Figure 5

A loop structure predicted by computational modeling in the VRA domain of FeLV-A is not sufficient to confer the binding phenotype of FeLV-945 SU. A. Ribbon diagram of homology models of the receptor binding domain in FeLV-A/61E and FeLV-945 SU proteins. Homology modeling was performed using the SwissModel Program and the known crystal structure of the receptor binding domain of FeLV-B SU (FeLV-B 1LCS) as a modeling template. A prominent loop (circled) was predicted by the models within the VRA domain of FeLV-A/61E and FeLV-945 proteins, and is distinct from the structure of FeLV-B in the same region. B. Comparison of amino acids sequences of FeLV-A/61E and FeLV-945 in the predicted VRA domain loop. The five amino acid differences between the sequences are indicated by shading. C. Comparative flow cytometric binding assays of SU proteins encoded by FeLV-A/61E, FeLV-945 and 61E/945-5, a mutant in which the FeLV-945 sequence at all of the five highlighted residues shown in Figure 5B was substituted by site-directed mutagenesis into FeLV-A/61E. Binding assays were performed using feline 3201 cells as described in Figure 2. A representative histogram is shown (left panel), demonstrating the binding activity of FeLV-A/61E SU (gray shaded), FeLV-945 SU (black shaded) and 61E/945-5 SU (open histogram, solid line). Negative controls (open histograms, broken lines) include supernatants from pCS2/Ctrl-transfected cells and 61E/945-5 SU with isotype control antibody. Right 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.