Single-particle kinetics of influenza virus membrane fusion

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.enveloped viruses ͉ lipid bilayer ͉ single molecule ͉ virus entry

Relating influenza virus membrane fusion kinetics to stoichiometry of neutralizing antibodies at the single-particle level

“…Our data reveal a need for two to three HA trimers for fusion and strongly support a kinetic model of HA-mediated fusion requiring simultaneous action of multiple, neighboring trimers (15,16). This model requires only the stochastic behavior of the HA trimers in combination with a high HA surface density.…”

“…Fluorescence intensity from individual IgG/Fab molecules adsorbed to clean glass in pH 5.0 buffer was acquired under identical illumination conditions. Data analysis was performed using custom MATLAB (Mathworks, Inc.) scripts, similar to those previously described (15,29). Briefly, viruses were identified in the red channel and paired with separately identified locations in the green channel.…”

“…In this manner, we obtain information about the speed of the rate-limiting step along the fusion pathway and the number of rate-limiting steps. This latter value has been shown to represent the number of HA required for fusion (15). This kinetic analysis requires at least 50 events to have the statistical power to determine the number of HA trimers involved (39) and because increasing IgG/Fab concentrations results in fewer fusing virions, not all concentrations could be analyzed (SI Materials and Methods).…”

“…Refolding of the protein brings the viral and endosomal membranes close together and catalyzes their fusion (11,12). Several biophysical studies indicate that multiple HA trimers must work together by coordinating their conformational changes for membrane fusion to occur (13)(14)(15)(16).…”

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

“…This modification also resulted in SNAP25-dependent fusion 24 . Bilayers cushioned on a soft polymer layer had been known to better preserve transmembrane protein mobility and function 32 , and had been used in fusion studies with viruses 33 . In addition, PEGylated bilayers seem to retain some ability to self-heal and are very robust 34,35 .…”