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. 2003 Sep;77(18):9922-30.
doi: 10.1128/jvi.77.18.9922-9930.2003.

Human T-cell leukemia virus type 1 envelope glycoprotein gp46 interacts with cell surface heparan sulfate proteoglycans

Josefina D Piñon  1 , P J Klasse, Sushma R Jassal, Sandy Welson, Jonathan Weber, David W Brighty, Quentin J Sattentau
Affiliations

Human T-cell leukemia virus type 1 envelope glycoprotein gp46 interacts with cell surface heparan sulfate proteoglycans

Josefina D Piñon et al. J Virol. 2003 Sep.

Abstract

The major receptors required for attachment and entry of the human T-cell leukemia virus type 1 (HTLV-1) remain to be identified. Here we demonstrate that a functional, soluble form of the HTLV-1 surface envelope glycoprotein, gp46, fused to an immunoglobulin Fc region (gp46-Fc) binds to heparan sulfate proteoglycans (HSPGs) on mammalian cells. Substantial binding of gp46-Fc to HeLa and Chinese hamster ovary (CHO) K1 cells that express HSPGs was detected, whereas binding to the sister CHO lines 2244, which expresses no HSPGs, and 2241, which expresses no glycosaminoglycans (GAGs), was much reduced. Enzymatic removal of HSPGs from HeLa and CHO K1 cells also reduced gp46-Fc binding. Dextran sulfate inhibited gp46-Fc binding to HSPG-expressing cells in a dose-dependent manner, whereas chondroitin sulfate was less effective. By contrast, dextran sulfate inhibited gp46-Fc binding to GAG-negative cells such as CHO 2244, CHO 2241, and Jurkat T cells weakly or not at all. Dextran sulfate inhibited HTLV-1 envelope glycoprotein (Env)-pseudotyped virus infection of permissive, HSPG-expressing target cells and blocked syncytium formation between HTLV-1 Env-expressing cells and HSPG-expressing permissive target cells. Finally, HSPG-expressing cells were more permissive for HTLV-1 Env-pseudotyped virus infection than HSPG-negative cells. Thus, similar to other pathogenic viruses, HTLV-1 may have evolved to use HSPGs as cellular attachment receptors to facilitate its propagation.

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Figures

FIG. 1.
FIG. 1.
HTLV-1 gp46-Fc binds to HSPGs on CHO and HeLa cells. HeLa and CHO K1 cells and the 2241 and 2244 variants were detached, stained for 2 h at 4°C with gp46-Fc or MAb F58-10E4, specific for HSPG, washed, labeled with the appropriate secondary conjugated antibody, and fixed. Cells were analyzed by flow cytometry: 104 events gated for forward and side scatter were accumulated and analyzed in the FL2 channel. (A) Staining of CHO K1 and variants with F58-10E4 (left) and gp46-Fc (right) expressed as histograms in CellQuest. Solid curve, anti-human IgG-phycoerythrin conjugate alone; dotted curve, irrelevant IgM control antibody (anti-trinitrophenol). (B) Staining intensities (after subtraction of negative control values) for F58-10E4 (left) and gp46-Fc (right) of HeLa and CHO cells expressed as mean fluorescence intensities of 104 gated events. Each value is the mean of three replicates. Error bars represent +1 standard deviation.
FIG. 1.
FIG. 1.
HTLV-1 gp46-Fc binds to HSPGs on CHO and HeLa cells. HeLa and CHO K1 cells and the 2241 and 2244 variants were detached, stained for 2 h at 4°C with gp46-Fc or MAb F58-10E4, specific for HSPG, washed, labeled with the appropriate secondary conjugated antibody, and fixed. Cells were analyzed by flow cytometry: 104 events gated for forward and side scatter were accumulated and analyzed in the FL2 channel. (A) Staining of CHO K1 and variants with F58-10E4 (left) and gp46-Fc (right) expressed as histograms in CellQuest. Solid curve, anti-human IgG-phycoerythrin conjugate alone; dotted curve, irrelevant IgM control antibody (anti-trinitrophenol). (B) Staining intensities (after subtraction of negative control values) for F58-10E4 (left) and gp46-Fc (right) of HeLa and CHO cells expressed as mean fluorescence intensities of 104 gated events. Each value is the mean of three replicates. Error bars represent +1 standard deviation.
FIG. 2.
FIG. 2.
Titration of gp46-Fc onto CHO cells. CHO K1 cells and the 2241 and 2244 variants were detached, labeled with serial threefold dilutions of gp46-Fc, and stained as described above. Data are expressed as mean fluorescent intensities of 104 gated events: each datum point represents the mean of triplicate samples, with negative control values subtracted. Error bars represent ±1 standard deviation.
FIG. 3.
FIG. 3.
Enzymatic removal of HSPG reduces gp46-Fc binding. (A) HeLa cells were treated with HS lyase or chondroitinase ABC or not treated prior to labeling with MAb F58-10E4 (left), F69-3G10 (middle), or gp46-Fc (right). Cells were stained and fixed as described in the legend to Fig. 1, and data are expressed as histograms in CellQuest. (B) Data from HeLa cell analysis expressed as percentages of inhibition of gp46-Fc binding normalized to 0% for gp46-Fc binding to untreated HeLa cells. Each value is the mean of triplicates, and values were derived from the mean fluorescence intensities of 104 accumulated events. Error bars represent +1 standard deviation. Chond, chondroitinase ABC; lyase, HS lyase; Hep-III, heparitinase III.
FIG. 3.
FIG. 3.
Enzymatic removal of HSPG reduces gp46-Fc binding. (A) HeLa cells were treated with HS lyase or chondroitinase ABC or not treated prior to labeling with MAb F58-10E4 (left), F69-3G10 (middle), or gp46-Fc (right). Cells were stained and fixed as described in the legend to Fig. 1, and data are expressed as histograms in CellQuest. (B) Data from HeLa cell analysis expressed as percentages of inhibition of gp46-Fc binding normalized to 0% for gp46-Fc binding to untreated HeLa cells. Each value is the mean of triplicates, and values were derived from the mean fluorescence intensities of 104 accumulated events. Error bars represent +1 standard deviation. Chond, chondroitinase ABC; lyase, HS lyase; Hep-III, heparitinase III.
FIG. 4.
FIG. 4.
Soluble polyanions inhibit gp46-Fc binding to HeLa cells. gp46-Fc was treated with serial dilutions of DexS or CS A, B, or C for 30 min at 4°C prior to addition to HeLa cells. After 2 h at 4°C with agitation cells were washed, stained, and analyzed as described for Fig. 1. Each datum point is the mean of triplicates representing the percentage of inhibition of gp46-Fc binding normalized to 0% for untreated gp46-Fc. Error bars represent ±1 standard deviation.
FIG. 5.
FIG. 5.
DexS inhibits HTLV-1 gp46-Fc binding to HSPG-expressing cells. (A) gp46-Fc was treated with serial dilutions of DexS or dextran for 30 min at 4°C prior to addition to HeLa or CHO K1 cells (top) or CHO 2241 or 2244 cells (bottom). (B) gp46-Fc was treated with serial dilutions of DexS or dextran for 30 min at 4°C prior to addition to Jurkat cells. After 2 h at 4°C with agitation cells were washed, stained, and analyzed as described for Fig. 1. Each datum point is the mean of triplicate samples representing the percentage inhibition of gp46-Fc binding normalized to 0% for untreated gp46-Fc. Error bars represent ±1 standard deviation.
FIG. 5.
FIG. 5.
DexS inhibits HTLV-1 gp46-Fc binding to HSPG-expressing cells. (A) gp46-Fc was treated with serial dilutions of DexS or dextran for 30 min at 4°C prior to addition to HeLa or CHO K1 cells (top) or CHO 2241 or 2244 cells (bottom). (B) gp46-Fc was treated with serial dilutions of DexS or dextran for 30 min at 4°C prior to addition to Jurkat cells. After 2 h at 4°C with agitation cells were washed, stained, and analyzed as described for Fig. 1. Each datum point is the mean of triplicate samples representing the percentage inhibition of gp46-Fc binding normalized to 0% for untreated gp46-Fc. Error bars represent ±1 standard deviation.
FIG. 6.
FIG. 6.
GAG expression increases HTLV-1 Env pseudotype transduction of CHO cells. Serial twofold dilutions of HTLV-1 Env-pseudotyped virus were incubated with CHO K1 or 2241 cells and then incubated for 24 h prior to lysis, addition of substrate, and measurement of luciferase activity. Results are expressed as percentages of transduction normalized to 100% for undiluted supernatant on CHO K1 cells.
FIG. 7.
FIG. 7.
GAG expression subtly decreases HTLV-1 Env-mediated syncytium formation in CHO cells. HeLa cells transiently transfected with the HTLV-1 env gene were mixed with an equivalent number of untransfected HeLa cells (positive control normalized to 100%) or CHO K1, 2241, or 2244 cells and cocultured overnight. Syncytia in 10 low-power fields per replicate were counted, and there were three replicate cultures for each experiment. Data from a representative experiment are shown; error bars represent +1 standard deviation.
FIG. 8.
FIG. 8.
DexS inhibition of HTLV-1 Env-pseudotyped virus transduction of HeLa cells. Serial dilutions of DexS or CS A were preincubated with HTLV-1 Env-pseudotyped virus prior to plating onto HOS cells. Luciferase activity was evaluated 24 h later, and results are expressed as percentages of virus transduction normalized to 100% for the signal obtained from untreated virus. Each datum point represents the mean of triplicates, and the error bars represent ±1 standard deviation.
FIG. 9.
FIG. 9.
DexS inhibits HTLV-1 Env-mediated syncytium formation. HeLa cells were transfected with the HTLV-1 env gene and cocultured with an equivalent number of untransfected HeLa cells for 18 h in the presence or absence of CS A, B, or C or DexS at 500 μg/ml. After fixation, syncytia were visualized by low-power microscopy. Syncytia in 10 fields per replicate were counted, and there were three replicate cultures for each experiment. Bars represent the means of triplicate experiments, and the error bars represent +1 standard deviation.
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