The main protease (Mpro) remains an essential therapeutic target for COVID-19 post infection intervention given its critical role in processing the majority of viral proteins encoded by the genome of SARS-CoV-2. Upon viral entry, the +ssRNA genome is translated into two long polyproteins (pp1a or the frameshift-dependent pp1ab) containing all the non-structural proteins (nsps) required by the virus for immune modulation, replication, and ultimately, virion assembly. Included among these nsps is the cysteine protease Mpro (nsp5) which self-excises from the polyprotein, dimerizes, then consecutively cleaves 11 of the 15 cut site junctions found between each nsp within the polyprotein. Many structures of Mpro (often bound to various inhibitor or peptide inhibitors) have been detailed recently, including structures of Mpro bound to each of the polyprotein cleavage sequences, showing that Mpro can accommodate a wide range of targets within its active site. However, to date, kinetic characterization of the interaction of Mpro with each of its native cut site sequences remains incomplete. Here we present a robust and cost-effective FRET based system that benefits from a more consistent presentation of the substrate that is also closer in organization to the native polyprotein environment compared to previously reported FRET systems that employ chemically modified peptides. Using this system, we were able to show that while each site maintains a similar Michaelis constant, the catalytic efficiency of Mpro varies greatly between cut site sequences, suggesting a clear preference for the order of nsp processing.

© 2024 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology.