Figure 8

A single amino acid residue at position 186 accounts for the ability of a VRB-containing domain to confer increased receptor binding affinity to FeLV-A/61E SU. A. A representative histogram from a comparative flow cytometric binding assay of SU proteins encoded by FeLV-A/61E (green), 61E/945-VRB (orange) and I186V (blue) is shown (left). Binding assays were performed as described in Figure 2 using anti-SU antibody C11D8. Negative controls include supernatants from pCS2/Ctrl-transfected cells (gray) and I186V SU with isotype control antibody (gold). Inset shows chemiluminescent western blot analysis with C11D8 antibody to validate equivalent mass amounts of the SU proteins used in the binding assay. Replicate binding assays were performed (right) using six (I186V) or four (FeLV-A/61E and 61E/945-VRB) independently generated and titered batches of SU proteins (p < 0.05). B. A representative histogram (left) and geometric mean fluorescence of replicate flow cytometric binding assays (right) is shown demonstrating the binding activity of SU proteins encoded by FeLV-A/61E (green), I186V (blue) or the reciprocal mutant V186I (red). Negative controls included supernatants from pCS2/Ctrl-transfected cells (gray) and V186I SU with isotype control antibody (gold). Replicate binding assays were performed as in A using four independently generated and titered batches of each SU protein (p < 0.05). Inset shows chemiluminescent western blot analysis with C11D8 antibody to validate equivalent mass amounts of the SU proteins used in the binding assay. C. Geometric mean fluorescence of replicate binding assays using soluble SU proteins expressed from FeLV-A/61E, 61E/945-VRB, I186V, and V186I is shown (p < 0.05). Negative control is supernatant of pCS2/Ctrl-transfected cells. Comparative flow cytometric binding assays were performed as in A except using anti-HA antibody to detect SU protein. Assays were performed with three (61E/945-VRB) or four (FeLV-A/61E, I186V, V186I) independently generated and titered batches of SU proteins.