Biochemical Characterization of Middle East Respiratory Syndrome Coronavirus Helicase

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 propert...

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Autores principales:	Adeyemi O. Adedeji, Hilary Lazarus
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2016
Materias:	ATP hydrolysis DNA RNA coronavirus enzyme kinetics helicase Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/96f954f4cd944f5990ade34557681cc0
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spelling	oai:doaj.org-article:96f954f4cd944f5990ade34557681cc02021-11-15T15:21:30ZBiochemical Characterization of Middle East Respiratory Syndrome Coronavirus Helicase10.1128/mSphere.00235-162379-5042https://doaj.org/article/96f954f4cd944f5990ade34557681cc02016-10-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00235-16https://doaj.org/toc/2379-5042ABSTRACT 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.Adeyemi O. AdedejiHilary LazarusAmerican Society for MicrobiologyarticleATP hydrolysisDNARNAcoronavirusenzyme kineticshelicaseMicrobiologyQR1-502ENmSphere, Vol 1, Iss 5 (2016)
institution	DOAJ
collection	DOAJ
language	EN
topic	ATP hydrolysis DNA RNA coronavirus enzyme kinetics helicase Microbiology QR1-502
spellingShingle	ATP hydrolysis DNA RNA coronavirus enzyme kinetics helicase Microbiology QR1-502 Adeyemi O. Adedeji Hilary Lazarus Biochemical Characterization of Middle East Respiratory Syndrome Coronavirus Helicase
description	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.
format	article
author	Adeyemi O. Adedeji Hilary Lazarus
author_facet	Adeyemi O. Adedeji Hilary Lazarus
author_sort	Adeyemi O. Adedeji
title	Biochemical Characterization of Middle East Respiratory Syndrome Coronavirus Helicase
title_short	Biochemical Characterization of Middle East Respiratory Syndrome Coronavirus Helicase
title_full	Biochemical Characterization of Middle East Respiratory Syndrome Coronavirus Helicase
title_fullStr	Biochemical Characterization of Middle East Respiratory Syndrome Coronavirus Helicase
title_full_unstemmed	Biochemical Characterization of Middle East Respiratory Syndrome Coronavirus Helicase
title_sort	biochemical characterization of middle east respiratory syndrome coronavirus helicase
publisher	American Society for Microbiology
publishDate	2016
url	https://doaj.org/article/96f954f4cd944f5990ade34557681cc0
work_keys_str_mv	AT adeyemioadedeji biochemicalcharacterizationofmiddleeastrespiratorysyndromecoronavirushelicase AT hilarylazarus biochemicalcharacterizationofmiddleeastrespiratorysyndromecoronavirushelicase
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Biochemical Characterization of Middle East Respiratory Syndrome Coronavirus Helicase

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