title: Mutations in the Leucine Zipper-Like Motif of the Human Parainfluenza Virus 3 Fusion Protein Impair Fusion Activity.
abstract: 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.
  
  
  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.
  
  
  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.
authors.affiliation: Department of Virology, School of Public Health, Shandong University, Jinan, PR China.
authors.forename: Wenyan
authors.initials: W
authors.lastname: Xie
authors.forename: Hongling
authors.initials: H
authors.lastname: Wen
authors.forename: Fulu
authors.initials: F
authors.lastname: Chu
authors.forename: Shaofeng
authors.initials: S
authors.lastname: Yan
authors.forename: Wenli
authors.initials: W
authors.lastname: Xie
authors.forename: Bin
authors.initials: B
authors.lastname: Lin
authors.forename: Yuzhen
authors.initials: Y
authors.lastname: Chen
authors.forename: Zhenmei
authors.initials: Z
authors.lastname: Li
authors.forename: Guijie
authors.initials: G
authors.lastname: Ren
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Song
authors.forename: Li
authors.initials: L
authors.lastname: Zhao
authors.forename: Zhiyu
authors.initials: Z
authors.lastname: Wang
mesh_terms: D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D004252:DNA Mutational Analysis; D004912:Erythrocytes; D006801:Humans; D016350:Leucine Zippers; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010224:Parainfluenza Virus 3, Human / Q000502:physiology*; D014760:Viral Fusion Proteins / Q000235:genetics* / Q000378:metabolism*; D053586:Virus Internalization
chemical_list: D050505:Mutant Proteins; D014760:Viral Fusion Proteins; C072983:F protein, parainfluenza virus 3