Figure 6

Substitution of both FeLV-945 VRA and VRB is sufficient to confer the enhanced binding phenotype to FeLV-A/61E SU. A. Diagram of the 412-amino acid FeLV-A/61E SU protein and mutants into which FeLV-945 sequences were substituted. Positions of the VRA, VRB, and PRR domains are indicated (shaded boxes). FeLV-945 sequences that have been substituted into FeLV-A/61E SU to construct each mutant are indicated (black boxes), and vertical lines represent the relative locations of amino acid sequence differences between the two SU proteins. B. - D. Comparative flow cytometric binding assays of SU proteins encoded by FeLV-A/61E, FeLV-945 and substitution mutant SU proteins as indicated. Binding assays were performed using feline 3201 cells as described in Figure 2. Representative histograms are shown, demonstrating the binding activity of FeLV-A/61E SU (green), FeLV-945 SU (pink) and the substitution mutant indicated in each case (blue). Negative controls included supernatants from pCS2/Ctrl-transfected or mock-transfected cells (gray) and each mutant SU with isotype control antibody (gold). Inset in each panel shows chemiluminescent western blot analysis to validate equivalent mass amounts of the SU proteins used in the binding assay as previously quantified by infrared dye-based densitometry. C. and D., Right panels. Replicate binding assays were performed using four (61E/945-VRA/VRB) or two (945/61E-VRA/VRB) independently generated and infrared-quantified batches of SU proteins. The geometric mean fluorescence from individual assays is shown, as is the mean of four independent replicate experiments (horizontal bar). Asterisk indicates statistical significance (*; C: p < 0.05; D: p < 0.01).