Membrane fusion is the key molecular event during the entry of enveloped viruses into cells, but it is not understood how the activity of fusion proteins is coordinated to catalyze this kinetically unfavorable process. Knowledge of the kinetics of the fusion reaction is largely derived from studies where many fusion events are observed simultaneously, thereby making a straightforward quantitative Analysis difficult. The objective of this application is to study viral membrane fusion by a fundamentally new strategy: reconstituting viral fusion in vitro with only the bare minimum of molecular components and monitoring the kinetics of individual fusion events. The resulting 'molecular movies' will allow us to dissect the reaction kinetics at a level of detail inaccessible to conventional ensemble experiments. As a model system, the hemagglutinin (HA) fusion machinery of influenza virus will be used. HA- containing virosomes will be labeled with different fluorescent probes and fluorescence microscopy used to monitor the kinetics effusion and hemifusion of the individual particles with a planar, supported bilayer.
The Specific Aims are: 1) to determine the kinetics of the transitions between the various bilayer fusion intermediates and establish the roles of the different hemifusion states, and 2) to determine the molecular nature of the cooperative behavior in HA-mediated fusion. The long-term goal of the proposed work is to combine the kinetic information as obtained from single- particle studies with known structural and biochemical properties into one complete molecular picture that accurately describes the mechanism of membrane fusion in influenza infection. Furthermore, the proposed single-particle techniques will be directly applicable to studying molecular mechanisms of membrane fusion in other systems. Public health relevance: Enveloped viruses invade cells by merging their membrane with the lipid bilayer surrounding the target cell. A detailed understanding of the molecular details of this fusion process is a necessary tool in the identification of drug targets and the rational design of therapeutics. The proposed development and use of single-particle imaging techniques to study the fusion mechanism of influenza will lead to a more complete and dynamic picture of the various pathways involved. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21AI072346-01
Application #
7187143
Study Section
Special Emphasis Panel (ZRG1-IDM-G (90))
Program Officer
Salomon, Rachelle
Project Start
2007-01-15
Project End
2008-12-31
Budget Start
2007-01-15
Budget End
2007-12-31
Support Year
1
Fiscal Year
2007
Total Cost
$211,875
Indirect Cost
Name
Harvard University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
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Ivanovic, Tijana; Rozendaal, Rutger; Floyd, Daniel L et al. (2012) Kinetics of proton transport into influenza virions by the viral M2 channel. PLoS One 7:e31566
Floyd, Daniel L; Harrison, Stephen C; van Oijen, Antoine M (2009) Method for measurement of viral fusion kinetics at the single particle level. J Vis Exp :
Floyd, Daniel L; Ragains, Justin R; Skehel, John J et al. (2008) Single-particle kinetics of influenza virus membrane fusion. Proc Natl Acad Sci U S A 105:15382-7