Influenza A virus is a paradigm for understanding viral entry of host cells via receptor-mediated endocytosis. We seek to deepen our understanding of viral membrane fusion in order to identify possible strategies for interfering with the process and arresting the infection cycle. The hemagglutinin (HA) protein mediates fusion of the virus and host ceil membranes. HA shares core elements with other type-1 fusion proteins such as those found in Ebola virus and HIV. While certain facets of HA-mediated fusion have been characterized, significant gaps remain in the characterization of the molecular conformations that actually drive membrane fusion, in our understanding of how HA changes conformation, and in our understanding of the interplay between fusion protein and membrane deformation. We propose to use,solution X-ray scattering with ab initio shape reconstruction to determine the structure and dynamics of the hemagglutinin fusion protein. By docking known high resolution structures of components into the solution scattering-derived shape envelopes, pseudoatomic models will be composed. This type of integrative approach is necessary for characterization of complex macromolecular machines. The experiments will provide unique insight into the conformational trajectory traversed by HA as it converts from metastable to fusion-active form. In addition, cryo-eiectron tomography will be used to elucidate the lipidic and proteinaceous architecture of the HAmediated fusion pore and to determine the higher-order organization of HA arrayed on membranes. Lastly, single-particle fluorescence microscopy will be used to reveal the dynamic fusion-activation of HA when it is arrayed on the viral membrane. This approach will identify whether the hundreds of HA spikes on the virus surface become fusion-active in a cooperative fashion.

Public Health Relevance

Influenza virus is a major human pathogen that in a typical year results in more than 200,000 hospitalizations and approximately 36,000 deaths in the United States. The proposed research seeks to gain a detailed understanding of the function of the hemagglutinin machinery that influenza uses to invade new host cells. Such an understanding is necessary to identify how best to interfere with the cycle of viral infection.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Transition Award (R00)
Project #
5R00GM080352-04
Application #
7798167
Study Section
Special Emphasis Panel (NSS)
Program Officer
Flicker, Paula F
Project Start
2007-04-15
Project End
2012-03-31
Budget Start
2010-04-01
Budget End
2011-03-31
Support Year
4
Fiscal Year
2010
Total Cost
$246,509
Indirect Cost
Name
University of Washington
Department
Type
Schools of Pharmacy
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Gui, Long; Ebner, Jamie L; Mileant, Alexander et al. (2016) Visualization and Sequencing of Membrane Remodeling Leading to Influenza Virus Fusion. J Virol 90:6948-62
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, Miklós; Váradi, Csaba; Lee, Kelly K et al. (2015) Comparative glycoprofiling of HIV gp120 immunogens by capillary electrophoresis and MALDI mass spectrometry. Electrophoresis 36:1305-13
Guttman, Miklos; Garcia, Natalie K; Cupo, Albert et al. (2014) CD4-induced activation in a soluble HIV-1 Env trimer. Structure 22:974-84
Taylor, Andrew F; Amundsen, Susan K; Guttman, Miklos et al. (2014) Control of RecBCD enzyme activity by DNA binding- and Chi hotspot-dependent conformational changes. J Mol Biol 426:3479-99
Guttman, Miklos; Weis, David D; Engen, John R et al. (2013) Analysis of overlapped and noisy hydrogen/deuterium exchange mass spectra. J Am Soc Mass Spectrom 24:1906-12
Guttman, Miklos; Weinkam, Patrick; Sali, Andrej et al. (2013) All-atom ensemble modeling to analyze small-angle x-ray scattering of glycosylated proteins. Structure 21:321-31
Guttman, Miklos; Lee, Kelly K (2013) A functional interaction between gp41 and gp120 is observed for monomeric but not oligomeric, uncleaved HIV-1 Env gp140. J Virol 87:11462-75
Davenport, Thaddeus M; Guttman, Miklos; Guo, Wenjin et al. (2013) Isolate-specific differences in the conformational dynamics and antigenicity of HIV-1 gp120. J Virol 87:10855-73
Guttman, Miklos; Kahn, Maria; Garcia, Natalie K et al. (2012) Solution structure, conformational dynamics, and CD4-induced activation in full-length, glycosylated, monomeric HIV gp120. J Virol 86:8750-64

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