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. 2001 May;75(10):4528-39.
doi: 10.1128/JVI.75.10.4528-4539.2001.

Binding of recombinant feline immunodeficiency virus surface glycoprotein to feline cells: role of CXCR4, cell-surface heparans, and an unidentified non-CXCR4 receptor

A de Parseval  1 , J H Elder
Affiliations

Binding of recombinant feline immunodeficiency virus surface glycoprotein to feline cells: role of CXCR4, cell-surface heparans, and an unidentified non-CXCR4 receptor

A de Parseval et al. J Virol. 2001 May.

Abstract

To address the role of CXCR4 in the cell-surface attachment of the feline immunodeficency virus (FIV), a soluble fusion protein, gp95-Fc, consisting of the surface glycoprotein (SU, gp95) of either a primary (PPR) or cell line-adapted (34TF10) FIV strain was fused in frame with the Fc domain of human immunoglobulin G1. The recombinant SU-immunoadhesins were used as probes to investigate the cellular binding of FIV SU. In agreement with the host cell range properties of both viruses, binding of 34TF10 gp95-Fc was observed for all cell lines tested, whereas PPR gp95-Fc bound only to primary feline T cells. 34TF10 gp95-Fc also bound to Jurkat and HeLa cells, consistent with the ability of FIV to use human CXCR4 as a fusion receptor. As expected, 34TF10 gp95-Fc binding to Jurkat cells was blocked by addition of stromal cell-derived factor 1alpha (SDF-1alpha), as was binding to the 3201 feline lymphoma cell line. However, SDF-1alpha, RANTES, macrophage inflammatory protein 1beta, and heparin all failed to inhibit the binding of either gp95-Fc to primary T cells, suggesting that a non-CXCR4 receptor is involved in the binding of FIV SU. In this regard, an unidentified 40-kDa protein species from the surface of primary T cells but not Jurkat and 3201 cells specifically coprecipitated with both gp95-Fc. Yet another type of binding of 34TF10 gp95-Fc to adherent kidney cells was noted. SDF-1alpha failed to block the binding of 34TF10 gp95-Fc to either HeLa, Crandel feline leukemia, or G355-5 cells. However, binding was severely impaired in the presence of soluble heparin, as well as after enzymatic removal of surface heparans or on cells deficient in heparan expression. These overall findings suggest that in addition to CXCR4, a non-CXCR4 receptor and cell-surface heparans also play an important role in FIV gp95 cell surface interactions on specific target cells.

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Figures

FIG. 1
FIG. 1
Inhibition of FIV-PPR infection of 104-C1 cells by SDF- 1α. 104-C1 cells were preincubated with either SDF-1α, MIP-1β, or RANTES at the indicated concentrations and infected with FIV-PPR at a multiplicity of infection of 0.01. Virus production was monitored 7 days after the initiation of infection by RT assay as described in Materials and Methods. Results are means and standard deviations for triplicate determinations.
FIG. 2
FIG. 2
Construction of an FIV SU-immunoadhesin. (A) The FIV genome is represented at the top. The FIV Env region contains a leader (L) sequence followed by the surface (gp95) and transmembrane (gp36) subunits. FIV gp95, including the leader sequence, was cloned in frame to the hinge (H) region of human IgG Fc. The recombinant immunoadhesin proteins were stably transfected in CHO cells and batch purified from cell supernatants by affinity chromatography over protein A. (B) Samples (500 ng) of PPR (lanes PP) or 34TF10 (lanes 34) gp95-Fc were subjected to SDS-PAGE in the presence or absence of β-mercaptoethanol (β-ME) and stained by Coomassie blue. (C) Samples of PPR (lanes PP) and 34TF10 (lanes 34) gp95-Fc were subjected to SDS-PAGE under reducing conditions and immunoblotted with an antibody specific for human IgG1 Fc (α-Fc) or FIV Env (α-Env).
FIG. 3
FIG. 3
Flow cytometry analysis of gp95-Fc binding to lymphoid and nonlymphoid cells. Cells were incubated with either Fc or gp95-Fc for 1 h at 4°C and washed, and cell-surface binding was detected by using fluorescein isothiocyanate-conjugated goat anti-human IgG1 Fc antibody. Fluorescence was analyzed by flow cytometry on 2.5 × 103 gated events.
FIG. 4
FIG. 4
Inhibition of gp95-Fc binding to lymphoid cells. 104-C1, Jurkat, and 3201 cells were pretreated with the indicated chemokines at 0.3 or 1.0 μg/ml for 1 h at 4°C before incubation with gp95-Fc for another hour at 4°C. After washing, gp95-Fc binding was monitored by FACS analysis as described in the legend to Fig. 2. Results are means and standard deviations for triplicate determinations.
FIG. 5
FIG. 5
Inhibition of 34TF10 gp95-Fc binding to adherent cells. Cells were preincubated with the indicated chemokines (1 μg/ml) at 4°C for 1 h before incubation with gp95-Fc (A) or coincubated with gp95-Fc and heparin at 1 and 10 μg/ml at 4°C for 1 h (B). After washing, gp95-Fc binding was monitored by FACS analysis. Results are means and standard deviations for triplicate determinations.
FIG. 6
FIG. 6
Cell-surface heparans mediate binding of 34TF10 gp95-Fc to adherent cells. (A) Cells were incubated with the glycosaminoglycan-specific enzymes heparinase I and chondroitinase ABC at 10 U/ml for 1 h at 37°C. Cells were washed and preincubated in the absence or presence of SDF-1α (1 μg/ml) at 4°C for 1 h prior to incubation with gp95-Fc. After washing, gp95-Fc binding was monitored by FACS analysis. Efficient removal of cell-surface heparans was monitored by using an antibody specific for heparan sulfate chains (α-HS; bold lines). Background controls include Fc for the detection of gp95-Fc binding and an isotype-matched antibody (thin lines) for the expression of cell-surface heparans. (B) Fc and 34TF10 gp95-Fc binding as well as the levels of expression of cell-surface heparans were assessed by flow cytometry on CHO wild-type (WT) cells and on a mutant cell line, pgsA745, deficient in the biosynthesis of cell-surface heparans.
FIG. 7
FIG. 7
Heparin interferes with gp95-Fc binding to CXCR4. (A) Cell were coincubated with 34TF10 gp95-Fc and heparin at 1 and 10 μg/ml for 1 h at 4°C. After washing, gp95-Fc binding was detected as described for Fig. 2. Results are means and standard deviations for triplicate determinations. (B) Jurkat cells were treated in the absence or presence of heparinase I at 10 U/ml. 34TF10 gp95-Fc binding was monitored by FACS analysis as described for Fig. 2. For inhibition studies, cells were preincubated with SDF-1α (1 μg/ml) at 4°C for 1 h before incubation with gp95-Fc or coincubated with gp95-Fc and heparin (1 μg/ml). After washing, gp95-Fc binding was monitored by FACS analysis. Percent inhibition of gp95-Fc binding is indicated in the top right corner of each histogram. Efficient removal of heparan sulfate chains by heparinase was monitored by FACS using an anti-heparan sulfate antibody (α-HS, bold lines; isotype-matched antibody, thin lines).
FIG. 8
FIG. 8
Comparative cell surface expression of heparans and CXCR4. Expression of heparans and CXCR4 (bold lines) was assessed by flow cytometry on HeLa, CrFK, G355-5, Jurkat, 104-C1, and 3201 cells. Background controls include isotype-matched antibodies (thin lines).
FIG. 9
FIG. 9
gp95-Fc interacts with a 40-kDa protein species from the surface of feline primary T cells but not from Jurkat and 3201 cells. Cells were cell surface biotinylated and lysed, and precleared lysate supernatants were incubated with either Fc (lane 1), PPR gp95-Fc (PP; lane 2), or 34TF10 gp95-Fc (34; lane 3). Complexes were immunoprecipitated with protein A, resolved by SDS-PAGE, and immunoblotted with neutravidin. Bound neutravidin was revealed by an enhanced chemiluminescence procedure.
FIG. 10
FIG. 10
Amino acid sequences and overall charge of FIV V3 loop. Residues differing between the clones are indicated. Amino acids (positions 407 and 409) that are important for CrFK tropism are boxed.
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