Spike Processing and Protease Usage Effects on SARS-CoV-2 Pathogenesis

The emergence of SARS-CoV-2 and its subsequent variants have ignited a multitude of studies on the spike protein and its domains. While most studies focus on the impact of receptor binding domain changes, mutations in the C-terminus of S1 (CTS1) have largely been overlooked. The SARS-CoV-2 CTS1 contains various features that are unusual to sarbecoviruses – the furin cleavage site (FCS) and the QTQTN motif. In this dissertation, we demonstrate that the CTS1 is more complex than previously thought. We have established the aspects of cleavage site efficiency, differential protease usage, loop length, and post translational modifications that all contribute to SARS-CoV-2 pathogenesis. Using our reverse genetics system, we generated several infectious clones to examine the various features of the CTS1: ΔQTQTN demonstrated the importance of loop length and glycosylation. PQQA established that the integrity of the FCS is necessary. Omicron CTS1 mutants YKH (H655Y, N679K, and P681H) and N679K demonstrated the interplay between mutations with different contributions and expanded on the impact of post translational modifications. These studies show that alterations to any of these aspects greatly affect the pathogenicity of the virus. Together, we demonstrate that the mutations in the CTS1 are key determinants of coronavirus pathogenicity and should be especially considered in surveillance for the next coronavirus outbreak.