FIG 4 .

FCT residues Phe-Ser-Asn are sufficient for filamentous assembly at the cell surface. HEp-2 cells were transfected with the indicated FCT construct and RSV M, N, and P (A to L). Column 1 shows images of the whole cell (A, E, and I). Columns 2 to 4 (B to D, F to H, and J to L) show an enlargement of the corresponding inset in column 1. Column 2 shows actin staining only (B, F, and J); column 3 shows RSV F staining only (C, G, and K); and column 4 shows the overlay, with RSV F in green and actin in red (D, H, and L). (M and N) HEp-2 cells were transfected as described for panels A to L. Filament length was determined by measuring the lengths of individual filaments using Zeiss LSM software as described in Materials and Methods (M). Percent transfected cells was determined by counting RSV F-transfected cells with filaments and then dividing that number by the total number of RSV F-transfected cells (N). (O) Total cell lysate was collected from HEp-2 cells transfected with the indicated FCT construct, and RSV F and actin were detected by immunoblotting. The RSV F band was normalized against actin, and the expression (exp) ratio represents a normalization to FCT wt. (P) Total surface expression of RSV F was determined by flow cytometry using HEp-2 cells transfected with the indicated FCT construct and RSV M, N, and P. Data are plotted as means, and error bars represent standard deviations. Weighted MFI is mean fluorescence intensity (MFI) × the frequency of positive cells. MPV F wt is used as a specificity control.