A split luciferase system for studying coronavirus Mpro dimerization in vitro and in living cells

The main protease enzyme (M^pro) of coronaviruses cleaves the viral polyprotein into functional units essential for virus replication. Prior work has demonstrated that M^pro functions as a homodimer. However, studies on the mechanism of dimerization have been challenging because the purified protease is mostly dimeric, dimerization-defective mutants lack proteolytic activity, and robust cell-based assays have yet to be reported. To enable work on M^pro dimerization, we have developed a quantitative luciferase-based SARS-CoV-2 (SARS2) M^pro biosensor that accurately reports protein dimerization in living cells and, upon purification, also in vitro. Co-transfection of cells with a construct expressing M^pro fused to the 18 kDa LargeBiT of luciferase (LgBiT) and a second construct with M^pro fused to the 1 kDa SmallBiT of luciferase (SmBiT) results in a reconstitution of luciferase activity in a dose-dependent manner that requires conserved residues within the dimerization interface. Proteolytic activity is dispensable for dimerization and, uniquely, a C145A catalytically inactive mutant exhibits enhanced dimerization signal likely due to lower cytotoxicity. M^pro enzymes from multiple different coronaviruses also dimerize in this system, indicating mechanistic conservation. Interestingly, this dimerization biosensor also provides a quantitative read-out of inhibitor-facilitated dimerization. Covalent SARS2 M^pro inhibitors such as nirmatrelvir cause a 3- to 5-fold increase in luciferase activity. Together with corroborating structural, biophysical, and molecular dynamics experiments, our studies support a model in which covalent M^pro inhibitors such as nirmatrelvir simultaneously block catalytic activity and induce allosteric stabilization of the dimeric complex.