The gene encoding nonstructural protein Npro of BVDV was amplified by PCR,then was inserted into pEGFP-C1 vector.The 293 cells were transfected in vitro with recombinant plasmid by liposome.Npro-EGFP fusion protein wa...The gene encoding nonstructural protein Npro of BVDV was amplified by PCR,then was inserted into pEGFP-C1 vector.The 293 cells were transfected in vitro with recombinant plasmid by liposome.Npro-EGFP fusion protein was viewed directly with fluorensce microscope and mRNA expression of Npro was detected by reverse transcription-polymerase chain reaction（RT-PCR）.Results showed that recombinant plasmid was confirmed to be constructed correctly by PCR,restriction enzyme digestion and DNA sequencing;meanwhile,the gene carried was expressed in 293 cells.The fusion protein had properties of both the two Npro and enhanced green fluorescent proteins（EGFP） and distributed in the cytoplasm.This research lays a foundation for further researches which explored Npro gene function in the process of viral replication and synthesis.展开更多
The first part of this report describes the data reduction of non-merohedrally twinned crystals measured on Bruker and Agilent area-detector diffractometers. The image frames of methyl-2-aminopyrazine-3-carboxylate we...The first part of this report describes the data reduction of non-merohedrally twinned crystals measured on Bruker and Agilent area-detector diffractometers. The image frames of methyl-2-aminopyrazine-3-carboxylate were processed with APEX2 to furnish a set of overlapping diffraction indices that were used for solution and refinement. CrysAlisPRO was used for processing the frames of bis（diethyldicarbamato）nickel, which exists in monoclinic and tetragonal polymorphs, and in untwinned and twinned forms. In the second part, the crystal structure of [（3-formyl-4- hydroxyphenyl）methyl]triphenylphosphanium chloride was refined through the ‘HKLF 5＇（based on a combined set of diffraction indices） and PLATON（based on one set of diffraction indices） routes to give identical outcomes because the amount of overlap of the twin domains is small. For the third part, in a proof-of-concept investigation, the diffraction pattern of untwinned and twinned 4-{（E）-（4-aminophenyl）diazenyl]phenylamine was recorded simultaneously in one run; the three domains could be indexed and the crystal structure satisfactorily refined. The refinement was identical to those derived from independent measurements; the crystal structure features two independent centrosymmetric molecules, one of which is ordered and the other whole-molecule-disordered. This two-in-one run opens up the possibility that two or more crystals having different atomic compositions can be measured simultaneously if their reciprocal lattices do not overlap significantly.展开更多
SARS CoV 3CL^pro is known to be a promising target for development of therapeutic agents against the severe acute respiratory syndrome （SARS）. A quinolinone compound 1 was selected via virtual screening, and it was ...SARS CoV 3CL^pro is known to be a promising target for development of therapeutic agents against the severe acute respiratory syndrome （SARS）. A quinolinone compound 1 was selected via virtual screening, and it was syn- thetized and tested for enzymatic inhibition in vitro. Compound 1 showed potent inhibitory activity （ICs0= 0.44 μmol/L） toward SARS CoV 3CL^pro. Further work on a series of quinolinone derivatives resulted in the discovery of the most potent compound 23, inhibiting SARS CoV 3CL^pro with an IC50 of 36.86 μmol/L. The structure-activity relationships were also discussed.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
文摘The gene encoding nonstructural protein Npro of BVDV was amplified by PCR,then was inserted into pEGFP-C1 vector.The 293 cells were transfected in vitro with recombinant plasmid by liposome.Npro-EGFP fusion protein was viewed directly with fluorensce microscope and mRNA expression of Npro was detected by reverse transcription-polymerase chain reaction（RT-PCR）.Results showed that recombinant plasmid was confirmed to be constructed correctly by PCR,restriction enzyme digestion and DNA sequencing;meanwhile,the gene carried was expressed in 293 cells.The fusion protein had properties of both the two Npro and enhanced green fluorescent proteins（EGFP） and distributed in the cytoplasm.This research lays a foundation for further researches which explored Npro gene function in the process of viral replication and synthesis.
文摘The first part of this report describes the data reduction of non-merohedrally twinned crystals measured on Bruker and Agilent area-detector diffractometers. The image frames of methyl-2-aminopyrazine-3-carboxylate were processed with APEX2 to furnish a set of overlapping diffraction indices that were used for solution and refinement. CrysAlisPRO was used for processing the frames of bis（diethyldicarbamato）nickel, which exists in monoclinic and tetragonal polymorphs, and in untwinned and twinned forms. In the second part, the crystal structure of [（3-formyl-4- hydroxyphenyl）methyl]triphenylphosphanium chloride was refined through the ‘HKLF 5＇（based on a combined set of diffraction indices） and PLATON（based on one set of diffraction indices） routes to give identical outcomes because the amount of overlap of the twin domains is small. For the third part, in a proof-of-concept investigation, the diffraction pattern of untwinned and twinned 4-{（E）-（4-aminophenyl）diazenyl]phenylamine was recorded simultaneously in one run; the three domains could be indexed and the crystal structure satisfactorily refined. The refinement was identical to those derived from independent measurements; the crystal structure features two independent centrosymmetric molecules, one of which is ordered and the other whole-molecule-disordered. This two-in-one run opens up the possibility that two or more crystals having different atomic compositions can be measured simultaneously if their reciprocal lattices do not overlap significantly.
文摘SARS CoV 3CL^pro is known to be a promising target for development of therapeutic agents against the severe acute respiratory syndrome （SARS）. A quinolinone compound 1 was selected via virtual screening, and it was syn- thetized and tested for enzymatic inhibition in vitro. Compound 1 showed potent inhibitory activity （ICs0= 0.44 μmol/L） toward SARS CoV 3CL^pro. Further work on a series of quinolinone derivatives resulted in the discovery of the most potent compound 23, inhibiting SARS CoV 3CL^pro with an IC50 of 36.86 μmol/L. The structure-activity relationships were also discussed.
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).
