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Kenward, Calem Austin

University of British Columbia

The emergence and global dissemination of SARS-CoV-2, the virus responsible for COVID-19, stands as the most significant public health crisis in recent memory. Despite significant advances in therapeutics and vaccines, ongoing emergence of variants with enhanced infectivity and immune evasion continues to burden healthcare systems worldwide. Much of the foundational work for current treatments and vaccines stems from studying previous viral outbreaks, emphasizing the importance of understanding the molecular mechanisms of SARS-CoV-2 in addressing both current and future viral threats. This thesis aimed to characterize essential viral proteins and mechanisms crucial to the lifecycle of SARS-CoV-2 using various structural biology and biochemical assay methods. First, the structural interactions of Mᴾʳº with its native polyprotein cut-site sequences were investigated. Through x-ray crystallography of C-terminal substituted Mᴾʳº chimeras, 10 of the 11 specificity sequences were captured in enzyme- product and acyl-intermediate like complexes with catalytically inactive Mᴾʳº, providing insights into the interactions between the viral cysteine protease and its native targets. These structural analyses revealed core conserved features within the Mpro active site and highlighted the plasticity of Mᴾʳº through variations in subsite conformations and cleavage sequence approaches. In collaboration with a multidisciplinary research team, this approach helped inform the design and structural characterization of potent direct-acting antivirals resistant to proposed mutational hotspots in Mᴾʳº. Next, the kinetic interaction of Mᴾʳº with each of its cut-site sequences was evaluated using a linked-peptide FRET system. Fluorescent assays demonstrated similar affinity but significantly different catalytic efficiencies between Mᴾʳº and each cut-site sequence, likely influencing polyprotein processing order and viral protein complex lifetime within infected cells. Lastly, routine cryo-EM characterization of the SARS-CoV-2 spike glycoprotein revealed the presence of linoleic acid bound within a cryptic binding site. A series of compounds designed to target this fatty acid binding site and induce the compact inactive form of spike observed when linoleic acid was bound. Screening using surface plasmon resonance showed that only analogues closely resembling linoleic acid adequately interact with this site and inhibit the spike-ACE2 interaction. This work furthers our understanding of the dynamic proteins involved in SARS-CoV-2 lifecycle and pathogenicity.

Text

2024-08-15

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/88954

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/