Differential Kinetics of SARS-CoV-2 Proteases Revealed by a Dual-Color, BRET-based Protease Biosensor, DuProSense

While SARS-CoV-2 Mpro and PLpro proteases are known to cleave polyproteins pp1a and pp1ab at multiple sites, these have not been comprehensively characterized in living cells. Here we engineered a two-color Bioluminescence Resonance Energy Transfer (BRET)-based, dual protease (DuProSense) biosensor platform relying on a proximity-dependent energy transfer from a luciferase donor to two spectrally separated fluorescent protein acceptors enabling simultaneous monitoring of processing of two cleavage sites in a single assay with high specificity. DuProSense revealed a similar Mpro and PLpro cleavage kinetics for their N-terminal autocleavage sites. Importantly, systematic characterization of various Mpro and PLpro cleavage sites using DuProSense revealed significant differences in cleavage rates and nirmatrelvir potency of Mpro cleavage sites but no correlation between the cleavage rates and nirmatrelvir IC50 values. Overall, our results provide deeper insights into the proteolytic processing of SARS-CoV-2 polyproteins and the dual color BRET platform will find wider applications in the future. HighlightsO_LIEngineered a two-color BRET-based, dual protease biosensor (DuProSense) C_LIO_LIDuProSense biosensor enabled simultaneous and specific monitoring of Mpro and PLpro activities C_LIO_LIDuProSense platform revealed differential cleavage kinetics of Mpro cleavage sites in live cells C_LIO_LIDuProSense platform revealed Mpro cleavage site-dependent nirmatrelvir potency in live cells C_LI