title: Soluble respiratory syncytial virus fusion protein in the fully cleaved, pretriggered state is triggered by exposure to low-molarity buffer.
abstract: The paramyxovirus fusion (F) glycoprotein is anchored in the virion membrane in a metastable, pretriggered form. Once triggered, the F protein undergoes a dramatic conformational extension that inserts its hydrophobic fusion peptide into the target cell membrane, then folds back on itself to bring the membranes together and initiate fusion. Unlike most other paramyxoviruses, the respiratory syncytial virus (RSV) F protein alone is sufficient to mediate membrane fusion and virus infection. To study the triggering mechanism of the RSV F protein, we have generated a soluble F (sF) protein by replacing the transmembrane and cytoplasmic tail domains with a 6His tag. The sF protein is secreted efficiently from 293T cells in a fully cleaved form. It is recognized by neutralizing monoclonal antibodies, appears spherical by electron microscopic analysis, and is not aggregated, all consistent with a native, pretriggered trimer. The sF protein was purified on a Ni(+2) column and eluted with 50 mM phosphate buffer containing 500 mM NaCl and 250 mM imidazole. Dialysis against 10 mM buffer caused the sF protein to trigger, forming "hat pin"-shaped molecules that aggregated as rosettes, characteristic of the posttriggered form. Further dialysis experiments indicated that the efficiency of triggering correlated well with the reduction of buffer molarity. Reduction of buffer molarity by dilution also resulted in exposure of the fusion peptide, as detected by liposome association, confirming sF protein triggering. Mutation of the furin cleavage site adjacent to the fusion peptide prevented liposome association, further confirming that association is via the fusion peptide.
authors.affiliation: The Research Institute at Nationwide Children's Hospital, 700 Children's Dr., Columbus, OH 43205, USA.
authors.forename: Supranee
authors.initials: S
authors.lastname: Chaiwatpongsakorn
authors.forename: Raquel F
authors.initials: RF
authors.lastname: Epand
authors.forename: Peter L
authors.initials: PL
authors.lastname: Collins
authors.forename: Richard M
authors.initials: RM
authors.lastname: Epand
authors.forename: Mark E
authors.initials: ME
authors.lastname: Peeples
mesh_terms: D002021:Buffers; D006801:Humans; D009997:Osmotic Pressure; D012136:Respiratory Syncytial Viruses / Q000235:genetics / Q000502:physiology*; D014760:Viral Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D053586:Virus Internalization
chemical_list: D002021:Buffers; C526850:F protein, human respiratory syncytial virus; D014760:Viral Fusion Proteins