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Dissertation/Thesis Abstract

Visualizing influenza virus membrane fusion: Inhibition and kinetics
by Otterstrom, Jason John, Ph.D., Harvard University, 2013, 267; 3611569
Abstract (Summary)

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.

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Indexing (document details)
Advisor: van Oijen, Antoine M.
Commitee: Hogle, James M., Loparo, Joseph J., Zhuang, Xiaowei
School: Harvard University
Department: Biophysics
School Location: United States -- Massachusetts
Source: DAI-B 75/05(E), Dissertation Abstracts International
Subjects: Virology, Biophysics
Keywords: Hemagglutinin, Influenza, Membrane fusion, Neutralization stoichiometry, Neutralizing antibodies, Single molecule biophysics
Publication Number: 3611569
ISBN: 978-1-303-72453-4
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