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URL: https://dx.doi.org/DOI:10.17504/protocols.io.bfrtjm6n

Authors: Ronald W.A.L. Limpens, Eric J. Snijder, Abraham J. Koster, Montserrat Bárcena

Group: Coronavirus Method Development Community

Summary: This protocol allowed the subcellular localisation of viral RNA synthesis in coronavirus-infected cells by EM autoradiography, though it is largely applicable to other types of EM autoradiography samples. Newly synthesized viral RNA is metabolically labelled by providing the infected cells with tritiated uridine while arresting cellular transcription. Subsequently, the samples are processed for EM, sectioned and incubated with a photographic emulsion. Radioactive disintegrations from the labelled viral RNA generate signal around the radioactive source that, after development, becomes apparent as electron-dense grains that can be detected by transmission electron microscopy. The advantages of this approach are its high sensitivity and its compatibility with high-contrast EM sample preparation protocols. The protocol presented here is an adaptation of different methods and tips to EM autoradiography discussed in detail by Williams, in Glauert, 1977, Practical Methods in Electron Microscopy, Volume 6, Chapter 4.

Associated Publications: Snijder EJ, Limpens RWAL, Wilde AHd, Jong AWMd, Zevenhoven-Dobbe JC, Maier HJ, Faas FFGA, Koster AJ, Bárcena M (2020) A unifying structural and functional model of the coronavirus replication organelle: Tracking down RNA synthesis. PLoS Biol 18(6): e3000715. doi: 10.1371/journal.pbio.3000715

Affiliations: Section Electron Microscopy, Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, The Netherlands, Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands, Section Electron Microscopy, Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, The Netherlands, Section Electron Microscopy, Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, The Netherlands

External URL: https://www.kosterlab.nl/virus-replication-18

Version: 1

Publication Date: 2020

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