epidemic, cause grave international public health concern. In infected cells, the CoV RNA-23 synthesizing machinery associates with modified endoplasmic reticulum membranes that are 24 transformed into the viral replication organelle (RO). While double-membrane vesicles 25 (DMVs) appear to be a pan-coronavirus RO element, studies to date describe an assortment RO [15, 20, 21] , was entirely possible and started to attract 71 attention. Notably, DMVs can be also formed in the absence of vRNA synthesis by 72 expression of key transmembrane nsps [22] [23] [24] [25] [26] . Moreover, several studies suggested a lack of 73 direct correlation between the number of DMVs and the level of CoV replication in the 74 infected cell [27, 28] . 75 The interpretation of the CoV RO structure and function was further compounded by the 76 discovery of different RO 
106 double-membrane spherules (DMSs) 107 We first set out to analyse the ultrastructure of MERS-CoV-infected Huh7 cells under sample 108 preparation conditions favourable for autoradiography (see Materials and Methods) (Fig 1, S1 109 Video). Strikingly, in addition to the DMVs and CM that are well established hallmarks of 110 beta-CoV infections, the presence of small spherules, occasionally in large numbers, was 111 readily apparent (Fig 1A and 1B) . These spherules were notably similar to the DMSs 112 previously described for the gamma-CoV IBV [29] . Their remarkably regular size of ~80 nm 113 (average diameter 79.8 ± 2.5 nm, n=58), a delimiting double membrane and their electron-114 dense content, made these spherules clearly distinct from other structures, including progeny 115 virions, which had comparable diameter (Fig 1C and 1D ). The double-membrane spherules 116 (DMSs) generated during IBV infection were previously described as invaginations of the 117 zippered ER that remain open to the cytosol [29] . In MERS-CoV-infected cells, the DMSs 118 were connected to the CM from which they seemed to derive ( Fig 1E) . Clear openings to the 119 cytosol could not be detected for the large majority (~80%, n=54) of the fully reconstructed 120 DMSs, which suggests that the original invagination may eventually transform into a sealed 121 compartment. This type of apparently closed DMSs were also present, though in a lower 122 proportion (~50%, n=39), in IBV-infected cell samples processed in an identical manner (S1 The 3D architecture of MERS-CoV-induced RO aligned with previous observations for other 156 CoV [15, 29] . No clear openings connecting the interior of the DMVs and the cytosol could 157 be detected. All three types of MERS-CoV-induced membrane modifications appeared to be 158 interconnected, either directly or indirectly through the ER. While DMSs were connected to 159 CM, and CM to ER, ER membranes were often continuous with DMVs (Fig 1F, arrowheads) . 160 Therefore, like other CoVs, MERS-CoV infection appears to induce a network of largely 161 interconnected modified ER membranes that, as a whole, can be considered the CoV RO. Quantification of the autoradiography signal per subcellular structure (see also S1 Table) . Labelling 214 densities and relative labelling indexes (RLI) in different subcellular regions of (C) Vero E6 cells 215 infected with SARS-CoV (MOI 10) or (D) Huh7 cells infected with MERS-CoV (MOI 5). 216
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. 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 ). 356 Previously, the CM induced by SARS-CoV and MHV were shown by IEM to accumulate 357 viral nsps, while dsRNA signal was primarily found inside the DMVs [15, 20] . Similarly, 358 nsp3 mapped to the CM induced in MERS-CoV infection, but also to the DMSs to a 359 comparable extent (Fig 6G) . Our attempts to combine dsRNA antibody labelling with thawed 360 cryo-sections were unsuccessful, which made us resort to HPF-FS samples. In these, 361 however, while DMVs were easily detected, the morphology of CM and DMSs was less 362 clearly defined. Nevertheless, dsRNA signal was clearly associated with DMVs, while the 363 dark membranous regions between DMVs that we interpreted as CM and DMSs clusters 364 appeared devoid of signal (Fig 6H and 6I ). 365 In summary, for the antibodies tested (recognizing N, M, S, nsp3, and dsRNA), the labelling 366 pattern in MERS-CoV-induced DMSs closely resembled that of the CM, from which they 367 seem to derive. The absence of labelling for key proteins in virus assembly, like the M and S 368 proteins, strongly suggest that DMSs do not represent (spurious) virus assembly events.
The comprehensive analysis presented here demonstrates that viruses across different CoV 371 genera induce essentially the same type of membrane structures. After somewhat disparate 372 observations [15, 20, 21, 29, 30, 47] , the unifying model that emerges from our study is that Our results add to studies that, in the last years and after much speculation, have started to 419 provide experimental evidence that the DMVs induced by +RNA viruses are active sites of 420 vRNA synthesis [11, [58] [59] [60] . However, it is not clear that DMVs always play the primary role 421 in virus replication that we demonstrate here for CoV. For picornaviruses, for example, virus- were freeze-substituted in a Leica AFS2 system with 0.1% (wt/vol) uranyl acetate as 576 previously described [71] , with the only modification that acetone was replaced by ethanol 577 from the last washing step before Lowycril infiltration onwards. Cell sections (75 nm thick) 578 were incubated with the primary mouse antibody, then with a bridging rabbit anti-mouse-IgG 579 antibody (Dako Cytomation), and finally with protein A coupled to 15-nm gold particles. 580 After immunolabelling, samples were additionally stained with 7% uranyl acetate and 581 Reynold's lead citrate. Large mosaic EM maps containing dozens of cell profiles were used for the quantitative 608 analysis of the newly-synthesized RNA autoradiography 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 621 labelling indexes (RLI) were then calculated from the annotated points [39] . 622 For the analysis of the association of vRNA synthesis with each of the different ROs motifs, 623 the specific DMVs, DMSs and CM included in the analysis were carefully selected. Only 624 individual DMVs that were at least one micron away from any other virus-induced membrane 625 modification were included in the analysis. For every grain present in an area of 750 nm 626 radius around each DMV, the distance to the DMV centre was measured. In the case of 627 DMSs, which were always part of clusters of virus-induced membrane structures, only DMSs 628 in the periphery of these clusters were selected. The quantified signal was limited to sub-629 areas devoid of other RO motifs, which were defined by circular arcs (typically 30 o to 100 o , 630 radius 500 nm) opposite to the RO clusters. CMs are irregular structures that appear partially 631 28 or totally surrounded by DMVs. Only large CM (> 0.6 µm across) were selected in order to 632 make more apparent (if present) any decay of the autoradiography signal as the distance to 633 the surrounding DMVs increased. For each autoradiography grain, both the distance to the 634 closest CM boundary (d 1 ) and the distance to the opposite CM edge (d 2 ) were measured. The 635 relative distance to the CM edge was then calculated as d 1 /(d 1 +d 2 ) and expressed in 636 percentages. All the measurements in different DMVs, DMSs and CM were made using 637 Aperio Imagescope software (Leica) and pooled together into three single data sets. 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 2 that take this factor into account, like those presented in this study, become indispensable to 25 maximize the information that autoradiography can provide. as for MER-CoV-infected cells (Fig 1) . Tomographic slices through two regions containing 32