Year of Publication

2021

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department/School/Program

Molecular and Cellular Biochemistry

First Advisor

Dr. Rebecca Dutch

Abstract

Enveloped viruses must bind target cells and then fuse the viral membrane with a cell membrane to enter a host cell. These viruses use one or more surface glycoproteins to carry out these critical functions. The surface glycoprotein that carries out the fusion function, termed a fusion protein, is divided into three classes based on structural similarities. Some of the most studied human viral pathogens, such as human immunodeficiency virus (HIV), Ebola virus, influenza, measles, and the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), possess class I fusion proteins. Following synthesis, class I fusion proteins associate as non-covalently linked homo-trimers, and remain as trimers throughout the fusion process. Key proteolytic processing events and subsequent receipt of a triggering signal, drive the fusion protein to undergo large, irreversible conformational changes to facilitate the merging of the viral and host cell membranes. Using this same fusion process, several class I fusion proteins can also promote cell-cell fusion.

Previous work has demonstrated that protein-protein interactions within the transmembrane region of some fusion proteins may play a role in the overall trimeric association. Utilizing fusion protein from viruses in the family Paramyxoviridae and Pneumoviridae, we targeted interactions in the transmembrane (TM) regions, disrupting overall protein stability and fusion function, thus demonstrating a novel target for antiviral therapeutic development. To further delineate the role of residues within the Hendra virus fusion protein TM domain, we performed alanine scanning mutagenesis of the N-terminal end of that region, demonstrating that residues M491/L492 appear to play a role in the protein fusion process. In early 2020, we shifted to investigating the fusion protein of SARS-CoV-2 (spike) and examined spike protein stability, proteolytic processing, and factors involved in cell-cell fusion. Through this work we assessed cleavage patterns, identified residues that modulate the fusion process, and showed that protein processing impacts the trimer stability. Finally, we examined the fusion protein of respiratory syncytial virus (RSV). In this study we characterized protein trafficking, cleavage, and post translational modification differences between the fusion proteins from the two RSV subtypes, A and B. Using mutagenesis, we investigated the role of the two cleavage sites that are present and conserved between these subtypes to better understand the cleavage and subsequent fusion processes of the RSV fusion protein. Since these fusion proteins are located on the viral surface and are crucial for viral entry, they are key therapeutic targets. Therefore, understanding the mechanisms and interactions that drive protein function and novel ways to target this protein are critical.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2021.074

Funding Information

National Institutes of Health, National Institute of Allergy and Infectious Diseases, Grant R01AI051517 to Rebecca Dutch 2016-2020.

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