A cell-free platform to measure coronavirus membrane fusion.

Here we present a protocol to measure coronavirus-mediated membrane fusion, an essential event in coronavirus cell entry. The approach uses nanoluciferase (Nluc) "HiBiT"-tagged corona virus-like particles (VLPs) and Nluc "LgBiT"-containing extracellular vesicles (EVs) as proxies for virus and cell, respectively. VLP-EV membrane fusion allows HiBiT and LgBiT to combine into measurable Nluc, which signifies virus fusion with target cell membranes. We highlight assay utility with methods to assess coronavirus-mediated fusion and its inhibition by antibodies and antiviral agents. For complete details on the use and execution of this protocol, please refer to Qing et al. (2021),1 Qing et al. (2022),2 and Marcink et al. (2022).3.