Carboxypeptidase D (gp180), a Golgi-resident protein, functions in the attachment and entry of avian hepatitis B viruses

Abstract

Carboxypeptidase D (gp180), one of many candidate receptors proposed for hepatitis B viruses (HBVs), was examined and found to be the actual cellular receptor for avian HBVs. This conclusion was based on the following observations: (i) gp180 was the only host protein that bound with high affinity to the pre-S ectodomain of the large duck hepatitis B virus (DHBV) envelope protein, which is known to be essential for virus infection; (ii) a pre-S subdomain which determines physical binding to gp180 was found to coincide with a domain functionally defined in infection competition experiments as a receptor binding domain; (iii) soluble gp180, lacking the membrane anchor, efficiently inhibited DHBV infection; (iv) efficient interspecies gp180-pre-S interaction was limited to the natural hosts of avian hepadnaviruses; and (v) expression of gp180 in a heterologous hepatoma cell line mediated cellular attachment and subsequent internalization of fluorescently labeled viral particles into vesicular structures. However, gp180 expression did not render transfected heterologous cells permissive for productive infection, suggesting that a species-specific coreceptor is required for fusion to complete viral entry. In contrast to the case for known virus receptors, gp180 was not detected on the hepatocyte cell surface but was found to be concentrated in the Golgi apparatus, from where it functions by cycling to and from the plasma membrane.

FIG. 1

gp180 is the dominant DHBV pre-S binding protein in duck liver. (A) Duck liver proteins eluted from a DHBV pre-S affinity matrix were analyzed by SDS-PAGE and silver staining. Binding had been carried out in the absence (−) or the presence (+) of 100 pmol of free DHBV pre-S polypeptide as a competitor. Molecular masses (in kilodaltons) are indicated on the right. (B) An immunoblot from a similar experiment with an anti-gp180 antiserum as the probe.

FIG. 2

Mapping of the region within the DHBV pre-S sequence required for gp180 interaction. (A) Schematic representation of a series of His-tagged pre-S polypeptides used to map the gp180 binding domain. The numbers on the left correspond to the lanes in panel B. The borders of each deletion are given at the right. The dashed lines reflect the borders of the minimal domain determined to be required for receptor interaction on PDHs competing DHBV infection (30). (B) Western blot probed with anti-gp180 antiserum, detecting gp180 bound to immobilized DHBV pre-S with or without (−) the presence of the respective competing pre-S polypeptide (1 to 10 in panel A).

FIG. 3

sgp180 efficiently inhibits DHBV infection. PDHs were incubated with a DHBV-containing duck serum (10² DNA-containing particles/cell; 14 h, 37°C) in the presence of increasing amounts of sgp180. On day 6 postinfection, the cells were harvested and assayed by DNA dot blotting for intracellular DHBV DNA. Values from two independent measurements were normalized to the value obtained in the absence of sgp180; bars indicate standard deviations. One microgram of sgp180 per milliliter corresponds to approximately 50 molecules of sgp180 per viral particle, assuming 10³ SVPs per DNA-containing virion.

FIG. 4

gp180 is concentrated in the Golgi apparatus. (A) PDHs prepared from a duckling were immunostained with an anti-gp180 antiserum and analyzed by confocal microscopy. (B) HuH7 cells were transfected with Myc epitope-tagged gp180. At 1 day posttransfection, the cells were fixed and coimmunostained for gp180 (tetramethylrhodamine) (left panel) and for endogenous TGN46 (fluorescein) (right panel). Fluorescence was analyzed by confocal microscopy (sequential scanning of the same cells).

FIG. 5

gp180 mediates uptake of DHBV particles or pre-S polypeptide in nonpermissive cells. HuH7 cells were transfected with gp180 (A, B, and C) or with gp180^Tm, a mutant lacking the cytoplasmic tail (D). After incubation with fluorescein-labeled DHBV particles (A and B) or pre-S (C and D), the cells were fixed and immunostained for the transfected gp180 (red fluorescence). Nuclei were stained with DAPI (A) (blue fluorescence). Fluorescence was analyzed either by conventional fluorescence microscopy, using sequential multiple exposures of the same cells (A), or by confocal fluorescence microscopy (B, C, and D) (simultaneous scans).

FIG. 6

Efficient gp180–pre-S interaction is restricted to the hosts of avian hepadnaviruses. (A) Proteins from heron liver, bound to HHBV pre-S–Sepharose in the absence (−) or the presence (+) of 250 pmol of free HHBV pre-S as a competitor, were eluted and analyzed by SDS-PAGE and silver staining. (B) Anti-gp180 immunoblot from a similar experiment comparing the affinities of binding of duck liver gp180 and the homologous protein from chicken liver to a DHBV pre-S–matrix. Free DHBV pre-S was used as a competitor to demonstrate specific binding (+). Similar levels of protein from either species were present during the binding reaction, and the antibody used detected the proteins from duck and chicken equally well. Molecular masses (in kilodaltons) are indicated on the right.