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Hong, Christopher Yee

University of British Columbia

Since the onset of the pandemic, the proteolytic activation of SARS-CoV-2 spike glycoprotein has been a central topic of discussion. The evolution of the S1/S2 furin cleavage site reinvigorated the investigation of coronavirus spike cleavage at both S1/S2 and S2’ cleavage sites. In this study, we aimed to characterize the S2’ cleavage for both SARS-CoV-2 and hCoV-229E by developing a mass-spectrometry (MS) based quantification assay. To this end, we setup a series of biochemical assays using recombinant spike glycoproteins and various type II transmembrane proteases. These systems are then utilized to evaluate the currently available therapeutic candidates targeting the S2’ cleavage event. Remarkably, we observed the poor inhibition of S2’ activation by the widely used camostat and nafamostat in contrast to N-0385. Furthermore, we hypothesized that the evolution of the S1/S2 furin cleavage site enhances the activation of coronavirus at the S2’ site. To test this hypothesis, we established a set of transfection models to evaluate the activation state of the coronavirus spike. Both recombinant proteins and transfection systems are then utilized for the development of a MS-based assay. We explored techniques including N-terminal acetylation and various alternative proteases. In the end, LysC based methodology allowed for the distinguishing between cleaved and uncleaved S2’, revealing the role of TMPRSS2 in the activation of delta and BA.5 spike. Additionally, with the observation of neurological implications of SARS-CoV-2 infection, we explored the proteome of human astrocytes infected with hCoV-229E. The comparative proteomics analysis identified various dysregulated proteins and pathways providing the basis for future investigation in these potential pathogenic mechanisms.

Text

2024-01-12

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/87224

Microbiology and Immunology

University of British Columbia

UBCV

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