siRNA targeting the Leader sequence of SARS-CoV inhibits virus replication
Identifieur interne : 000E42 ( Ncbi/Merge ); précédent : 000E41; suivant : 000E43siRNA targeting the Leader sequence of SARS-CoV inhibits virus replication
Auteurs : T. Li [République populaire de Chine] ; Y. Zhang [République populaire de Chine] ; L. Fu [République populaire de Chine] ; C. Yu [République populaire de Chine] ; X. Li [République populaire de Chine] ; Y. Li [République populaire de Chine] ; X. Zhang [République populaire de Chine] ; Z. Rong [République populaire de Chine] ; Y. Wang [République populaire de Chine] ; H. Ning [République populaire de Chine] ; R. Liang ; W. Chen [République populaire de Chine] ; L A Babiuk ; Z. Chang [République populaire de Chine]Source :
- Gene Therapy [ 0969-7128 ] ; 2005.
Descripteurs français
- KwdFr :
- ARN messager (génétique), ARN viral (génétique), Animaux, Cellules Vero, Cellules cultivées, Ciblage de gène, Données de séquences moléculaires, Expression des gènes, Extinction de l'expression des gènes, Gènes rapporteurs, Interférence par ARN, Microscopie de fluorescence, Oligonucléotides antisens (génétique), Petit ARN interférent (génétique), Régions 5' non traduites (génétique), Réplication virale (génétique), Séquence nucléotidique, Technique de Western, Transfection, Virus du SRAS (génétique), Virus du SRAS (physiologie).
- MESH :
- génétique : ARN messager, ARN viral, Oligonucléotides antisens, Petit ARN interférent, Régions 5' non traduites, Réplication virale, Virus du SRAS.
- physiologie : Virus du SRAS.
- Animaux, Cellules Vero, Cellules cultivées, Ciblage de gène, Données de séquences moléculaires, Expression des gènes, Extinction de l'expression des gènes, Gènes rapporteurs, Interférence par ARN, Microscopie de fluorescence, Séquence nucléotidique, Technique de Western, Transfection.
English descriptors
- KwdEn :
- 5' Untranslated Regions (genetics), Animals, Base Sequence, Blotting, Western, Cells, Cultured, Chlorocebus aethiops, Gene Expression, Gene Silencing, Gene Targeting, Genes, Reporter, Microscopy, Fluorescence, Molecular Sequence Data, Oligonucleotides, Antisense (genetics), RNA Interference, RNA, Messenger (genetics), RNA, Small Interfering (genetics), RNA, Viral (genetics), SARS Virus (genetics), SARS Virus (physiology), Transfection, Vero Cells, Virus Replication (genetics).
- MESH :
- chemical , genetics : 5' Untranslated Regions, Oligonucleotides, Antisense, RNA, Messenger, RNA, Small Interfering, RNA, Viral.
- genetics : SARS Virus, Virus Replication.
- physiology : SARS Virus.
- Animals, Base Sequence, Blotting, Western, Cells, Cultured, Chlorocebus aethiops, Gene Expression, Gene Silencing, Gene Targeting, Genes, Reporter, Microscopy, Fluorescence, Molecular Sequence Data, RNA Interference, Transfection, Vero Cells.
Abstract
SARS-CoV (the SARS-Associated Coronavirus) was reported as a novel virus member in the coronavirus family, which was the cause of severe acute respiratory syndrome. Coronavirus replication occurs through a unique mechanism employing Leader sequence in the transcripts when initiating transcription from the genome. Therefore, we cloned the Leader sequence from SARS-CoV(BJ01), which is identical to that identified from SARS-CoV(HKU-39849), and constructed specific siRNA targeting the Leader sequence. Using EGFP and RFP reporter genes fused with the cloned SARS-CoV Leader sequence, we demonstrated that the siRNA targeting the Leader sequence decreased the mRNA abundance and protein expression levels of the reporter genes in 293T cells. By stably expressing the siRNA in Vero E6 cells, we provided data that the siRNA could effectively and specifically decrease the mRNA abundance of SARS-CoV genes as analyzed by RT-PCR and Northern blot. Our data indicated that the siRNA targeting the Leader sequence inhibited the replication of SARS-CoV in Vero E6 cells by silencing gene expression. We further demonstrated, via transient transfection experiments, that the siRNA targeting the Leader sequence had a much stronger inhibitory effect on SARS-CoV replication than the siRNAs targeting the Spike gene or the antisense oligodeoxynucleotides did. This report provides evidence that targeting Leader sequence using siRNA could be a powerful tool in inhibiting SARS-CoV replication.
Url:
DOI: 10.1038/sj.gt.3302479
PubMed: 15772689
PubMed Central: 7091583
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PMC:7091583Le document en format XML
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Li</name><affiliation wicri:level="1"><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.12527.33</institution-id><institution-id institution-id-type="ISNI">0000 0001 0662 3178</institution-id><institution>Department of Biological Sciences and Biotechnology, Institute of Biomedicine,</institution><institution>Tsinghua University,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Zhang, Y" sort="Zhang, Y" uniqKey="Zhang Y" first="Y" last="Zhang">Y. 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Fu</name><affiliation wicri:level="1"><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.410740.6</institution-id><institution-id institution-id-type="ISNI">0000 0004 1803 4911</institution-id><institution>Institute of Microbiology and Epidemiology, AMMS,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Yu, C" sort="Yu, C" uniqKey="Yu C" first="C" last="Yu">C. Yu</name><affiliation wicri:level="1"><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.410740.6</institution-id><institution-id institution-id-type="ISNI">0000 0004 1803 4911</institution-id><institution>Institute of Microbiology and Epidemiology, AMMS,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Li, X" sort="Li, X" uniqKey="Li X" first="X" last="Li">X. Li</name><affiliation wicri:level="1"><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.12527.33</institution-id><institution-id institution-id-type="ISNI">0000 0001 0662 3178</institution-id><institution>Department of Biological Sciences and Biotechnology, Institute of Biomedicine,</institution><institution>Tsinghua University,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Li, Y" sort="Li, Y" uniqKey="Li Y" first="Y" last="Li">Y. Li</name><affiliation wicri:level="1"><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.12527.33</institution-id><institution-id institution-id-type="ISNI">0000 0001 0662 3178</institution-id><institution>Department of Biological Sciences and Biotechnology, Institute of Biomedicine,</institution><institution>Tsinghua University,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Zhang, X" sort="Zhang, X" uniqKey="Zhang X" first="X" last="Zhang">X. Zhang</name><affiliation wicri:level="1"><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.12527.33</institution-id><institution-id institution-id-type="ISNI">0000 0001 0662 3178</institution-id><institution>Department of Biological Sciences and Biotechnology, Institute of Biomedicine,</institution><institution>Tsinghua University,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Rong, Z" sort="Rong, Z" uniqKey="Rong Z" first="Z" last="Rong">Z. Rong</name><affiliation wicri:level="1"><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.12527.33</institution-id><institution-id institution-id-type="ISNI">0000 0001 0662 3178</institution-id><institution>Department of Biological Sciences and Biotechnology, Institute of Biomedicine,</institution><institution>Tsinghua University,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Wang, Y" sort="Wang, Y" uniqKey="Wang Y" first="Y" last="Wang">Y. Wang</name><affiliation wicri:level="1"><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.12527.33</institution-id><institution-id institution-id-type="ISNI">0000 0001 0662 3178</institution-id><institution>Department of Biological Sciences and Biotechnology, Institute of Biomedicine,</institution><institution>Tsinghua University,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Ning, H" sort="Ning, H" uniqKey="Ning H" first="H" last="Ning">H. Ning</name><affiliation wicri:level="1"><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.12527.33</institution-id><institution-id institution-id-type="ISNI">0000 0001 0662 3178</institution-id><institution>Department of Biological Sciences and Biotechnology, Institute of Biomedicine,</institution><institution>Tsinghua University,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Liang, R" sort="Liang, R" uniqKey="Liang R" first="R" last="Liang">R. Liang</name><affiliation><nlm:aff id="Aff3"><institution-wrap><institution-id institution-id-type="GRID">grid.25152.31</institution-id><institution-id institution-id-type="ISNI">0000 0001 2154 235X</institution-id><institution>Vaccine & Infectious Disease Organization (VIDO),</institution><institution>University of Saskatchewan,</institution></institution-wrap>Saskatoon, SK Canada</nlm:aff><wicri:noCountry code="subfield">SK Canada</wicri:noCountry></affiliation></author><author><name sortKey="Chen, W" sort="Chen, W" uniqKey="Chen W" first="W" last="Chen">W. Chen</name><affiliation wicri:level="1"><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.410740.6</institution-id><institution-id institution-id-type="ISNI">0000 0004 1803 4911</institution-id><institution>Institute of Microbiology and Epidemiology, AMMS,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Babiuk, L A" sort="Babiuk, L A" uniqKey="Babiuk L" first="L A" last="Babiuk">L A Babiuk</name><affiliation><nlm:aff id="Aff3"><institution-wrap><institution-id institution-id-type="GRID">grid.25152.31</institution-id><institution-id institution-id-type="ISNI">0000 0001 2154 235X</institution-id><institution>Vaccine & Infectious Disease Organization (VIDO),</institution><institution>University of Saskatchewan,</institution></institution-wrap>Saskatoon, SK Canada</nlm:aff><wicri:noCountry code="subfield">SK Canada</wicri:noCountry></affiliation></author><author><name sortKey="Chang, Z" sort="Chang, Z" uniqKey="Chang Z" first="Z" last="Chang">Z. Chang</name><affiliation wicri:level="1"><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.12527.33</institution-id><institution-id institution-id-type="ISNI">0000 0001 0662 3178</institution-id><institution>Department of Biological Sciences and Biotechnology, Institute of Biomedicine,</institution><institution>Tsinghua University,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author></analytic><series><title level="j">Gene Therapy</title><idno type="ISSN">0969-7128</idno><idno type="eISSN">1476-5462</idno><imprint><date when="2005">2005</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>5' Untranslated Regions (genetics)</term><term>Animals</term><term>Base Sequence</term><term>Blotting, Western</term><term>Cells, Cultured</term><term>Chlorocebus aethiops</term><term>Gene Expression</term><term>Gene Silencing</term><term>Gene Targeting</term><term>Genes, Reporter</term><term>Microscopy, Fluorescence</term><term>Molecular Sequence Data</term><term>Oligonucleotides, Antisense (genetics)</term><term>RNA Interference</term><term>RNA, Messenger (genetics)</term><term>RNA, Small Interfering (genetics)</term><term>RNA, Viral (genetics)</term><term>SARS Virus (genetics)</term><term>SARS Virus (physiology)</term><term>Transfection</term><term>Vero Cells</term><term>Virus Replication (genetics)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ARN messager (génétique)</term><term>ARN viral (génétique)</term><term>Animaux</term><term>Cellules Vero</term><term>Cellules cultivées</term><term>Ciblage de gène</term><term>Données de séquences moléculaires</term><term>Expression des gènes</term><term>Extinction de l'expression des gènes</term><term>Gènes rapporteurs</term><term>Interférence par ARN</term><term>Microscopie de fluorescence</term><term>Oligonucléotides antisens (génétique)</term><term>Petit ARN interférent (génétique)</term><term>Régions 5' non traduites (génétique)</term><term>Réplication virale (génétique)</term><term>Séquence nucléotidique</term><term>Technique de Western</term><term>Transfection</term><term>Virus du SRAS (génétique)</term><term>Virus du SRAS (physiologie)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>5' Untranslated Regions</term><term>Oligonucleotides, Antisense</term><term>RNA, Messenger</term><term>RNA, Small Interfering</term><term>RNA, Viral</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>SARS Virus</term><term>Virus Replication</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>ARN messager</term><term>ARN viral</term><term>Oligonucléotides antisens</term><term>Petit ARN interférent</term><term>Régions 5' non traduites</term><term>Réplication virale</term><term>Virus du SRAS</term></keywords><keywords scheme="MESH" qualifier="physiologie" xml:lang="fr"><term>Virus du SRAS</term></keywords><keywords scheme="MESH" qualifier="physiology" xml:lang="en"><term>SARS Virus</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Animals</term><term>Base Sequence</term><term>Blotting, Western</term><term>Cells, Cultured</term><term>Chlorocebus aethiops</term><term>Gene Expression</term><term>Gene Silencing</term><term>Gene Targeting</term><term>Genes, Reporter</term><term>Microscopy, Fluorescence</term><term>Molecular Sequence Data</term><term>RNA Interference</term><term>Transfection</term><term>Vero Cells</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Animaux</term><term>Cellules Vero</term><term>Cellules cultivées</term><term>Ciblage de gène</term><term>Données de séquences moléculaires</term><term>Expression des gènes</term><term>Extinction de l'expression des gènes</term><term>Gènes rapporteurs</term><term>Interférence par ARN</term><term>Microscopie de fluorescence</term><term>Séquence nucléotidique</term><term>Technique de Western</term><term>Transfection</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p id="Par1">SARS-CoV (the SARS-Associated Coronavirus) was reported as a novel virus member in the coronavirus family, which was the cause of severe acute respiratory syndrome. Coronavirus replication occurs through a unique mechanism employing Leader sequence in the transcripts when initiating transcription from the genome. Therefore, we cloned the Leader sequence from SARS-CoV(BJ01), which is identical to that identified from SARS-CoV(HKU-39849), and constructed specific siRNA targeting the Leader sequence. Using EGFP and RFP reporter genes fused with the cloned SARS-CoV Leader sequence, we demonstrated that the siRNA targeting the Leader sequence decreased the mRNA abundance and protein expression levels of the reporter genes in 293T cells. By stably expressing the siRNA in Vero E6 cells, we provided data that the siRNA could effectively and specifically decrease the mRNA abundance of SARS-CoV genes as analyzed by RT-PCR and Northern blot. Our data indicated that the siRNA targeting the Leader sequence inhibited the replication of SARS-CoV in Vero E6 cells by silencing gene expression. We further demonstrated, via transient transfection experiments, that the siRNA targeting the Leader sequence had a much stronger inhibitory effect on SARS-CoV replication than the siRNAs targeting the Spike gene or the antisense oligodeoxynucleotides did. This report provides evidence that targeting Leader sequence using siRNA could be a powerful tool in inhibiting SARS-CoV replication.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Blackmore, T" uniqKey="Blackmore T">T Blackmore</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Parry, J" uniqKey="Parry J">J Parry</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Holmes, Kv" uniqKey="Holmes K">KV Holmes</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Drosten, C" uniqKey="Drosten C">C Drosten</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ksiazek, Tg" uniqKey="Ksiazek T">TG Ksiazek</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Peiris, Js" uniqKey="Peiris J">JS Peiris</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lai, 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sortKey="Jia, Q" uniqKey="Jia Q">Q Jia</name></author><author><name sortKey="Sun, R" uniqKey="Sun R">R Sun</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gallagher, Tm" uniqKey="Gallagher T">TM Gallagher</name></author><author><name sortKey="Buchmeier, Mj" uniqKey="Buchmeier M">MJ Buchmeier</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Guan, Y" uniqKey="Guan Y">Y Guan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Vogel, G" uniqKey="Vogel G">G Vogel</name></author></analytic></biblStruct></listBibl></div1></back></TEI><double pmid="15772689"><pmc><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">siRNA targeting the Leader sequence of SARS-CoV inhibits virus replication</title><author><name sortKey="Li, T" sort="Li, T" uniqKey="Li T" first="T" last="Li">T. Li</name><affiliation wicri:level="1"><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.12527.33</institution-id><institution-id institution-id-type="ISNI">0000 0001 0662 3178</institution-id><institution>Department of Biological Sciences and Biotechnology, Institute of Biomedicine,</institution><institution>Tsinghua University,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Zhang, Y" sort="Zhang, Y" uniqKey="Zhang Y" first="Y" last="Zhang">Y. Zhang</name><affiliation wicri:level="1"><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.12527.33</institution-id><institution-id institution-id-type="ISNI">0000 0001 0662 3178</institution-id><institution>Department of Biological Sciences and Biotechnology, Institute of Biomedicine,</institution><institution>Tsinghua University,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Fu, L" sort="Fu, L" uniqKey="Fu L" first="L" last="Fu">L. Fu</name><affiliation wicri:level="1"><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.410740.6</institution-id><institution-id institution-id-type="ISNI">0000 0004 1803 4911</institution-id><institution>Institute of Microbiology and Epidemiology, AMMS,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Yu, C" sort="Yu, C" uniqKey="Yu C" first="C" last="Yu">C. Yu</name><affiliation wicri:level="1"><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.410740.6</institution-id><institution-id institution-id-type="ISNI">0000 0004 1803 4911</institution-id><institution>Institute of Microbiology and Epidemiology, AMMS,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Li, X" sort="Li, X" uniqKey="Li X" first="X" last="Li">X. Li</name><affiliation wicri:level="1"><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.12527.33</institution-id><institution-id institution-id-type="ISNI">0000 0001 0662 3178</institution-id><institution>Department of Biological Sciences and Biotechnology, Institute of Biomedicine,</institution><institution>Tsinghua University,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Li, Y" sort="Li, Y" uniqKey="Li Y" first="Y" last="Li">Y. Li</name><affiliation wicri:level="1"><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.12527.33</institution-id><institution-id institution-id-type="ISNI">0000 0001 0662 3178</institution-id><institution>Department of Biological Sciences and Biotechnology, Institute of Biomedicine,</institution><institution>Tsinghua University,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Zhang, X" sort="Zhang, X" uniqKey="Zhang X" first="X" last="Zhang">X. Zhang</name><affiliation wicri:level="1"><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.12527.33</institution-id><institution-id institution-id-type="ISNI">0000 0001 0662 3178</institution-id><institution>Department of Biological Sciences and Biotechnology, Institute of Biomedicine,</institution><institution>Tsinghua University,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Rong, Z" sort="Rong, Z" uniqKey="Rong Z" first="Z" last="Rong">Z. Rong</name><affiliation wicri:level="1"><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.12527.33</institution-id><institution-id institution-id-type="ISNI">0000 0001 0662 3178</institution-id><institution>Department of Biological Sciences and Biotechnology, Institute of Biomedicine,</institution><institution>Tsinghua University,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Wang, Y" sort="Wang, Y" uniqKey="Wang Y" first="Y" last="Wang">Y. Wang</name><affiliation wicri:level="1"><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.12527.33</institution-id><institution-id institution-id-type="ISNI">0000 0001 0662 3178</institution-id><institution>Department of Biological Sciences and Biotechnology, Institute of Biomedicine,</institution><institution>Tsinghua University,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Ning, H" sort="Ning, H" uniqKey="Ning H" first="H" last="Ning">H. Ning</name><affiliation wicri:level="1"><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.12527.33</institution-id><institution-id institution-id-type="ISNI">0000 0001 0662 3178</institution-id><institution>Department of Biological Sciences and Biotechnology, Institute of Biomedicine,</institution><institution>Tsinghua University,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Liang, R" sort="Liang, R" uniqKey="Liang R" first="R" last="Liang">R. Liang</name><affiliation><nlm:aff id="Aff3"><institution-wrap><institution-id institution-id-type="GRID">grid.25152.31</institution-id><institution-id institution-id-type="ISNI">0000 0001 2154 235X</institution-id><institution>Vaccine & Infectious Disease Organization (VIDO),</institution><institution>University of Saskatchewan,</institution></institution-wrap>Saskatoon, SK Canada</nlm:aff><wicri:noCountry code="subfield">SK Canada</wicri:noCountry></affiliation></author><author><name sortKey="Chen, W" sort="Chen, W" uniqKey="Chen W" first="W" last="Chen">W. Chen</name><affiliation wicri:level="1"><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.410740.6</institution-id><institution-id institution-id-type="ISNI">0000 0004 1803 4911</institution-id><institution>Institute of Microbiology and Epidemiology, AMMS,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Babiuk, L A" sort="Babiuk, L A" uniqKey="Babiuk L" first="L A" last="Babiuk">L A Babiuk</name><affiliation><nlm:aff id="Aff3"><institution-wrap><institution-id institution-id-type="GRID">grid.25152.31</institution-id><institution-id institution-id-type="ISNI">0000 0001 2154 235X</institution-id><institution>Vaccine & Infectious Disease Organization (VIDO),</institution><institution>University of Saskatchewan,</institution></institution-wrap>Saskatoon, SK Canada</nlm:aff><wicri:noCountry code="subfield">SK Canada</wicri:noCountry></affiliation></author><author><name sortKey="Chang, Z" sort="Chang, Z" uniqKey="Chang Z" first="Z" last="Chang">Z. Chang</name><affiliation wicri:level="1"><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.12527.33</institution-id><institution-id institution-id-type="ISNI">0000 0001 0662 3178</institution-id><institution>Department of Biological Sciences and Biotechnology, Institute of Biomedicine,</institution><institution>Tsinghua University,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">15772689</idno><idno type="pmc">7091583</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7091583</idno><idno type="RBID">PMC:7091583</idno><idno type="doi">10.1038/sj.gt.3302479</idno><date when="2005">2005</date><idno type="wicri:Area/Pmc/Corpus">000376</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000376</idno><idno type="wicri:Area/Pmc/Curation">000376</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">000376</idno><idno type="wicri:Area/Pmc/Checkpoint">001186</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Checkpoint">001186</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">siRNA targeting the Leader sequence of SARS-CoV inhibits virus replication</title><author><name sortKey="Li, T" sort="Li, T" uniqKey="Li T" first="T" last="Li">T. Li</name><affiliation wicri:level="1"><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.12527.33</institution-id><institution-id institution-id-type="ISNI">0000 0001 0662 3178</institution-id><institution>Department of Biological Sciences and Biotechnology, Institute of Biomedicine,</institution><institution>Tsinghua University,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Zhang, Y" sort="Zhang, Y" uniqKey="Zhang Y" first="Y" last="Zhang">Y. Zhang</name><affiliation wicri:level="1"><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.12527.33</institution-id><institution-id institution-id-type="ISNI">0000 0001 0662 3178</institution-id><institution>Department of Biological Sciences and Biotechnology, Institute of Biomedicine,</institution><institution>Tsinghua University,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Fu, L" sort="Fu, L" uniqKey="Fu L" first="L" last="Fu">L. Fu</name><affiliation wicri:level="1"><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.410740.6</institution-id><institution-id institution-id-type="ISNI">0000 0004 1803 4911</institution-id><institution>Institute of Microbiology and Epidemiology, AMMS,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Yu, C" sort="Yu, C" uniqKey="Yu C" first="C" last="Yu">C. Yu</name><affiliation wicri:level="1"><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.410740.6</institution-id><institution-id institution-id-type="ISNI">0000 0004 1803 4911</institution-id><institution>Institute of Microbiology and Epidemiology, AMMS,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Li, X" sort="Li, X" uniqKey="Li X" first="X" last="Li">X. Li</name><affiliation wicri:level="1"><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.12527.33</institution-id><institution-id institution-id-type="ISNI">0000 0001 0662 3178</institution-id><institution>Department of Biological Sciences and Biotechnology, Institute of Biomedicine,</institution><institution>Tsinghua University,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Li, Y" sort="Li, Y" uniqKey="Li Y" first="Y" last="Li">Y. Li</name><affiliation wicri:level="1"><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.12527.33</institution-id><institution-id institution-id-type="ISNI">0000 0001 0662 3178</institution-id><institution>Department of Biological Sciences and Biotechnology, Institute of Biomedicine,</institution><institution>Tsinghua University,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Zhang, X" sort="Zhang, X" uniqKey="Zhang X" first="X" last="Zhang">X. Zhang</name><affiliation wicri:level="1"><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.12527.33</institution-id><institution-id institution-id-type="ISNI">0000 0001 0662 3178</institution-id><institution>Department of Biological Sciences and Biotechnology, Institute of Biomedicine,</institution><institution>Tsinghua University,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Rong, Z" sort="Rong, Z" uniqKey="Rong Z" first="Z" last="Rong">Z. Rong</name><affiliation wicri:level="1"><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.12527.33</institution-id><institution-id institution-id-type="ISNI">0000 0001 0662 3178</institution-id><institution>Department of Biological Sciences and Biotechnology, Institute of Biomedicine,</institution><institution>Tsinghua University,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Wang, Y" sort="Wang, Y" uniqKey="Wang Y" first="Y" last="Wang">Y. Wang</name><affiliation wicri:level="1"><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.12527.33</institution-id><institution-id institution-id-type="ISNI">0000 0001 0662 3178</institution-id><institution>Department of Biological Sciences and Biotechnology, Institute of Biomedicine,</institution><institution>Tsinghua University,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Ning, H" sort="Ning, H" uniqKey="Ning H" first="H" last="Ning">H. Ning</name><affiliation wicri:level="1"><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.12527.33</institution-id><institution-id institution-id-type="ISNI">0000 0001 0662 3178</institution-id><institution>Department of Biological Sciences and Biotechnology, Institute of Biomedicine,</institution><institution>Tsinghua University,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Liang, R" sort="Liang, R" uniqKey="Liang R" first="R" last="Liang">R. Liang</name><affiliation><nlm:aff id="Aff3"><institution-wrap><institution-id institution-id-type="GRID">grid.25152.31</institution-id><institution-id institution-id-type="ISNI">0000 0001 2154 235X</institution-id><institution>Vaccine & Infectious Disease Organization (VIDO),</institution><institution>University of Saskatchewan,</institution></institution-wrap>Saskatoon, SK Canada</nlm:aff><wicri:noCountry code="subfield">SK Canada</wicri:noCountry></affiliation></author><author><name sortKey="Chen, W" sort="Chen, W" uniqKey="Chen W" first="W" last="Chen">W. Chen</name><affiliation wicri:level="1"><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.410740.6</institution-id><institution-id institution-id-type="ISNI">0000 0004 1803 4911</institution-id><institution>Institute of Microbiology and Epidemiology, AMMS,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author><author><name sortKey="Babiuk, L A" sort="Babiuk, L A" uniqKey="Babiuk L" first="L A" last="Babiuk">L A Babiuk</name><affiliation><nlm:aff id="Aff3"><institution-wrap><institution-id institution-id-type="GRID">grid.25152.31</institution-id><institution-id institution-id-type="ISNI">0000 0001 2154 235X</institution-id><institution>Vaccine & Infectious Disease Organization (VIDO),</institution><institution>University of Saskatchewan,</institution></institution-wrap>Saskatoon, SK Canada</nlm:aff><wicri:noCountry code="subfield">SK Canada</wicri:noCountry></affiliation></author><author><name sortKey="Chang, Z" sort="Chang, Z" uniqKey="Chang Z" first="Z" last="Chang">Z. Chang</name><affiliation wicri:level="1"><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.12527.33</institution-id><institution-id institution-id-type="ISNI">0000 0001 0662 3178</institution-id><institution>Department of Biological Sciences and Biotechnology, Institute of Biomedicine,</institution><institution>Tsinghua University,</institution></institution-wrap>Beijing, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Beijing</wicri:regionArea></affiliation></author></analytic><series><title level="j">Gene Therapy</title><idno type="ISSN">0969-7128</idno><idno type="eISSN">1476-5462</idno><imprint><date when="2005">2005</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p id="Par1">SARS-CoV (the SARS-Associated Coronavirus) was reported as a novel virus member in the coronavirus family, which was the cause of severe acute respiratory syndrome. Coronavirus replication occurs through a unique mechanism employing Leader sequence in the transcripts when initiating transcription from the genome. Therefore, we cloned the Leader sequence from SARS-CoV(BJ01), which is identical to that identified from SARS-CoV(HKU-39849), and constructed specific siRNA targeting the Leader sequence. Using EGFP and RFP reporter genes fused with the cloned SARS-CoV Leader sequence, we demonstrated that the siRNA targeting the Leader sequence decreased the mRNA abundance and protein expression levels of the reporter genes in 293T cells. By stably expressing the siRNA in Vero E6 cells, we provided data that the siRNA could effectively and specifically decrease the mRNA abundance of SARS-CoV genes as analyzed by RT-PCR and Northern blot. Our data indicated that the siRNA targeting the Leader sequence inhibited the replication of SARS-CoV in Vero E6 cells by silencing gene expression. We further demonstrated, via transient transfection experiments, that the siRNA targeting the Leader sequence had a much stronger inhibitory effect on SARS-CoV replication than the siRNAs targeting the Spike gene or the antisense oligodeoxynucleotides did. This report provides evidence that targeting Leader sequence using siRNA could be a powerful tool in inhibiting SARS-CoV replication.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Blackmore, T" uniqKey="Blackmore T">T Blackmore</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Parry, J" uniqKey="Parry J">J Parry</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Holmes, Kv" uniqKey="Holmes K">KV Holmes</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Drosten, C" uniqKey="Drosten C">C Drosten</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ksiazek, Tg" uniqKey="Ksiazek T">TG Ksiazek</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Peiris, Js" uniqKey="Peiris J">JS Peiris</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lai, 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sortKey="Jia, Q" uniqKey="Jia Q">Q Jia</name></author><author><name sortKey="Sun, R" uniqKey="Sun R">R Sun</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gallagher, Tm" uniqKey="Gallagher T">TM Gallagher</name></author><author><name sortKey="Buchmeier, Mj" uniqKey="Buchmeier M">MJ Buchmeier</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Guan, Y" uniqKey="Guan Y">Y Guan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Vogel, G" uniqKey="Vogel G">G Vogel</name></author></analytic></biblStruct></listBibl></div1></back></TEI></pmc><pubmed><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">siRNA targeting the leader sequence of SARS-CoV inhibits virus replication.</title><author><name sortKey="Li, T" sort="Li, T" uniqKey="Li T" first="T" last="Li">T. Li</name><affiliation wicri:level="3"><nlm:affiliation>Department of Biological Sciences and Biotechnology, Institute of Biomedicine, Tsinghua University, Beijing, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Department of Biological Sciences and Biotechnology, Institute of Biomedicine, Tsinghua University, Beijing</wicri:regionArea><placeName><settlement type="city">Pékin</settlement></placeName></affiliation></author><author><name sortKey="Zhang, Y" sort="Zhang, Y" uniqKey="Zhang Y" first="Y" last="Zhang">Y. Zhang</name></author><author><name sortKey="Fu, L" sort="Fu, L" uniqKey="Fu L" first="L" last="Fu">L. Fu</name></author><author><name sortKey="Yu, C" sort="Yu, C" uniqKey="Yu C" first="C" last="Yu">C. Yu</name></author><author><name sortKey="Li, X" sort="Li, X" uniqKey="Li X" first="X" last="Li">X. Li</name></author><author><name sortKey="Li, Y" sort="Li, Y" uniqKey="Li Y" first="Y" last="Li">Y. Li</name></author><author><name sortKey="Zhang, X" sort="Zhang, X" uniqKey="Zhang X" first="X" last="Zhang">X. Zhang</name></author><author><name sortKey="Rong, Z" sort="Rong, Z" uniqKey="Rong Z" first="Z" last="Rong">Z. Rong</name></author><author><name sortKey="Wang, Y" sort="Wang, Y" uniqKey="Wang Y" first="Y" last="Wang">Y. Wang</name></author><author><name sortKey="Ning, H" sort="Ning, H" uniqKey="Ning H" first="H" last="Ning">H. Ning</name></author><author><name sortKey="Liang, R" sort="Liang, R" uniqKey="Liang R" first="R" last="Liang">R. Liang</name></author><author><name sortKey="Chen, W" sort="Chen, W" uniqKey="Chen W" first="W" last="Chen">W. Chen</name></author><author><name sortKey="Babiuk, L A" sort="Babiuk, L A" uniqKey="Babiuk L" first="L A" last="Babiuk">L A Babiuk</name></author><author><name sortKey="Chang, Z" sort="Chang, Z" uniqKey="Chang Z" first="Z" last="Chang">Z. Chang</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2005">2005</date><idno type="RBID">pubmed:15772689</idno><idno type="pmid">15772689</idno><idno type="doi">10.1038/sj.gt.3302479</idno><idno type="wicri:Area/PubMed/Corpus">002839</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">002839</idno><idno type="wicri:Area/PubMed/Curation">002839</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Curation">002839</idno><idno type="wicri:Area/PubMed/Checkpoint">002339</idno><idno type="wicri:explorRef" wicri:stream="Checkpoint" wicri:step="PubMed">002339</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">siRNA targeting the leader sequence of SARS-CoV inhibits virus replication.</title><author><name sortKey="Li, T" sort="Li, T" uniqKey="Li T" first="T" last="Li">T. Li</name><affiliation wicri:level="3"><nlm:affiliation>Department of Biological Sciences and Biotechnology, Institute of Biomedicine, Tsinghua University, Beijing, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Department of Biological Sciences and Biotechnology, Institute of Biomedicine, Tsinghua University, Beijing</wicri:regionArea><placeName><settlement type="city">Pékin</settlement></placeName></affiliation></author><author><name sortKey="Zhang, Y" sort="Zhang, Y" uniqKey="Zhang Y" first="Y" last="Zhang">Y. Zhang</name></author><author><name sortKey="Fu, L" sort="Fu, L" uniqKey="Fu L" first="L" last="Fu">L. Fu</name></author><author><name sortKey="Yu, C" sort="Yu, C" uniqKey="Yu C" first="C" last="Yu">C. Yu</name></author><author><name sortKey="Li, X" sort="Li, X" uniqKey="Li X" first="X" last="Li">X. Li</name></author><author><name sortKey="Li, Y" sort="Li, Y" uniqKey="Li Y" first="Y" last="Li">Y. Li</name></author><author><name sortKey="Zhang, X" sort="Zhang, X" uniqKey="Zhang X" first="X" last="Zhang">X. Zhang</name></author><author><name sortKey="Rong, Z" sort="Rong, Z" uniqKey="Rong Z" first="Z" last="Rong">Z. Rong</name></author><author><name sortKey="Wang, Y" sort="Wang, Y" uniqKey="Wang Y" first="Y" last="Wang">Y. Wang</name></author><author><name sortKey="Ning, H" sort="Ning, H" uniqKey="Ning H" first="H" last="Ning">H. Ning</name></author><author><name sortKey="Liang, R" sort="Liang, R" uniqKey="Liang R" first="R" last="Liang">R. Liang</name></author><author><name sortKey="Chen, W" sort="Chen, W" uniqKey="Chen W" first="W" last="Chen">W. Chen</name></author><author><name sortKey="Babiuk, L A" sort="Babiuk, L A" uniqKey="Babiuk L" first="L A" last="Babiuk">L A Babiuk</name></author><author><name sortKey="Chang, Z" sort="Chang, Z" uniqKey="Chang Z" first="Z" last="Chang">Z. Chang</name></author></analytic><series><title level="j">Gene therapy</title><idno type="ISSN">0969-7128</idno><imprint><date when="2005" type="published">2005</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>5' Untranslated Regions (genetics)</term><term>Animals</term><term>Base Sequence</term><term>Blotting, Western</term><term>Cells, Cultured</term><term>Chlorocebus aethiops</term><term>Gene Expression</term><term>Gene Silencing</term><term>Gene Targeting</term><term>Genes, Reporter</term><term>Microscopy, Fluorescence</term><term>Molecular Sequence Data</term><term>Oligonucleotides, Antisense (genetics)</term><term>RNA Interference</term><term>RNA, Messenger (genetics)</term><term>RNA, Small Interfering (genetics)</term><term>RNA, Viral (genetics)</term><term>SARS Virus (genetics)</term><term>SARS Virus (physiology)</term><term>Transfection</term><term>Vero Cells</term><term>Virus Replication (genetics)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ARN messager (génétique)</term><term>ARN viral (génétique)</term><term>Animaux</term><term>Cellules Vero</term><term>Cellules cultivées</term><term>Ciblage de gène</term><term>Données de séquences moléculaires</term><term>Expression des gènes</term><term>Extinction de l'expression des gènes</term><term>Gènes rapporteurs</term><term>Interférence par ARN</term><term>Microscopie de fluorescence</term><term>Oligonucléotides antisens (génétique)</term><term>Petit ARN interférent (génétique)</term><term>Régions 5' non traduites (génétique)</term><term>Réplication virale (génétique)</term><term>Séquence nucléotidique</term><term>Technique de Western</term><term>Transfection</term><term>Virus du SRAS (génétique)</term><term>Virus du SRAS (physiologie)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>5' Untranslated Regions</term><term>Oligonucleotides, Antisense</term><term>RNA, Messenger</term><term>RNA, Small Interfering</term><term>RNA, Viral</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>SARS Virus</term><term>Virus Replication</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>ARN messager</term><term>ARN viral</term><term>Oligonucléotides antisens</term><term>Petit ARN interférent</term><term>Régions 5' non traduites</term><term>Réplication virale</term><term>Virus du SRAS</term></keywords><keywords scheme="MESH" qualifier="physiologie" xml:lang="fr"><term>Virus du SRAS</term></keywords><keywords scheme="MESH" qualifier="physiology" xml:lang="en"><term>SARS Virus</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Animals</term><term>Base Sequence</term><term>Blotting, Western</term><term>Cells, Cultured</term><term>Chlorocebus aethiops</term><term>Gene Expression</term><term>Gene Silencing</term><term>Gene Targeting</term><term>Genes, Reporter</term><term>Microscopy, Fluorescence</term><term>Molecular Sequence Data</term><term>RNA Interference</term><term>Transfection</term><term>Vero Cells</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Animaux</term><term>Cellules Vero</term><term>Cellules cultivées</term><term>Ciblage de gène</term><term>Données de séquences moléculaires</term><term>Expression des gènes</term><term>Extinction de l'expression des gènes</term><term>Gènes rapporteurs</term><term>Interférence par ARN</term><term>Microscopie de fluorescence</term><term>Séquence nucléotidique</term><term>Technique de Western</term><term>Transfection</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">SARS-CoV (the SARS-Associated Coronavirus) was reported as a novel virus member in the coronavirus family, which was the cause of severe acute respiratory syndrome. Coronavirus replication occurs through a unique mechanism employing Leader sequence in the transcripts when initiating transcription from the genome. Therefore, we cloned the Leader sequence from SARS-CoV(BJ01), which is identical to that identified from SARS-CoV(HKU-39849), and constructed specific siRNA targeting the Leader sequence. Using EGFP and RFP reporter genes fused with the cloned SARS-CoV Leader sequence, we demonstrated that the siRNA targeting the Leader sequence decreased the mRNA abundance and protein expression levels of the reporter genes in 293T cells. By stably expressing the siRNA in Vero E6 cells, we provided data that the siRNA could effectively and specifically decrease the mRNA abundance of SARS-CoV genes as analyzed by RT-PCR and Northern blot. Our data indicated that the siRNA targeting the Leader sequence inhibited the replication of SARS-CoV in Vero E6 cells by silencing gene expression. We further demonstrated, via transient transfection experiments, that the siRNA targeting the Leader sequence had a much stronger inhibitory effect on SARS-CoV replication than the siRNAs targeting the Spike gene or the antisense oligodeoxynucleotides did. This report provides evidence that targeting Leader sequence using siRNA could be a powerful tool in inhibiting SARS-CoV replication.</div></front></TEI></pubmed></double></record>Pour manipuler ce document sous Unix (Dilib)
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