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 : 000A68 ( Ncbi/Curation ); précédent : 000A67; suivant : 000A69Characterization 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 [République populaire de Chine] ; 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.
Descripteurs français
- KwdFr :
- MESH :
- enzymologie : Virus du SRAS.
- métabolisme : Endopeptidases, Protéines virales.
- pharmacologie : Antiviraux.
- Amorces ADN, Animaux, Cellules Vero, Cysteine endopeptidases, Hydrolyse, Séquence nucléotidique, Virus du SRAS.
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
Links toward previous steps (curation, corpus...)
- to stream PubMed, to step Corpus: Pour aller vers cette notice dans l'étape Curation :002B59
- to stream PubMed, to step Curation: Pour aller vers cette notice dans l'étape Curation :002B59
- to stream PubMed, to step Checkpoint: Pour aller vers cette notice dans l'étape Curation :002D90
- to stream Ncbi, to step Merge: Pour aller vers cette notice dans l'étape Curation :000A68
Links to Exploration step
pubmed:15358553Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Characterization of trans- and cis-cleavage activity of the SARS coronavirus 3CLpro protease: basis for the in vitro screening of anti-SARS drugs.</title>
<author><name sortKey="Lin, Cheng Wen" sort="Lin, Cheng Wen" uniqKey="Lin C" first="Cheng-Wen" last="Lin">Cheng-Wen Lin</name>
<affiliation wicri:level="1"><nlm: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</nlm:affiliation>
<country xml:lang="fr" wicri:curation="lc">République populaire de Chine</country>
<wicri:regionArea>Department of Medical Laboratory Science and Biotechnology, China Medical University, No. 91, Hsueh-Shih Road, Taichung 404, Taiwan</wicri:regionArea>
<wicri:noRegion>Taiwan</wicri:noRegion>
</affiliation>
</author>
<author><name sortKey="Tsai, Chang Hai" sort="Tsai, Chang Hai" uniqKey="Tsai C" first="Chang-Hai" last="Tsai">Chang-Hai Tsai</name>
</author>
<author><name sortKey="Tsai, Fuu Jen" sort="Tsai, Fuu Jen" uniqKey="Tsai F" first="Fuu-Jen" last="Tsai">Fuu-Jen Tsai</name>
</author>
<author><name sortKey="Chen, Pei Jer" sort="Chen, Pei Jer" uniqKey="Chen P" first="Pei-Jer" last="Chen">Pei-Jer Chen</name>
</author>
<author><name sortKey="Lai, Chien Chen" sort="Lai, Chien Chen" uniqKey="Lai C" first="Chien-Chen" last="Lai">Chien-Chen Lai</name>
</author>
<author><name sortKey="Wan, Lei" sort="Wan, Lei" uniqKey="Wan L" first="Lei" last="Wan">Lei Wan</name>
</author>
<author><name sortKey="Chiu, Hua Hao" sort="Chiu, Hua Hao" uniqKey="Chiu H" first="Hua-Hao" last="Chiu">Hua-Hao Chiu</name>
</author>
<author><name sortKey="Lin, Kuan Hsun" sort="Lin, Kuan Hsun" uniqKey="Lin K" first="Kuan-Hsun" last="Lin">Kuan-Hsun Lin</name>
</author>
</titleStmt>
<publicationStmt><idno type="wicri:source">PubMed</idno>
<date when="2004">2004</date>
<idno type="RBID">pubmed:15358553</idno>
<idno type="pmid">15358553</idno>
<idno type="doi">10.1016/j.febslet.2004.08.017</idno>
<idno type="wicri:Area/PubMed/Corpus">002B59</idno>
<idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">002B59</idno>
<idno type="wicri:Area/PubMed/Curation">002B59</idno>
<idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Curation">002B59</idno>
<idno type="wicri:Area/PubMed/Checkpoint">002D90</idno>
<idno type="wicri:explorRef" wicri:stream="Checkpoint" wicri:step="PubMed">002D90</idno>
<idno type="wicri:Area/Ncbi/Merge">000A68</idno>
<idno type="wicri:Area/Ncbi/Curation">000A68</idno>
</publicationStmt>
<sourceDesc><biblStruct><analytic><title xml:lang="en">Characterization of trans- and cis-cleavage activity of the SARS coronavirus 3CLpro protease: basis for the in vitro screening of anti-SARS drugs.</title>
<author><name sortKey="Lin, Cheng Wen" sort="Lin, Cheng Wen" uniqKey="Lin C" first="Cheng-Wen" last="Lin">Cheng-Wen Lin</name>
<affiliation wicri:level="1"><nlm: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</nlm:affiliation>
<country xml:lang="fr" wicri:curation="lc">République populaire de Chine</country>
<wicri:regionArea>Department of Medical Laboratory Science and Biotechnology, China Medical University, No. 91, Hsueh-Shih Road, Taichung 404, Taiwan</wicri:regionArea>
<wicri:noRegion>Taiwan</wicri:noRegion>
</affiliation>
</author>
<author><name sortKey="Tsai, Chang Hai" sort="Tsai, Chang Hai" uniqKey="Tsai C" first="Chang-Hai" last="Tsai">Chang-Hai Tsai</name>
</author>
<author><name sortKey="Tsai, Fuu Jen" sort="Tsai, Fuu Jen" uniqKey="Tsai F" first="Fuu-Jen" last="Tsai">Fuu-Jen Tsai</name>
</author>
<author><name sortKey="Chen, Pei Jer" sort="Chen, Pei Jer" uniqKey="Chen P" first="Pei-Jer" last="Chen">Pei-Jer Chen</name>
</author>
<author><name sortKey="Lai, Chien Chen" sort="Lai, Chien Chen" uniqKey="Lai C" first="Chien-Chen" last="Lai">Chien-Chen Lai</name>
</author>
<author><name sortKey="Wan, Lei" sort="Wan, Lei" uniqKey="Wan L" first="Lei" last="Wan">Lei Wan</name>
</author>
<author><name sortKey="Chiu, Hua Hao" sort="Chiu, Hua Hao" uniqKey="Chiu H" first="Hua-Hao" last="Chiu">Hua-Hao Chiu</name>
</author>
<author><name sortKey="Lin, Kuan Hsun" sort="Lin, Kuan Hsun" uniqKey="Lin K" first="Kuan-Hsun" last="Lin">Kuan-Hsun Lin</name>
</author>
</analytic>
<series><title level="j">FEBS letters</title>
<idno type="ISSN">0014-5793</idno>
<imprint><date when="2004" type="published">2004</date>
</imprint>
</series>
</biblStruct>
</sourceDesc>
</fileDesc>
<profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Animals</term>
<term>Antiviral Agents (pharmacology)</term>
<term>Base Sequence</term>
<term>Chlorocebus aethiops</term>
<term>Cysteine Endopeptidases</term>
<term>DNA Primers</term>
<term>Endopeptidases (metabolism)</term>
<term>Hydrolysis</term>
<term>SARS Virus (drug effects)</term>
<term>SARS Virus (enzymology)</term>
<term>Vero Cells</term>
<term>Viral Proteins (metabolism)</term>
</keywords>
<keywords scheme="KwdFr" xml:lang="fr"><term>Amorces ADN</term>
<term>Animaux</term>
<term>Antiviraux (pharmacologie)</term>
<term>Cellules Vero</term>
<term>Cysteine endopeptidases</term>
<term>Endopeptidases (métabolisme)</term>
<term>Hydrolyse</term>
<term>Protéines virales (métabolisme)</term>
<term>Séquence nucléotidique</term>
<term>Virus du SRAS ()</term>
<term>Virus du SRAS (enzymologie)</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Endopeptidases</term>
<term>Viral Proteins</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="pharmacology" xml:lang="en"><term>Antiviral Agents</term>
</keywords>
<keywords scheme="MESH" qualifier="drug effects" xml:lang="en"><term>SARS Virus</term>
</keywords>
<keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Virus du SRAS</term>
</keywords>
<keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>SARS Virus</term>
</keywords>
<keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Endopeptidases</term>
<term>Protéines virales</term>
</keywords>
<keywords scheme="MESH" qualifier="pharmacologie" xml:lang="fr"><term>Antiviraux</term>
</keywords>
<keywords scheme="MESH" xml:lang="en"><term>Animals</term>
<term>Base Sequence</term>
<term>Chlorocebus aethiops</term>
<term>Cysteine Endopeptidases</term>
<term>DNA Primers</term>
<term>Hydrolysis</term>
<term>Vero Cells</term>
</keywords>
<keywords scheme="MESH" xml:lang="fr"><term>Amorces ADN</term>
<term>Animaux</term>
<term>Cellules Vero</term>
<term>Cysteine endopeptidases</term>
<term>Hydrolyse</term>
<term>Séquence nucléotidique</term>
<term>Virus du SRAS</term>
</keywords>
</textClass>
</profileDesc>
</teiHeader>
<front><div type="abstract" xml:lang="en">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.</div>
</front>
</TEI>
</record>
Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Sante/explor/SrasV1/Data/Ncbi/Curation
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 000A68 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Ncbi/Curation/biblio.hfd -nk 000A68 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien |wiki= Sante |area= SrasV1 |flux= Ncbi |étape= Curation |type= RBID |clé= pubmed:15358553 |texte= Characterization of trans- and cis-cleavage activity of the SARS coronavirus 3CLpro protease: basis for the in vitro screening of anti-SARS drugs. }}
Pour générer des pages wiki
HfdIndexSelect -h $EXPLOR_AREA/Data/Ncbi/Curation/RBID.i -Sk "pubmed:15358553" \ | HfdSelect -Kh $EXPLOR_AREA/Data/Ncbi/Curation/biblio.hfd \ | NlmPubMed2Wicri -a SrasV1
![]() | This area was generated with Dilib version V0.6.33. | ![]() |