Fig. 3

Effect of alanine substitutions in gp51p16-C of pBLV-IF on viral expression and fusogenicity, and conservation. After transfection, pBLV-IF, mutated pBLV-IFs with the single alanine mutations (R7A, R9A, F10A, V16A, and Y18A), or a negative control vector and the GFP expression vector pEGFP-N1 were performed Western blotting (a) and syncytium (b) analyses. GFP was used as the reporter molecule for discrimination between transfected and untransfected cells. a For Western blotting, a fraction of 23CLN cells were harvested 20 h following transfection and were analyzed for the ratio of GFP-expressing cells by flow cytometry. The remaining 23CLN cells were lysed 48 h after transfection, and lysates with equal numbers of GFP-expressing cells were subjected to Western blotting using anti-BLV Env protein and anti-Gag protein monoclonal antibodies and HRP-conjugated goat antibodies. FLK-BLV cells, which were productively infected with BLV, were used as the positive control. Positions of protein marker and BLV glycoprotein (gp51) were indicated and BLV Gag protein (p24). b For syncytium formation, 293T cells were visualized by confocal laser scanning microscopy. c Distribution of polymorphic and frequencies of antibody binding amino acid residues in gp51p16-C. BLV nucleotide sequences (n = 517) were collected from GenBank, and amino acid sequences corresponding to gp51p16-C were aligned. The amino acid positions 7, 9, 10, 16, and 18 are indicated by triangle. A single letter above gp51p16-C sequences indicates a polymorphic amino acid residue