The influenza virus hemagglutinin (HA) surface protein is a primary antigenic target for neutralization of viral infection. HA also mediates membrane fusion between the virus and a cell, which is the first critical step during infection. Traditional techniques to study infection neutralization by antibodies or the membrane fusion process rely on ensemble measurements, confounding the precise mechanism of infection neutralization and obscuring transient conformational intermediates. This dissertation describes advances made in a fluorescence microscopy-based single-particle fusion assay to overcome the limitations of ensemble measurements in these types of studies. Virus particles are labeled to visualize lipid mixing between a virus and a target membrane formed upon a glass or polymer support. Optionally, the viral lumen can be labeled to visualize the subsequent release of viral contents. Recently isolated antibodies recognizing highly conserved epitopes on the HA protein that are critical for the protein's fusogenic capacity are able to neutralize infection from a broad range of influenza subtypes. Binding of these antibodies to a virus prior to inducing fusion with a target membrane resulted in inhibition of the fusion process, directly confirming one mechanism of infection neutralization. Fluorescently labeling the antibodies allowed for functional stoichiometric measurements that indicate a virion can be rendered non-fusogenic without the need for antibodies to bind and inactivate every HA present on the viral surface. A molecular model of fusion inhibition is proposed wherein coordination between neighboring HA is disrupted that leads to neutralization of the entire particle. Though polyunsaturated lipids are critical components of biological membranes that frequently undergo membrane fusion, their precise effects on the fusion process are poorly characterized. A fully automated data extraction program was designed and implemented to enhance the reproducibility of selecting fusing and non-fusing virions. Analysis of the resulting fusion kinetics revealed that the rate of fusion pore expansion is enhanced by high degrees of lipid unsaturation. During fusion at low pH, lower degrees of saturation resulted in the onset of a restricted hemifusion state. It is postulated that cholesterol and the HA fusion peptide create a molecular fence that confines lipid diffusion between the virus and target membranes.
Some files may require a special program or browser plug-in. More Information
|Advisor:||van Oijen, Antoine M.|
|Commitee:||Hogle, James M., Loparo, Joseph J., Zhuang, Xiaowei|
|School Location:||United States -- Massachusetts|
|Source:||DAI-B 75/05(E), Dissertation Abstracts International|
|Keywords:||Hemagglutinin, Influenza, Membrane fusion, Neutralization stoichiometry, Neutralizing antibodies, Single molecule biophysics|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be