Figure 2

Cell surface binding assays of SU and truncated proteins. A) Schematic representation of SU and truncated proteins. Gray boxes indicate signal peptide (SP) and 15-amino-acid AAARVHRGS-H6 sequence (Tag). Residues are numbered starting from the initiation methionine. ISKP, THTS, NFRP and VSLF sequences correspond to the carboxy-terminal amino acid residues of the native SU included within each construct, SU, Env233, Env197 and Env168, respectively. The underlined amino acids correspond to the numbered residue located just upstream from the tag. B) Detection of SU and truncated proteins in culture medium. Culture supernatants were collected from HEK293T cells transfected with either the SU domain or truncated envelope expression vectors as described in fig 1B. 20 μl of supernatant was denatured (0.5% sodium dodecyl sulfate [SDS], 1% β-mercaptoethanol) at 100°C for 10 min and analyzed by SDS-10% polyacrylamide gel electrophoresis. Blots were probed with with an anti-histidine antibody (anti-RGS(H4)-Mab; Qiagen). Blots were developed using horseradish peroxidase-conjugated antibodies (Jackson) together with an enhanced chemiluminescence kit (Amersham Pharmacia). * indicates the position of Env168 SU protein. C) Identification of the receptor binding domain. SU and truncated soluble proteins were incubated at 37°C for 1 h with XC-hASCT2 cells (shaded) or with parental XC cells (white). Binding assays were performed as described above. The binding capacity of each recombinant protein onto hASCT2 receptor is depicted with a green (efficient) or red (inefficient) highlighted name.