Single-particle kinetics of influenza virus membrane fusion
- Daniel L. Floyd*,
- Justin R. Ragains*,
- John J. Skehel†,
- Stephen C. Harrison†,‡,§, and
- Antoine M. van Oijen*,§
- *Department of Biological Chemistry and Molecular Pharmacology, and
- ‡Jack and Eileen Connors Structural Biology Laboratory and Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115; and
- †MRC National Institute of Medical Research, Mill Hill, London NW7 1AA, United Kingdom
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Contributed by Stephen C. Harrison, August 13, 2008 (sent for review July 24, 2008)
Abstract
Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays are generally limited to observation of ensembles of multiple fusion events, confounding more detailed analysis of the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support. Analysis of lipid- and content-mixing trajectories on a particle-by-particle basis provides evidence for multiple, long-lived kinetic intermediates leading to hemifusion, followed by a single, rate-limiting step to pore formation. We interpret the series of intermediates preceding hemifusion as a result of the requirement that multiple copies of the trimeric hemagglutinin fusion protein be activated to initiate the fusion process.
Footnotes
- §To whom correspondence may be addressed. E-mail: harrison{at}crystal.harvard.edu or antoine_van_oijen{at}hms.harvard.edu
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Author contributions: D.L.F., J.J.S., S.C.H., and A.M.v.O. designed research; D.L.F. and J.R.R. performed research; J.J.S. contributed new reagents/analytic tools; D.L.F., S.C.H., and A.M.v.O. analyzed data; and D.L.F., S.C.H., and A.M.v.O. wrote the paper.
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The authors declare no conflict of interest.
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Freely available online through the PNAS open access option.
- © 2008 by The National Academy of Sciences of the USA
