Alphaviruses and flaviviruses cause severe human and animal illnesses such as encephalitis, polyarthritis, and dengue fever, with millions of cases in humans per year. These viruses include many potential bioterrorist agents that are category A, B, or C priority pathogens, such as the encephalitic alphaviruses and the flaviviruses West Nile, yellow fever, Japanese encephalitis and dengue virus (DV). Alphaviruses and flaviviruses infect cells through a low pH-triggered membrane fusion reaction mediated by their structurally similar fusion proteins. These """"""""class II fusion proteins"""""""" rearrange to a target-membrane inserted homotrimer (HT) to drive the fusion reaction. In collaboration with Dr. Felix Rey, we have recently determined the structure of the HT of the fusion protein E1 from the alphavirus Semliki Forest virus (SFV). This structure reveals that the HT is a trimeric hairpin in which domain III (DIII) and the stem region fold back against the trimer core, positioning the fusion peptide loops and transmembrane (TM) domains at the same side of the molecule. We have developed recombinant DIll proteins that block refolding to the final hairpin and inhibit SFV and DV fusion and infection. Based on this progress, we will now address the molecular mechanism of class II membrane fusion using the highly developed SFV experimental system. We will characterize the critical features of trimerization using expressed E1 domains and structure-based mutagenesis studies. DIll proteins and a wide variety of available virus fusion intermediates will be used to define key conformational changes and correlate them with steps in fusion. All of the post-fusion structures of viral fusion proteins are missing the TM domain, and thus its interaction with the fusion loops is undefined. Through our ongoing collaboration we will determine the structure of the full-length E1 HT. We will use cryo- and negative stain electron microscopy to define the interaction of the fusion loop with the target membrane and the specific contacts between HTs. We will test the role of HT-interactions both at the fusion site and outside the fusion site to address the functions of HT cooperativity and """"""""bystander"""""""" fusion proteins. Understanding the molecular mechanism of class II membrane fusion will provide insights into virus disease mechanisms, enable the design of specific antiviral therapies, and advance our knowledge of viral and cellular membrane fusion reactions.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
3R01AI075647-15S1
Application #
7919163
Study Section
Virology - A Study Section (VIRA)
Program Officer
Repik, Patricia M
Project Start
2009-09-12
Project End
2010-08-31
Budget Start
2009-09-12
Budget End
2010-08-31
Support Year
15
Fiscal Year
2009
Total Cost
$60,584
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
110521739
City
Bronx
State
NY
Country
United States
Zip Code
10461
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Brown, Rebecca S; Wan, Judy J; Kielian, Margaret (2018) The Alphavirus Exit Pathway: What We Know and What We Wish We Knew. Viruses 10:
Frei, Julia C; Wirchnianski, Ariel S; Govero, Jennifer et al. (2018) Engineered Dengue Virus Domain III Proteins Elicit Cross-Neutralizing Antibody Responses in Mice. J Virol 92:
Byrd, Emily A; Kielian, Margaret (2017) An Alphavirus E2 Membrane-Proximal Domain Promotes Envelope Protein Lateral Interactions and Virus Budding. MBio 8:
Bauve, Elisa La; Vernon, Briana C; Ye, Dongmei et al. (2016) Method for measuring the unbinding energy of strongly-bound membrane-associated proteins. Biochim Biophys Acta 1858:2753-2762
Dubé, Mathieu; Etienne, Loïc; Fels, Maximilian et al. (2016) Calcium-Dependent Rubella Virus Fusion Occurs in Early Endosomes. J Virol 90:6303-6313
Kielian, Margaret; Saphire, Erica Ollmann (2015) Potent Antibody Protection against an Emerging Alphavirus Threat. Cell 163:1053-1054
Frei, Julia C; Kielian, Margaret; Lai, Jonathan R (2015) Comprehensive mapping of functional epitopes on dengue virus glycoprotein E DIII for binding to broadly neutralizing antibodies 4E11 and 4E5A by phage display. Virology 485:371-82
Fields, Whitney; Kielian, Margaret (2015) Interactions involved in pH protection of the alphavirus fusion protein. Virology 486:173-9
Dubé, Mathieu; Rey, Felix A; Kielian, Margaret (2014) Rubella virus: first calcium-requiring viral fusion protein. PLoS Pathog 10:e1004530

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