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

Abstract

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 .

Three-dimensional domain swapping as a mechanism to lock the active conformation in a super-active octamer of SARS-CoV main protease

“…Almost all these studies are carried out with mature M pro , and little is clearly known about M pro in the viral polyproteins and its maturation process. Recently, Chen et al reported that two monomeric mutants of M pro can still perform N-terminal autocleavage, while the dimerization of mature protease and trans-cleavage activity following auto-processing are completely lost (Chen et al, 2010). They proposed that the auto-processing of M pro from polyproteins is through an "intermediate dimer" structure which does not strictly depend on the active conformation existing in mature M pro , and the possible "intermediate dimer" may be formed through Cterminal domain dimerization.…”

Activation and maturation of SARS-CoV main protease

“…In another study, Chen et al reported an interesting observation that the expression of GST or Trx tagged M pro mutants E290R or R298E with an N-terminal M pro cleavage site only results in the production of mature proteins as WT-M pro does, even though no trans-cleavage activity could be detected for the two mutants (Chen et al, 2010). In vitro proteolysis assay revealed that M pro mutant E290R still possesses obvious enzymatic activity towards the inactive GST tagged M pro mutant C145A/E290R with an N-terminal M pro cleavage site, but not for the effector domain of influenza A virus non-structural protein 1 with the same N-terminal cleavage site, while WT-M pro can cleave both.…”

“…These are consistent with the previously mentioned observation by Shan et al (Shan et al, 2004), suggesting that the active dimer conformation of M pro is unnecessary for the N-terminal auto-cleavage of M pro since the E290R mutant is unable to form dimer. It was thus proposed that the N-terminal autocleavage might only need two "immature" M pro in monomeric polyproteins to form an "intermediate" dimer that is not related to the active dimer of the mature M pro (Chen et al, 2010).…”

Chimeric Exchange of Coronavirus nsp5 Proteases (3CLpro) Identifies Common and Divergent Regulatory Determinants of Protease Activity

“…However, the role of nsp5 in regulating replicase polyprotein processing during virus infection remains much less well understood. Specifically, following translation of ORF1a/b, nsp5 must properly fold within the context of the replicase polyprotein and an nsp4-10 intermediate, orchestrate its autoproteolytic processing, dimerize, and recognize and process up to 9 additional cleavage sites (5,9,14,34,39,40). Thus, while the general specificity of nsp5 is known for specific cleavage site peptides in vitro, the accessibility, activity, and hierarchy of events during infection are not known.…”