Enveloped viruses use specialized proteins present at the virus surface to translocate their genetic material across the host cell membrane during infection. For coronaviruses, homotrimers of the spike glycoprotein promote host cell attachment and fusion of the viral and host membranes. Although coronaviruses have a significant pandemic potential, the lack of high-resolution data for any coronavirus spike trimer limits our mechanistic understanding of infection by this family of viruses. The objective of the proposed work is to obtain high-resolution snapshots corresponding to the various stages of the fusion reaction mediated by coronavirus spikes and to study the structural determinants associated with antibody inhibition of viral infection.
In Aim I, we propose to elucidate the architecture of the Mouse Hepatitis Virus (MHV) pre-fusion spike using cryoEM.
Aim II will be dedicated to studying the conformational changes associated with the fusion reaction with an emphasis on the first intermediate (extended intermediate) and the post-fusion spike.
In Aim III, we will characterize the 3D organization of human coronavirus spikes to understand how these viruses overcome the species barrier and to identify structurally conserved regions that could be potential targets for therapeutic initiatives.
The final aim will rely on structure-guided protein design to engineer antibodies targeting human coronavirus spikes with the goal of identifying immunogens for raising broadly-neutralizing antibodies.

Public Health Relevance

The tremendous pandemic potential of coronaviruses has already been demonstrated twice in the last decade as two global outbreaks of deadly pneumonia. Coronavirus spike glycoproteins are responsible for virus entry into host cells, by promoting fusion of the viral and host membranes, and represent important therapeutic targets. We are exploiting MHV as a model system to grasp the structural and thermodynamic parameters of the membrane fusion process. This work is extended to the spikes of human pathogenic coronaviruses to identify structurally conserved regions that could be potential targets for vaccinology initiatives.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM120553-03S1
Application #
9701554
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Flicker, Paula F
Project Start
2016-08-01
Project End
2021-07-31
Budget Start
2018-08-01
Budget End
2019-07-31
Support Year
3
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Washington
Department
Biochemistry
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Borst, Andrew J; Weidle, Connor E; Gray, Matthew D et al. (2018) Germline VRC01 antibody recognition of a modified clade C HIV-1 envelope trimer and a glycosylated HIV-1 gp120 core. Elife 7:
Snijder, Joost; Ortego, Michael S; Weidle, Connor et al. (2018) An Antibody Targeting the Fusion Machinery Neutralizes Dual-Tropic Infection and Defines a Site of Vulnerability on Epstein-Barr Virus. Immunity 48:799-811.e9
Ting, See-Yeun; Bosch, Dustin E; Mangiameli, Sarah M et al. (2018) Bifunctional Immunity Proteins Protect Bacteria against FtsZ-Targeting ADP-Ribosylating Toxins. Cell 175:1380-1392.e14
Smith, F Donelson; Esseltine, Jessica L; Nygren, Patrick J et al. (2017) Local protein kinase A action proceeds through intact holoenzymes. Science 356:1288-1293
Frenz, Brandon; Walls, Alexandra C; Egelman, Edward H et al. (2017) RosettaES: a sampling strategy enabling automated interpretation of difficult cryo-EM maps. Nat Methods 14:797-800
Walls, Alexandra; Tortorici, M Alejandra; Bosch, Berend-Jan et al. (2017) Crucial steps in the structure determination of a coronavirus spike glycoprotein using cryo-electron microscopy. Protein Sci 26:113-121
Walls, Alexandra C; Tortorici, M Alejandra; Snijder, Joost et al. (2017) Tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion. Proc Natl Acad Sci U S A 114:11157-11162
Snijder, Joost; Borst, Andrew J; Dosey, Annie et al. (2017) Vitrification after multiple rounds of sample application and blotting improves particle density on cryo-electron microscopy grids. J Struct Biol 198:38-42
Xiong, Xiaoli; Tortorici, M Alejandra; Snijder, Joost et al. (2017) Glycan shield and fusion activation of a deltacoronavirus spike glycoprotein fine-tuned for enteric infections. J Virol :
Borst, Andrew J; James, Zachary M; Zagotta, William N et al. (2017) The Therapeutic Antibody LM609 Selectively Inhibits Ligand Binding to Human ?V?3 Integrin via Steric Hindrance. Structure 25:1732-1739.e5

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