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

The main protease (Mpro) and the papain-like protease (PLpro) 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 Mpro and PLpro cleavage kinetics for their N-terminal autocleavage sites. Importantly, systematic characterization of all Mpro and PLpro cleavage sites individually using the DuProSense platform revealed significant differences in their cleavage rates. Further, in vitro assays using purified Mpro cleavage-site-containing DuProSense biosensors revealed significant differences in their catalytic processing by Mpro. Additionally, Mpro cleavage-site-containing DuProSense biosensors revealed Mpro 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.