A CHARACTERIZATION OF KEY RESIDUES IN CLASS I VIRAL FUSION PROTEINS IMPORTANT FOR FUSOGENIC ACTIVITY
Author ORCID Identifier
Date Available
6-5-2023
Year of Publication
2023
Document Type
Doctoral Dissertation
Degree Name
Doctor of Philosophy (PhD)
College
Medicine
Department/School/Program
Molecular and Cellular Biochemistry
Advisor
Dr. Rebecca Dutch
Abstract
Viral fusion proteins are critical for viral entry and subsequent infection. Class I fusion proteins are characterized by synthesis as an inactive precursor requiring cleavage by a host cell protease to become fusion competent. Though vaccine and antiviral therapeutic developments often target the fusion protein, questions surrounding cleavage dynamics and protein stability remain. The work presented in this dissertation investigates specific regions of three class I viral fusion proteins in an effort to identify key residues involved in proteolytic processing and membrane fusion.
The trimeric severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S) mediates receptor binding, facilitates fusion needed for viral entry, and drives cell–cell fusion. We demonstrate that S must be proteolytically processed at the S1/S2 border and within the S2 subunit in order to become fusion competent. We also identify residues within the internal fusion peptide and the cytoplasmic tail that modulate S-mediated cell–cell fusion.
The transmembrane (TM) region of the HeV hendra virus fusion protein (F) has been shown to play a role in F protein stability and the overall trimeric association of F. Previous work classified β-branched residues within the C-terminal TM domain as important for F protein endocytosis, proteolytic processing, and protein stability. The work presented here completes the analysis of the HeV F TM and identifies specific residues that alter F protein function, suggesting a role for these residues in the fusion process.
The respiratory syncytial virus (RSV) fusion protein (F) requires cleavage at two sites, separated by 27 amino acids. Cleavage at both sites results in a 27 amino acid fragment, termed Pep27. Previous work has provided conflicting results on the relative timing of when the two cleavage events occur. In addition, the fate of Pep27 is unclear. Examination of F cleavage kinetics in both infected and transfected systems over time determined that cleavage of both sites occurs within the secretory pathway as F is transported to the cell surface. We found that the deletion of Pep27 does not alter F function, but the mutation of N-linked glycosylation sites within Pep27 reduces both F surface expression and cell-cell fusion activity. This work clarifies the timing of RSV F proteolytic cleavage and offers insight into the crucial role the N-linked glycosylation sites within the Pep27 play in the biological function of F. The work presented in this dissertation identifies residues within distinct regions of class I viral fusion proteins critical for fusion protein cleavage and stability, therefore impacting infection.
Digital Object Identifier (DOI)
https://doi.org/10.13023/etd.2023.278
Recommended Citation
Neal, Hadley E., "A CHARACTERIZATION OF KEY RESIDUES IN CLASS I VIRAL FUSION PROTEINS IMPORTANT FOR FUSOGENIC ACTIVITY" (2023). Theses and Dissertations--Molecular and Cellular Biochemistry. 67.
https://uknowledge.uky.edu/biochem_etds/67
