Figure 1

Cell surface binding assays of soluble SU. A) Schematic representation of HERV-W envelope protein (Env-W) and SU protein (EnvSU). Surface (SU, 1–313) and transmembrane (TM, 318–538) domains and the consensus furin cleavage site (RNKR, 314–317) are indicated. (^|) N-glycosylation sites) [5]. Gray boxes indicate the signal peptide (SP) and a 15-amino-acid AAARVHRGS-H6 sequence (Tag). Residues are numbered starting from the initiation methionine. The ISKP sequence corresponds to the carboxy-terminal amino acid residues of the native SU included within the construct, and the P underlined amino acid corresponds to numbered residue located just upstream from the tag. B) Interaction of the soluble SU with the type D mammalian receptor. Soluble SU protein was secreted from HEK293T cells transfected with the SU domain expression vector and cultured for 24 h in medium without serum. Parental TE671, TE671 RD (hASCT2-blocked cells) and TE671 GALV (PiT1-blocked cells) cells were incubated at 37°C for 1 h in supernatants with (shaded) or without (white) soluble SU protein, collected from transfected or native HEK293T cells, respectively. Binding of the tagged soluble SU onto the cells was detected by incubating 1 h at 4°C the cell-protein mixture with an anti-histidine antibody (anti-RGS(H4)-Mab; Qiagen) in PBA (PBS with 2% fetal calf serum and 0.1% sodium azide). Cells were washed once and incubated with fluorescein isothiocyanate-conjugated antibody (DAKO) for 1 h at 4°C in PBA. The viable cells were analyzed by flow cytometry. C) Binding assay of SU soluble protein with target cells expressing various levels of hASCT1 and hASCT2 receptors. TE671, HeLa, HEK293T, XC-hASCT2 and XC-hASCT1 cells were incubated at 37°C for 1 h in HEK293T cell culture supernatants with (shaded) or without (white) soluble SU protein as indicated above. Binding assays were performed as described in 1B. D) Binding of SU soluble protein is restricted to human ASCT receptors. Soluble SU protein were incubated at 37°C for 1 h with parental XC rat cells (white) and XC-hASCT2 or XC-hASCT1 (shaded) stable cells Binding assays were performed as described in 1B.