Three-Dimensional Remodeling of SARS-CoV2-Infected Cells Revealed by Cryogenic Soft X-ray Tomography

Plus-strand RNA viruses are proficient at remodeling host cell membranes for optimal viral genome replication and the production of infectious progeny. These ultrastructural alterations result in the formation of viral membranous organelles and may be observed by different imaging techniques, provid...

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Autores: Castro, Victoria, Pérez-Berna, Ana Joaquina, Calvo, Gema, Pereiro, Eva, Gastaminza, Pablo
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:digital.csic.es:10261/339150
Acceso en línea:http://hdl.handle.net/10261/339150
Access Level:acceso abierto
Palabra clave:SARS-CoV-2
Microscopy
Cryo-SXT
Viral replication
Vesiculo-tubular network
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network_name_str España
repository_id_str
dc.title.none.fl_str_mv Three-Dimensional Remodeling of SARS-CoV2-Infected Cells Revealed by Cryogenic Soft X-ray Tomography
title Three-Dimensional Remodeling of SARS-CoV2-Infected Cells Revealed by Cryogenic Soft X-ray Tomography
spellingShingle Three-Dimensional Remodeling of SARS-CoV2-Infected Cells Revealed by Cryogenic Soft X-ray Tomography
Castro, Victoria
SARS-CoV-2
Microscopy
Cryo-SXT
Viral replication
Vesiculo-tubular network
title_short Three-Dimensional Remodeling of SARS-CoV2-Infected Cells Revealed by Cryogenic Soft X-ray Tomography
title_full Three-Dimensional Remodeling of SARS-CoV2-Infected Cells Revealed by Cryogenic Soft X-ray Tomography
title_fullStr Three-Dimensional Remodeling of SARS-CoV2-Infected Cells Revealed by Cryogenic Soft X-ray Tomography
title_full_unstemmed Three-Dimensional Remodeling of SARS-CoV2-Infected Cells Revealed by Cryogenic Soft X-ray Tomography
title_sort Three-Dimensional Remodeling of SARS-CoV2-Infected Cells Revealed by Cryogenic Soft X-ray Tomography
dc.creator.none.fl_str_mv Castro, Victoria
Pérez-Berna, Ana Joaquina
Calvo, Gema
Pereiro, Eva
Gastaminza, Pablo
author Castro, Victoria
author_facet Castro, Victoria
Pérez-Berna, Ana Joaquina
Calvo, Gema
Pereiro, Eva
Gastaminza, Pablo
author_role author
author2 Pérez-Berna, Ana Joaquina
Calvo, Gema
Pereiro, Eva
Gastaminza, Pablo
author2_role author
author
author
author
dc.contributor.none.fl_str_mv ALBA Synchrotron
Consejo Superior de Investigaciones Científicas (España)
Agencia Estatal de Investigación (España)
Ministerio de Ciencia, Innovación y Universidades (España)
European Commission
Castro, Victoria [0000-0001-9151-5138]
Pereiro, Eva [0000-0001-7626-5935]
Gastaminza, Pablo [0000-0002-7873-5491]
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv SARS-CoV-2
Microscopy
Cryo-SXT
Viral replication
Vesiculo-tubular network
topic SARS-CoV-2
Microscopy
Cryo-SXT
Viral replication
Vesiculo-tubular network
description Plus-strand RNA viruses are proficient at remodeling host cell membranes for optimal viral genome replication and the production of infectious progeny. These ultrastructural alterations result in the formation of viral membranous organelles and may be observed by different imaging techniques, providing nanometric resolution. Guided by confocal and electron microscopy, this study describes the generation of wide-field volumes using cryogenic soft-X-ray tomography (cryo-SXT) on SARS-CoV-2-infected human lung adenocarcinoma cells. Confocal microscopy showed accumulation of double-stranded RNA (dsRNA) and nucleocapsid (N) protein in compact perinuclear structures, preferentially found around centrosomes at late stages of the infection. Transmission electron microscopy (TEM) showed accumulation of membranous structures in the vicinity of the infected cell nucleus, forming a viral replication organelle containing characteristic double-membrane vesicles and virus-like particles within larger vesicular structures. Cryo-SXT revealed viral replication organelles very similar to those observed by TEM but indicated that the vesicular organelle observed in TEM sections is indeed a vesiculo-tubular network that is enlarged and elongated at late stages of the infection. Overall, our data provide additional insight into the molecular architecture of the SARS-CoV-2 replication organelle.
publishDate 2023
dc.date.none.fl_str_mv 2023
2023
2023
dc.type.none.fl_str_mv info:eu-repo/semantics/article
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Publisher's version
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/339150
url http://hdl.handle.net/10261/339150
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
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info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/PID2020-115476RB-I00
info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/SAF2017-87846-R
info:eu-repo/grantAgreement/EC/H2020/1010171162
info:eu-repo/grantAgreement/EC/H2020/ 871029
Castro, Victoria; Pérez-Berna, Ana Joaquina; Calvo, Gema; Pereiro, Eva; Gastaminza, Pablo; 2023; Supporting Material to Three-dimensional remodeling of SARS-CoV2- infected cells revealed by cryogenic soft X-ray tomography [Dataset]; American Chemical Society; https://doi.org/10.1021/acsnano.3c07265
https://doi.org/10.1021/acsnano.3c07265
Sí
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dc.publisher.none.fl_str_mv American Chemical Society
publisher.none.fl_str_mv American Chemical Society
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
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spelling Three-Dimensional Remodeling of SARS-CoV2-Infected Cells Revealed by Cryogenic Soft X-ray TomographyCastro, VictoriaPérez-Berna, Ana JoaquinaCalvo, GemaPereiro, EvaGastaminza, PabloSARS-CoV-2MicroscopyCryo-SXTViral replicationVesiculo-tubular networkPlus-strand RNA viruses are proficient at remodeling host cell membranes for optimal viral genome replication and the production of infectious progeny. These ultrastructural alterations result in the formation of viral membranous organelles and may be observed by different imaging techniques, providing nanometric resolution. Guided by confocal and electron microscopy, this study describes the generation of wide-field volumes using cryogenic soft-X-ray tomography (cryo-SXT) on SARS-CoV-2-infected human lung adenocarcinoma cells. Confocal microscopy showed accumulation of double-stranded RNA (dsRNA) and nucleocapsid (N) protein in compact perinuclear structures, preferentially found around centrosomes at late stages of the infection. Transmission electron microscopy (TEM) showed accumulation of membranous structures in the vicinity of the infected cell nucleus, forming a viral replication organelle containing characteristic double-membrane vesicles and virus-like particles within larger vesicular structures. Cryo-SXT revealed viral replication organelles very similar to those observed by TEM but indicated that the vesicular organelle observed in TEM sections is indeed a vesiculo-tubular network that is enlarged and elongated at late stages of the infection. Overall, our data provide additional insight into the molecular architecture of the SARS-CoV-2 replication organelle.This study was funded by ALBA Synchrotron standard proposals 2022065884 and 2021024899, the Spanish National Research Council (grant number PIE-RD-COVID-19 ref E202020E079) and PID2020-115476RB-I00 funded by MCIN/AEI/10.13039/501100011033, and by “ERDF A way of making Europe” to the CNB-CSIC. This research work was also funded by the European Commission – NextGenerationEU (Regulation EU 2020/2094), through CSIC’s Global Health Platform (PTI Salud Global). G.C. was initially supported by SAF2017-87846-R from the Spanish Ministry of Science, and V.C. was initially supported by PIE-RD-COVID-19. A.J.P.-B., G.C., V.C., E.P., and P.G. are part of the CoCID Consortium (cocid.eu), which has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 1010171162. We acknowledge R. Molenkamp (Erasmus University Medical Center, Rotterdam, The Netherlands; participant of the EU-funded EVA-GLOBAL project, grant agreement 871029) for the SARS-CoV-2 strain NL/2020 virus, Ralf Bartenschlager lab for the SARS-CoV-2 reporter construct, L. Enjuanes (Department of Molecular and Cell Biology, CNB-CSIC) for the Vero-E6 cells, and Urtzi Garaigorta (Department of Molecular and Cell Biology, CNB-CSIC) for critically reading the manuscript. We acknowledge the outstanding technical support from the BSL3 laboratory, Advanced Microscopy, Electron Microscopy and Cryoelectron Microscopy Core facilities personnel at the CNB as well as the support of ALBA staff, in particular Andrea Sorrentino and Ricardo Valcarcel from the MISTRAL beamline.Peer reviewedAmerican Chemical SocietyALBA SynchrotronConsejo Superior de Investigaciones Científicas (España)Agencia Estatal de Investigación (España)Ministerio de Ciencia, Innovación y Universidades (España)European CommissionCastro, Victoria [0000-0001-9151-5138]Pereiro, Eva [0000-0001-7626-5935]Gastaminza, Pablo [0000-0002-7873-5491]Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]202320232023info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionapplication/pdfhttp://hdl.handle.net/10261/339150reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Inglés#PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE#info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/PID2020-115476RB-I00info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/SAF2017-87846-Rinfo:eu-repo/grantAgreement/EC/H2020/1010171162info:eu-repo/grantAgreement/EC/H2020/ 871029Castro, Victoria; Pérez-Berna, Ana Joaquina; Calvo, Gema; Pereiro, Eva; Gastaminza, Pablo; 2023; Supporting Material to Three-dimensional remodeling of SARS-CoV2- infected cells revealed by cryogenic soft X-ray tomography [Dataset]; American Chemical Society; https://doi.org/10.1021/acsnano.3c07265https://doi.org/10.1021/acsnano.3c07265Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/3391502026-05-22T06:33:51Z
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