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

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in IBV-infected cell samples processed in an identical manner (S1 The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.24.005298 doi: bioRxiv preprint (right) of a representative area containing the different types of MERS-CoV-induced membrane 130 modifications: DMSs (orange), convoluted membranes (CM, blue) and DMVs (yellow and lilac, outer 131 and inner membranes). The model also highlights ER membranes (green) and a vesicle (silver) 132