A Kinome-Wide Small Interfering RNA Screen Identifies Proviral and Antiviral Host Factors in Severe Acute Respiratory Syndrome Coronavirus Replication, Including Double-Stranded RNA-Activated Protein Kinase and Early Secretory Pathway Proteins.
Identifieur interne : 001381 ( Main/Exploration ); précédent : 001380; suivant : 001382A Kinome-Wide Small Interfering RNA Screen Identifies Proviral and Antiviral Host Factors in Severe Acute Respiratory Syndrome Coronavirus Replication, Including Double-Stranded RNA-Activated Protein Kinase and Early Secretory Pathway Proteins.
Auteurs : Adriaan H. De Wilde [Pays-Bas] ; Kazimier F. Wannee [Pays-Bas] ; Florine E M. Scholte [Pays-Bas] ; Jelle J. Goeman [Pays-Bas] ; Peter Ten Dijke [Pays-Bas] ; Eric J. Snijder [Pays-Bas] ; Marjolein Kikkert [Pays-Bas] ; Martijn J. Van Hemert [Pays-Bas]Source :
- Journal of virology [ 1098-5514 ] ; 2015.
Descripteurs français
- KwdFr :
- MESH :
- génétique : Petit ARN interférent, Protein kinases.
- métabolisme : Protein kinases.
- pathogénicité : Coronavirus.
- virologie : Syndrome respiratoire aigu sévère.
- Animaux, Cellules HEK293, Cellules Vero, Humains, Réplication virale.
English descriptors
- KwdEn :
- MESH :
- chemical , genetics : Protein Kinases, RNA, Small Interfering.
- chemical , metabolism : Protein Kinases.
- pathogenicity : Coronavirus.
- virology : Severe Acute Respiratory Syndrome.
- Animals, Chlorocebus aethiops, HEK293 Cells, Humans, Vero Cells, Virus Replication.
Abstract
To identify host factors relevant for severe acute respiratory syndrome-coronavirus (SARS-CoV) replication, we performed a small interfering RNA (siRNA) library screen targeting the human kinome. Protein kinases are key regulators of many cellular functions, and the systematic knockdown of their expression should provide a broad perspective on factors and pathways promoting or antagonizing coronavirus replication. In addition to 40 proteins that promote SARS-CoV replication, our study identified 90 factors exhibiting an antiviral effect. Pathway analysis grouped subsets of these factors in specific cellular processes, including the innate immune response and the metabolism of complex lipids, which appear to play a role in SARS-CoV infection. Several factors were selected for in-depth validation in follow-up experiments. In cells depleted for the β2 subunit of the coatomer protein complex (COPB2), the strongest proviral hit, we observed reduced SARS-CoV protein expression and a >2-log reduction in virus yield. Knockdown of the COPB2-related proteins COPB1 and Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1) also suggested that COPI-coated vesicles and/or the early secretory pathway are important for SARS-CoV replication. Depletion of the antiviral double-stranded RNA-activated protein kinase (PKR) enhanced virus replication in the primary screen, and validation experiments confirmed increased SARS-CoV protein expression and virus production upon PKR depletion. In addition, cyclin-dependent kinase 6 (CDK6) was identified as a novel antiviral host factor in SARS-CoV replication. The inventory of pro- and antiviral host factors and pathways described here substantiates and expands our understanding of SARS-CoV replication and may contribute to the identification of novel targets for antiviral therapy.
DOI: 10.1128/JVI.01029-15
PubMed: 26041291
Affiliations:
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De Wilde</name><affiliation wicri:level="3"><nlm:affiliation>Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands.</nlm:affiliation><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden</wicri:regionArea><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author><author><name sortKey="Wannee, Kazimier F" sort="Wannee, Kazimier F" uniqKey="Wannee K" first="Kazimier F" last="Wannee">Kazimier F. Wannee</name><affiliation wicri:level="3"><nlm:affiliation>Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands.</nlm:affiliation><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden</wicri:regionArea><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author><author><name sortKey="Scholte, Florine E M" sort="Scholte, Florine E M" uniqKey="Scholte F" first="Florine E M" last="Scholte">Florine E M. 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Snijder</name><affiliation wicri:level="3"><nlm:affiliation>Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands.</nlm:affiliation><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden</wicri:regionArea><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author><author><name sortKey="Kikkert, Marjolein" sort="Kikkert, Marjolein" uniqKey="Kikkert M" first="Marjolein" last="Kikkert">Marjolein Kikkert</name><affiliation wicri:level="3"><nlm:affiliation>Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands m.kikkert@lumc.nl m.j.van_hemert@lumc.nl.</nlm:affiliation><country wicri:rule="url">Pays-Bas</country><wicri:regionArea>Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden</wicri:regionArea><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author><author><name sortKey="Van Hemert, Martijn J" sort="Van Hemert, Martijn J" uniqKey="Van Hemert M" first="Martijn J" last="Van Hemert">Martijn J. Van Hemert</name><affiliation wicri:level="3"><nlm:affiliation>Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands m.kikkert@lumc.nl m.j.van_hemert@lumc.nl.</nlm:affiliation><country wicri:rule="url">Pays-Bas</country><wicri:regionArea>Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden</wicri:regionArea><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author></analytic><series><title level="j">Journal of virology</title><idno type="eISSN">1098-5514</idno><imprint><date when="2015" type="published">2015</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Animals</term><term>Chlorocebus aethiops</term><term>Coronavirus (pathogenicity)</term><term>HEK293 Cells</term><term>Humans</term><term>Protein Kinases (genetics)</term><term>Protein Kinases (metabolism)</term><term>RNA, Small Interfering (genetics)</term><term>Severe Acute Respiratory Syndrome (virology)</term><term>Vero Cells</term><term>Virus Replication</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Animaux</term><term>Cellules HEK293</term><term>Cellules Vero</term><term>Coronavirus (pathogénicité)</term><term>Humains</term><term>Petit ARN interférent (génétique)</term><term>Protein kinases (génétique)</term><term>Protein kinases (métabolisme)</term><term>Réplication virale</term><term>Syndrome respiratoire aigu sévère (virologie)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Protein Kinases</term><term>RNA, Small Interfering</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Protein Kinases</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Petit ARN interférent</term><term>Protein kinases</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Protein kinases</term></keywords><keywords scheme="MESH" qualifier="pathogenicity" xml:lang="en"><term>Coronavirus</term></keywords><keywords scheme="MESH" qualifier="pathogénicité" xml:lang="fr"><term>Coronavirus</term></keywords><keywords scheme="MESH" qualifier="virologie" xml:lang="fr"><term>Syndrome respiratoire aigu sévère</term></keywords><keywords scheme="MESH" qualifier="virology" xml:lang="en"><term>Severe Acute Respiratory Syndrome</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Animals</term><term>Chlorocebus aethiops</term><term>HEK293 Cells</term><term>Humans</term><term>Vero Cells</term><term>Virus Replication</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Animaux</term><term>Cellules HEK293</term><term>Cellules Vero</term><term>Humains</term><term>Réplication virale</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">To identify host factors relevant for severe acute respiratory syndrome-coronavirus (SARS-CoV) replication, we performed a small interfering RNA (siRNA) library screen targeting the human kinome. Protein kinases are key regulators of many cellular functions, and the systematic knockdown of their expression should provide a broad perspective on factors and pathways promoting or antagonizing coronavirus replication. In addition to 40 proteins that promote SARS-CoV replication, our study identified 90 factors exhibiting an antiviral effect. Pathway analysis grouped subsets of these factors in specific cellular processes, including the innate immune response and the metabolism of complex lipids, which appear to play a role in SARS-CoV infection. Several factors were selected for in-depth validation in follow-up experiments. In cells depleted for the β2 subunit of the coatomer protein complex (COPB2), the strongest proviral hit, we observed reduced SARS-CoV protein expression and a >2-log reduction in virus yield. Knockdown of the COPB2-related proteins COPB1 and Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1) also suggested that COPI-coated vesicles and/or the early secretory pathway are important for SARS-CoV replication. Depletion of the antiviral double-stranded RNA-activated protein kinase (PKR) enhanced virus replication in the primary screen, and validation experiments confirmed increased SARS-CoV protein expression and virus production upon PKR depletion. In addition, cyclin-dependent kinase 6 (CDK6) was identified as a novel antiviral host factor in SARS-CoV replication. The inventory of pro- and antiviral host factors and pathways described here substantiates and expands our understanding of SARS-CoV replication and may contribute to the identification of novel targets for antiviral therapy.</div></front></TEI><affiliations><list><country><li>Pays-Bas</li></country><region><li>Hollande-Méridionale</li></region><settlement><li>Leyde</li></settlement></list><tree><country name="Pays-Bas"><region name="Hollande-Méridionale"><name sortKey="De Wilde, Adriaan H" sort="De Wilde, Adriaan H" uniqKey="De Wilde A" first="Adriaan H" last="De Wilde">Adriaan H. De Wilde</name></region><name sortKey="Goeman, Jelle J" sort="Goeman, Jelle J" uniqKey="Goeman J" first="Jelle J" last="Goeman">Jelle J. Goeman</name><name sortKey="Kikkert, Marjolein" sort="Kikkert, Marjolein" uniqKey="Kikkert M" first="Marjolein" last="Kikkert">Marjolein Kikkert</name><name sortKey="Scholte, Florine E M" sort="Scholte, Florine E M" uniqKey="Scholte F" first="Florine E M" last="Scholte">Florine E M. Scholte</name><name sortKey="Snijder, Eric J" sort="Snijder, Eric J" uniqKey="Snijder E" first="Eric J" last="Snijder">Eric J. Snijder</name><name sortKey="Ten Dijke, Peter" sort="Ten Dijke, Peter" uniqKey="Ten Dijke P" first="Peter" last="Ten Dijke">Peter Ten Dijke</name><name sortKey="Van Hemert, Martijn J" sort="Van Hemert, Martijn J" uniqKey="Van Hemert M" first="Martijn J" last="Van Hemert">Martijn J. Van Hemert</name><name sortKey="Wannee, Kazimier F" sort="Wannee, Kazimier F" uniqKey="Wannee K" first="Kazimier F" last="Wannee">Kazimier F. Wannee</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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