Inhibition of SARS-CoV replication cycle by small interference RNAs silencing specific sars proteins, 7a/7b, 3a/3b and s
Identifieur interne : 000369 ( PascalFrancis/Checkpoint ); précédent : 000368; suivant : 000370Inhibition of SARS-CoV replication cycle by small interference RNAs silencing specific sars proteins, 7a/7b, 3a/3b and s
Auteurs : Sara Akerström [Suède] ; Ali Mirazimi [Suède] ; Yee-Joo Tan [Singapour]Source :
- Antiviral research [ 0166-3542 ] ; 2007.
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Abstract
The severe acute respiratory syndrome coronavirus (SARS CoV) genome has 14 potential open reading frames (ORFs). The first ORF is translated from the full-length genomic mRNA while the remaining ORFs are translated from eight subgeomic RNAs (sgRNAs). In this study, we designed small interference RNAs (siRNAs) targeting sgRNA 2, 3 and 7 and tested their efficiency and specificity in silencing the protein translated from the targeted sgRNA. Our results demonstrated that siRNA 7 could inhibit sgRNA 7, which showed 19/19 nucleotides (nt) matching, and sgRNA 8, which showed 18/19 nt matching; but, it did not inhibit the full-length genomic mRNA which showed 17/19 nt matching. Overall, each of the siRNAs can inhibit the targeted sgRNA without affecting the full-length genomic mRNA or the other sgRNAs that showed mismatch of two or more nt. Thus, siRNA could be designed so as to knockdown the expression of viral protein(s) from a targeted sgRNA during viral infection, thereby allowing the contribution of individual viral proteins to viral infection to be delineated. When Vero E6 cells expressing siRNA 2, 3 or 7 were infected with SARS-CoV, a significant reduction in the yield of progeny virus was observed. Indirect immunofluorescence assays showed that in the infected cells expressing each of the siRNAs, there was aspecific silencing of S, 3a and 7a, respectively, but the expression of nucleocapsid protein was not affected. Thus, our data suggests that the accessory proteins, i.e. 3a and 7a, could play an important role during the replication cycle of the SARS-CoV.
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en" level="a">Inhibition of SARS-CoV replication cycle by small interference RNAs silencing specific sars proteins, 7a/7b, 3a/3b and s</title><author><name sortKey="Akerstrom, Sara" sort="Akerstrom, Sara" uniqKey="Akerstrom S" first="Sara" last="Akerström">Sara Akerström</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Center for Microbiological Preparedness, Swedish Institute for Infectious Disease Control</s1><s2>171 82 Solna</s2><s3>SWE</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ></inist:fA14><country>Suède</country><wicri:noRegion>171 82 Solna</wicri:noRegion></affiliation></author><author><name sortKey="Mirazimi, Ali" sort="Mirazimi, Ali" uniqKey="Mirazimi A" first="Ali" last="Mirazimi">Ali Mirazimi</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Center for Microbiological Preparedness, Swedish Institute for Infectious Disease Control</s1><s2>171 82 Solna</s2><s3>SWE</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ></inist:fA14><country>Suède</country><wicri:noRegion>171 82 Solna</wicri:noRegion></affiliation></author><author><name sortKey="Tan, Yee Joo" sort="Tan, Yee Joo" uniqKey="Tan Y" first="Yee-Joo" last="Tan">Yee-Joo Tan</name><affiliation wicri:level="1"><inist:fA14 i1="02"><s1>Institute of Molecular and Cell Biology, 61 Biopolis Drive</s1><s2>138673 Singapore</s2><s3>SGP</s3><sZ>3 aut.</sZ></inist:fA14><country>Singapour</country><wicri:noRegion>138673 Singapore</wicri:noRegion></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">INIST</idno><idno type="inist">07-0183200</idno><date when="2007">2007</date><idno type="stanalyst">PASCAL 07-0183200 INIST</idno><idno type="RBID">Pascal:07-0183200</idno><idno type="wicri:Area/PascalFrancis/Corpus">000383</idno><idno type="wicri:Area/PascalFrancis/Curation">000606</idno><idno type="wicri:Area/PascalFrancis/Checkpoint">000369</idno><idno type="wicri:explorRef" wicri:stream="PascalFrancis" wicri:step="Checkpoint">000369</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a">Inhibition of SARS-CoV replication cycle by small interference RNAs silencing specific sars proteins, 7a/7b, 3a/3b and s</title><author><name sortKey="Akerstrom, Sara" sort="Akerstrom, Sara" uniqKey="Akerstrom S" first="Sara" last="Akerström">Sara Akerström</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Center for Microbiological Preparedness, Swedish Institute for Infectious Disease Control</s1><s2>171 82 Solna</s2><s3>SWE</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ></inist:fA14><country>Suède</country><wicri:noRegion>171 82 Solna</wicri:noRegion></affiliation></author><author><name sortKey="Mirazimi, Ali" sort="Mirazimi, Ali" uniqKey="Mirazimi A" first="Ali" last="Mirazimi">Ali Mirazimi</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Center for Microbiological Preparedness, Swedish Institute for Infectious Disease Control</s1><s2>171 82 Solna</s2><s3>SWE</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ></inist:fA14><country>Suède</country><wicri:noRegion>171 82 Solna</wicri:noRegion></affiliation></author><author><name sortKey="Tan, Yee Joo" sort="Tan, Yee Joo" uniqKey="Tan Y" first="Yee-Joo" last="Tan">Yee-Joo Tan</name><affiliation wicri:level="1"><inist:fA14 i1="02"><s1>Institute of Molecular and Cell Biology, 61 Biopolis Drive</s1><s2>138673 Singapore</s2><s3>SGP</s3><sZ>3 aut.</sZ></inist:fA14><country>Singapour</country><wicri:noRegion>138673 Singapore</wicri:noRegion></affiliation></author></analytic><series><title level="j" type="main">Antiviral research</title><title level="j" type="abbreviated">Antivir. res.</title><idno type="ISSN">0166-3542</idno><imprint><date when="2007">2007</date></imprint></series></biblStruct></sourceDesc><seriesStmt><title level="j" type="main">Antiviral research</title><title level="j" type="abbreviated">Antivir. res.</title><idno type="ISSN">0166-3542</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Antiviral</term><term>Coronavirus</term><term>Gene silencing</term><term>Protein</term><term>RNA interference</term><term>Replication</term><term>Severe acute respiratory syndrome</term><term>Severe acute respiratory syndrome virus</term><term>Small Interference RNA</term></keywords><keywords scheme="Pascal" xml:lang="fr"><term>Coronavirus</term><term>Syndrome respiratoire aigu sévère</term><term>Réplication</term><term>siRNA</term><term>Protéine</term><term>Silence expression génique</term><term>Virus syndrome respiratoire aigu sévère</term><term>Antiviral</term><term>Interférence ARN</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The severe acute respiratory syndrome coronavirus (SARS CoV) genome has 14 potential open reading frames (ORFs). The first ORF is translated from the full-length genomic mRNA while the remaining ORFs are translated from eight subgeomic RNAs (sgRNAs). In this study, we designed small interference RNAs (siRNAs) targeting sgRNA 2, 3 and 7 and tested their efficiency and specificity in silencing the protein translated from the targeted sgRNA. Our results demonstrated that siRNA 7 could inhibit sgRNA 7, which showed 19/19 nucleotides (nt) matching, and sgRNA 8, which showed 18/19 nt matching; but, it did not inhibit the full-length genomic mRNA which showed 17/19 nt matching. Overall, each of the siRNAs can inhibit the targeted sgRNA without affecting the full-length genomic mRNA or the other sgRNAs that showed mismatch of two or more nt. Thus, siRNA could be designed so as to knockdown the expression of viral protein(s) from a targeted sgRNA during viral infection, thereby allowing the contribution of individual viral proteins to viral infection to be delineated. When Vero E6 cells expressing siRNA 2, 3 or 7 were infected with SARS-CoV, a significant reduction in the yield of progeny virus was observed. Indirect immunofluorescence assays showed that in the infected cells expressing each of the siRNAs, there was aspecific silencing of S, 3a and 7a, respectively, but the expression of nucleocapsid protein was not affected. Thus, our data suggests that the accessory proteins, i.e. 3a and 7a, could play an important role during the replication cycle of the SARS-CoV.</div></front></TEI><inist><standard h6="B"><pA><fA01 i1="01" i2="1"><s0>0166-3542</s0></fA01><fA02 i1="01"><s0>ARSRDR</s0></fA02><fA03 i2="1"><s0>Antivir. res.</s0></fA03><fA05><s2>73</s2></fA05><fA06><s2>3</s2></fA06><fA08 i1="01" i2="1" l="ENG"><s1>Inhibition of SARS-CoV replication cycle by small interference RNAs silencing specific sars proteins, 7a/7b, 3a/3b and s</s1></fA08><fA11 i1="01" i2="1"><s1>AKERSTRÖM (Sara)</s1></fA11><fA11 i1="02" i2="1"><s1>MIRAZIMI (Ali)</s1></fA11><fA11 i1="03" i2="1"><s1>TAN (Yee-Joo)</s1></fA11><fA14 i1="01"><s1>Center for Microbiological Preparedness, Swedish Institute for Infectious Disease Control</s1><s2>171 82 Solna</s2><s3>SWE</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ></fA14><fA14 i1="02"><s1>Institute of Molecular and Cell Biology, 61 Biopolis Drive</s1><s2>138673 Singapore</s2><s3>SGP</s3><sZ>3 aut.</sZ></fA14><fA20><s1>219-227</s1></fA20><fA21><s1>2007</s1></fA21><fA23 i1="01"><s0>ENG</s0></fA23><fA43 i1="01"><s1>INIST</s1><s2>18839</s2><s5>354000147008440100</s5></fA43><fA44><s0>0000</s0><s1>© 2007 INIST-CNRS. 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