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

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a pan-coronavirus RO element, studies to date describe an assortment The copyright holder for this preprint (which was not peer-reviewed) is the .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 4 DMVs would pose the conundrum of how metabolites and newly made genomic and 64 subgenomic mRNAs could be transported across the double-lipid bilayer. Nevertheless, 65 experimental evidence for the location of vRNA synthesis within the RO was lacking. 66 Importantly, dsRNA is not a bona fide marker for vRNA synthesis as it may no longer be 67 associated with the active enzymatic replication complexes in which most of the 16 viral non-68 structural proteins (nsps) come together. Thus, the possibility of vRNA synthesis taking place 69 in alternative locations, like the convoluted membranes (CM) that are also prominent 70 elements of the beta-CoV 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 elements, never reported in beta-CoV infections, that set apart 77 other distantly-related CoVs genera. In particular, zippered ER (instead of CM) and double-78 membrane spherules (DMSs) were detected for the avian gamma-CoV infectious bronchitis 79 virus (IBV) [29] and, recently, for the porcine deltacoronavirus [30] . The size and topology 80 of these DMSs, invaginations in the zippered ER, were remarkably similar to those of the 81 spherular invaginations induced by other +RNA viruses and, consequently, DMSs were 82 suggested to be sites of vRNA synthesis [29, 31] . 83 In this work, we provide an in-depth analysis of the structure and function of the RO induced 84 by members of different CoV genera, with a special focus on beta-CoVs like SARS-CoV and 85 MERS-CoV (Middle East respiratory syndrome-CoV) [32, 33]