Kinetic comparison of all eleven viral polyprotein cleavage site processing events by SARS-CoV-2 main protease using...

The main protease (M^pro) 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 severe acute respiratory syndrome related coronavirus 2 (SARS-CoV-2). Upon viral entry, the +ssRNA genome is translated into two long polyproteins (pp1a or the frameshift-dependent pp1ab) containing all the nonstructural proteins (nsps) required by the virus for immune modulation, replication, and ultimately, virion assembly. Included among these nsps is the cysteine protease M^pro (nsp5) which self-excises from the polyprotein, dimerizes, then sequentially cleaves 11 of the 15 cut-site junctions found between each nsp within the polyprotein. Many structures of M^pro (often bound to various small molecule inhibitors or peptides) have been detailed recently, including structures of M^pro bound to each of the polyprotein cleavage sequences, showing that M^pro can accommodate a wide range of targets within its active site. However, to date, kinetic characterization of the interaction of M^pro with each of its native cleavage 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 use chemically modified peptides. Using this system, we were able to show that while each site maintains a similar Michaelis constant, the catalytic efficiency of M^pro varies greatly between cut-site sequences, suggesting a clear preference for the order of nsp processing.