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A validated protocol to UV-inactivate SARS-CoV-2 and herpesvirus-infected cells
Downstream analysis of virus-infected cell samples, such as reverse transcription polymerase chain reaction (RT PCR) or mass spectrometry, often needs to be performed at lower biosafety levels than their actual cultivation, and thus the samples require inactivation before they can be transferred. Co...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10171616/ https://www.ncbi.nlm.nih.gov/pubmed/37163509 http://dx.doi.org/10.1371/journal.pone.0274065 |
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author | Soh, Timothy K. Pfefferle, Susanne Wurr, Stephanie von Possel, Ronald Oestereich, Lisa Rieger, Toni Uetrecht, Charlotte Rosenthal, Maria Bosse, Jens B. |
author_facet | Soh, Timothy K. Pfefferle, Susanne Wurr, Stephanie von Possel, Ronald Oestereich, Lisa Rieger, Toni Uetrecht, Charlotte Rosenthal, Maria Bosse, Jens B. |
author_sort | Soh, Timothy K. |
collection | PubMed |
description | Downstream analysis of virus-infected cell samples, such as reverse transcription polymerase chain reaction (RT PCR) or mass spectrometry, often needs to be performed at lower biosafety levels than their actual cultivation, and thus the samples require inactivation before they can be transferred. Common inactivation methods involve chemical crosslinking with formaldehyde or denaturing samples with strong detergents, such as sodium dodecyl sulfate. However, these protocols destroy the protein quaternary structure and prevent the analysis of protein complexes, albeit through different chemical mechanisms. This often leads to studies being performed in over-expression or surrogate model systems. To address this problem, we generated a protocol that achieves the inactivation of infected cells through ultraviolet (UV) irradiation. UV irradiation damages viral genomes and crosslinks nucleic acids to proteins but leaves the overall structure of protein complexes mostly intact. Protein analysis can then be performed from intact cells without biosafety containment. While UV treatment protocols have been established to inactivate viral solutions, a protocol was missing to inactivate crude infected cell lysates, which heavily absorb light. In this work, we develop and validate a UV inactivation protocol for SARS-CoV-2, HSV-1, and HCMV-infected cells. A fluence of 10,000 mJ/cm(2) with intermittent mixing was sufficient to completely inactivate infected cells, as demonstrated by the absence of viral replication even after three sequential passages of cells inoculated with the treated material. The herein described protocol should serve as a reference for inactivating cells infected with these or similar viruses and allow for the analysis of protein quaternary structure from bona fide infected cells. |
format | Online Article Text |
id | pubmed-10171616 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-101716162023-05-11 A validated protocol to UV-inactivate SARS-CoV-2 and herpesvirus-infected cells Soh, Timothy K. Pfefferle, Susanne Wurr, Stephanie von Possel, Ronald Oestereich, Lisa Rieger, Toni Uetrecht, Charlotte Rosenthal, Maria Bosse, Jens B. PLoS One Lab Protocol Downstream analysis of virus-infected cell samples, such as reverse transcription polymerase chain reaction (RT PCR) or mass spectrometry, often needs to be performed at lower biosafety levels than their actual cultivation, and thus the samples require inactivation before they can be transferred. Common inactivation methods involve chemical crosslinking with formaldehyde or denaturing samples with strong detergents, such as sodium dodecyl sulfate. However, these protocols destroy the protein quaternary structure and prevent the analysis of protein complexes, albeit through different chemical mechanisms. This often leads to studies being performed in over-expression or surrogate model systems. To address this problem, we generated a protocol that achieves the inactivation of infected cells through ultraviolet (UV) irradiation. UV irradiation damages viral genomes and crosslinks nucleic acids to proteins but leaves the overall structure of protein complexes mostly intact. Protein analysis can then be performed from intact cells without biosafety containment. While UV treatment protocols have been established to inactivate viral solutions, a protocol was missing to inactivate crude infected cell lysates, which heavily absorb light. In this work, we develop and validate a UV inactivation protocol for SARS-CoV-2, HSV-1, and HCMV-infected cells. A fluence of 10,000 mJ/cm(2) with intermittent mixing was sufficient to completely inactivate infected cells, as demonstrated by the absence of viral replication even after three sequential passages of cells inoculated with the treated material. The herein described protocol should serve as a reference for inactivating cells infected with these or similar viruses and allow for the analysis of protein quaternary structure from bona fide infected cells. Public Library of Science 2023-05-10 /pmc/articles/PMC10171616/ /pubmed/37163509 http://dx.doi.org/10.1371/journal.pone.0274065 Text en © 2023 Soh et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Lab Protocol Soh, Timothy K. Pfefferle, Susanne Wurr, Stephanie von Possel, Ronald Oestereich, Lisa Rieger, Toni Uetrecht, Charlotte Rosenthal, Maria Bosse, Jens B. A validated protocol to UV-inactivate SARS-CoV-2 and herpesvirus-infected cells |
title | A validated protocol to UV-inactivate SARS-CoV-2 and herpesvirus-infected cells |
title_full | A validated protocol to UV-inactivate SARS-CoV-2 and herpesvirus-infected cells |
title_fullStr | A validated protocol to UV-inactivate SARS-CoV-2 and herpesvirus-infected cells |
title_full_unstemmed | A validated protocol to UV-inactivate SARS-CoV-2 and herpesvirus-infected cells |
title_short | A validated protocol to UV-inactivate SARS-CoV-2 and herpesvirus-infected cells |
title_sort | validated protocol to uv-inactivate sars-cov-2 and herpesvirus-infected cells |
topic | Lab Protocol |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10171616/ https://www.ncbi.nlm.nih.gov/pubmed/37163509 http://dx.doi.org/10.1371/journal.pone.0274065 |
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