Single-particle kinetics of influenza virus membrane fusion.
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.
Floyd
Daniel L
DL
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Ragains
Justin R
JR
Skehel
John J
JJ
Harrison
Stephen C
SC
van Oijen
Antoine M
AM
D006863:Hydrogen-Ion Concentration; D009980:Influenza A virus / Q000378:metabolism*; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014760:Viral Fusion Proteins / Q000378:metabolism; D053586:Virus Internalization
D008051:Lipid Bilayers; D014760:Viral Fusion Proteins