Insights into the mechanism of SARS-CoV-2 main protease autocatalytic maturation from model precursors

A critical step for SARS-CoV-2 assembly and maturation involves the autoactivation of the main protease (MPro(WT)) from precursor polyproteins. Upon expression, a model precursor of MPro(WT) mediates its own release at its termini rapidly to yield a mature dimer. A construct with an E290A mutation within MPro exhibits time dependent autoprocessing of the accumulated precursor at the N-terminal nsp4/nsp5 site followed by the C-terminal nsp5/nsp6 cleavage. In contrast, a precursor containing E290A and R298A mutations (MPro(M)) displays cleavage only at the nsp4/nsp5 site to yield an intermediate monomeric product, which is cleaved at the nsp5/nsp6 site only by MPro(WT). MPro(M) and the catalytic domain (MPro(1-199)) fused to the truncated nsp4 region also show time-dependent conversion in vitro to produce MPro(M) and MPro(1-199), respectively. The reactions follow first-order kinetics indicating that the nsp4/nsp5 cleavage occurs via an intramolecular mechanism. These results support a mechanism involving an N-terminal intramolecular cleavage leading to an increase in the dimer population and followed by an intermolecular cleavage at the C-terminus. Thus, targeting the predominantly monomeric MPro precursor for inhibition may lead to the identification of potent drugs for treatment.