Glycogen synthase kinase-3 regulates the phosphorylation of severe acute respiratory syndrome coronavirus nucleocapsid protein and viral replication.
Identifieur interne : 002B10 ( Main/Exploration ); précédent : 002B09; suivant : 002B11Glycogen synthase kinase-3 regulates the phosphorylation of severe acute respiratory syndrome coronavirus nucleocapsid protein and viral replication.
Auteurs : Chia-Hsin Wu [Taïwan] ; Shiou-Hwei Yeh ; Yeou-Guang Tsay ; Ya-Hsiung Shieh ; Chuan-Liang Kao ; Yen-Shun Chen ; Sheng-Han Wang ; Ti-Jung Kuo ; Ding-Shinn Chen ; Pei-Jer ChenSource :
- The Journal of biological chemistry [ 0021-9258 ] ; 2009.
Descripteurs français
- KwdFr :
- Animaux, Cartographie peptidique (), Cellules Vero, Effet cytopathogène viral (), Effet cytopathogène viral (physiologie), Glycogen Synthase Kinases, Humains, Inhibiteurs de protéines kinases (pharmacologie), Motifs d'acides aminés (physiologie), Mutation, Phosphorylation (), Phosphorylation (physiologie), Protéines nucléocapside (génétique), Protéines nucléocapside (métabolisme), Réplication virale (), Réplication virale (physiologie), Souris, Séquence consensus (physiologie), Virus de l'hépatite murine (physiologie), Virus du SRAS (physiologie).
- MESH :
- génétique : Protéines nucléocapside.
- métabolisme : Protéines nucléocapside.
- pharmacologie : Inhibiteurs de protéines kinases.
- physiologie : Effet cytopathogène viral, Motifs d'acides aminés, Phosphorylation, Réplication virale, Séquence consensus, Virus de l'hépatite murine, Virus du SRAS.
- Animaux, Cartographie peptidique, Cellules Vero, Effet cytopathogène viral, Glycogen Synthase Kinases, Humains, Mutation, Phosphorylation, Réplication virale, Souris.
English descriptors
- KwdEn :
- Amino Acid Motifs (physiology), Animals, Chlorocebus aethiops, Consensus Sequence (physiology), Cytopathogenic Effect, Viral (drug effects), Cytopathogenic Effect, Viral (physiology), Glycogen Synthase Kinases, Humans, Mice, Murine hepatitis virus (physiology), Mutation, Nucleocapsid Proteins (genetics), Nucleocapsid Proteins (metabolism), Peptide Mapping (methods), Phosphorylation (drug effects), Phosphorylation (physiology), Protein Kinase Inhibitors (pharmacology), SARS Virus (physiology), Vero Cells, Virus Replication (drug effects), Virus Replication (physiology).
- MESH :
- chemical , genetics : Nucleocapsid Proteins.
- chemical , metabolism : Nucleocapsid Proteins.
- chemical , pharmacology : Protein Kinase Inhibitors.
- chemical : Glycogen Synthase Kinases.
- drug effects : Cytopathogenic Effect, Viral, Phosphorylation, Virus Replication.
- methods : Peptide Mapping.
- physiology : Amino Acid Motifs, Consensus Sequence, Cytopathogenic Effect, Viral, Murine hepatitis virus, Phosphorylation, SARS Virus, Virus Replication.
- Animals, Chlorocebus aethiops, Humans, Mice, Mutation, Vero Cells.
Abstract
Coronavirus (CoV) nucleocapsid (N) protein is a highly phosphorylated protein required for viral replication, but whether its phosphorylation and the related kinases are involved in the viral life cycle is unknown. We found the severe acute respiratory syndrome CoV N protein to be an appropriate system to address this issue. Using high resolution PAGE analysis, this protein could be separated into phosphorylated and unphosphorylated isoforms. Mass spectrometric analysis and deletion mapping showed that the major phosphorylation sites were located at the central serine-arginine (SR)-rich motif that contains several glycogen synthase kinase (GSK)-3 substrate consensus sequences. GSK-3-specific inhibitor treatment dephosphorylated the N protein, and this could be recovered by the constitutively active GSK-3 kinase. Immunoprecipitation brought down both N and GSK-3 proteins in the same complex, and the N protein could be phosphorylated directly at its SR-rich motif by GSK-3 using an in vitro kinase assay. Mutation of the two priming sites critical for GSK-3 phosphorylation in the SR-rich motif abolished N protein phosphorylation. Finally, GSK-3 inhibitor was found to reduce N phosphorylation in the severe acute respiratory syndrome CoV-infected VeroE6 cells and decrease the viral titer and cytopathic effects. The effect of GSK-3 inhibitor was reproduced in another coronavirus, the neurotropic JHM strain of mouse hepatitis virus. Our results indicate that GSK-3 is critical for CoV N protein phosphorylation and suggest that it plays a role in regulating the viral life cycle. This study, thus, provides new avenues to further investigate the specific role of N protein phosphorylation in CoV replication.
DOI: 10.1074/jbc.M805747200
PubMed: 19106108
Affiliations:
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Jer" sort="Chen, Pei Jer" uniqKey="Chen P" first="Pei-Jer" last="Chen">Pei-Jer Chen</name></author></analytic><series><title level="j">The Journal of biological chemistry</title><idno type="ISSN">0021-9258</idno><imprint><date when="2009" type="published">2009</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Amino Acid Motifs (physiology)</term><term>Animals</term><term>Chlorocebus aethiops</term><term>Consensus Sequence (physiology)</term><term>Cytopathogenic Effect, Viral (drug effects)</term><term>Cytopathogenic Effect, Viral (physiology)</term><term>Glycogen Synthase Kinases</term><term>Humans</term><term>Mice</term><term>Murine hepatitis virus (physiology)</term><term>Mutation</term><term>Nucleocapsid Proteins (genetics)</term><term>Nucleocapsid Proteins (metabolism)</term><term>Peptide Mapping (methods)</term><term>Phosphorylation (drug effects)</term><term>Phosphorylation 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peptidique</term><term>Cellules Vero</term><term>Effet cytopathogène viral</term><term>Glycogen Synthase Kinases</term><term>Humains</term><term>Mutation</term><term>Phosphorylation</term><term>Réplication virale</term><term>Souris</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Coronavirus (CoV) nucleocapsid (N) protein is a highly phosphorylated protein required for viral replication, but whether its phosphorylation and the related kinases are involved in the viral life cycle is unknown. We found the severe acute respiratory syndrome CoV N protein to be an appropriate system to address this issue. Using high resolution PAGE analysis, this protein could be separated into phosphorylated and unphosphorylated isoforms. Mass spectrometric analysis and deletion mapping showed that the major phosphorylation sites were located at the central serine-arginine (SR)-rich motif that contains several glycogen synthase kinase (GSK)-3 substrate consensus sequences. GSK-3-specific inhibitor treatment dephosphorylated the N protein, and this could be recovered by the constitutively active GSK-3 kinase. Immunoprecipitation brought down both N and GSK-3 proteins in the same complex, and the N protein could be phosphorylated directly at its SR-rich motif by GSK-3 using an in vitro kinase assay. Mutation of the two priming sites critical for GSK-3 phosphorylation in the SR-rich motif abolished N protein phosphorylation. Finally, GSK-3 inhibitor was found to reduce N phosphorylation in the severe acute respiratory syndrome CoV-infected VeroE6 cells and decrease the viral titer and cytopathic effects. The effect of GSK-3 inhibitor was reproduced in another coronavirus, the neurotropic JHM strain of mouse hepatitis virus. Our results indicate that GSK-3 is critical for CoV N protein phosphorylation and suggest that it plays a role in regulating the viral life cycle. This study, thus, provides new avenues to further investigate the specific role of N protein phosphorylation in CoV replication.</div></front></TEI><affiliations><list><country><li>Taïwan</li></country></list><tree><noCountry><name sortKey="Chen, Ding Shinn" sort="Chen, Ding Shinn" uniqKey="Chen D" first="Ding-Shinn" last="Chen">Ding-Shinn Chen</name><name sortKey="Chen, Pei Jer" sort="Chen, Pei Jer" uniqKey="Chen P" first="Pei-Jer" last="Chen">Pei-Jer Chen</name><name sortKey="Chen, Yen Shun" sort="Chen, Yen Shun" uniqKey="Chen Y" first="Yen-Shun" last="Chen">Yen-Shun Chen</name><name sortKey="Kao, Chuan Liang" sort="Kao, Chuan Liang" uniqKey="Kao C" first="Chuan-Liang" last="Kao">Chuan-Liang Kao</name><name sortKey="Kuo, Ti Jung" sort="Kuo, Ti Jung" uniqKey="Kuo T" first="Ti-Jung" last="Kuo">Ti-Jung Kuo</name><name sortKey="Shieh, Ya Hsiung" sort="Shieh, Ya Hsiung" uniqKey="Shieh Y" first="Ya-Hsiung" last="Shieh">Ya-Hsiung Shieh</name><name sortKey="Tsay, Yeou Guang" sort="Tsay, Yeou Guang" uniqKey="Tsay Y" first="Yeou-Guang" last="Tsay">Yeou-Guang Tsay</name><name sortKey="Wang, Sheng Han" sort="Wang, Sheng Han" uniqKey="Wang S" first="Sheng-Han" last="Wang">Sheng-Han Wang</name><name sortKey="Yeh, Shiou Hwei" sort="Yeh, Shiou Hwei" uniqKey="Yeh S" first="Shiou-Hwei" last="Yeh">Shiou-Hwei Yeh</name></noCountry><country name="Taïwan"><noRegion><name sortKey="Wu, Chia Hsin" sort="Wu, Chia Hsin" uniqKey="Wu C" first="Chia-Hsin" last="Wu">Chia-Hsin Wu</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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