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 #
5R01GM099989-02
Application #
8449270
Study Section
Virology - A Study Section (VIRA)
Program Officer
Sakalian, Michael
Project Start
2012-04-01
Project End
2017-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
2
Fiscal Year
2013
Total Cost
$280,626
Indirect Cost
$97,276
Name
University of Washington
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Lee, Kelly K; Gui, Long (2016) Dissecting Virus Infectious Cycles by Cryo-Electron Microscopy. PLoS Pathog 12:e1005625
Davenport, Thaddeus M; Gorman, Jason; Joyce, M Gordon et al. (2016) Somatic Hypermutation-Induced Changes in the Structure and Dynamics of HIV-1 Broadly Neutralizing Antibodies. Structure :
Guttman, Miklos; Lee, Kelly K (2016) Isotope Labeling of Biomolecules: Structural Analysis of Viruses by HDX-MS. Methods Enzymol 566:405-26
Garcia, Natalie K; Lee, Kelly K (2016) Dynamic Viral Glycoprotein Machines: Approaches for Probing Transient States That Drive Membrane Fusion. Viruses 8:
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
Guttman, Miklos; Wales, Thomas E; Whittington, Dale et al. (2016) Tuning a High Transmission Ion Guide to Prevent Gas-Phase Proton Exchange During H/D Exchange MS Analysis. J Am Soc Mass Spectrom 27:662-8
Guttman, Miklos; Lee, Kelly K (2016) Site-Specific Mapping of Sialic Acid Linkage Isomers by Ion Mobility Spectrometry. Anal Chem 88:5212-7
Garcia, Natalie K; Guttman, Miklos; Ebner, Jamie L et al. (2015) Dynamic changes during acid-induced activation of influenza hemagglutinin. Structure 23:665-76
Guttman, Miklos; Cupo, Albert; Julien, Jean-Philippe et al. (2015) Antibody potency relates to the ability to recognize the closed, pre-fusion form of HIV Env. Nat Commun 6:6144
Gupta, Sayan; Guttman, Miklos; Leverenz, Ryan L et al. (2015) Local and global structural drivers for the photoactivation of the orange carotenoid protein. Proc Natl Acad Sci U S A 112:E5567-74

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