Asma Fatima, Anupriya M. Geethakumari, Shahid Uddin, Angelin M. Philip, Asfia Sultana, Zeyaul Islam, Mustapha Aouida, Prasanna R. Kolatkar, Kabir H. Biswas
Summary While SARS-CoV-2 M pro and PL pro 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 M pro and PL pro cleavage kinetics for their N-terminal autocleavage sites. Importantly, systematic characterization of various M pro and PL pro cleavage sites using DuProSense revealed significant differences in cleavage rates and nirmatrelvir potency of M pro cleavage sites but no correlation between the cleavage rates and nirmatrelvir IC 50 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. Highlights Engineered a two-color BRET-based, dual protease biosensor (DuProSense) DuProSense biosensor enabled simultaneous and specific monitoring of M pro and PL pro activities DuProSense platform revealed differential cleavage kinetics of M pro cleavage sites in live cells DuProSense platform revealed M pro cleavage site-dependent nirmatrelvir potency in live cells