Defective Strand-Displacement DNA Synthesis Due to Accumulation of Thymidine Analogue Resistance Mutations in HIV-2 Reverse Transcriptase

Retroviral reverse transcriptases (RTs) have the ability to carry out strand displacement DNA synthesis in the absence of accessory proteins. Although studies with RTs and other DNA polymerases suggest that fingers subdomain residues participate in strand displacement, molecular determinants of this...

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Autores: Martín-Alonso, Samara, Álvarez, Mar, Nevot, María, Martínez, Miguel, A., Menéndez-Arias, Luis
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2020
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/228592
Acceso en línea:http://hdl.handle.net/10261/228592
Access Level:acceso abierto
Palabra clave:Strand displacement
HIV
Reverse transcriptase
Antiretroviral drugs
DNA polymerases
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spelling Defective Strand-Displacement DNA Synthesis Due to Accumulation of Thymidine Analogue Resistance Mutations in HIV-2 Reverse TranscriptaseMartín-Alonso, SamaraÁlvarez, MarNevot, MaríaMartínez, Miguel, A.Menéndez-Arias, LuisStrand displacementHIVReverse transcriptaseAntiretroviral drugsDNA polymerasesRetroviral reverse transcriptases (RTs) have the ability to carry out strand displacement DNA synthesis in the absence of accessory proteins. Although studies with RTs and other DNA polymerases suggest that fingers subdomain residues participate in strand displacement, molecular determinants of this activity are still unknown. A mutant human immunodeficiency virus type 2 (HIV-2) RT (M41L/D67N/K70R/S215Y) with low strand displacement activity was identified after screening a panel of purified enzymes, including several antiretroviral drug-resistant HIV-1 and HIV-2 RTs. In HIV-1, resistance to zidovudine and other thymidine analogues is conferred by different combinations of M41L, D67N, K70R, L210W, T215F/Y, and K219E/Q (designated as thymidine analogue resistance-associated mutations (TAMs)). However, those changes are rarely selected in HIV-2. We show that the strand displacement activity of HIV-2ROD mutants M41L/S215Y and D67N/K70R was only slightly reduced compared to the wild-type RT. In contrast, mutants D67N/K70R/S215Y and M41L/D67N/K70R/S215Y were the most defective RTs in reactions carried out with nicked and gapped substrates. Moreover, these enzymes showed the lowest nucleotide incorporation rates in assays carried out with strand displacement substrates. Unlike in HIV-2, substitutions M41L/T215Y and D67N/K70R/T215Y/K219Q had no effect on the strand displacement activity of HIV-1BH10 RT. The strand displacement efficiencies of HIV-2ROD RTs were consistent with the lower replication capacity of HIV-2 strains bearing the four major TAMs in their RT. Our results highlight the role of the fingers subdomain in strand displacement. These findings might be important for the development of strand-displacement defective RTs.Ministry of Science, Innovation and Universities of Spain through grants BIO2016-76716-R (AEI/FEDER, UE) to L.M.-A. and SAF2016-75277-R to M.A.M.; S.M.-A. is a predoctoral fellow of the Ministry of Science, Innovation and Universities of Spain (BES-2017-079836). M.N. was supported by the Instituto de Salud Carlos III through the Spanish AIDS network (RD16-0025/0041). An institutional grant of the Fundación Ramón Areces to the CBMSO is also acknowledgedMinisterio de Ciencia, Innovación y Universidades (España)Instituto de Salud Carlos IIIFundación Ramón ArecesConsejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]2021202120202021info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionhttp://hdl.handle.net/10261/228592reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Ingléshttp://dx.doi.org/10.1021/acsinfecdis.9b00512Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/2285922026-05-22T06:33:51Z
dc.title.none.fl_str_mv Defective Strand-Displacement DNA Synthesis Due to Accumulation of Thymidine Analogue Resistance Mutations in HIV-2 Reverse Transcriptase
title Defective Strand-Displacement DNA Synthesis Due to Accumulation of Thymidine Analogue Resistance Mutations in HIV-2 Reverse Transcriptase
spellingShingle Defective Strand-Displacement DNA Synthesis Due to Accumulation of Thymidine Analogue Resistance Mutations in HIV-2 Reverse Transcriptase
Martín-Alonso, Samara
Strand displacement
HIV
Reverse transcriptase
Antiretroviral drugs
DNA polymerases
title_short Defective Strand-Displacement DNA Synthesis Due to Accumulation of Thymidine Analogue Resistance Mutations in HIV-2 Reverse Transcriptase
title_full Defective Strand-Displacement DNA Synthesis Due to Accumulation of Thymidine Analogue Resistance Mutations in HIV-2 Reverse Transcriptase
title_fullStr Defective Strand-Displacement DNA Synthesis Due to Accumulation of Thymidine Analogue Resistance Mutations in HIV-2 Reverse Transcriptase
title_full_unstemmed Defective Strand-Displacement DNA Synthesis Due to Accumulation of Thymidine Analogue Resistance Mutations in HIV-2 Reverse Transcriptase
title_sort Defective Strand-Displacement DNA Synthesis Due to Accumulation of Thymidine Analogue Resistance Mutations in HIV-2 Reverse Transcriptase
dc.creator.none.fl_str_mv Martín-Alonso, Samara
Álvarez, Mar
Nevot, María
Martínez, Miguel, A.
Menéndez-Arias, Luis
author Martín-Alonso, Samara
author_facet Martín-Alonso, Samara
Álvarez, Mar
Nevot, María
Martínez, Miguel, A.
Menéndez-Arias, Luis
author_role author
author2 Álvarez, Mar
Nevot, María
Martínez, Miguel, A.
Menéndez-Arias, Luis
author2_role author
author
author
author
dc.contributor.none.fl_str_mv Ministerio de Ciencia, Innovación y Universidades (España)
Instituto de Salud Carlos III
Fundación Ramón Areces
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv Strand displacement
HIV
Reverse transcriptase
Antiretroviral drugs
DNA polymerases
topic Strand displacement
HIV
Reverse transcriptase
Antiretroviral drugs
DNA polymerases
description Retroviral reverse transcriptases (RTs) have the ability to carry out strand displacement DNA synthesis in the absence of accessory proteins. Although studies with RTs and other DNA polymerases suggest that fingers subdomain residues participate in strand displacement, molecular determinants of this activity are still unknown. A mutant human immunodeficiency virus type 2 (HIV-2) RT (M41L/D67N/K70R/S215Y) with low strand displacement activity was identified after screening a panel of purified enzymes, including several antiretroviral drug-resistant HIV-1 and HIV-2 RTs. In HIV-1, resistance to zidovudine and other thymidine analogues is conferred by different combinations of M41L, D67N, K70R, L210W, T215F/Y, and K219E/Q (designated as thymidine analogue resistance-associated mutations (TAMs)). However, those changes are rarely selected in HIV-2. We show that the strand displacement activity of HIV-2ROD mutants M41L/S215Y and D67N/K70R was only slightly reduced compared to the wild-type RT. In contrast, mutants D67N/K70R/S215Y and M41L/D67N/K70R/S215Y were the most defective RTs in reactions carried out with nicked and gapped substrates. Moreover, these enzymes showed the lowest nucleotide incorporation rates in assays carried out with strand displacement substrates. Unlike in HIV-2, substitutions M41L/T215Y and D67N/K70R/T215Y/K219Q had no effect on the strand displacement activity of HIV-1BH10 RT. The strand displacement efficiencies of HIV-2ROD RTs were consistent with the lower replication capacity of HIV-2 strains bearing the four major TAMs in their RT. Our results highlight the role of the fingers subdomain in strand displacement. These findings might be important for the development of strand-displacement defective RTs.
publishDate 2020
dc.date.none.fl_str_mv 2020
2021
2021
2021
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/10261/228592
url http://hdl.handle.net/10261/228592
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv http://dx.doi.org/10.1021/acsinfecdis.9b00512
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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