Characterization of trans- and cis-cleavage activity of the SARS coronavirus 3CLpro protease: basis for the in vitro screening of anti-SARS drugs.
Identifieur interne : 002B59 ( PubMed/Corpus ); précédent : 002B58; suivant : 002B60Characterization of trans- and cis-cleavage activity of the SARS coronavirus 3CLpro protease: basis for the in vitro screening of anti-SARS drugs.
Auteurs : Cheng-Wen Lin ; Chang-Hai Tsai ; Fuu-Jen Tsai ; Pei-Jer Chen ; Chien-Chen Lai ; Lei Wan ; Hua-Hao Chiu ; Kuan-Hsun LinSource :
- FEBS letters [ 0014-5793 ] ; 2004.
English descriptors
- KwdEn :
- MESH :
- chemical , metabolism : Endopeptidases, Viral Proteins.
- chemical , pharmacology : Antiviral Agents.
- drug effects : SARS Virus.
- enzymology : SARS Virus.
- Animals, Base Sequence, Chlorocebus aethiops, Cysteine Endopeptidases, DNA Primers, Hydrolysis, Vero Cells.
Abstract
Severe acute respiratory syndrome (SARS) has been globally reported. A novel coronavirus (CoV), SARS-CoV, was identified as the etiological agent of the disease. SARS-CoV 3C-like protease (3CLpro) mediates the proteolytic processing of replicase polypeptides 1a and 1ab into functional proteins, playing an important role in viral replication. In this study, we demonstrated the expression of the SARS-CoV 3CLpro in Escherichia coli and Vero cells, and then characterized the in vitro trans-cleavage and the cell-based cis-cleavage by the 3CLpro. Mutational analysis of the 3CLpro demonstrated the importance of His41, Cys145, and Glu166 in the substrate-binding subsite S1 for keeping the proteolytic activity. In addition, alanine substitution of the cleavage substrates indicated that Gln-(P1) in the substrates mainly determined the cleavage efficiency. Therefore, this study not only established the quantifiable and reliable assay for the in vitro and cell-based measurement of the 3CLpro activity, but also characterized the molecular interaction of the SARS-CoV 3CLpro with the substrates. The results will be useful for the rational development of the anti-SARS drugs.
DOI: 10.1016/j.febslet.2004.08.017
PubMed: 15358553
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pubmed:15358553Le document en format XML
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A novel coronavirus (CoV), SARS-CoV, was identified as the etiological agent of the disease. SARS-CoV 3C-like protease (3CLpro) mediates the proteolytic processing of replicase polypeptides 1a and 1ab into functional proteins, playing an important role in viral replication. In this study, we demonstrated the expression of the SARS-CoV 3CLpro in Escherichia coli and Vero cells, and then characterized the in vitro trans-cleavage and the cell-based cis-cleavage by the 3CLpro. Mutational analysis of the 3CLpro demonstrated the importance of His41, Cys145, and Glu166 in the substrate-binding subsite S1 for keeping the proteolytic activity. In addition, alanine substitution of the cleavage substrates indicated that Gln-(P1) in the substrates mainly determined the cleavage efficiency. Therefore, this study not only established the quantifiable and reliable assay for the in vitro and cell-based measurement of the 3CLpro activity, but also characterized the molecular interaction of the SARS-CoV 3CLpro with the substrates. The results will be useful for the rational development of the anti-SARS drugs.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">15358553</PMID><DateCompleted><Year>2004</Year><Month>11</Month><Day>02</Day></DateCompleted><DateRevised><Year>2020</Year><Month>04</Month><Day>18</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Print">0014-5793</ISSN><JournalIssue CitedMedium="Print"><Volume>574</Volume><Issue>1-3</Issue><PubDate><Year>2004</Year><Month>Sep</Month><Day>10</Day></PubDate></JournalIssue><Title>FEBS letters</Title><ISOAbbreviation>FEBS Lett.</ISOAbbreviation></Journal><ArticleTitle>Characterization of trans- and cis-cleavage activity of the SARS coronavirus 3CLpro protease: basis for the in vitro screening of anti-SARS drugs.</ArticleTitle><Pagination><MedlinePgn>131-7</MedlinePgn></Pagination><Abstract><AbstractText>Severe acute respiratory syndrome (SARS) has been globally reported. A novel coronavirus (CoV), SARS-CoV, was identified as the etiological agent of the disease. SARS-CoV 3C-like protease (3CLpro) mediates the proteolytic processing of replicase polypeptides 1a and 1ab into functional proteins, playing an important role in viral replication. In this study, we demonstrated the expression of the SARS-CoV 3CLpro in Escherichia coli and Vero cells, and then characterized the in vitro trans-cleavage and the cell-based cis-cleavage by the 3CLpro. Mutational analysis of the 3CLpro demonstrated the importance of His41, Cys145, and Glu166 in the substrate-binding subsite S1 for keeping the proteolytic activity. In addition, alanine substitution of the cleavage substrates indicated that Gln-(P1) in the substrates mainly determined the cleavage efficiency. Therefore, this study not only established the quantifiable and reliable assay for the in vitro and cell-based measurement of the 3CLpro activity, but also characterized the molecular interaction of the SARS-CoV 3CLpro with the substrates. The results will be useful for the rational development of the anti-SARS drugs.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Lin</LastName><ForeName>Cheng-Wen</ForeName><Initials>CW</Initials><AffiliationInfo><Affiliation>Department of Medical Laboratory Science and Biotechnology, China Medical University, No. 91, Hsueh-Shih Road, Taichung 404, Taiwan, ROC. cwlin@mail.cmu.edu.tw</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Tsai</LastName><ForeName>Chang-Hai</ForeName><Initials>CH</Initials></Author><Author ValidYN="Y"><LastName>Tsai</LastName><ForeName>Fuu-Jen</ForeName><Initials>FJ</Initials></Author><Author ValidYN="Y"><LastName>Chen</LastName><ForeName>Pei-Jer</ForeName><Initials>PJ</Initials></Author><Author ValidYN="Y"><LastName>Lai</LastName><ForeName>Chien-Chen</ForeName><Initials>CC</Initials></Author><Author ValidYN="Y"><LastName>Wan</LastName><ForeName>Lei</ForeName><Initials>L</Initials></Author><Author ValidYN="Y"><LastName>Chiu</LastName><ForeName>Hua-Hao</ForeName><Initials>HH</Initials></Author><Author ValidYN="Y"><LastName>Lin</LastName><ForeName>Kuan-Hsun</ForeName><Initials>KH</Initials></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList></Article><MedlineJournalInfo><Country>England</Country><MedlineTA>FEBS Lett</MedlineTA><NlmUniqueID>0155157</NlmUniqueID><ISSNLinking>0014-5793</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D000998">Antiviral Agents</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D017931">DNA Primers</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D014764">Viral Proteins</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 3.4.-</RegistryNumber><NameOfSubstance UI="D010450">Endopeptidases</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 3.4.22.-</RegistryNumber><NameOfSubstance UI="C099456">3C-like proteinase, Coronavirus</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 3.4.22.-</RegistryNumber><NameOfSubstance UI="D003546">Cysteine Endopeptidases</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D000818" MajorTopicYN="N">Animals</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D000998" MajorTopicYN="N">Antiviral Agents</DescriptorName><QualifierName UI="Q000494" MajorTopicYN="Y">pharmacology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D001483" MajorTopicYN="N">Base Sequence</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D002522" MajorTopicYN="N">Chlorocebus aethiops</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D003546" MajorTopicYN="N">Cysteine Endopeptidases</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D017931" MajorTopicYN="N">DNA Primers</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D010450" MajorTopicYN="N">Endopeptidases</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D006868" MajorTopicYN="N">Hydrolysis</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D045473" MajorTopicYN="N">SARS Virus</DescriptorName><QualifierName UI="Q000187" MajorTopicYN="Y">drug effects</QualifierName><QualifierName UI="Q000201" MajorTopicYN="N">enzymology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D014709" MajorTopicYN="N">Vero Cells</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D014764" MajorTopicYN="N">Viral Proteins</DescriptorName><QualifierName UI="Q000378" 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