Expression of glucose transporter 1 confers susceptibility to human T-cell leukemia virus envelope-mediated fusion

Abstract

Human T-cell leukemia virus type 1 (HTLV-1) was the first human retrovirus identified and causes both adult T-cell leukemia/lymphoma and tropical spastic paraparesis/HTLV-1-associated myelopathy, among other disorders. In vitro, HTLV-1 has an extremely broad host cell tropism in that it is capable of infecting most mammalian cell types, although at the same time viral titers remain relatively low. Despite years of study, only recently has a bona fide candidate cellular receptor, glucose transporter 1 (glut-1), been identified. Although glut-1 was shown to bind specifically to the ectodomain of HTLV-1 and HTLV-2 envelope glycoproteins, which was reversible with small interfering RNA directed against glut-1, cellular susceptibility to HTLV upon expression of glut-1 was not established. Here we show that expression of glut-1 in relatively resistant MDBK cells conferred increased susceptibility to both HTLV-1- and HTLV-2-pseudotyped particles. glut-1 also markedly increased syncytium formation in MDBK cells after exposure to HTLV-1. Another assay also demonstrated HTLV-1 envelope-cell fusion in the presence of glut-1. Taken together, these results provide additional evidence that glut-1 is a receptor for HTLV.

FIG. 1.

MDBK cells are relatively resistant to BIV (HTLV) particles. MDBK and 293T cells were separately transduced with BIV-eGFP (HTLV-1) (gray bars) or BIV-eGFP (VSV G) (black bars) and subjected to flow cytometry 3 days later. Shown are the transduction efficiencies and mean fluorescence intensities (base of each bar) of eGFP-positive cells.

FIG. 2.

Immunoblot assay of glut-1 in MDBK cells. MDBK cells were lysed, and proteins were separated by SDS-PAGE, blotted onto nitrocellulose, and probed with rabbit anti-glut-1 antiserum, followed by goat anti-rabbit antiserum conjugated to horseradish peroxidase. Lanes: 1, MDBK.cycT1 cells; 2, MDBK.empty cells; 3, MDBK.glut-1 cells; 4, MDBK.cycT1.empty cells; 5, MDBK.cycT1.glut-1 cells. Note the heterogeneous reactivity at 55 kDa; other bands are presumably background or perhaps reflect endogenous glut-1 reactivity.

FIG. 3.

Immunofluorescence assay of glut-1 in MDBK cells. Cells were fixed and permeabilized and stained for glut-1 as described in the text. Panels: A, HOS cells (positive control); B, MDBK.empty cells; C, MDBK.glut-1 cells; D, MDBK.cycT1.empty cells; E, MDBK.cycT.glut-1 cells. Note both cytoplasmic and plasma membrane staining in panels C and E.

FIG. 4.

Cocultivation of HIV (HTLV) with MDBK cells. 293T cells were transfected with both pHIV-neo and pSV-HTLV-1 env rre plasmids, treated with mitomycin C, and then cocultured with the indicated cells. After 48 h, cells were passaged into selective medium containing Geneticin at 2.0 mg/ml. Colonies were enumerated 12 days later. Cocultivation of both cell types with 293T cells transfected with both pHIV-neo and pME VSV G gave approximately equivalent titers of 50,000/2 × 10⁶ 293T cells, whereas when envelope glycoprotein was omitted no colonies were observed. Note the logarithmic scale.

FIG. 5.

glut-1-eYFP fusion also allows HIV (HTLV) infection. (A) Cartoon of fusion proteins with the autofluorescent protein portion shown in light grey and the plasma membrane shown as two curved lines. (B) Cell-free pseudotyping results obtained with either 6 ml of HIV-cycT1-IRES-bsd (HTLV-2) (II) or 1 ml of HIV-cycT1-IRES-bsd (VSV G) (G). Closed bars indicate MDBK.cycT1.LESTREGFP cells, and open bars indicate MDBK.cycT1.GLUT1EYFP cells. Average ± standard deviation is shown. *, P = 0.004 compared to HTLV-2 titers on MDBK.cycT1.LESTREGFP cells (Student's t test). Note the logarithmic scale.

FIG. 6.

Syncytium formation in the presence of glut-1. Cells were cocultured with either Jurkat T cells (panels A, C, and E) or C91/PL cells (panels B, D, and F) and then fixed and stained with crystal violet. Panels: A and B, HOS cells; C and D, MDBK.empty cells; E and F, MDBK.glut-1 cells; G, quantitation of syncytia. Values are the average number of syncytia per 10× field. Downward arrows indicate that less than one syncytium was present per field. Open boxes, 10⁵ C91/PL cells per 12-well plate; closed boxes, 2 × 10⁵ C91/PL cells per 12-well plate. In the absence of C91/PL cells or in the presence of Jurkat T cells, only rare syncytia were observed for any of the cell lines. For HOS cells in the presence of C91/PL cells, values exceeded 100 (not shown).

FIG. 7.

HTLV-induced cell fusion in the presence of glut-1. 293T Ω12 cells (stably express LacZω protein) were transfected with the indicated glycoproteins and then cocultured with the indicated cells stably expressing LacZα peptide. After 48 h, cells were lysed and soluble β-galactosidase activity was measured. Note the increase in the activity of HTLV-1 env in the presence of glut-1; no increase was observed when 293T cells were transfected or when LacZα peptide was absent.