Fig 1.

Characterization of fusion protein mutants by using cell-based assays. (A) Schematic of the NDV fusion protein and the PSA activation mutants. (B) Fold change in cell surface expression, measured quantitatively by flow cytometry. The percentages of fluorescent cells were determined by setting the gates using mock-transfected cells as a negative control and wild-type F as a positive control. (C) Immunofluorescent staining of Vero cells cotransfected with NDV F and HN plasmids, using an antibody against F protein (magnification, ×10). (D) Activation of mutant F proteins with an exogenous PSA overlay (100 ng/ml) (at 12, 24, and 48 h posttransfection) compared to the activation of wild-type F protein with no overlay. Lysates collected after 48 h posttransfection were used to detect the F1 peptide using a polyclonal anti-NDV chicken serum. (E) Determination of the optimal PSA overlay concentration. The exogenous PSA overlay concentration was calculated based on the percentage of cell viability and the fusion index in PC-3 cells. Cotransfection of expression plasmids (mutant pCAGGS-KLQL and wt pCAGGS-HN) in PC-3 cells, followed by an exogenous PSA overlay (1 ng/ml to 400 ng/ml), resulted in variable fusion indices and cell viabilities. Results are means ± standard deviations (SD) obtained from five independent experiments. (F) Syncytium formation in BSR-T7/5 cells 72 h posttransfection (blue arrows).