Compelling work in the last decade has shown that transmembrane domains (TMDs), once thought to function solely as hydrophobic anchors, are central players driving cellular protein oligomerization, cell signaling, and regulation of channel function. However, the role of TMDs in viral glycoprotein stability and function remains poorly understood. During the previous funding period, our studies of the Hendra virus (HeV) fusion (F) protein identified key roles for TMDs in regulation of F protein stability, cellular trafficking, ad fusion function. Our novel studies utilizing sedimentation equilibrium (SE) analysis established that F protein TMDs efficiently trimerize separate from the rest of the protein. Altered TM-TM association was found for multiple functionally important mutations, implicating TM-TM interactions in regulation of F protein folding and function. The long-term goal of our research is to understand the molecular details and regulation of membrane fusion promoted by the paramyxovirus F proteins. The overall hypothesis of this proposal is that interactions of the TMD with itself and the lipid environment are critical for fusion protein stability and function, makin these interactions potential targets for antiviral therapeutics. To test this important hypothesis, we will pursue three specific aims. First, we will define key elements driving F protein TM-TM interactions including defining the role of a L/I heptad in TM-TM association and Hendra F protein stability. Second, we will determine how the TMD affects fusion triggering and promotion of membrane fusion, with a focus on the role of C-terminal -branched residues and a determination of the conformational changes that can occur when TM-TM interactions cannot be released. Third, we will dissect the role of the lipid environment in prefusion F stability in the context of cellular and viral membranes. These important experiments will provide critical new insight into the role of TMDs in regulation of F protein stability and fusion promotion.

Public Health Relevance

Viral fusion proteins promote fusion between the viral membrane and a membrane of the target cell, a critical first step in the infection of enveloped viruses. Studies to determine factors regulating the stability and function of these proteins are important to the development of novel antiviral approaches targeting these viral proteins. This proposal addresses the role of the paramyxovirus fusion protein transmembrane domain is control of fusion protein stability and membrane fusion activity, potentially identifying a novel target for antivirals.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI051517-15
Application #
9606425
Study Section
Virology - B Study Section (VIRB)
Program Officer
Park, Eun-Chung
Project Start
2002-05-15
Project End
2019-12-31
Budget Start
2019-01-01
Budget End
2019-12-31
Support Year
15
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Kentucky
Department
Biochemistry
Type
Schools of Medicine
DUNS #
939017877
City
Lexington
State
KY
Country
United States
Zip Code
40526
Webb, Stacy R; Smith, Stacy E; Fried, Michael G et al. (2018) Transmembrane Domains of Highly Pathogenic Viral Fusion Proteins Exhibit Trimeric Association In Vitro. mSphere 3:
Cifuentes-Muñoz, Nicolás; Dutch, Rebecca Ellis; Cattaneo, Roberto (2018) Direct cell-to-cell transmission of respiratory viruses: The fast lanes. PLoS Pathog 14:e1007015
Webb, Stacy; Nagy, Tamas; Moseley, Hunter et al. (2017) Hendra virus fusion protein transmembrane domain contributes to pre-fusion protein stability. J Biol Chem 292:5685-5694
Klimyte, Edita M; Smith, Stacy E; Oreste, Pasqua et al. (2016) Inhibition of Human Metapneumovirus Binding to Heparan Sulfate Blocks Infection in Human Lung Cells and Airway Tissues. J Virol 90:9237-50
Chai, Qian; Wang, Zhaoshuai; Webb, Stacy R et al. (2016) The ssrA-Tag Facilitated Degradation of an Integral Membrane Protein. Biochemistry 55:2301-4
Chai, Qian; Webb, Stacy R; Wang, Zhaoshuai et al. (2016) Study of the degradation of a multidrug transporter using a non-radioactive pulse chase method. Anal Bioanal Chem 408:7745-7751
El Najjar, Farah; Cifuentes-Muñoz, Nicolás; Chen, Jing et al. (2016) Human metapneumovirus Induces Reorganization of the Actin Cytoskeleton for Direct Cell-to-Cell Spread. PLoS Pathog 12:e1005922
El Najjar, Farah; Lampe, Levi; Baker, Michelle L et al. (2015) Analysis of cathepsin and furin proteolytic enzymes involved in viral fusion protein activation in cells of the bat reservoir host. PLoS One 10:e0115736
El Najjar, Farah; Schmitt, Anthony P; Dutch, Rebecca Ellis (2014) Paramyxovirus glycoprotein incorporation, assembly and budding: a three way dance for infectious particle production. Viruses 6:3019-54
Masante, Cyril; El Najjar, Farah; Chang, Andres et al. (2014) The human metapneumovirus small hydrophobic protein has properties consistent with those of a viroporin and can modulate viral fusogenic activity. J Virol 88:6423-33

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