a molecule cycling between the cell surface and the trans-Golgi network, targets DHBV particles to the secretary pathway for proteolytic cleavage of viral envelope protein. DGD represents the functional equivalent of other virus receptors in its interaction with processed viral particles.

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Figures

FIG. 1.
FIG. 1.
Concordant decline of DHBV infectivity and DGD expression in cultured duck hepatocytes. (A) DHBV DNA replication. PDH were infected overnight with DHBV at different days after plating as indicated. Cells were harvested immediately (day 0) or 2 or 6 days later. DHBV DNA was detected by Southern blot. RC, linear (L), and single-stranded (SS) viral DNA forms are indicated. The signal at day 6 relative to that at day 0 indicates the degree of productive infection. (B and C) Western blot analysis of the expression of DGD (B) and DCPD (C) from the same batch of uninfected PDH at the same time points postplating. Bound antibodies were revealed by 125I-labeled protein A. (D and E) DHBV binding (D) and endocytosis (E). Following overnight infection of PDH at the same time points as shown in panels A to C, cells were harvested immediately by scraping and divided into two parts. One part was treated with trypsin to remove cell surface-bound virus (E). Cell-associated DHBV DNA was analyzed by Southern blotting. The weak DHBV signal shown in lane 1 of panel E could reflect enhanced permeability of the cells to trypsin, since the experiment was performed immediately following attachment of collagenase-treated hepatocytes.
FIG. 2.
FIG. 2.
Effect of exogenous DGD on DHBV infection in aged PDH. (A) PDH cultured for 17 days were transfected with pcDNA vector or pcDNA-based DCPD or DGD construct. Cells were infected with DHBV 2 days later and harvested 6 days postinfection for analysis of total viral DNA (left panel) and protein-free DNA (right panel) forms. Transfection efficiency as measured by cotransfected GFP was found to be approximately 10%. RC, RC DNA; L, linear DNA; SS, single-stranded DNA; CCC, cccDNA. (B) PDH cultured for 3 weeks were infected with empty adenovirus or adenovirus expressing DCPD or DGD. Cells were incubated with viremic duck serum and harvested 4 and 9 days later. Both viral core protein and total viral DNA were analyzed.
FIG. 3.
FIG. 3.
Inhibition of DGD expression and DHBV infectivity by adenovirus-mediated antisense constructs. (A) Schematic representation of two DGD antisense constructs (AS1/2 and AS3/2) and one sense construct lacking the N-terminal 94 residues (tr8/2). The DGD open reading frame (ORF) and untranslated regions (UTR) are shown at the top. (B) Effect of DGD antisense constructs on endogenous DGD and DCPD expression. Freshly cultured PDH were infected overnight with adenovirus vector or the two antisense constructs at an MOI of 100 or 500 as indicated. Cells were harvested 48 h postinfection, and both DGD and DCPD proteins were analyzed in Western blots. (C) Effect of antisense constructs on DHBV infectivity. Cells were infected with adenovirus for 2 days, incubated with viremic duck serum for 6 h, and harvested 7 days later. Viral large envelope protein and core protein, as well as viral DNA, were analyzed. (D) Effects of a DCPD antisense construct (BamDCPD) and DGD sense construct (tr8/2) on DHBV infectivity and DGD expression. Experimental conditions were the same as described for panel C, and DHBV DNA at day 7 postinfection is shown. Western blot analysis of GAPDH expression serves as a loading control.
FIG. 4.
FIG. 4.
Effects of adenovirus infection (A) and DGD antiserum (B) on PDH cell viability. (A) Cells were infected with adenoviruses (Ad) overnight at an MOI of 100 or 500. Cell viability was analyzed at days 1, 2, and 3 postinfection using a modified MTT (WST-8) assay. Shown are data from day 2, but similar results were obtained from cells at day 1 and day 3. (B) PDH were incubated with various dilutions of preimmune or immune serum for 48 h, and cell viability was measured by WST-8 reagent 2, 3, and 5 days later. Shown are data from cells at day 2 following antibody (Ab) incubation. O.D., optical density.
FIG. 5.
FIG. 5.
(A) Effect of DGD and DCPD antibodies on DGD binding to a truncated pre-S peptide. Duck liver lysate was incubated with pre-S peptide 80-102 immobilized on beads via the GST tag. DGD or DCPD antibodies (Ab) in various dilutions were present during the incubation, and DGD retained on the beads was detected by Western blot. (B) Antibody binding to duck hepatocytes and internalization. PDH were incubated with a 1:20 dilution of preimmune or immune serum for 1, 2, or 3 days, washed, and harvested with (+) or without (−) trypsin treatment. Cell-associated immunoglobulin was detected. (C) Specificity of antibodies attached to PDH. PDH were metabolically labeled and subsequently incubated for 6, 24, or 48 h with or without a 1:20 dilution of DGD antiserum. Cells were harvested, and the cell lysate was incubated with 30 μl of protein A beads. Bound proteins were revealed by SDS-PAGE and fluorography. The 120-kDa DGD protein was pulled down only in cells preincubated with DGD immune serum.
FIG. 6.
FIG. 6.
Reduction of DGD protein levels and impairment of DHBV infection by a 1:20 dilution of DGD antibodies added soon after virus binding. (A) Effect of rabbit serum added immediately following DHBV attachment. Left panels, PDH were incubated with a low dose of viremic duck serum for 6 h, washed, and immediately incubated with DGD preimmune or immune serum at various dilutions for 48 h. Cells were harvested 1 week postinfection. Viral DNA replication (upper left panel) and DGD levels (lower left panel) were measured. Right panel, cells infected with DHBV for 6 h were incubated with a 1:20 dilution of preimmune or immune serum for 24 h and harvested immediately. The protein-free DNA forms were analyzed. (B) Temporal effect of DGD antibodies. PDH were infected with viremic duck serum for 6 h and harvested 1 week postinfection. Rabbit serum (preimmune, immune, or nonrelevant) at a 1:20 dilution was present during the 6-h DHBV infection (left panels) or at different points following infection for 48 h (right panels). DGD protein levels at the time of harvest are shown in the lower panels, while markers for productive infection, such as viral DNA and core and envelope proteins, are shown in the upper panels. (C) Effect of DGD antibodies on DGD protein levels in DHBV-free cells. PDH were incubated with various dilutions of DGD immune serum for 1 or 2 days and harvested immediately for analysis of DGD levels. The GAPDH levels were measured to control for protein loading.
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References

    1. Bour, S., R. Geleziunas, and M. Wainberg. 1995. The human immunodeficiency virus type 1 (HIV-1) CD4 and its central role in promotion of HIV-1 infection. Microbiol. Rev. 59:63-93. - PMC - PubMed
    1. Breiner, K., S. Urban, and H. Schaller. 1998. Carboxypeptidase D (gp180), a Golgi-resident protein, functions in the attachment and entry of avian hepatitis B viruses. J. Virol. 72:8098-8104. - PMC - PubMed
    1. Bruss, V. 1997. A short linear sequence in the pre-S domain of the large hepatitis B virus envelope protein required for virion formation. J. Virol. 71:9350-9357. - PMC - PubMed
    1. Bruss, V., X. Lu, R. Thomssen, and W. Gerlich. 1994. Post-translational alterations in transmembrane topology of the hepatitis B virus large envelope protein. EMBO J. 13:2273-2279. - PMC - PubMed
    1. Chassot, S., V. Lambert, A. Kay, C. Godinot, B. Roux, C. Trepo, and L. Cova. 1993. Fine mapping of neutralization epitopes on duck hepatitis B virus (DHBV) pre-S protein using monoclonal antibodies and overlapping peptides. Virology 192:217-223. - PubMed

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