Figure 3

Validation of RBD functionality. A) Cell-cell fusion inhibition by the RBD. The wild-type HERV-W plasmid was transfected in TELCeB6 wild-type envelope producer cells phenotypically characterized by a nucleus expressing β-galactosidase. The HEK293T indicator target cells were transfected with antisense env gene (fusion control), Env71 (non-binding domain), Env144 (RBD) and EnvSU (inhibition control) plasmids. Producer cells were overlaid with HEK293T indicator cells transiently transfected with truncation mutants. Coculture were stained with X-Gal substrate then with May-Grünwald and Giemsa solutions. The determination of the fusion activity of the transfected envelope was performed after 20 h of coculture. The total number of syncytia in one well and the number of nuclei in each syncytia were determined. B) Binding inhibition by an anti-RBD antibody. The Env 144 protein was pre-incubated at 37°C for 1 h with the anti-SU and anti-TM antibodies (dotted line) [5]. Then, XC-hASCT2 cells were incubated at 37°C for 1 h with the protein-antibody complex. The shaded histogram (Env144 incubated with XC-hASCT2) and white histogram (Env144 incubated with parental XC cells) were indicated as controls. The binding was analyzed by flow cytometry with an anti-histidine antibody (anti-RGS(H4)-Mab; Qiagen). C) Receptor binding domain. Variable amino acids (aa) (green dot) among hominoids (upper-case), human (lower case) and neutral insertions preserving the envelope functions (white dots) are indicated above the sequence [23]. Strictly conserved aa (grey boxes) and similar aa (straight line) within the D interference group are underlined. Mutations altering spleen necrosis virus infectivity are indicated as black dots [24]. Blue arrows covering aa 21–69 and 117–144 indicate deletions detrimental to hASCT2 binding. The red arrow corresponds to the SU-EnvW peptide used for rabbit immunization and affinity purification [5].