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Method for Measurement of Viral Fusion Kinetics at the Single Particle Level
14:59

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Published on: September 7, 2009

Refolding of a paramyxovirus F protein from prefusion to postfusion conformations observed by liposome binding and

Sarah A Connolly1, George P Leser, Hsien-Shen Yin

  • 1Howard Hughes Medical Institute, Northwestern University, Evanston, IL 60208-3500, USA.

Proceedings of the National Academy of Sciences of the United States of America
|November 10, 2006
PubMed

Insights

Heat triggers paramyxovirus fusion protein (F) refolding and membrane insertion, revealing its conformational changes. This study elucidates the triggering mechanism essential for viral entry.

Area of Science:

  • Virology
  • Structural Biology
  • Biochemistry

Background:

  • Paramyxoviruses utilize attachment and fusion (F) proteins for cell entry.
  • The F protein undergoes conformational changes from a metastable prefusion state to a stable postfusion state.
  • The precise triggering mechanism for paramyxovirus fusion remains unclear.

Purpose of the Study:

  • To correlate the molecular structure of a soluble prefusion F protein (PIV5 F-GCNt) with its biological function.
  • To elucidate the triggering mechanism controlling paramyxovirus F protein conformational changes.

Main Methods:

  • Utilized a soluble form of the paramyxovirus F protein (PIV5 F-GCNt).
  • Employed heat as a surrogate trigger in the absence of the HN attachment protein.
  • Applied liposome association, sucrose density gradients, and electron microscopy for structural analysis.

Main Results:

  • Heat successfully triggered refolding of the soluble F protein.
  • Triggered F protein associated with liposomes and formed aggregates.
  • Electron microscopy revealed triggered F protein formed rosettes, suggesting fusion peptide release and membrane insertion.
  • Heating induced conversion to a postfusion form, characterized by "golf tee" morphology.

Conclusions:

  • Cleavage and heat are crucial for F protein's hydrophobic fusion peptide release and membrane insertion.
  • Soluble and membrane-bound F proteins likely refold via similar pathways.
  • Heat serves as a viable trigger to study F protein conformational dynamics.

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