A unifying structural and functional model of the coronavirus replication organelle: tracking down RNA synthesis

Eric J. Snijder; Ronald W.A.L. Limpens; Adriaan H. de Wilde; Anja W. M. de Jong; Jessika C. Zevenhoven-Dobbe; Helena J. Maier; F.G.A. Faas; Abraham J. Koster; Montserrat Bárcena

y signal (see S1 Table) . For each 609 CoV, different conditions (infected and mock-infected cells, plus different labelling times) 610 were compared using only samples developed after the same period of time. The analysis of 611 the signal in different subcellular regions was carried out using home-built software. Areas of 612 4 µm 2 were randomly selected from the mosaic EM maps and the autoradiography grains 613 present in those areas were manually assigned to the underlying cellular structures. The 614 abundance of the different types of subcellular structures was estimated through virtual points 615 in a 5×5 lattice superimposed to each selected area, which were also assigned to the different 616 subcellular classes. Regularly along the process, the annotated data per condition was split 617 into two random groups and the Kendall and Spearman coefficients, which measure the 618 concordance between two data sets [73], were calculated. New random regions were added 619 until the average Kendall and Spearman coefficients resulting from 10 random splits were 620 higher than 0.8 and 0.9, respectively (maximum value, 1). Labelling densities and relative The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.24.005298 doi: bioRxiv preprint Autoradiography is a classic technique that allows the EM visualization of a radioactive 3 marker, usually targeting a certain process, and thus reveals the subcellular localization of 4 that process [1, 2] . Tritiated uridine, for example, can be used to locate active RNA synthesis 5 [3] [4] [5] , as shown also in this study. A clear advantage over the use of alternatives for metabolic 6 labelling of newly-synthesized RNA (e.g. Br-uridine, Br-UTP, 5-ethynil uridine) is that the 7 radioactive precursor is chemically identical to the natural substrate. 8 After labelling, the samples are immediately fixed and processed for EM. The location of the 9 radioactive marker can then be made apparent by applying a highly-sensitive photographic 10 emulsion (a nuclear emulsion) on top of the cell sections and exposing it for several weeks to 11 months. The beta particles that are emitted as a result of tritium disintegrations generate 12 electrons that get trapped in the silver halide emulsion and create a "latent image". When the 13 emulsion is developed, these negative charges promote the reduction to metallic silver, 14 generating electron-dense grains that are visible by EM. In principle, given enough time to 15 accumulate enough radioactive disintegrations, even low levels of the radioactive marker 16 could be detected. In practice, other factors (e.g. background radiation, emulsion aging) set 17 some limits to autoradiography, which is nonetheless a very sensitive technique. 18 The resolution of EM autoradiography is limited by the fact that radioactive disintegrations 19 generate beta particles that are emitted in random directions. Importantly, the probability of 20 giving rise to signal degreases with the distance from the radioactive source; however, some 21 beta particles may travel up to a few hundred nanometers before striking the photographic 22 emulsion [2] . Therefore, it is important to keep in mind that the silver grains may not directly 23 overlay the structure containing the radioactive source. Quantitative analyses of the signal 24 . CC-BY 4.0 International license author/funder. It is made available under a The copyright holder for this pr