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

Radioactively-labelled uridine was provided for the indicated periods of time immediately before 217 fixation at 7 hpi and 12 hpi, respectively. Control mock-infected cells are excluded from the RLI 218 plots, as RLI comparisons between conditions require the same number of classes (subcellular 219 regions) and these cells lack ROs and virions. The copyright holder for this preprint (which was not peer-reviewed) is the .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 11 up to a few hundred nanometers from the original radioactive source (see S1 Appendix). This 233 type of analysis was used to compare CoV-and mock-infected cells labelled for different 234 periods of time. To this end, we analysed the autoradiography signal present in hundreds of 235 regions that were randomly picked from large EM mosaic images [38] and calculated 236 labelling densities and relative labelling indexes (RLI) per compartment [39] (see Materials 237 and Methods, S1 Table) . The results for SARS-CoV did not show association of vRNA 238 synthesis with any subcellular structure other than ROs ( Fig 3C) . In infected cells labelled for 239 a short period of time (20 min), the RO labelling densities were one order of magnitude The copyright holder for this preprint (which was not peer-reviewed) is the .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 13 (n DMVs = 36, see Material and Methods for selection criteria and details). The data is plotted (C) as a 282 histogram, or (D) normalized by the radius to the DMV centre to account for the increase in the 283 screened area. Scale bars, (A) 5 µm, (B) 500 nm. 284 285 Next, we specifically investigated the possible involvement in vRNA synthesis of CM and/or 286 DMSs, which always were present in membrane-modification clusters that also contained 287 DMVs. A close inspection of CM showed that these structures were mainly devoid of signal 288 and that the occasional silver grains present primarily appeared in the periphery of CM, 289 therefore likely stemming from surrounding labelled DMVs (Fig 5A and B) . budding vesicles (Fig 6A-C) .The M and S proteins also localized to the Golgi complex, 348 aligning with previous observations for other CoVs [20, 44, 48, 49] . The MERS-CoV N 349 protein was found in regions with CM and DMSs, though the distribution of signal was 350 homogenous and DMSs were not particularly densely labelled (Fig 6D) . The presence of the assembly, or the S protein (Fig 6E and 6F ).