Kinetics of Membrane Fusion of Individual Virus Particles with Lipid Bilayers
With total internal reflection fluorescence microscopy we resolve the fusion of individual virus particles with supported lipid bilayers. The single particle approach allowed us 1) to observe the dynamics of the fusion reaction, and 2) to determine the interval between virus binding and fusion, the residence time. We observed systematic variation in the residence time as a function of target bilayer composition and buffer conditions. In particular, the combination of cholesterol and pH below 5 dramatically increased the delay between binding and fusion for Sindbis virus. These results will constrain modeling of the mechanism of membrane fusion.
We use TIRFM to image R18 dye molecules incorporated into the viral membrane. Since these dye molecules are lipids they should be able to move freely within the viral envelope, and once fusion between the viral envelope and the lipid bilayer occurs, the lipid dye molecules will be able to move freely within the lipid bilayer. They will disperse via diffusion which will decrease their intensity along with photobleaching.
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A histogram of dequenching times (10%-90%) for 49 of 51 fusion events taken over 10 movies from 5 slides (2 were longer than 200 ms). Most events occur in two time steps. Some events that are fast enough to be completed within one time bin will be counted in the group requiring 2 time bins because those events may begin toward the end of one time bin and finish during the next bin. An exponential fit omitting the first time bin yields a time constant of 28 msec. | |||||||||||||||||||||||||
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Residence times for Sindbis and influenza virus as a function of pH. The median residence time accumulated from many experiments for Sindbis virus increases sharply by a factor of ten at low pH compared to the near-constant residence time for influenza. Note that most fusion events occur quickly after virus binds to the bilayer. | |||||||||||||||||||||||||
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Fusion of Sindbis virus with bilayers formed from mixtures of purified lipids. Liposomes of the indicated mixtures of lipids (CH=cholesterol, SM= Sphingomyelin) were used in bulk fusion assays or to form supported lipid bilayers for single particle fusion studies. The final extent of fusion from bulk fusion experiments illustrates the strict cholesterol requirement for fusion with Sindbis virus. The essential nature of sphingomyelin is evident at pH 5.5 but is reduced at pH 4.6. The column of residence times is from single particle bilayer experiments at pH 4.6.. The residence time increased in the presence of cholesterol only for experiments with pH below 5. |
In conclusion, sphingomyelin (SM) and cholesterol (CH) are both required for low-pH triggered membrane fusion of Sindbis virus. Using a single particle assay we found that for pH below 5 the requirement for SM is decreased. At these lowest pH the presence of cholesterol increased the interval between binding and fusion. Under all conditions, the dynamics of the fusion transition were faster than our instrument limit of 30 msec.
Future aims
- Extend our assay to observe both content and lipid mixing during fusion
- Apply these methods to investigate fusion of Sindbis virus in living cells.
- Integrate a microfluidic platform to improve our time resolution and throughput.
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