Charge Engineering of the Nucleic Acid Binding Cleft of a Thermostable HIV-1 Reverse Transcriptase Reveals Key Interactions and a Novel Mechanism of RNase H Inactivation

Coupled with PCR, reverse transcriptases (RTs) have been widely used for RNA detection and gene expression analysis. Increased thermostability and nucleic acid binding affinity are desirable RT properties to improve yields and sensitivity of these applications. The effects of amino acid substitution...

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Autores: Martínez del Río, Javier, López-Carrobles, Nerea, Mendieta-Moreno, Jesús I., Herrera-Chacón, Óscar, Sánchez-Ibáñez, Adrián, Menéndez-Arias, Luis
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2023
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:dnet:digitalcsic_::cb4563b4c96b986de2878a264ee4845d
Acceso en línea:http://hdl.handle.net/10261/342287
Access Level:acceso abierto
Palabra clave:Reverse transcriptase
RNase H
Polymerase engineering
RT-PCR
Template-primer binding
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spelling Charge Engineering of the Nucleic Acid Binding Cleft of a Thermostable HIV-1 Reverse Transcriptase Reveals Key Interactions and a Novel Mechanism of RNase H InactivationMartínez del Río, JavierLópez-Carrobles, NereaMendieta-Moreno, Jesús I.Herrera-Chacón, ÓscarSánchez-Ibáñez, AdriánMendieta-Moreno, Jesús I.Menéndez-Arias, LuisReverse transcriptaseRNase HPolymerase engineeringRT-PCRTemplate-primer bindingCoupled with PCR, reverse transcriptases (RTs) have been widely used for RNA detection and gene expression analysis. Increased thermostability and nucleic acid binding affinity are desirable RT properties to improve yields and sensitivity of these applications. The effects of amino acid substitutions in the RT RNase H domain were tested in an engineered HIV-1 group O RT, containing mutations K358R/A359G/S360A and devoid of RNase H activity due to the presence of E478Q (O3MQ RT). Twenty mutant RTs with Lys or Arg at positions interacting with the template-primer (i.e., at positions 473–477, 499–502 and 505) were obtained and characterized. Most of them produced significant amounts of cDNA at 37, 50 and 65 °C, as determined in RT-PCR reactions. However, a big loss of activity was observed with mutants A477K/R, S499K/R, V502K/R and Y505K/R, particularly at 65 °C. Binding affinity experiments confirmed that residues 477, 502 and 505 were less tolerant to mutations. Amino acid substitutions Q500K and Q500R produced a slight increase of cDNA synthesis efficiency at 50 and 65 °C, without altering the K for model DNA/DNA and RNA/DNA heteroduplexes. Interestingly, molecular dynamics simulations predicted that those mutations inactivate the RNase H activity by altering the geometry of the catalytic site. Proof of this unexpected effect was obtained after introducing Q500K or Q500R in the wild-type HIV-1 RT and mutant K358R/A359G/S360A RT. Our results reveal a novel mechanism of RNase H inactivation that preserves RT DNA binding and polymerization efficiency without substituting RNase H active site residues.his work was supported in part by the Ministry of Science and Innovation of Spain through grants PID2019-104176RB-I00/AEI/MCI/10.13039/501100011033 awarded to L.M.-A, and PID2021-125604NB-I00/AEI/MCI/10.13039/501100011033 (supporting J.M. and J.I.M.-M.). J.M.R. is a predoctoral fellow of the Spanish Ministry of Universities (Formación de Profesorado Universitario, FPU19/01653). N.L.-C. was supported by a contract (PEJ-2020-AI/BMD-19429) of the Youth Guarantee programme of the European Union, with the participation of the Comunidad de Madrid (Consejería de Educación, Universidades, Ciencia y Portavocía). A.S.-I. received a short-term fellowship from CSIC (JAE-Intro program, JAEINT_20_00736/JAEINT_20_EX_0774). An institutional grant of Fundación Ramón Areces to the CBMSO is also acknowledged. The CBMSO has been certified since 2023 as Severo Ochoa Center of Excellence by AEI/MCI/10.13039/501100011033. The team at CBMSO is member of the Global Virus Network.Ministerio de Ciencia e Innovación (España)Ministerio de Universidades (España)European CommissionComunidad de MadridFundación Ramón ArecesConsejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]2024202420232024info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionhttp://hdl.handle.net/10261/342287reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Inglés#PLACEHOLDER_PARENT_METADATA_VALUE#info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/PID2019-104176RB-I00http://dx.doi.org/10.1016/j.jmb.2023.168219Síinfo:eu-repo/semantics/openAccessoai:dnet:digitalcsic_::cb4563b4c96b986de2878a264ee4845d2026-05-22T06:33:51Z
dc.title.none.fl_str_mv Charge Engineering of the Nucleic Acid Binding Cleft of a Thermostable HIV-1 Reverse Transcriptase Reveals Key Interactions and a Novel Mechanism of RNase H Inactivation
title Charge Engineering of the Nucleic Acid Binding Cleft of a Thermostable HIV-1 Reverse Transcriptase Reveals Key Interactions and a Novel Mechanism of RNase H Inactivation
spellingShingle Charge Engineering of the Nucleic Acid Binding Cleft of a Thermostable HIV-1 Reverse Transcriptase Reveals Key Interactions and a Novel Mechanism of RNase H Inactivation
Martínez del Río, Javier
Reverse transcriptase
RNase H
Polymerase engineering
RT-PCR
Template-primer binding
title_short Charge Engineering of the Nucleic Acid Binding Cleft of a Thermostable HIV-1 Reverse Transcriptase Reveals Key Interactions and a Novel Mechanism of RNase H Inactivation
title_full Charge Engineering of the Nucleic Acid Binding Cleft of a Thermostable HIV-1 Reverse Transcriptase Reveals Key Interactions and a Novel Mechanism of RNase H Inactivation
title_fullStr Charge Engineering of the Nucleic Acid Binding Cleft of a Thermostable HIV-1 Reverse Transcriptase Reveals Key Interactions and a Novel Mechanism of RNase H Inactivation
title_full_unstemmed Charge Engineering of the Nucleic Acid Binding Cleft of a Thermostable HIV-1 Reverse Transcriptase Reveals Key Interactions and a Novel Mechanism of RNase H Inactivation
title_sort Charge Engineering of the Nucleic Acid Binding Cleft of a Thermostable HIV-1 Reverse Transcriptase Reveals Key Interactions and a Novel Mechanism of RNase H Inactivation
dc.creator.none.fl_str_mv Martínez del Río, Javier
López-Carrobles, Nerea
Mendieta-Moreno, Jesús I.
Herrera-Chacón, Óscar
Sánchez-Ibáñez, Adrián
Mendieta-Moreno, Jesús I.
Menéndez-Arias, Luis
author Martínez del Río, Javier
author_facet Martínez del Río, Javier
López-Carrobles, Nerea
Mendieta-Moreno, Jesús I.
Herrera-Chacón, Óscar
Sánchez-Ibáñez, Adrián
Menéndez-Arias, Luis
author_role author
author2 López-Carrobles, Nerea
Mendieta-Moreno, Jesús I.
Herrera-Chacón, Óscar
Sánchez-Ibáñez, Adrián
Menéndez-Arias, Luis
author2_role author
author
author
author
author
dc.contributor.none.fl_str_mv Ministerio de Ciencia e Innovación (España)
Ministerio de Universidades (España)
European Commission
Comunidad de Madrid
Fundación Ramón Areces
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv Reverse transcriptase
RNase H
Polymerase engineering
RT-PCR
Template-primer binding
topic Reverse transcriptase
RNase H
Polymerase engineering
RT-PCR
Template-primer binding
description Coupled with PCR, reverse transcriptases (RTs) have been widely used for RNA detection and gene expression analysis. Increased thermostability and nucleic acid binding affinity are desirable RT properties to improve yields and sensitivity of these applications. The effects of amino acid substitutions in the RT RNase H domain were tested in an engineered HIV-1 group O RT, containing mutations K358R/A359G/S360A and devoid of RNase H activity due to the presence of E478Q (O3MQ RT). Twenty mutant RTs with Lys or Arg at positions interacting with the template-primer (i.e., at positions 473–477, 499–502 and 505) were obtained and characterized. Most of them produced significant amounts of cDNA at 37, 50 and 65 °C, as determined in RT-PCR reactions. However, a big loss of activity was observed with mutants A477K/R, S499K/R, V502K/R and Y505K/R, particularly at 65 °C. Binding affinity experiments confirmed that residues 477, 502 and 505 were less tolerant to mutations. Amino acid substitutions Q500K and Q500R produced a slight increase of cDNA synthesis efficiency at 50 and 65 °C, without altering the K for model DNA/DNA and RNA/DNA heteroduplexes. Interestingly, molecular dynamics simulations predicted that those mutations inactivate the RNase H activity by altering the geometry of the catalytic site. Proof of this unexpected effect was obtained after introducing Q500K or Q500R in the wild-type HIV-1 RT and mutant K358R/A359G/S360A RT. Our results reveal a novel mechanism of RNase H inactivation that preserves RT DNA binding and polymerization efficiency without substituting RNase H active site residues.
publishDate 2023
dc.date.none.fl_str_mv 2023
2024
2024
2024
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Publisher's version
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/342287
url http://hdl.handle.net/10261/342287
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv #PLACEHOLDER_PARENT_METADATA_VALUE#
info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/PID2019-104176RB-I00
http://dx.doi.org/10.1016/j.jmb.2023.168219
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
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