Liberation of SARS-CoV main protease from the viral polyprotein:N-terminal autocleavage does not depend on the mature dimerization mode

The main protease(M^(pro))plays a vital role in proteolytic processing of the polyproteins in the replicative cycle of SARS coronavirus(SARS-CoV).Dimerization of this enzyme has been shown to be indispensable for transcleavage activity.However,the auto-processing mechanism of M^(pro),i.e.its own release from the polyproteins through autocleavage,remains unclear.This study elucidates the relationship between the N-terminal autocleavage activity and the dimerization of“immature”M^(pro).Three residues(Arg4,Glu290,and Arg298),which contribute to the active dimer conformation of mature M^(pro),are selected for mutational analyses.Surprisingly,all three mutants still perform N-terminal autocleavage,while the dimerization of mature protease and transcleavage activity following auto-processing are completely inhibited by the E290R and R298E mutations and partially so by the R4E mutation.Furthermore,the mature E290R mutant can resume N-terminal autocleavage activity when mixed with the“immature”C145A/E290R double mutant whereas its trans-cleavage activity remains absent.Therefore,the N-terminal auto-processing of M^(pro) appears to require only two“immature”monomers approaching one another to form an“intermediate”dimer structure and does not strictly depend on the active dimer conformation existing in mature protease.In conclusion,an auto-release model of M^(pro) from the polyproteins is proposed,which will help understand the auto-processing mechanism and the difference between the autocleavage and trans-cleavage proteolytic activities of SARS-CoV M^(pro).