Protein-mediated membrane fusion is a fundamental biological process that lies at the heart of enveloped virus infection, synaptic signaling, intracellular vesicle trafficking, gamete fertilization, and cell-cell fusion. Despite intensive study, at present we have a limited mechanistic understanding of how fusion protein machinery manipulates lipid membranes in order to induce their fusion. Enveloped viruses use specialized protein machinery to fuse their membrane with the membrane of host cells and deliver their genetic material for replication. In influenza virus, the trimeric hemagglutinin (HA) glycoprotein spike is responsible for host cell attachment and membrane fusion. While structures of a subset of conformations and parts of the fusion machinery have been characterized, the states that drive the fusion process have proven to be refractory to classical structure determination. In addition, the nature of membrane deformations during fusion has largely eluded characterization. In essence, we lack a mechanistic understanding of membrane fusion and hence viral entry. This proposal focuses on determining the structural and functional interplay between hemagglutinin and membranes during fusion. A combined approach including fluorescence spectroscopy, electron cryo-tomography (ECT), small-angle X-ray scattering (SAXS) with 3-D shape reconstruction, and Hydrogen-Deuterium-exchange with mass spectrometry analysis (HD-MS) will be employed. An integrative approach is needed to reveal the mechanics of membrane fusion from the level of understanding how individual HA spikes perturb membranes to mapping the architecture of whole virion-membrane fusion complexes. In this proposal, ECT will be used to image the 3-dimensional architecture of pre-fusion, hemi-fusion and fusion pores between whole virions and target membranes. Target membranes will be chosen to test the role of lipid composition, leaflet distribution, and geometry on the fusion process. We will also use ECT to dissect the membrane-bending activity of soluble HA ectodomain and full-length HA. SAXS and HD-MS will be combined to determine the structure of the fusogenic, prehairpin HA intermediate state. The model will be validated by chemical crosslinking with mass spectrometry analysis.

Public Health Relevance

This proposal will address significant gaps in our understanding of how influenza virus invades new host cells. This stage of the virus life cycle is gaining interest as a target for new antiviral therapeutics, however progress is hindered by a lack of understanding of the structure and function of the viral entry machinery. We will use state-of-the-art structural and biophysical methods to image components and whole influenza virus particles undergoing the process of membrane fusion in order to gain unprecedented mechanistic insight into this critical process.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM099989-03S1
Application #
8901345
Study Section
Virology - A Study Section (VIRA)
Program Officer
Sakalian, Michael
Project Start
2012-04-01
Project End
2017-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
3
Fiscal Year
2014
Total Cost
$97,233
Indirect Cost
$32,414
Name
University of Washington
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Benhaim, Mark; Lee, Kelly K; Guttman, Miklos (2018) Tracking Higher Order Protein Structure by Hydrogen-Deuterium Exchange Mass Spectrometry. Protein Pept Lett :
Pardi, Norbert; Hogan, Michael J; Naradikian, Martin S et al. (2018) Nucleoside-modified mRNA vaccines induce potent T follicular helper and germinal center B cell responses. J Exp Med 215:1571-1588
Gui, Long; Lee, Kelly K (2018) Influenza Virus-Liposome Fusion Studies Using Fluorescence Dequenching and Cryo-electron Tomography. Methods Mol Biol 1836:261-279
Williams, James A; Gui, Long; Hom, Nancy et al. (2017) Dissection of epitope-specific mechanisms of neutralization of influenza virus by intact IgG and Fab fragments. J Virol :
Strauch, Eva-Maria; Bernard, Steffen M; La, David et al. (2017) Computational design of trimeric influenza-neutralizing proteins targeting the hemagglutinin receptor binding site. Nat Biotechnol 35:667-671
Liang, Yu; Guttman, Miklos; Davenport, Thaddeus M et al. (2016) Probing the Impact of Local Structural Dynamics of Conformational Epitopes on Antibody Recognition. Biochemistry 55:2197-213
Gui, Long; Ebner, Jamie L; Mileant, Alexander et al. (2016) Visualization and Sequencing of Membrane Remodeling Leading to Influenza Virus Fusion. J Virol 90:6948-6962
Lee, Kelly K; Gui, Long (2016) Dissecting Virus Infectious Cycles by Cryo-Electron Microscopy. PLoS Pathog 12:e1005625
Guttman, Miklos; Lee, Kelly K (2016) Isotope Labeling of Biomolecules: Structural Analysis of Viruses by HDX-MS. Methods Enzymol 566:405-26
Guttman, Miklos; Lee, Kelly K (2016) Site-Specific Mapping of Sialic Acid Linkage Isomers by Ion Mobility Spectrometry. Anal Chem 88:5212-7

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