Viral envelope fusion with the host plasma membrane (PM) for genome release is a hallmark step in the life cycle of many enveloped viruses. This process is regulated by a complex network of biomolecules on the PM, but robust tools to precisely elucidate the dynamic mechanisms of virus-PM fusion events are still lacking. Here, we developed a quantitative single-virus tracking approach based on highly efficient dual-color labelling of viruses and batch trajectory analysis to achieve the spatiotemporal quantification of fusion events. This approach allows us to comprehensively analyze the membrane fusion mechanism utilized by pseudotyped severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the single-virus level and precisely elucidate how the relevant biomolecules synergistically regulate the fusion process. Our results revealed that SARS-CoV-2 may promote the formation of supersaturated clusters of cholesterol to facilitate the initiation of the membrane fusion process and accelerate the viral genome release.
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