FIGURE 4.

HPIV3 HN associates with F on the cell surface before receptor interaction. A, current models of HN and F interaction are shown. B, 293T cells co-transfected with constructs encoding HN N-Venus and F C-CFP were treated overnight with 10 mm zanamivir, washed, then 1 h before analysis replaced with 10 mm zanamivir and cycloheximide. Evenly distributed fluorescence was visualized across the cell surface. C, 293T cells co-transfected with constructs encoding HN N-Venus and F C-CFP were treated overnight with 10 mm zanamivir, washed, then 1 h before analysis the zanamivir was removed to allow HN to engage receptor and replaced with cycloheximide and 10 μm HRC peptides to inhibit progression of fusion beyond the stage of fusion peptide insertion. BiFC clustering of HN/F complexes was evident. D, 293T cells co-transfected with constructs encoding HN N-Venus and F C-CFP were treated overnight with 10 mm zanamivir, washed, then 1 h before analysis treated with 10 mm zanamivir and cycloheximide. Fusion was recorded over 90 min.