Fig. 6.

Recombinant MeV expressing the hyperfusogenic F mutants from each of the four sites exhibit the expected phenotype. Recombinant MeV with wt F, T461I, and representative F mutations (L137F, S262N, G464W, and H297Y) from sites I to IV (Fig. 4) were rescued in Bsr-T7 cells. Virus growth was monitored via fluorescence microscopy. (A) At day 6 postrescue (with one passage at day 3), cytosolic RNA was collected, and genome copy number was quantified by RT-qPCR. (B) Genome copy numbers for all the mutants were normalized to that for wt MeV, which was set to 1. (B and C) Growth of H297Y mutants was further evaluated for 8 d incubation period (with one passage at day 4). (C) Data shown are mean relative genome copy numbers (± SD) from five independent experiments. (B and C) Statistically significant differences from wt were determined by Dunnet’s multiple comparison test (B) and t test (C). Syncytia images were generated by the imaging cytometer as in previous figures. (Scale Bar, 2 mm.) MeV-specific FIP inhibited the F activity of all the hyperfusogenic mutants except the G464W mutant, as evaluated by our QIFA. (D) Data shown are mean (± SD) from five independent experiments. Representative images of the F inhibitory data are shown in E. P < 0.05 or <0.01 for the indicated comparisons (Student’s t test). Images generated on the image cytometer as before. (Scale Bar, 500 μm.) (F and G) Cell surface biotinylation experiments show that the L137F, S262N, G464W, and H297Y hyperfusogenic mutants were expressed at lower levels than wt F or F-T461I. At 48 hpt, biotinylated cell surface proteins on MeV-F transfected 293T cells were pulled down by streptavidin beads and Western blotted with an MeV-F–specific antibody. Cadherin and GAPDH served as respective cell surface and cytosolic protein controls. The upper and lower blots in F show the input and surface protein (streptavidin pull-down) fraction, respectively. The full-length F₀ and cleaved F₁ products are marked (arrowhead). * indicates the nonspecific band from the cytosol (upper blot), which disappeared in the cell surface pull-down fraction (lower blot). G shows the relative F₁ surface expression levels of the various F proteins normalized to wt F set at 1 (based on densitometric measurements). H shows the F₁/F₀ ratio of cell surface F, which indicates cleavage efficiency. Data shown are the average and range of two independent experiments.