Biochemical Characterization of Middle East Respiratory Syndrome Coronavirus Helicase.
Identifieur interne : 000F66 ( PubMed/Corpus ); précédent : 000F65; suivant : 000F67Biochemical Characterization of Middle East Respiratory Syndrome Coronavirus Helicase.
Auteurs : Adeyemi O. Adedeji ; Hilary LazarusSource :
- mSphere [ 2379-5042 ]
Abstract
Middle East respiratory syndrome coronavirus (MERS-CoV) helicase is a superfamily 1 helicase containing seven conserved motifs. We have cloned, expressed, and purified a Strep-fused recombinant MERS-CoV nonstructural protein 13 (M-nsp13) helicase. Characterization of its biochemical properties showed that it unwound DNA and RNA similarly to severe acute respiratory syndrome CoV nsp13 (S-nsp13) helicase. We showed that M-nsp13 unwound in a 5'-to-3' direction and efficiently unwound the partially duplex RNA substrates with a long loading strand relative to those of the RNA substrates with a short or no loading strand. Moreover, the Km of ATP for M-nsp13 is inversely proportional to the length of the 5' loading strand of the partially duplex RNA substrates. Finally, we also showed that the rate of unwinding (ku) of M-nsp13 is directly proportional to the length of the 5' loading strand of the partially duplex RNA substrate. These results provide insights that enhance our understanding of the biochemical properties of M-nsp13. IMPORTANCE Coronaviruses are known to cause a wide range of diseases in humans and animals. Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel coronavirus discovered in 2012 and is responsible for acute respiratory syndrome in humans in the Middle East, Europe, North Africa, and the United States of America. Helicases are motor proteins that catalyze the processive separation of double-stranded nucleic acids into two single-stranded nucleic acids by utilizing the energy derived from ATP hydrolysis. MERS-CoV helicase is one of the most important viral replication enzymes of this coronavirus. Herein, we report the first bacterial expression, enzyme purification, and biochemical characterization of MERS-CoV helicase. The knowledge obtained from this study might be used to identify an inhibitor of MERS-CoV replication, and the helicase might be used as a therapeutic target.
DOI: 10.1128/mSphere.00235-16
PubMed: 27631026
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Biochemical Characterization of Middle East Respiratory Syndrome Coronavirus Helicase.</title><author><name sortKey="Adedeji, Adeyemi O" sort="Adedeji, Adeyemi O" uniqKey="Adedeji A" first="Adeyemi O" last="Adedeji">Adeyemi O. Adedeji</name><affiliation><nlm:affiliation>Department of Pathology and Population Medicine, College of Veterinary Medicine, Midwestern University, Glendale, Arizona, USA.</nlm:affiliation></affiliation></author><author><name sortKey="Lazarus, Hilary" sort="Lazarus, Hilary" uniqKey="Lazarus H" first="Hilary" last="Lazarus">Hilary Lazarus</name><affiliation><nlm:affiliation>Department of Pathology and Population Medicine, College of Veterinary Medicine, Midwestern University, Glendale, Arizona, USA.</nlm:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="????"><PubDate><MedlineDate>2016 Sep-Oct</MedlineDate></PubDate></date><idno type="RBID">pubmed:27631026</idno><idno type="pmid">27631026</idno><idno type="doi">10.1128/mSphere.00235-16</idno><idno type="wicri:Area/PubMed/Corpus">000F66</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">000F66</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Biochemical Characterization of Middle East Respiratory Syndrome Coronavirus Helicase.</title><author><name sortKey="Adedeji, Adeyemi O" sort="Adedeji, Adeyemi O" uniqKey="Adedeji A" first="Adeyemi O" last="Adedeji">Adeyemi O. Adedeji</name><affiliation><nlm:affiliation>Department of Pathology and Population Medicine, College of Veterinary Medicine, Midwestern University, Glendale, Arizona, USA.</nlm:affiliation></affiliation></author><author><name sortKey="Lazarus, Hilary" sort="Lazarus, Hilary" uniqKey="Lazarus H" first="Hilary" last="Lazarus">Hilary Lazarus</name><affiliation><nlm:affiliation>Department of Pathology and Population Medicine, College of Veterinary Medicine, Midwestern University, Glendale, Arizona, USA.</nlm:affiliation></affiliation></author></analytic><series><title level="j">mSphere</title><idno type="ISSN">2379-5042</idno></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Middle East respiratory syndrome coronavirus (MERS-CoV) helicase is a superfamily 1 helicase containing seven conserved motifs. We have cloned, expressed, and purified a Strep-fused recombinant MERS-CoV nonstructural protein 13 (M-nsp13) helicase. Characterization of its biochemical properties showed that it unwound DNA and RNA similarly to severe acute respiratory syndrome CoV nsp13 (S-nsp13) helicase. We showed that M-nsp13 unwound in a 5'-to-3' direction and efficiently unwound the partially duplex RNA substrates with a long loading strand relative to those of the RNA substrates with a short or no loading strand. Moreover, the Km of ATP for M-nsp13 is inversely proportional to the length of the 5' loading strand of the partially duplex RNA substrates. Finally, we also showed that the rate of unwinding (ku) of M-nsp13 is directly proportional to the length of the 5' loading strand of the partially duplex RNA substrate. These results provide insights that enhance our understanding of the biochemical properties of M-nsp13. IMPORTANCE Coronaviruses are known to cause a wide range of diseases in humans and animals. Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel coronavirus discovered in 2012 and is responsible for acute respiratory syndrome in humans in the Middle East, Europe, North Africa, and the United States of America. Helicases are motor proteins that catalyze the processive separation of double-stranded nucleic acids into two single-stranded nucleic acids by utilizing the energy derived from ATP hydrolysis. MERS-CoV helicase is one of the most important viral replication enzymes of this coronavirus. Herein, we report the first bacterial expression, enzyme purification, and biochemical characterization of MERS-CoV helicase. The knowledge obtained from this study might be used to identify an inhibitor of MERS-CoV replication, and the helicase might be used as a therapeutic target. </div></front></TEI><pubmed><MedlineCitation Status="PubMed-not-MEDLINE" Owner="NLM"><PMID Version="1">27631026</PMID><DateCompleted><Year>2016</Year><Month>09</Month><Day>16</Day></DateCompleted><DateRevised><Year>2018</Year><Month>11</Month><Day>13</Day></DateRevised><Article PubModel="Electronic-eCollection"><Journal><ISSN IssnType="Print">2379-5042</ISSN><JournalIssue CitedMedium="Print"><Volume>1</Volume><Issue>5</Issue><PubDate><MedlineDate>2016 Sep-Oct</MedlineDate></PubDate></JournalIssue><Title>mSphere</Title><ISOAbbreviation>mSphere</ISOAbbreviation></Journal><ArticleTitle>Biochemical Characterization of Middle East Respiratory Syndrome Coronavirus Helicase.</ArticleTitle><ELocationID EIdType="doi" ValidYN="Y">10.1128/mSphere.00235-16</ELocationID><ELocationID EIdType="pii" ValidYN="Y">e00235-16</ELocationID><Abstract><AbstractText>Middle East respiratory syndrome coronavirus (MERS-CoV) helicase is a superfamily 1 helicase containing seven conserved motifs. We have cloned, expressed, and purified a Strep-fused recombinant MERS-CoV nonstructural protein 13 (M-nsp13) helicase. Characterization of its biochemical properties showed that it unwound DNA and RNA similarly to severe acute respiratory syndrome CoV nsp13 (S-nsp13) helicase. We showed that M-nsp13 unwound in a 5'-to-3' direction and efficiently unwound the partially duplex RNA substrates with a long loading strand relative to those of the RNA substrates with a short or no loading strand. Moreover, the Km of ATP for M-nsp13 is inversely proportional to the length of the 5' loading strand of the partially duplex RNA substrates. Finally, we also showed that the rate of unwinding (ku) of M-nsp13 is directly proportional to the length of the 5' loading strand of the partially duplex RNA substrate. These results provide insights that enhance our understanding of the biochemical properties of M-nsp13. IMPORTANCE Coronaviruses are known to cause a wide range of diseases in humans and animals. Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel coronavirus discovered in 2012 and is responsible for acute respiratory syndrome in humans in the Middle East, Europe, North Africa, and the United States of America. Helicases are motor proteins that catalyze the processive separation of double-stranded nucleic acids into two single-stranded nucleic acids by utilizing the energy derived from ATP hydrolysis. MERS-CoV helicase is one of the most important viral replication enzymes of this coronavirus. Herein, we report the first bacterial expression, enzyme purification, and biochemical characterization of MERS-CoV helicase. The knowledge obtained from this study might be used to identify an inhibitor of MERS-CoV replication, and the helicase might be used as a therapeutic target. </AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Adedeji</LastName><ForeName>Adeyemi O</ForeName><Initials>AO</Initials><AffiliationInfo><Affiliation>Department of Pathology and Population Medicine, College of Veterinary Medicine, Midwestern University, Glendale, Arizona, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Lazarus</LastName><ForeName>Hilary</ForeName><Initials>H</Initials><AffiliationInfo><Affiliation>Department of Pathology and Population Medicine, College of Veterinary Medicine, Midwestern University, Glendale, Arizona, USA.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2016</Year><Month>09</Month><Day>07</Day></ArticleDate></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>mSphere</MedlineTA><NlmUniqueID>101674533</NlmUniqueID><ISSNLinking>2379-5042</ISSNLinking></MedlineJournalInfo><KeywordList Owner="NOTNLM"><Keyword MajorTopicYN="N">ATP hydrolysis</Keyword><Keyword MajorTopicYN="N">DNA</Keyword><Keyword MajorTopicYN="N">RNA</Keyword><Keyword MajorTopicYN="N">coronavirus</Keyword><Keyword MajorTopicYN="N">enzyme kinetics</Keyword><Keyword MajorTopicYN="N">helicase</Keyword></KeywordList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="received"><Year>2016</Year><Month>08</Month><Day>12</Day></PubMedPubDate><PubMedPubDate PubStatus="accepted"><Year>2016</Year><Month>08</Month><Day>19</Day></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2016</Year><Month>9</Month><Day>16</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate 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