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Tam, Wen-Xi (Donald)

University of British Columbia

Currently, the deadliest viral outbreak of the 21st century was caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). In total, the coronavirus disease 19 (COVID-19) pandemic has exceeded seven million deaths and led to over 700 hundred million infections worldwide. What has made this virus more infectious than its predecessors, SARS-CoV and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), is the accumulation of sporadic mutations in its viral proteins. These genetic alterations have allowed the virus to infect its target more effectively, efficiently replicate within the host and disrupt the host’s innate immune response. The current literature regarding the function of each viral protein encoded by SARS-CoV-2 is incomplete. The function of some viral proteins, specifically the structural proteins, is well documented. However, the function of some nonstructural and accessory proteins is either vague or missing entirely. Although we can compare the genome of SARS-CoV-2 with other coronaviruses to deduce function, this method is insufficient. The main issue is that we cannot predict how mutational changes to the DNA will affect protein function. Additionally, these mutational differences found in SARS-CoV-2 must be the reason why this novel coronavirus is significantly more pathogenic and deadlier than its predecessors. The goal of this thesis was to identify a novel protein-protein interaction between the nonstructural protein 15 (NSP15) of SARS-CoV-2 and the host. This was accomplished by incubating NSP15 with Vero-E6 cell lysates to facilitate protein binding. The interactions were identified using mass spectrometry and the identity of the unknown protein was found to be keratin. Binding was validated by immunoprecipitation and Western blot. Immunofluorescence microscopy was used to visualize how the profection of NSP15 into Vero-E6 cells affects the keratin cytoskeleton. It was found that NSP15 specifically disrupts the keratin cytoskeleton. A kinetic cytotoxicity assay was then performed to show that the profection of NSP15 also resulted in a statistically significant reduction in cell viability. This suggests that the binding of NSP15 to keratin results in cytoskeletal depolymerization and cell lysis. This would aid in the release of the newly synthesized viral progeny into the surrounding environment to infect new targets.

Text

2024-08-02

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/88867

Experimental Medicine

University of British Columbia

UBCV

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