Conclusions
This motif has an important influence on fusion activity, and its integrality is indispensable for membrane fusion.
Methods
Site-directed mutagenesis was utilized to substitute the heptadic residues at 257, 264, 271, 278, 285, 292, and 299 in this motif with alanine. Additionally, 3 middle heptadic leucine residues at 271, 278, and 285 were replaced with alanine singly or in combination. A vaccinia virus-T7 RNA polymerase transient expression system was employed to express the wild-type or mutated fusion (F) proteins. Three different types of membrane fusion assays were performed to analyze the fusogenic activity, fluorescence-activated cell sorting (FACS) analysis was executed to examine the cell surface expression level, and a coimmunoprecipitation assay was conducted to probe the hemagglutinin-neuraminidase (HN)-F interaction at the cell surface.
Objective
To investigate the effect of the leucine zipper-like motif between HRA and HRB of the human parainfluenza virus 3 fusion protein on fusion activity.
Results
All of the substitutions in this motif exhibited diminished or even lost fusion activity in all stages of fusion, although they all had no effect on cell surface expression. In the coimmunoprecipitation assay, all mutants resulted in decreased detection of the HN-F complexes compared with that of the wild-type F protein. Conclusions: This motif has an important influence on fusion activity, and its integrality is indispensable for membrane fusion.