Differential kinetics of SARS-CoV-2 proteases revealed by a two-color, BRET-based dual protease biosensor, DuProSense

The main protease (M^pro) and the papain-like protease (PL^pro) are critical for SARS-CoV-2 replication due to their roles in proteolytic processing of viral pp1a and pp1ab polyproteins into functional proteins. While the two proteases and their cleavage sites have been studied extensively, a comprehensive understanding of their cleavage kinetics and inhibitor potency in living cells is lacking in the literature. Here, we report the engineering of a two-color Bioluminescence Resonance Energy Transfer (BRET)-based, dual protease biosensor (DuProSense) platform that revealed differential cleavage kinetics and inhibitor potency of SARS-CoV-2 proteases. Briefly, we incorporated two spectrally distinct fluorescent acceptor proteins, along with SARS-CoV-2 protease cleavage peptides at the N- and C-termini of a BRET donor luciferase protein for simultaneous, highly specific monitoring of two proteolytic cleavage events. Live-cell experiments with DuProSense revealed similar M^pro and PL^pro cleavage kinetics for their N-terminal autocleavage sites. Importantly, systematic characterization of all M^pro and PL^pro cleavage sites individually using the DuProSense platform revealed significant differences in their cleavage rates. Further, in vitro assays using purified M^pro cleavage-site-containing DuProSense biosensors revealed significant differences in their catalytic processing by M^pro. Additionally, M^pro cleavage-site-containing DuProSense biosensors revealed M^pro cleavage-site-specific nirmatrelvir potency, which did not correlate with their cleavage rates in living cells. Overall, the DuProSense platform developed here provided deeper insights into SARS-CoV-2 polyprotein processing and will find wider applications that require simultaneous monitoring of two proteolytic events.