Document: . CC-BY 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the .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 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. [15, 20, 21, 29, 30, 47] , the unifying model that emerges from our study is that thus be a by-product of viral protein overexpression. Although DMSs seem to be derived 408 from CM, it is harder to imagine that these highly-regular structures would also lack a 409 specific function. However, their possible role remains elusive. The idea that DMSs could be 410 DMV precursors seems unlikely, considering that no intermediate structures between the two 411 were found, and that DMV formation precedes the appearance of CM, from which DMSs 412 seem to originate. A suggestive possibility was that DMSs represented non-productive virus 413 assembly events, but the lack of DMS labelling for key structural proteins seems to rule out 414 this option. In fact, no differences were apparent in the labelling patterns of CM 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- 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 20 formation would appear spurious, it cannot be completely discarded at this stage. The most 444 appealing scenario is that in which vRNA synthesis only takes place inside the DMVs. This 445 would provide the compartmentalization of vRNA synthesis that may be most beneficial for 446 viral replication, although it would require the existence of a yet unidentified import/export 447 mechanism. Notice that a transport mechanism would also be needed in the third possible 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 Quantitative analysis of autoradiography samples 607 Large mosaic EM maps containing dozens of cell profiles were used for the quantitative 608 analysis of the newly-synthesized RNA autoradiograph
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