A Comparison of Coronavirus Fusion Mechanisms

Coronavirus spike glycoproteins play a crucial role in attachment to and subsequent fusion with target cells. The single-virus fusion assay was used to explore the effects of factors including concentration of exogenous fusion-triggering protease, spike conformation, and spike sequence on fusion of SARS-CoV-2 and other related coronaviruses. SARS-CoV-2 fusion was studied in-depth. The virus was found to have invariable kinetics across a wide range of concentrations of spike-activating protease and between different proteases, suggesting that subsequent conformational changes in spike protein structure, rather than proteolytic processing of spike, is the rate-determining step of membrane fusion. Additionally, it was found that the simultaneous addition of soluble ACE2 receptor along with protease sped up the viral fusion process, suggesting that binding of spike to ACE2 receptor promotes a conformational state that is more fusion-ready than unbound spike. Fusion kinetics of viruses sampled across the coronavirus family were then compared to SARSCoV-2. Kinetics were indistinguishable between samples except in particles expressing HCoV-NL63 spike, which was found to undergo fusion significantly faster. Examination of spike sequences revealed that a network of cooperating amino acids, rather than a single point mutation, is likely responsible for this increase in kinetics. Gel electrophoresis was used to determine if differences in proteolytic processing are contributing factors to the observed differences in fusion kinetics. It was found that SARS-CoV-2 spike was more susceptible to proteolysis than that of HCoV-NL63 despite undergoing fusion at a slower rate, further suggesting that conformational changes taking place post-proteolysis are the rate-limiting step of fusion.