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Dissertation
Influenza hemagglutinin mediated membrane fusion: from kinetics to architecture of the fusion site
Doctor of Philosophy (Ph.D.), Drexel University
Feb 2002
DOI:
https://doi.org/10.17918/00009965
Abstract
This work focuses on understanding molecular architecture of the fusion site of influenza hemagglutinin (HA) mediated membrane fusion from kinetic data. Some key HA-mediated fusion data has been analyzed using a mass-action kinetic model (Bentz, 2000a). In Chapters 3 and 4, the comprehensive model has brought together kinetic data for fusion of HA expressing cells with erythrocytes, and fusion of the virus with liposomes, despite differences in assays and experimental design. Consensus of all the analyses shows that of the 8 or more HAs at the fusion site, only 2 need to undergo a slow "essential" conformational change to initiate bilayer destabilization. In Chapter 3, we find that HAs bound to sialates on glycophorin can participate in fusion as members of the fusogenic aggregate, but they cannot undergo the essential conformational change, thus solving a long standing debate about the role of receptor-bound HAs. In Chapter 4, the model is extended to include inactivation and activation of HA following protonation. Various experiments involving A/PR/8/34, X31 and A/Japan/305/57 strains of HA are analyzed. We find that "pKa" for the final protonation site on each monomer of the trimer molecule is 5.6-5.7, irrespective of the strain. We also find that "pKi" for the PR/8 strain is 4.8-4.9. Inactivation rate constants for HA, measured from experiments done with PR/8 virions fusing with liposomes and X31 HA-expressing cells fusing with RBCs, are both found to be of the order of 10-4 s-1. In Chapter 5, kinetics of lipid mixing between HA expressing cells and R18 labeled RBCs are compared using three different experimental approaches. In Chapter 6, an automated video fluorescence microscopy technique has been developed that allows us to monitor kinetics of large numbers of single cell-cell fusion events and enables us to separate fusion from hemifusion. For cell pairs showing any lipid mixing, the onset to completion kinetics of lipid mixing measured by the technique suggest more than one subsets of events. We believe that the fate of a fusion event is decided during the delay time before onset of lipid mixing, however it is independent of the delay time.
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Details
- Title
- Influenza hemagglutinin mediated membrane fusion
- Creators
- Aditya Mittal
- Contributors
- Joseph Edward Bentz (Advisor) - Drexel University, Drexel University (1970-)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- x, 183 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- College of Arts and Sciences; Drexel University
- Other Identifier
- 991021889094204721