Objective: To study whether 2 hepatitis delta virus genomic ribozymes(g. Rz) have trans-cleaving activity. Methods: g. Rz74 and g. Rz55 were transcribed from their templates which were synthesized and cloned into plas...Objective: To study whether 2 hepatitis delta virus genomic ribozymes(g. Rz) have trans-cleaving activity. Methods: g. Rz74 and g. Rz55 were transcribed from their templates which were synthesized and cloned into plasmid pGEM-4Z; Homologous substrate RNA was transcribed for pRz277B and labelled withα-32 P-UTP. The trans-cleaving activity was studied by mixing the ribozymes and substrate in appropriate who under certain conditions. Results: The trans-cleaved products were generated after mixing the ribozyme with the substrate for 30 min, and accumulated more for 90 min. Conclusion: Both g. Rz74 and g. Rz55 possess trans-cleaving activity.展开更多
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 tran...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).展开更多
基金Supported by National Natural Science Foundation of China, NO.3960003l
文摘Objective: To study whether 2 hepatitis delta virus genomic ribozymes(g. Rz) have trans-cleaving activity. Methods: g. Rz74 and g. Rz55 were transcribed from their templates which were synthesized and cloned into plasmid pGEM-4Z; Homologous substrate RNA was transcribed for pRz277B and labelled withα-32 P-UTP. The trans-cleaving activity was studied by mixing the ribozymes and substrate in appropriate who under certain conditions. Results: The trans-cleaved products were generated after mixing the ribozyme with the substrate for 30 min, and accumulated more for 90 min. Conclusion: Both g. Rz74 and g. Rz55 possess trans-cleaving activity.
基金This work was supported,in part,by the Sino-European Project on SARS Diagnostics and Antivirals(SEPSDA,contract NO.SP22-CT-2004-003831)by VIZIER(contract no.LSHG-CT-2004-511960)+1 种基金both funded by the European Commission.We acknowledge support from the Sino-German Center for the Promotion of Research,Beijing(grant no.233(202/6))the Schleswig-Holstein Innovation Fund,and the DFG(Hi 611/4 and Cluster of Excellence“Inflammation at Interfaces”).
文摘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).
