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; Frank F G A Faas; Abraham J Koster; Montserrat Bárcena

doi:10.1371/journal.pbio.3000715

. 2020 Jun 8;18(6):e3000715. doi: 10.1371/journal.pbio.3000715

A unifying structural and functional model of the coronavirus replication organelle: Tracking down RNA synthesis

Eric J Snijder ^1,^*, Ronald W A L Limpens ², Adriaan H de Wilde ^1,^¤, Anja W M de Jong ², Jessika C Zevenhoven-Dobbe ¹, Helena J Maier ³, Frank F G A Faas ², Abraham J Koster ², Montserrat Bárcena ^2,^*

Editor: Andrea Cimarelli⁴

PMCID: PMC7302735 PMID: 32511245

Abstract

Zoonotic coronavirus (CoV) infections, such as those responsible for the current severe acute respiratory syndrome-CoV 2 (SARS-CoV-2) pandemic, cause grave international public health concern. In infected cells, the CoV RNA-synthesizing machinery associates with modified endoplasmic reticulum membranes that are transformed into the viral replication organelle (RO). Although double-membrane vesicles (DMVs) appear to be a pan-CoV RO element, studies to date describe an assortment of additional CoV-induced membrane structures. Despite much speculation, it remains unclear which RO element(s) accommodate viral RNA synthesis. Here we provide detailed 2D and 3D analyses of CoV ROs and show that diverse CoVs essentially induce the same membrane modifications, including the small open double-membrane spherules (DMSs) previously thought to be restricted to gamma- and delta-CoV infections and proposed as sites of replication. Metabolic labeling of newly synthesized viral RNA followed by quantitative electron microscopy (EM) autoradiography revealed abundant viral RNA synthesis associated with DMVs in cells infected with the beta-CoVs Middle East respiratory syndrome-CoV (MERS-CoV) and SARS-CoV and the gamma-CoV infectious bronchitis virus. RNA synthesis could not be linked to DMSs or any other cellular or virus-induced structure. Our results provide a unifying model of the CoV RO and clearly establish DMVs as the central hub for viral RNA synthesis and a potential drug target in CoV infection.

Coronaviruses drastically remodel the cellular endomembrane landscape, generating particularly complex replication organelles. This study shows that previous disparate observations converge on a unified model for the coronavirus replication organelle and establish which elements serve as central hubs for viral RNA synthesis.

Introduction

The RNA synthesis of all positive-stranded RNA (+RNA) viruses of eukaryotes occurs in the cytoplasm of the host cell, in conjunction with modified endomembranes that are often referred to as viral replication organelles (ROs) [1–3]. ROs are generally believed to provide tailored platforms that facilitate viral replication by concentrating relevant factors and spatially organizing distinct steps in the viral cycle. Additionally, ROs may contribute to the evasion of cellular innate immune defenses that detect viral RNA (vRNA) [4].

Two main RO prototypes have been discriminated: small spherular invaginations and large(r) vesiculotubular clusters consisting of single- and/or double-membrane structures, to which viral replicative proteins and specific host factors can be recruited. The formation of invaginations can occur at the membrane of various organelles, including endoplasmic reticulum (ER), endolysosomes, and mitochondria [5–9]. The lumen of the resulting microcompartment is connected with the cytosol by a “neck-like” channel that can mediate transport of metabolites and export of newly made positive-sense vRNAs to the cytosol for translation and packaging. In general, the morphological and functional characterization of ROs of the second, vesiculotubular type is lagging behind. Such structures, which always include double-membrane vesicles (DMVs), commonly derive from membranes of the secretory pathway and have been found in cells infected with, e.g., picornaviruses [10–11], noroviruses [12], hepatitis C virus (HCV) [13], and different nidoviruses, including the arterivirus and coronavirus (CoV) families [14–18].

The first electron tomography analysis of a CoV-induced RO, that of the severe acute respiratory syndrome-CoV [15] (SARS-CoV, genus Betacoronavirus), raised a variety of functional considerations. Intriguingly, while double-stranded (ds) RNA, a presumed intermediate and marker for vRNA synthesis [19], was found inside the virus-induced DMVs, these lacked visible connections to the cytosol [15]. vRNA synthesis inside fully closed DMVs would pose the conundrum of how metabolites and newly made genomic and subgenomic mRNAs could be transported across the double-lipid bilayer. Importantly, dsRNA is not a bona fide marker for vRNA synthesis because it may no longer be associated with the active enzymatic replication complexes in which most of the 16 viral nonstructural proteins (nsps) come together. Thus, the possibility of vRNA synthesis taking place in alternative locations, such as the convoluted membranes (CM) that are also prominent elements of the beta-CoV RO [15,20–21], was entirely possible and started to attract attention. Notably, DMVs can be also formed in the absence of vRNA synthesis by expression of key transmembrane nsps [22–23]. Moreover, several studies suggested a lack of direct correlation between the number of DMVs and the levels of CoV replication in the infected cell [24–25].

The interpretation of the CoV RO structure and function was further compounded by the discovery of different RO elements (never reported in beta-CoV infections) that set apart other distantly related CoVs genera. In particular, zippered ER (instead of CM) and double-membrane spherules (DMSs) were detected for the avian gamma-CoV infectious bronchitis virus (IBV) [26] and, recently, for the porcine deltacoronavirus [27]. The size and topology of these DMSs, which were invaginations in the zippered ER, were remarkably similar to those of the spherular invaginations induced by other +RNA viruses, and consequently, DMSs were suggested to be sites of vRNA synthesis [26].

In this work, we provide an in-depth analysis of the structure and function of the RO induced by members of different CoV genera, with a special focus on beta-CoVs such as SARS-CoV and Middle East respiratory syndrome-CoV (MERS-CoV) [28–29]. Although the SARS-CoV outbreak was contained in 2003, MERS-CoV has continued to pose a serious zoonotic threat to human health since 2012. A previously unknown beta-CoV (SARS-CoV-2), which emerged in China at the end of 2019 [30], is responsible for the current pandemic that is shaking societies and economies. The SARS-CoV-2 genome sequence is 79.5% identical to that of SARS-CoV [31], yielding 86% overall nsp sequence identity and suggesting strong functional similarities in the replication of both viruses.

Our observations on the 3D morphology of the MERS-CoV RO were compared with data from cells infected with other alpha-, beta-, and gamma-CoVs. This comparative analysis made it clear that all these CoV induce essentially the same membrane structures, including DMSs. Metabolic labeling of newly synthesized vRNA was used to determine the site(s) of vRNA synthesis within the CoV RO. To this end, we used a radiolabeled nucleoside ([³H]uridine) and applied the classic and highly sensitive technique of EM autoradiography [32–33] in combination with advanced quantitative analysis tools. This approach revealed that DMVs are the primary site of CoV RNA synthesis, with neither DMSs nor CM nor zippered ER being labeled to a significant extent. Our study provides a comprehensive and unifying model of the CoV RO structure. It also returns DMVs to center stage as the hub of CoV RNA synthesis and a potential antiviral drug target.

Results

MERS-CoV induces a membrane network of modified membranes that contains DMSs

We first set out to analyze the ultrastructure of MERS-CoV-infected Huh7 cells under sample preparation conditions favorable for autoradiography (see Materials and methods). To this end, Huh7 cells were infected with MERS-CoV at a multiplicity of infection (MOI) of 5, chemically fixed at 12 hours postinfection (hpi), and further processed for 2D EM and electron tomography (Fig 1, S1 Video). This time point, which represents the late exponential phase of viral replication [21], provided a variety of abundant virus-induced membrane structures. Strikingly, in addition to the DMVs and CM that are well-established hallmarks of beta-CoV infections, the presence of small spherules, occasionally in large numbers, was readily apparent (Fig 1A and 1B). These spherules were notably similar to the DMSs previously described for the gamma-CoV IBV [26]. Their remarkably regular size of approximately 80 nm (average diameter 79.8 ± 2.5 nm, n = 58), a delimiting double membrane, and their electron-dense content made these spherules clearly distinct from other structures, including progeny virions, which had comparable diameter (Fig 1C and 1D, S1 Data).

Fig 1 — Electron microscopy analysis of Huh7 cells infected with MERS-CoV (MOI 5, 12 hpi). (A) Electron micrograph of an area with abundant DMSs. DMVs (asterisks) are interspersed and surrounding the DMS cluster. (B) Slice through a tomogram (left) and corresponding surface-rendered model (right) of a representative area containing the different types of MERS-CoV-induced membrane modifications: CM (blue), DMSs (orange), and DMVs (yellow and lilac, outer and inner membranes, respectively). The model also highlights ER membranes (green) and a vesicle (silver) containing new virions (pink). (See also S1 Video.) (C) Comparison of DMSs and virions (arrowheads in left and right panels, respectively) in enlarged views of tomographic slices from the regions boxed in (B). The DMSs are similar in size but distinct in appearance from newly formed MERS-CoV particles. (D) Whisker plots of the size distribution of DMSs (n = 58), virions (n = 28), and DMVs (n = 109), as measured from the tomograms. DMSs and virions have a comparable size (median diameter, 80 nm), whereas the median diameter of the DMVs is 247 nm (S1 Data). (E) Models and tomographic slices through an open (left) and closed (right) DMS. Both types of DMSs are connected with the CM. In open DMSs, both the inner and outer membranes (dark blue and orange, respectively) are continuous with CM. Two slices approximately 8 nm apart in the reconstruction are shown. For closed DMSs, only the outer membrane is connected to CM, whereas the inner membrane seems to define a closed compartment. (F) Gallery of tomographic slices highlighting membrane connections between different elements of the MERS-CoV RO and of these with the ER. These include CM-ER (black arrowheads), DMV-ER (white arrowheads), CM-DMV (blue arrowheads), and CM-DMS (orange arrowhead) connections. Constrictions in the DMVs are indicated by arrows. Scale bars, 250 nm (A, B), and 100 nm (C-F). CM, convoluted membranes; DMS, double-membrane spherule; DMV, double-membrane vesicle; ER, endoplasmic reticulum; hpi, hours postinfection; MERS-CoV, Middle East respiratory syndrome-coronavirus; MOI, multiplicity of infection; RO, replication organelle.

The DMSs generated during IBV infection were previously described as invaginations of the zippered ER that remain open to the cytosol [26]. In MERS-CoV-infected cells, the DMSs were connected to the CM from which they seemed to derive (Fig 1E). Clear openings to the cytosol could not be detected for the large majority (around 80%, n = 54) of the fully reconstructed DMSs, which suggests that the original invagination may eventually transform into a sealed compartment. This type of apparently closed DMSs were also present, though in a lower proportion (around 50%, n = 39), in IBV-infected cell samples processed in an identical manner (Fig 2).

Fig 2 — Tomography of Vero cells infected with IBV, fixed at 16 hpi, and processed for EM following the same protocol as for MERS-CoV-infected cells (Fig 1). Tomographic slices through 2 regions containing IBV-induced membrane modifications. These include DMVs (asterisks), DMSs (white arrowheads), and zippered ER (white arrows). Most zippered ER consists of long stretches of ER-derived paired membranes (A), though branching zippered ER, closer to the CM described for beta-CoV, was also present. (B) Virus particles (black arrowheads) budding into the ER membranes were often observed. Scale bars, 250 nm. CM, convoluted membranes; CoV, coronavirus; DMS, double-membrane spherule; DMV, double-membrane vesicle; EM, electron microscopy; ER, endoplasmic reticulum; hpi, hours postinfection; IBV, infectious bronchitis virus; MERS-CoV, Middle East respiratory syndrome-CoV.

Our data suggest a functional analogy between zippered ER and CM, as both structures appear to provide membranes for DMS formation. In fact, both bore a striking resemblance under the same sample preparation conditions: MERS-CoV-induced CM noticeably consisted of zippered smooth membranes that were connected to the rough ER and branched and curved in intricate arrangements. Although unbranched zippered ER was most common in IBV-infected cells (Fig 2A), as described [26], we also detected zippered ER morphologically closer to CM (Fig 2B). These observations argue for zippered ER and CM representing alternative configurations of essentially the same type of virus-induced modification.

The 3D architecture of MERS-CoV-induced RO aligned with previous observations for other CoVs [15,26]. In our samples, the 2 delimiting membranes of the DMVs had a distended appearance, in contrast with the tight membrane apposition observed in samples prepared by high-pressure freezing, freeze-substitution (HPF-FS) [21]. Although constrictions in this distended pattern could be frequently observed (Fig 1F, arrows), no clear openings to the cytosol were detected. All 3 types of MERS-CoV-induced membrane modifications appeared to be interconnected, either directly or indirectly through the ER. While DMSs were connected to CM, and CM to ER, ER membranes were often continuous with the outer membrane of the DMVs (Fig 1F, arrowheads). Therefore, like other CoVs, MERS-CoV infection appears to induce a network of largely interconnected modified ER membranes that, as a whole, can be considered the CoV RO.

Diverse CoVs across different genera induce the same RO elements

Intriguingly, DMSs had never been reported for beta-CoV infections in previous characterizations (including ours) that used different sample preparation conditions and/or different cell lines. This prompted us to revisit those samples for a closer examination. A targeted search for DMSs in MERS-CoV-infected Vero cells [21] and SARS-CoV-infected Vero E6 cells [15] readily revealed similar DMSs, embedded and somewhat concealed in CM with a denser and more tangled appearance that can be attributed to sample preparation differences (S1 Fig, compare to Figs 1 and 3). To further explore whether these observations could be extended to other CoVs, we analyzed a third beta-CoV (murine hepatitis virus [MHV]) as well as a member of the genus Alphacoronavirus (human coronavirus 229E [HCoV-229E]). Both in MHV-infected 17Cl1 cells and in HCoV-229E-infected Huh7 cells, virus-induced DMSs could be detected, with their characteristic size, appearance, and spatial association with CM (Fig 3). These results demonstrate that virus-induced DMSs are not exclusive to some CoV genera or specific cell lines but are instead a common characteristic of CoV-infected cells.

Fig 3 — 2D-EM images from 100-nm-thick sections of different mammal cells infected with (from left to right) SARS-CoV (MOI 10, 9 hpi), MHV (MOI 10, 8 hpi), and HCoV-229E (MOI 5, 24 hpi). These time points represent intermediate to late stages in infection [34–36]. Both beta-CoVs (A,B) and the alpha-CoV (C) induce membrane modifications that include not only DMVs (asterisks) and CM but also DMSs (white arrowheads). Scale bars, 250 nm. CM, convoluted membranes; CoV, coronavirus; DMS, double-membrane spherule; DMV, double-membrane vesicle; EM, electron microscopy; ER, endoplasmic reticulum; HCoV-229E, human coronavirus 229E; hpi, hours postinfection; IBV, infectious bronchitis virus; MHV, murine hepatitis virus; MOI, multiplicity of infection; SARS-CoV, severe acute respiratory syndrome-CoV.

CoV RNA synthesis is confined to RO regions

To investigate the subcellular localization of vRNA synthesis in cells infected with different CoVs, we metabolically labeled newly synthesized vRNA by arresting cellular transcription with actinomycin D and using a radiolabeled nucleoside precursor ([5-³H]uridine) for subsequent detection by EM autoradiography [32–33] (see S1 Text). In CoV-infected cells, vRNA synthesis entails not only genome replication but also the production of a nested set of subgenomic RNAs encoding the structural and so-called “accessory” viral proteins. In terms of RNA copy number, genomic RNA represents only a small fraction of the total vRNA [21,37], but because of its much larger size, the relative amount of label incorporated per genome copy is significantly higher than for subgenomic mRNAs. For example, in MERS-CoV-infected cells, genomic RNA constitutes about 4% of the vRNA molecules [21]) but should incorporate 32% of the [³H]uridine label when compensating for its size and somewhat higher relative uracil content.

A key advantage of our approach over the use of modified precursors (e.g., Br-uridine) is that detection of the label does not rely on immunolabeling. This makes autoradiography compatible with high-contrast EM sample preparation protocols that provide excellent morphology at the price of epitope integrity. Moreover, as the signal derives from radioactive disintegrations, EM autoradiography is a very sensitive technique, which, in principle, should allow for short labeling pulses, essential to minimize the chance of migration of labeled vRNA products from their site of synthesis (Fig 4). Nevertheless, the pulse should be long enough to enable the internalization of the tritiated uridine, its conversion into ³H-UTP in the cell, and its incorporation into vRNA. In order to explore the practical limits of the approach, we tested different labeling pulses in Vero E6 cells infected with SARS-CoV, measuring the amount of radioactive label incorporated into RNA (Fig 4A). Although minimal labeling occurred within the first 10 minutes, a sharp increase in signal was observed between 10 and 20 minutes after label administration. Consequently, only samples labeled for 20 minutes or longer were prepared and processed for EM autoradiography analysis.

Abundant autoradiography signal was detected by EM in SARS-CoV-infected cells pulse-labeled for 20 minutes (Fig 4B). The signal accumulated in the regions that contained virus-induced membrane modifications, which aligned with the idea that these structures are the primary platforms for vRNA synthesis. This largely accepted notion, however, does not formally exclude the possibility that vRNA synthesis could also be associated with other cellular membranes, albeit to a lower extent. Such an association with morphologically intact membranes could be important, for example, in the first stages of infection, when the levels of the viral membrane-remodeling proteins are still low.

Importantly, establishing the association of autoradiography signal with specific subcellular structures requires a detailed quantitative analysis, as the autoradiography signal can spread up to a few hundred nanometers from the original radioactive source (see S1 Text). This type of analysis was used to compare CoV- and mock-infected cells labeled for different periods of time. To this end, we analyzed the autoradiography signal present in hundreds of regions that were randomly picked from large EM mosaic images [38] and calculated labeling densities and relative labeling indexes (RLIs) per compartment [39] (see Materials and methods, S3 Data). The results for SARS-CoV did not show association of vRNA synthesis with any subcellular structure other than ROs (Fig 4C). In infected cells labeled for a short period of time (20 minutes), the RO labeling densities were 1 order of magnitude higher than for any other subcellular structure. Even though the dispersion of signal around the radioactive source would inevitably cause “signal leakage” from these active ROs to neighboring organelles like the ER, none of those alternative locations showed an RLI significantly higher than 1 (RLI ≤ 1 indicates unspecific labeling), and their labeling densities were comparable to those in the control mock-infected cells. An increase in labeling densities in some subcellular regions was observed in infected cells when the labeling time was extended to 60 minutes. This could be explained by signal leakage in combination with the migration of vRNA from its site of synthesis, possibly toward the site of virus assembly on membranes of the ER-Golgi intermediate compartment (ERGIC) [40–42]. Indeed, virion-containing regions showed the sharpest increase in labeling density when extending the labeling time. Similar results followed from the analysis of MERS-CoV-infected Huh7 cells (Fig 4D), although no clear signs of RNA migration were observed in this case. Taken together, these results suggest that CoV RNA synthesis is restricted to the RO regions of the infected cell.

DMVs are the primary site of coronaviral RNA synthesis

Our next goal was to determine which elements of the coronaviral RO (DMVs, CM/zippered ER, and/or DMSs) are directly involved in vRNA synthesis. A first answer to this question became readily apparent as regions containing DMVs, but not either of the other RO structural elements, were densely labeled in cells infected with SARS-CoV (Fig 4B) or MERS-CoV (Fig 5). Interestingly, not all the DMV clusters in a sample, and sometimes even within a cell, appeared equally densely labeled, which suggests that the levels of vRNA synthesis in DMVs are variable and can change in time. The active role of DMVs in vRNA synthesis in MERS-CoV-infected cells was further corroborated by a detailed analysis of the distribution of autoradiography signal around isolated DMVs (Fig 5C and 5D). In case of a random distribution (i.e., if the DMVs were not a true radioactive source), the number of autoradiography grains around the DMVs would simply increase with the distance, as the perimeter of the screened area also increases and with it, the chances of detecting background signal. The signal around DMVs was clearly not random, showing maximum levels in the proximity of these structures (Fig 5C). Moreover, the distribution normalized by the distance made apparent a maximum around the average radius of the DMVs analyzed (133 ± 28 nm, n = 36) (Fig 5D), aligning with the idea that vRNA synthesis takes place in membrane-bound enzymatic complexes.

Fig 5 — Analysis of the association of autoradiography signal with DMVs in MERS-CoV-infected Huh7 cells (MOI 5). The cells were pretreated with actinomycin D at 10 hpi and labeled with tritiated uridine for 30 minutes immediately before fixation (12 hpi). (A) Overview of an infected cell in which regions with different virus-induced modifications are annotated in yellow (DMVs), blue (CM), and orange (DMSs). Several densely labeled regions containing DMVs (but not the other virus-induced structures) are apparent. A close-up of one of these regions (boxed area) is shown in (B), with DMVs highlighted by yellow asterisks. (C, D) Distribution of the autoradiography signal around DMVs (n_DMVs = 36, see Materials and methods for selection criteria and details, and S4 Data for the underlying numerical data). The data are plotted (C) as a histogram or (D) normalized by the radius to the DMV center to account for the increase in the perimeter of the screened area with the distance. Scale bars, (A) 5 μm, (B) 500 nm. CM, convoluted membranes; DMS, double-membrane spherule; DMV, double-membrane vesicle; hpi, hours postinfection; MERS-CoV, Middle East respiratory syndrome-coronavirus; MOI, multiplicity of infection; N, nucleus; vRNA, viral RNA.

Next, we specifically investigated the possible involvement in vRNA synthesis of CM and/or DMSs (Fig 6), which were always present in membrane-modification clusters that also contained DMVs. A close inspection of multiple CM showed that these structures were mainly devoid of signal and that the occasional silver grains present primarily appeared in the periphery of the CM, therefore likely stemming from surrounding labeled DMVs (Fig 6A and 6B). Similar observations were made for DMSs (Fig 6C–6E): most of them (83%) lacked signal, and the rest was close to abundantly labeled DMVs. Furthermore, no signs of DMSs acting as a signal source were apparent in the distribution of signal around them, which resembled that of a random pattern (Fig 6E, compare with Fig 5C).

To explore whether these observations could be extended to distantly related CoVs, we expanded this type of analysis to the gamma-CoV IBV. IBV-induced DMSs are particularly abundant, and a large proportion of them have an open configuration, which contributed to the hypothesis that these open DMSs could be engaged in vRNA synthesis [26]. However, no evidence supporting this hypothesis could be derived from our detailed analysis of the autoradiography signal in IBV-infected cells, which essentially produced the same results as for MERS-CoV (Fig 7). Taken together, our observations clearly point at coronaviral DMVs as the active site of vRNA synthesis and seem to indicate that neither CM/zippered ER nor DMSs are effectively involved in this process, at least not to a significant level above the detection limits of our method.

Fig 7 — Vero cells infected with IBV were pretreated with actinomycin D for 1 hour, then labeled for 30 or 60 minutes with tritiated uridine, immediately fixed at 16 and 17 hpi, respectively, and processed for autoradiography EM. These time points allow for a second cycle of infection and were chosen to increase the number of infected cells (see Materials and methods). (A) Overview of an IBV-infected Vero cell labeled for 60 minutes. The areas containing DMVs and zippered ER are outlined in yellow and blue, respectively, and other subcellular structures are also annotated. The autoradiography signal accumulates in areas of virus-induced membrane modifications that often only contain DMVs, in alignment with DMVs having an active role in vRNA synthesis. (B) Close-up of the area boxed in black in (A), which contains DMVs, zippered ER and DMSs (orange arrowheads). The contrast between the densely labeled DMVs and the zippered ER and DMSs largely lacking signal is apparent and suggests that the autoradiography grains sometimes present on the latter structures arose from radioactive disintegrations in the surrounding active DMVs. (C) In agreement with this possibility, most of the DMSs (96%) were devoid of signal, and most of those that contained label where close to an active DMV (n_DMS = 178). (D) Furthermore, the distribution of autoradiography grains around DMSs resembled that of a random distribution, in which the number of grains increase with the distance (n_DMS = 106). (E) In contrast, a similar analysis of the signal around the DMVs proved that these structures are associated with vRNA synthesis, as the signal reaches maximum values in the proximity of the DMVs (n_DMVs = 106). (C, D) See Materials and methods for the selection criteria and details and S6 Data for the underlying numerical data. Scale bars, 1 μm. Au, autophagosome; DMS, double-membrane spherule; DMV, double-membrane vesicle; EM, electron microscopy; ER, endoplasmic reticulum; hpi, hours postinfection; IBV, infectious bronchitis virus; m, mitochondrion; N, nucleus; VCR, virion-containing region; vRNA, viral RNA.

Viral markers in the MERS-CoV RO

If CM and DMSs are not involved in vRNA synthesis, what is the role (if any) of these RO structural elements in CoV replication? To investigate this, we analyzed the subcellular location of different viral markers in MERS-CoV-infected cells by immunoelectron microscopy (IEM) (Fig 8).

Fig 8 — (A-G) Immunogold labeling of thawed cryo-sections of MERS-CoV-infected Huh7 cells (12 hpi) for the detection of the indicated viral proteins. (A-C) Structural proteins were detected on virions (black arrowheads) and, for the M and S proteins, also on Golgi cisterna. While regions containing DMS (white arrowheads) and CM labeled for the N protein (D) and nsp3 (G), the M and S protein were not detected in these areas. (H-I) Immunogold labeling of dsRNA in HPF-FS samples of MERS-CoV-infected Huh7 cells (13 hpi). The label accumulated on DMVs, which could be easily detected in this type of samples (black arrows), whereas the regions with CM and DMSs, which appeared as dark areas among the DMV clusters, were devoid of dsRNA signal. Scale bars, 250 nm. CM, convoluted membranes; DMS, double-membrane spherule; dsRNA, double-stranded RNA; G, Golgi complex; HPF-FS, high-pressure freezing, freeze-substitution; hpi, hours postinfection; IEM, immunoelectron microscopy; m, mitochondrion; MERS-CoV, Middle East respiratory syndrome-CoV; nsp3, nonstructural protein 3.

CoV-induced DMSs, with their electron-dense content and their remarkably regular size, are particularly intriguing structures. Revealing DMSs in IEM samples, however, was challenging and, in our hands, required a modified protocol for the preparation of thawed cryo-sections [43] that, unfortunately, failed to make DMVs apparent (see Materials and methods). Given the similar size of DMSs and virus particles, we first considered the possibility that the DMSs would represent some kind of nonproductive virus assembly event on the CM. Although new CoV particles typically assemble in the ERGIC [40–42,44–45], virus budding from ER membranes, from which CM originate, can also occur [40,46–47], and we regularly observed it in MERS-CoV- and IBV-infected cells (e.g., Fig 2B). To investigate this possibility, we used antibodies against several of the structural proteins, namely, the nucleocapsid protein N, the envelope membrane protein M, and the spike protein S. As expected, all of them were detected in newly formed MERS-CoV particles present in budding vesicles (Fig 8A–8C). The M and S proteins also localized to the Golgi complex, aligning with previous observations for other CoVs [20,44,48–49]. The MERS-CoV N protein was found in regions with CM and DMSs, though the distribution of signal was homogenous and DMSs were not particularly densely labeled (Fig 8D). The presence of the N protein in the viral RO has also been shown for MHV [20] and suggested by a number of colocalization studies [42,50–53] and may be related to a possible role in vRNA synthesis of this multifunctional protein [54]. Importantly, neither DMSs nor CM labeled for the M protein (the most abundant viral envelope protein and the presumed orchestrator of virion assembly) or the S protein (Fig 8E and 8F).

Previously, the CM induced by SARS-CoV and MHV were shown by IEM to accumulate viral nsps, whereas dsRNA signal was primarily found inside the DMVs [15,20]. Similarly, nsp3 mapped to the CM induced in MERS-CoV infection but also to the DMSs to a comparable extent (Fig 8G). Our attempts to combine dsRNA antibody labeling with thawed cryo-sections were unsuccessful, which made us resort to HPF-FS samples. In these, however, while DMVs were easily detected, the morphology of CM and DMSs was less clearly defined. Nevertheless, dsRNA signal was clearly associated with DMVs, whereas the dark membranous regions between DMVs that we interpreted as CM and DMSs clusters appeared devoid of signal (Fig 8H and 8I).

In summary, for the antibodies tested (recognizing N, M, S, nsp3, and dsRNA), the labeling pattern in MERS-CoV-induced DMSs closely resembled that of the CM, from which they seem to derive. The absence of labeling for key proteins in virus assembly, like the M and S proteins, strongly suggest that DMSs do not represent (spurious) virus assembly events.

Discussion

The comprehensive analysis presented here demonstrates that viruses across different CoV genera induce essentially the same type of membrane structures. After somewhat disparate observations [15,20–21,26–27,47], the unifying model that emerges from our study is that of a CoV RO comprising 3 basic types of double-membrane structural elements: (1) CM or zippered ER, which would represent branched or unbranched configurations of paired-ER membranes; (2) small DMSs that appear to arise from CM or zippered ER; and (3) DMVs. These structural elements are largely interconnected and connected to the ER, together forming the reticulovesicular network that is typical of the coronaviral RO, including, in all likelihood, the RO of SARS-CoV-2, a close relative of SARS-CoV.

Our results clearly confirm and extend DMVs as the primary—if not only—site of vRNA synthesis within the coronaviral RO. This point has been subject to quite some speculation, due in part to the limited experimental data directly addressing this question. An early study using Br-UTP and IEM to detect newly synthesized vRNA mapped signal in DMV regions of MHV-infected cells [14], but the poor preservation of IEM samples did not allow the recognition of CM and DMSs, typically present in these regions. Later, a light microscopy study using 5-ethynil uridine labeling and click chemistry suggested that vRNA synthesis could take place, at least partially, in a different location than the DMVs [55]. Alternative interpretations of those results, like migration of vRNA from the DMVs during the relatively long labeling times used (60 minutes) appear now more plausible. Several other studies further contributed to question the idea of DMVs being the sites of vRNA synthesis by showing that higher numbers of DMVs did not necessarily translate into larger amounts of vRNA or provide a competitive advantage [24–25,56]. It should be noted, however, that the number of DMVs may not necessarily correlate with the number of active replication complexes that they contain at a given time, something that is also suggested by our observed variations in the level of autoradiography signal among DMVs in the same sample.

While the finding of open DMSs, similar to the invaginations that many +RNA viruses use as replication sites, made them attractive candidate sites for vRNA synthesis [26,57], we could not detect vRNA synthesis associated with them, nor with CM or any other subcellular structure using a highly sensitive technique like autoradiography. Although some level of vRNA synthesis in any of these structures cannot be completely discarded, our results suggest that, if present, this would only be marginal compared with the abundant DMV-associated activity.

Given the prevalence of CM and DMSs across different CoV genera, it is tempting to speculate that these structures must play a role in virus replication. In particular, for the highly regular DMSs, it is hard to imagine that they would lack a specific function; however, their role remains elusive. A suggestive possibility was that DMSs represent nonproductive virus assembly events, but the lack of DMS labeling for key structural proteins seems to rule out this option. In fact, for the viral markers tested, no differences were apparent in the labeling patterns of DMSs and the CM from which they seem to derive. Although their distinct morphology implies that DMSs likely contain specific host or viral proteins, these factors—which may give important clues about DMS function—remain to be identified. Another possibility is that CM and DMSs represent DMV precursors. In this scenario, ER membrane pairing would first give rise to CM, which would then produce DMSs that could eventually expand into DMVs. Pairing of ER membranes, which appears to be driven by 2 key transmembrane viral proteins (nsp3 and nsp4), indeed, is the likely first step in CoV DMV biogenesis, as suggested by studies in cells ectopically expressing these proteins [22–23,27,58]. CM, however, seem to appear later in infection than DMVs, as documented for SARS-CoV, MHV, and MERS-CoV [15,20–21]. While this may argue against CM and DMSs being DMV precursors, these virus-induced structures could still represent basic membrane-remodeling stages in DMV formation that, later in infection, do not progress adequately. It has been proposed, for example, that CM could be a form of cubic membranes [23,59], which are membrane aggregates resulting from ER protein overexpression [60]. CM, which abundantly label for viral nsps [15,20] and proliferate late in infection [15,20–21], could thus be a by-product of viral protein overexpression.

Our results add to studies that, in the last years and after much speculation, have started to provide experimental evidence that the DMVs induced by +RNA viruses are active sites of vRNA synthesis [11,61–63]. However, it is not clear that DMVs always play the primary role in virus replication that we demonstrate here for CoV. For picornaviruses, for example, virus-induced single-membrane structures, which are DMV precursors and also active sites of vRNA synthesis, could well be more relevant as they predominate at the peak of vRNA replication [10–11,63]. By clearly pointing to DMVs as the key sites for CoV replication, our results also bring back to center stage some of the challenges that CoV-induced DMVs pose, which extend to the distantly related arterivirus family and probably also to other members of the order Nidovirales. In contrast with the DMVs induced by other +RNA viruses [10–11,13,63], the DMVs in nidovirus-infected cells appear to lack membrane openings that would connect their inner compartment with the cytosol to allow import of precursors and export of genomic and subgenomic viral mRNAs [15–16,26]. This topological conundrum, however, starts with the assumption that vRNA synthesis takes place inside the DMVs, yet the evidence so far is insufficient to ascertain this point. Shielding of vRNA inside DMVs arguably provides the most straightforward explanation for the observation that intact membranes protected vRNA from nuclease treatment [64]. However, protection of vRNA could also be achieved in the outer DMV membrane through protein complexes that would rely on membranes for assembly/stability.

Although a definitive answer to this issue is still missing, our results allow narrowing down the possible scenarios. If the DMVs are closed structures lacking an import/export mechanism, vRNA synthesis on the outer DMV membrane appears as a necessity to provide vRNA for translation and encapsidation. Then, vRNA synthesis inside the DMVs would also be required to explain the observed accumulation of (presumably viral) dsRNA. Although this is not at all an attractive possibility, as vRNA synthesis inside the DMVs and even DMV formation would appear spurious, it cannot be completely discarded at this stage. The most appealing scenario is that in which vRNA synthesis only takes place inside the DMVs. This would provide the compartmentalization of vRNA synthesis that may be most beneficial for viral replication, although it would require the existence of a yet unidentified import/export mechanism. Notice that a transport mechanism would also be needed in the third possible scenario, i.e., if vRNA synthesis occurs only on the outer DMV membrane, to account for the accumulation of dsRNA inside the DMVs that could then perhaps serve to hide excess vRNA from detection by innate immune sensors [65].

Despite the lack of clear openings in the membranes of CoV-induced DMVs, a mechanism allowing exchange of material with the cytosol is conceivable. The openings of DMV may be extremely short-lived, and therefore, they may have eluded detection by EM. This may become apparent in the future, if mutant CoV-inducing DMVs with slower dynamics are found. An appealing alternative is the existence of molecular pores that may well be undetectable in conventional EM samples. Precedents of molecular complexes bridging double membranes include the large nuclear pore complex but also small transporters through the mitochondrial or chloroplast membranes. Visualizing such a putative small molecular pore on DMVs is a formidable challenge that would likely require the use of cryo-EM to preserve macromolecular components. Emerging techniques like in situ cryotomography, which allows the visualization of structures in their cellular context at macromolecular resolution, may be key to realize this next step and to understand how CoVs exploit the complex architecture of DMVs.

Materials and methods

Cells, viruses, and infections

Huh7 cells (kindly provided by Ralf Bartenschlager, Heidelberg University) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Lonza) supplemented with 8% (vol/vol) fetal calf serum (FCS; Bodinco), 2 mM L-glutamine (PAA Laboratories), and nonessential amino acids (PAA Laboratories). Vero cells (ECACC 84113001) were cultured in Eagle’s minimal essential medium (EMEM; Lonza) with 8% FCS and 2 mM L-glutamine. Vero E6 cells (ATCC CRL-1586) were maintained in DMEM supplemented with 8% FCS. Mouse 17 clone 1 (17Cl1) cells (gift from Stuart Siddell, University of Bristol) were grown in DMEM supplemented with 8% FCS and 8% (vol/vol) tryptose phosphate broth (Life Technologies). Penicillin and streptomycin (90 IU/ml, PAA Laboratories) were added to all media.

The CoVs used in this study include MERS-CoV (strain EMC/2012, [28–29], SARS-CoV (strain Frankfurt-1, [66]), MHV (ATCC VR-764), HCoV-229E ([67]), and IBV (strain Beau-R, [68]). All the infection experiments were carried out at 37°C, except for HCoV-229E infections, which were performed at 33°C. Cells were infected at high MOI (5–10), with the exception of IBV (MOI < 1). Times postinfection in the middle or late exponential phase of viral replication [21,34–36] were selected for analysis to favor both a good amount of label incorporation and a variety and abundant presence of virus-induced membrane structures. In the case of IBV, however, because of the low titer and to increase the number of infected cells, the times postinfection (16–17 hpi) extended beyond the first cycle of viral replication infection. For MHV infections, 1 μM HR2 peptide was added to the cell medium to prevent syncytia formation [69]. Control mock-infected cells were included in all the experiments. Infections were routinely assessed by immunofluorescence assays on parallel samples, essentially processed as previously described [51]. All work with live SARS-CoV and MERS-CoV was performed inside biosafety cabinets in the biosafety level 3 facility at Leiden University Medical Center.

Antibodies

For IEM of MERS-CoV-infected cells, the antibodies used included a previously described rabbit antiserum that recognizes SARS-CoV nsp3 protein and cross-reacts with MERS-CoV nsp3 [21,70], a polyclonal rabbit antibody generated against full-length MERS-CoV N protein (Sino Biological), and a mouse monoclonal antibody (J2) specific for dsRNA [71], purchased from Scicons. A MERS-CoV M-specific rabbit antiserum was ordered from Genscript and produced using as antigen a synthetic peptide representing the C-terminal 24 residues of the protein (CRYKAGNYRSPPITADIELALLRA). The specificity of the antiserum was verified by western blot analysis and immunofluorescence microscopy using samples from MERS-CoV-infected Vero or Huh7 cells, as described previously [21], while using preimmune serum and mock-infected cell lysates as negative controls. The human monoclonal antibody used against MERS-CoV spike protein was kindly provided by Dr. Berend Jan Bosch (Utrecht University) and has been described elsewhere as 1.6f9 [72].

Metabolic labeling and label incorporation measurements

To label newly synthesized RNA, CoV-infected cells and control mock-infected cells were incubated for different periods of time with tritiated uridine ([5-³H]uridine, 1 mCi/ml, Perkin Elmer), which was mixed in a 1:1 ratio with double-concentrated medium. Cellular transcription was blocked by providing the cells with 10 μg/ml actinomycin D both during labeling and in a preincubation step of 1 to 2 hours, depending on the specific set of samples. Directly after labeling, the cells were extensively washed with phosphate-buffered saline (PBS) and immediately fixed for EM autoradiography. A parallel set of samples was included in every experiment for label incorporation measurements. These cells were lysed using TriPure Isolation Reagent (Roche), and the RNA was isolated following the manufacturer’s instructions. The incorporation of radioactive label into RNA was then measured using scintillation counting.

Sample preparation for electron microscopy

For ultrastructural analysis, autoradiography, and tomography, EM samples of CoV-infected cells were prepared by chemical fixation. Chemical fixation was chosen over the alternative of HPF-FS to increase the yield of cells per EM grid and thus facilitate the autoradiography quantitative analysis, as it was observed that infected cells were easily washed away and lost during FS. At the desired times postinfection, the cells were fixed for 30 minutes with 1.5% (vol/vol) glutaraldehyde (GA) in 0.1 M cacodylate buffer (pH 7.4). Cells infected with SARS- or MERS-CoV were further maintained in the fixative overnight. After fixation, the samples were washed with 0.1 M cacodylate buffer either once or, in samples destined to autoradiography, 3 times (5 minutes each) to favor the elimination of unincorporated [5-³H]uridine. Next, the samples were treated with 1% (wt/vol) OsO₄ at 4°C for 1 hour, washed with 0.1 M cacodylate buffer and Milli-Q water, and stained at room temperature for 1 hour with 1% (wt/vol) low-molecular weight tannic acid (Electron Microscopy Science) in 0.1 M cacodylate buffer (SARS-CoV autoradiography experiments), or with 1% (wt/vol) uranyl acetate in Milli-Q (rest of the samples). Following a new washing step with Milli-Q water, the samples were dehydrated in increasing concentrations of ethanol (70%, 80%, 90%, 100%), embedded in epoxy resin (LX-112, Ladd Research), and polymerized at 60°C. Sections were collected on mesh-100 copper EM grids covered with a carbon-coated Pioloform layer, and poststained with 7% (wt/vol) uranyl acetate and Reynold’s lead citrate.

EM autoradiography samples

A step-by-step protocol for the preparation of autoradiography samples can be found in http://dx.doi.org/10.17504/protocols.io.bfrtjm6n. In short, EM grids with ultrathin cell sections (50-nm thick) were first attached to glass slides, including in each glass slide a grid for every condition tested within a given experiment, to be later developed simultaneously. In a dark room, a thin layer of nuclear emulsion ILFORD L4 was placed on top of the grids with the help of a wire loop [32]. Samples were maintained in the dark in a cold room for several weeks until development, which was performed as described in [73]. The progress in the exposure of the nuclear emulsion to radioactive disintegrations was evaluated regularly by EM until the number of autoradiography grains was sufficient for analysis (approximately 6 weeks for SARS-CoV-infected cells, 21 weeks for MERS-CoV-infected cells, and 12 to 13 weeks for IBV-infected cells).

IEM

Several types of MERS-CoV-infected cell samples were prepared for IEM. The labeling of viral proteins was performed on thawed cryo-sections, which are optimal for epitope preservation. To this end, at 12 hpi, infected and mock-infected cells were first chemically fixed for 1 hour at room temperature with 3% (wt/vol) paraformaldehyde (PFA) and 0.25% (vol/vol) GA in 0.1 M PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl₂ [pH 6.9]), briefly washed with 0.1 M PHEM buffer, and stored at 4°C in 3% (wt/vol) PFA in 0.1 M PHEM buffer until transfer from the biosafety level 3 facility for further processing. To prepare EM samples, the cells were first pelleted and embedded in 12% (wt/vol) gelatin. Cubes of approximately 1 mm³ in size were cut from these pellets, infiltrated with 2.3 M sucrose for cryo-protection, snap-frozen in liquid nitrogen, and sectioned by cryo-ultramicrotomy. Thawed cryo-sections (70-nm thick) deposited on EM grids were incubated first with the corresponding primary antibody and then with protein A coupled to colloidal 10-nm gold particles. Virus-induced membrane modifications, however, were not discernible in these samples, and clear signs of membrane extraction were present. To tackle this issue, we used a previously described modification of the protocol that includes sequential poststaining steps with 1% (wt/vol) OsO4, 2% (wt/vol) uranyl acetate, and Reynold’s lead citrate after immunogold labeling and prior to the final embedding in a thin layer of 1.8% methyl cellulose [43]. Both CM and DMSs were clearly recognizable in these samples, whereas DMVs were still not apparent and may have been extracted, as empty areas were often found in the vicinity of CM regions.

The detection of dsRNA required the preparation of HPF-FS samples. For biosafety considerations, Huh7 cells grown on sapphire disks and infected with MERS-CoV were first fixed overnight at 13 hpi with 3% (wt/vol) PFA and 0.25% (vol/vol) GA in 0.1 M PHEM buffer. Then, the samples were frozen with a Leica EM PACT2, after which they were freeze-substituted in a Leica AFS2 system with 0.1% (wt/vol) uranyl acetate as previously described [74], with the only modification that acetone was replaced by ethanol from the last washing step before Lowycril infiltration onwards. Cell sections (75-nm thick) were incubated with the primary mouse antibody, then with a bridging rabbit anti-mouse-IgG antibody (Dako Cytomation), and finally with protein A coupled to 15-nm gold particles. After immunolabeling, samples were additionally stained with 7% (wt/vol) uranyl acetate and Reynold’s lead citrate.

Electron microscopy imaging

Individual 2D-EM images were acquired in a Tecnai12 BioTwin or a Twin electron microscope, equipped with an Eagle 4k slow-scan change-couple device (CCD) camera (Thermo Fisher Scientific [formerly FEI]) or a OneView 4k high-frame rate CMOS camera (Gatan), respectively. Mosaic EM images of large grid areas were generated for the quantitative analysis of autoradiography samples, using overlapping automatically collected images (pixel size 2 nm, Tecnai 12 BioTwin) that were subsequently combined in a composite image as described in [38].

Electron tomography

Prior to the poststaining step, semithin sections (150-nm thick) of CoV-infected cells were incubated with protein A coupled to 10-nm colloidal gold particles that served later as fiducial markers for alignment. Dual-axis tilt series were collected in a Tecnai12 BioTwin microscope using Xplore 3D acquisition software (Thermo Fisher Scientific), covering each 120°–130° around the specimen with an angular sampling of 1° and a pixel size of 1.2 nm. The alignment of the tilt series and tomogram reconstruction by weighted back-projection was carried out in IMOD [75]. The diameters of DMVs, DMSs, and virions were measured at their equator. DMV profiles, often only roughly circular, were measured over their longest and shortest axes, and the diameter was estimated as the geometric mean of the 2 values. For visualization purposes, the tomograms were first mildly denoised and then processed in Amira 6.0.1 (Thermo Fisher Scientific) using a semiautomatic segmentation protocol as previously described [10].

Quantitative analysis of autoradiography samples

Large mosaic EM maps containing dozens of cell profiles were used for the quantitative analysis of the newly synthesized RNA autoradiography signal (see S3 Data). For each CoV, different conditions (infected and mock-infected cells, plus different labeling times) were compared using only samples developed after the same period of time. The analysis of the signal in different subcellular regions was carried out using home-built software. Areas of 4 μm² were randomly selected from the mosaic EM maps, and the autoradiography grains present in those areas were manually assigned to the underlying cellular structures. The abundance of the different types of subcellular structures was estimated through virtual points in a 5×5 lattice superimposed to each selected area, which were also assigned to the different subcellular classes. Regularly along the process, the annotated data per condition were split into 2 random groups, and the Kendall and Spearman coefficients, which measure the concordance between 2 data sets [76], were calculated. New random regions were added until the average Kendall and Spearman coefficients resulting from 10 random splits were higher than 0.8 and 0.9, respectively (maximum value, 1). Labeling densities and RLIs were then calculated from the annotated points [39].

For the analysis of the association of vRNA synthesis with each of the different ROs motifs, the specific DMVs, DMSs, and CM included in the analysis were carefully selected. Only individual DMVs that were at least 1 μm away from any other virus-induced membrane modification were selected. For every grain present in an area of 750-nm radius around each DMV, the distance to the DMV center was measured. In the case of DMSs, which were always part of clusters of virus-induced membrane structures, only DMSs in the periphery of these clusters were selected. The quantified signal was limited to subareas devoid of other RO motifs, which were defined by circular arcs (typically 30° to 100°, radius 500 nm) opposite to the RO clusters. CM are irregular structures that appear partially or totally surrounded by DMVs. Only large CM (>0.6 μm across) were selected in order to make more apparent (if present) any decay of the autoradiography signal as the distance to the surrounding DMVs increased. For each autoradiography grain, both the distance to the closest CM boundary (d₁) and the distance to the opposite CM edge (d₂) were measured. The relative distance to the CM edge was then calculated as d₁/(d₁+d₂) and expressed in percentages. All the measurements in different DMVs, DMSs, and CM were made using Aperio Imagescope software (Leica) and pooled together into 3 single data sets.

Supporting information

S1 Video. Electron tomography of the membrane structures induced in MERS-CoV infection.

Animation illustrating the tomography reconstruction and model presented in Fig 1B. The video first shows the tomographic slices (1.2-nm thick) through the reconstructed volume and then surface-rendered models of the different structures segmented from the tomogram: DMSs (orange), CM (blue), and DMVs (yellow and lilac, outer and inner membranes, respectively), ER (green), and a vesicle (silver) containing virions (pink). The movie highlights the DMS association with CM, which, in turn, connect to ER membranes, and these to DMVs. CM, convoluted membranes; DMS, double-membrane spherule; DMV, double-membrane vesicle; ER, endoplasmic reticulum; MERS-CoV, Middle East respiratory syndrome-coronavirus.

(MP4)

Click here for additional data file.^{(9.8MB, mp4)}

S1 Fig. Detection of DMSs in cryo-fixed and FS samples of CoV-infected cells.

Analysis of previously described samples of CoV-infected cells, prepared for EM either by HPF (A) or cryo-plunging (B). A targeted search revealed the presence of DMSs (white arrowheads) in close association with CM. In comparison with the chemically fixed samples used in this study, the superior ultrastructural preservation of cryo-fixation results in less distorted membranes, but also in a denser cytoplasm and darker CM that makes DMS less apparent. (A) Example from a MERS-CoV-infected Vero cell (16 hpi) in a sample used in [21]. (B) Region in a SARS-CoV-infected Vero E6 cell (8 hpi), adapted from [15]. Scale bars, 250 nm. CM, convoluted membranes; CoV, coronavirus; DMS, double-membrane spherule; EM, electron microscopy; FS, freeze-substituted; HPF, high-pressure freezing; hpi, hours postinfection; MERS-CoV, Middle East respiratory syndrome-coronavirus; SARS-CoV, severe acute respiratory syndrome-CoV.

(TIF)

Click here for additional data file.^{(6.1MB, tif)}

S1 Text. Autoradiography in electron microscopy.

(DOCX)

Click here for additional data file.^{(60.8KB, docx)}

S1 Data. Relative sizes of DMVs, DMSs, and virions.

DMS, double-membrane spherule; DMV, double-membrane vesicle.

(XLSX)

Click here for additional data file.^{(40.3KB, xlsx)}

S2 Data. Incorporation of radioactive label into RNA with different labeling times in SARS-CoV-infected cells.

SARS-CoV, severe acute respiratory syndrome-coronavirus.

(XLSX)

Click here for additional data file.^{(15.7KB, xlsx)}

S3 Data. Quantitative analysis of the autoradiography signal per subcellular structure in SARS-CoV and MERS-CoV-infected cells.

MERS-CoV, Middle East respiratory syndrome-coronavirus; SARS-CoV, severe acute respiratory syndrome-coronavirus.

(XLSX)

Click here for additional data file.^{(36.5KB, xlsx)}

S4 Data. Autoradiography signal distribution around DMVs in MERS-CoV-infected cells.

DMV, double-membrane vesicle; MERS-CoV, Middle East respiratory syndrome-coronavirus.

(XLSX)

Click here for additional data file.^{(48KB, xlsx)}

S5 Data. Autoradiography signal distribution in CM and DMSs in MERS-CoV-infected cells.

CM, convoluted membranes; DMS, double-membrane spherule; MERS-CoV, Middle East respiratory syndrome-coronavirus.

(XLSX)

Click here for additional data file.^{(45KB, xlsx)}

S6 Data. Analysis of the autoradiography signal distribution around DMVs and DMSs in IBV-infected cells.

DMS, double-membrane spherule; DMV, double-membrane vesicle; IBV, infectious bronchitis virus.

(XLSX)

Click here for additional data file.^{(73.4KB, xlsx)}

Acknowledgments

We are grateful to Yvonne van der Meer, Charlotte Melia, and Barbara van der Hoeven for their assistance.

Abbreviations

+RNA: positive-stranded RNA
CM: convoluted membranes
CoV: coronavirus
DMS: double-membrane spherule
DMV: double-membrane vesicle
ds: double-stranded
EM: electron microscopy
ER: endoplasmic reticulum
ERGIC: ER-Golgi intermediate compartment
HCoV-229E: human coronavirus 229E
HCV: hepatitis C virus
HPF-FS: high-pressure freezing and freeze-substitution
hpi: hours postinfection
IBV: infectious bronchitis virus
IEM: immunoelectron microscopy
MERS-CoV: Middle East respiratory syndrome-CoV
MHV: murine hepatitis virus
MOI: multiplicity of infection
nsp: nonstructural protein
RLI: relative labeling index
RO: replication organelle
SARS-CoV: severe acute respiratory syndrome-CoV
vRNA: viral RNA

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This research was partially funded by the Netherlands Organization for Scientific Research (NWO, https://www.nwo.nl/en) through grants to M.B. (NWO-MEERVOUD-863.10.003) and to E.J.S. and A.J.K. (NWO-CW-700.57.301). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1.Romero-Brey I, Bartenschlager R. Membranous replication factories induced by plus-strand RNA viruses. Viruses. 2014;6(7):2826–57. 10.3390/v6072826 . [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Harak C, Lohmann V. Ultrastructure of the replication sites of positive-strand RNA viruses. Virology. 2015;479–480:418–33. 10.1016/j.virol.2015.02.029 . [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Nagy PD, Strating JR, van Kuppeveld FJ. Building Viral Replication Organelles: Close Encounters of the Membrane Types. PLoS Pathog. 2016;12(10):e1005912 10.1371/journal.ppat.1005912 . [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Scutigliani EM, Kikkert M. Interaction of the innate immune system with positive-strand RNA virus replication organelles. Cytokine Growth Factor Rev. 2017;37:17–27. 10.1016/j.cytogfr.2017.05.007 . [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Schwartz M, Chen J, Janda M, Sullivan M, den Boon J, Ahlquist P. A positive-strand RNA virus replication complex parallels form and function of retrovirus capsids. Mol Cell. 2002;9(3):505–14. 10.1016/s1097-2765(02)00474-4 . [DOI] [PubMed] [Google Scholar]
6.Kopek BG, Perkins G, Miller DJ, Ellisman MH, Ahlquist P. Three-dimensional analysis of a viral RNA replication complex reveals a virus-induced mini-organelle. PLoS Biol. 2007;5(9):e220 10.1371/journal.pbio.0050220 . [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Welsch S, Miller S, Romero-Brey I, Merz A, Bleck CK, Walther P, et al. Composition and three-dimensional architecture of the dengue virus replication and assembly sites. Cell Host Microbe. 2009;5(4):365–75. 10.1016/j.chom.2009.03.007 . [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Kallio K, Hellstrom K, Balistreri G, Spuul P, Jokitalo E, Ahola T. Template RNA length determines the size of replication complex spherules for Semliki Forest virus. J Virol. 2013;87(16):9125–34. 10.1128/JVI.00660-13 . [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Fernandez de Castro I, Fernandez JJ, Barajas D, Nagy PD, Risco C. Three-dimensional imaging of the intracellular assembly of a functional viral RNA replicase complex. J Cell Sci. 2017;130(1):260–8. 10.1242/jcs.181586 . [DOI] [PubMed] [Google Scholar]
10.Limpens RW, van der Schaar HM, Kumar D, Koster AJ, Snijder EJ, van Kuppeveld FJ, et al. The transformation of enterovirus replication structures: a three-dimensional study of single- and double-membrane compartments. mBio. 2011;2(5). 10.1128/mBio.00166-11 . [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Belov GA, Nair V, Hansen BT, Hoyt FH, Fischer ER, Ehrenfeld E. Complex dynamic development of poliovirus membranous replication complexes. J Virol. 2012;86(1):302–12. 10.1128/JVI.05937-11 . [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Doerflinger SY, Cortese M, Romero-Brey I, Menne Z, Tubiana T, Schenk C, et al. Membrane alterations induced by nonstructural proteins of human norovirus. PLoS Pathog. 2017;13(10):e1006705 10.1371/journal.ppat.1006705 . [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Romero-Brey I, Merz A, Chiramel A, Lee JY, Chlanda P, Haselman U, et al. Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication. PLoS Pathog. 2012;8(12):e1003056 10.1371/journal.ppat.1003056 . [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Gosert R, Kanjanahaluethai A, Egger D, Bienz K, Baker SC. RNA replication of mouse hepatitis virus takes place at double-membrane vesicles. J Virol. 2002;76(8):3697–708. 10.1128/jvi.76.8.3697-3708.2002 . [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Knoops K, Kikkert M, Worm SH, Zevenhoven-Dobbe JC, van der Meer Y, Koster AJ, et al. SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum. PLoS Biol. 2008;6(9):e226 10.1371/journal.pbio.0060226 . [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Knoops K, Barcena M, Limpens RW, Koster AJ, Mommaas AM, Snijder EJ. Ultrastructural characterization of arterivirus replication structures: reshaping the endoplasmic reticulum to accommodate viral RNA synthesis. J Virol. 2012;86(5):2474–87. 10.1128/JVI.06677-11 . [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Maier HJ, Neuman BW, Bickerton E, Keep SM, Alrashedi H, Hall R, et al. Extensive coronavirus-induced membrane rearrangements are not a determinant of pathogenicity. Sci Rep. 2016;6:27126 10.1038/srep27126 . [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Zhang W, Chen K, Zhang X, Guo C, Chen Y, Liu X. An integrated analysis of membrane remodeling during porcine reproductive and respiratory syndrome virus replication and assembly. PLoS ONE. 2018;13(7):e0200919 10.1371/journal.pone.0200919 . [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Weber F, Wagner V, Rasmussen SB, Hartmann R, Paludan SR. Double-stranded RNA is produced by positive-strand RNA viruses and DNA viruses but not in detectable amounts by negative-strand RNA viruses. J Virol. 2006;80(10):5059–64. 10.1128/JVI.80.10.5059-5064.2006 . [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Ulasli M, Verheije MH, de Haan CA, Reggiori F. Qualitative and quantitative ultrastructural analysis of the membrane rearrangements induced by coronavirus. Cellular Microbiol. 2010;12(6):844–61. 10.1111/j.1462-5822.2010.01437.x . [DOI] [PMC free article] [PubMed] [Google Scholar]
21.de Wilde AH, Raj VS, Oudshoorn D, Bestebroer TM, van Nieuwkoop S, Limpens RW, et al. MERS-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin A or interferon-alpha treatment. J Gen Virol. 2013;94(Pt 8):1749–60. 10.1099/vir.0.052910-0 . [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Angelini MM, Akhlaghpour M, Neuman BW, Buchmeier MJ. Severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles. mBio. 2013;4(4):e00524–13. 10.1128/mBio.00524-13 . [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Oudshoorn D, Rijs K, Limpens R, Groen K, Koster AJ, Snijder EJ, et al. Expression and Cleavage of Middle East Respiratory Syndrome Coronavirus nsp3-4 Polyprotein Induce the Formation of Double-Membrane Vesicles That Mimic Those Associated with Coronaviral RNA Replication. mBio. 2017;8(6):e01658–17. 10.1128/mBio.01658-17 . [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Al-Mulla HM, Turrell L, Smith NM, Payne L, Baliji S, Zust R, et al. Competitive fitness in coronaviruses is not correlated with size or number of double-membrane vesicles under reduced-temperature growth conditions. mBio. 2014;5(2):e01107–13. 10.1128/mBio.01107-13 . [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Lundin A, Dijkman R, Bergstrom T, Kann N, Adamiak B, Hannoun C, et al. Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus. PLoS Pathog. 2014;10(5):e1004166 10.1371/journal.ppat.1004166 . [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Maier HJ, Hawes PC, Cottam EM, Mantell J, Verkade P, Monaghan P, et al. Infectious bronchitis virus generates spherules from zippered endoplasmic reticulum membranes. mBio. 2013;4(5):e00801–13. 10.1128/mBio.00801-13 . [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Doyle N, Hawes PC, Simpson J, Adams LH, Maier HJ. The Porcine Deltacoronavirus Replication Organelle Comprises Double-Membrane Vesicles and Zippered Endoplasmic Reticulum with Double-Membrane Spherules. Viruses. 2019;11(11):1030 10.3390/v11111030 . [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus AD, Fouchier RA. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N Engl J Med. 2012;367(19):1814–20. 10.1056/NEJMoa1211721 . [DOI] [PubMed] [Google Scholar]
29.van Boheemen S, de Graaf M, Lauber C, Bestebroer TM, Raj VS, Zaki AM, et al. Genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans. mBio. 2012;3(6):e00473–12. 10.1128/mBio.00473-12 . [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, et al. A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med. 2020;382(8):727–733. 10.1056/NEJMoa2001017 . [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Lu R, Zhao X, Li J, Niu P, Yang B, Wu H, et al. Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet. 2020;395(10224):565–574. 10.1016/S0140-6736(20)30251-8 . [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Bienz KA. Techniques and applications of autoradiography in the light and electron microscope. Microsc Acta. 1977;79(1):1–22. . [PubMed] [Google Scholar]
33.Bozzola JJ, Russell LD. Autoradiography & Radioautography. Electron Microscopy: Principles and Techniques for Biologists. Sudbury (MA):Jones and Bartlett Publishers; 1999. p. 293–308. [Google Scholar]
34.Subissi L, Posthuma CC, Collet A, Zevenhoven-Dobbe JC, Gorbalenya AE, Decroly E, et al. One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities. Proc Natl Acad Sci U S A. 2014;111(37):E3900–9. 10.1073/pnas.1323705111 . [DOI] [PMC free article] [PubMed] [Google Scholar]
35.van den Worm SH, Knoops K, Zevenhoven-Dobbe JC, Beugeling C, van der Meer Y, Mommaas AM, et al. Development and RNA-synthesizing activity of coronavirus replication structures in the absence of protein synthesis. J Virol. 2011;85(11):5669–73. 10.1128/JVI.00403-11 . [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Muller C, Hardt M, Schwudke D, Neuman BW, Pleschka S, Ziebuhr J. Inhibition of Cytosolic Phospholipase A2alpha Impairs an Early Step of Coronavirus Replication in Cell Culture. J Virol. 2018;92(4):e01463–17. 10.1128/JVI.01463-17 . [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Ogando NS, Dalebout TJ, Zevenhoven-Dobbe JC, Limpens RW, van der Meer Y, Caly L, et al. SARS-coronavirus-2 replication in Vero E6 cells: replication kinetics, rapid adaptation and cytopathology. J Gen Virol. Forthcoming. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Faas FG, Avramut MC, van den Berg BM, Mommaas AM, Koster AJ, Ravelli RB. Virtual nanoscopy: generation of ultra-large high resolution electron microscopy maps. J Cell Biol. 2012;198(3):457–69. 10.1083/jcb.201201140 . [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Mayhew TM, Lucocq JM, Griffiths G. Relative labelling index: a novel stereological approach to test for non-random immunogold labelling of organelles and membranes on transmission electron microscopy thin sections. J Microsc. 2002;205(2):153–64. 10.1046/j.0022-2720.2001.00977.x . [DOI] [PubMed] [Google Scholar]
40.Tooze J, Tooze S, Warren G. Replication of coronavirus MHV-A59 in sac- cells: determination of the first site of budding of progeny virions. Eur J Cell Biol. 1984;33(2):281–93. . [PubMed] [Google Scholar]
41.Goldsmith CS, Tatti KM, Ksiazek TG, Rollin PE, Comer JA, Lee WW, et al. Ultrastructural characterization of SARS coronavirus. Emerg Infect Dis. 2004;10(2):320–6. 10.3201/eid1002.030913 . [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Stertz S, Reichelt M, Spiegel M, Kuri T, Martinez-Sobrido L, Garcia-Sastre A, et al. The intracellular sites of early replication and budding of SARS-coronavirus. Virology. 2007;361(2):304–15. 10.1016/j.virol.2006.11.027 . [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Karreman MA, Van Donselaar EG, Agronskaia AV, Verrips CT, Gerritsen HC. Novel contrasting and labeling procedures for correlative microscopy of thawed cryosections. J Histochem Cytochem. 2013;61(3):236–47. 10.1369/0022155412473756 . [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Klumperman J, Locker JK, Meijer A, Horzinek MC, Geuze HJ, Rottier PJ. Coronavirus M proteins accumulate in the Golgi complex beyond the site of virion budding. J Virol. 1994;68(10):6523–34. . [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Ng ML, Tan SH, See EE, Ooi EE, Ling AE. Proliferative growth of SARS coronavirus in Vero E6 cells. J Gen Virol. 2003;84(12):3291–303. 10.1099/vir.0.19505-0 . [DOI] [PubMed] [Google Scholar]
46.Becker WB, McIntosh K, Dees JH, Chanock RM. Morphogenesis of avian infectious bronchitis virus and a related human virus (strain 229E). J Virol. 1967;1(5):1019–27. . [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Zhou X, Cong Y, Veenendaal T, Klumperman J, Shi D, Mari M, et al. Ultrastructural Characterization of Membrane Rearrangements Induced by Porcine Epidemic Diarrhea Virus Infection. Viruses. 2017;9(9):251 10.3390/v9090251 . [DOI] [PMC free article] [PubMed] [Google Scholar]
48.Machamer CE, Mentone SA, Rose JK, Farquhar MG. The E1 glycoprotein of an avian coronavirus is targeted to the cis Golgi complex. Proc Natl Acad Sci U S A. 1990;87(18):6944–8. 10.1073/pnas.87.18.6944 . [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Opstelten DJ, Raamsman MJ, Wolfs K, Horzinek MC, Rottier PJ. Envelope glycoprotein interactions in coronavirus assembly. J Cell Biol. 1995;131(2):339–49. 10.1083/jcb.131.2.339 . [DOI] [PMC free article] [PubMed] [Google Scholar]
50.Denison MR, Spaan WJ, van der Meer Y, Gibson CA, Sims AC, Prentice E, et al. The putative helicase of the coronavirus mouse hepatitis virus is processed from the replicase gene polyprotein and localizes in complexes that are active in viral RNA synthesis. J Virol. 1999;73(8):6862–71. . [DOI] [PMC free article] [PubMed] [Google Scholar]
51.van der Meer Y, Snijder EJ, Dobbe JC, Schleich S, Denison MR, Spaan WJ, et al. Localization of mouse hepatitis virus nonstructural proteins and RNA synthesis indicates a role for late endosomes in viral replication. J Virol. 1999;73(9):7641–57. . [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Sims AC, Ostermann J, Denison MR. Mouse hepatitis virus replicase proteins associate with two distinct populations of intracellular membranes. J Virol. 2000;74(12):5647–54. 10.1128/jvi.74.12.5647-5654.2000 . [DOI] [PMC free article] [PubMed] [Google Scholar]
53.V'Kovski P, Gerber M, Kelly J, Pfaender S, Ebert N, Braga Lagache S, et al. Determination of host proteins composing the microenvironment of coronavirus replicase complexes by proximity-labeling. eLife. 2019;8:e42037 10.7554/eLife.42037 . [DOI] [PMC free article] [PubMed] [Google Scholar]
54.McBride R, van Zyl M, Fielding BC. The coronavirus nucleocapsid is a multifunctional protein. Viruses. 2014;6(8):2991–3018. 10.3390/v6082991 . [DOI] [PMC free article] [PubMed] [Google Scholar]
55.Hagemeijer MC, Vonk AM, Monastyrska I, Rottier PJ, de Haan CA. Visualizing coronavirus RNA synthesis in time by using click chemistry. J Virol. 2012;86(10):5808–16. 10.1128/JVI.07207-11 . [DOI] [PMC free article] [PubMed] [Google Scholar]
56.Beachboard DC, Anderson-Daniels JM, Denison MR. Mutations across murine hepatitis virus nsp4 alter virus fitness and membrane modifications. J Virol. 2015;89(4):2080–9. 10.1128/JVI.02776-14 . [DOI] [PMC free article] [PubMed] [Google Scholar]
57.Neuman BW. How the double spherules of infectious bronchitis virus impact our understanding of RNA virus replicative organelles. mBio. 2013;4(6):e00987–13. 10.1128/mBio.00987-13 . [DOI] [PMC free article] [PubMed] [Google Scholar]
58.Hagemeijer MC, Monastyrska I, Griffith J, van der Sluijs P, Voortman J, van Bergen en Henegouwen PM, et al. Membrane rearrangements mediated by coronavirus nonstructural proteins 3 and 4. Virology. 2014;458–459:125–35. 10.1016/j.virol.2014.04.027 . [DOI] [PMC free article] [PubMed] [Google Scholar]
59.Deng Y, Almsherqi ZA, Ng MM, Kohlwein SD. Do viruses subvert cholesterol homeostasis to induce host cubic membranes? Trends in cell biology. 2010;20(7):371–9. 10.1016/j.tcb.2010.04.001 . [DOI] [PMC free article] [PubMed] [Google Scholar]
60.Almsherqi ZA, Kohlwein SD, Deng Y. Cubic membranes: a legend beyond the Flatland* of cell membrane organization. J Cell Biol. 2006;173(6):839–44. 10.1083/jcb.200603055 . [DOI] [PMC free article] [PubMed] [Google Scholar]
61.Paul D, Hoppe S, Saher G, Krijnse-Locker J, Bartenschlager R. Morphological and biochemical characterization of the membranous hepatitis C virus replication compartment. J Virol. 2013;87(19):10612–27. 10.1128/JVI.01370-13 . [DOI] [PMC free article] [PubMed] [Google Scholar]
62.Melia CE, van der Schaar HM, Lyoo H, Limpens R, Feng Q, Wahedi M, et al. Escaping Host Factor PI4KB Inhibition: Enterovirus Genomic RNA Replication in the Absence of Replication Organelles. Cell Rep. 2017;21(3):587–99. 10.1016/j.celrep.2017.09.068 . [DOI] [PMC free article] [PubMed] [Google Scholar]
63.Melia CE, van der Schaar HM, de Jong AWM, Lyoo HR, Snijder EJ, Koster AJ, et al. The Origin, Dynamic Morphology, and PI4P-Independent Formation of Encephalomyocarditis Virus Replication Organelles. mBio. 2018;9(2):e00420–18. 10.1128/mBio.00420-18 . [DOI] [PMC free article] [PubMed] [Google Scholar]
64.van Hemert MJ, van den Worm SH, Knoops K, Mommaas AM, Gorbalenya AE, Snijder EJ. SARS-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro. PLoS Pathog. 2008;4(5):e1000054 10.1371/journal.ppat.1000054 . [DOI] [PMC free article] [PubMed] [Google Scholar]
65.Kindler E, Gil-Cruz C, Spanier J, Li Y, Wilhelm J, Rabouw HH, et al. Early endonuclease-mediated evasion of RNA sensing ensures efficient coronavirus replication. PLoS Pathog. 2017;13(2):e1006195 10.1371/journal.ppat.1006195 . [DOI] [PMC free article] [PubMed] [Google Scholar]
66.Thiel V, Ivanov KA, Putics A, Hertzig T, Schelle B, Bayer S, et al. Mechanisms and enzymes involved in SARS coronavirus genome expression. J Gen Virol. 2003;84(Pt 9):2305–15. 10.1099/vir.0.19424-0 . [DOI] [PubMed] [Google Scholar]
67.Hamre D, Procknow JJ. A new virus isolated from the human respiratory tract. Proc Soc Exp Biol Med. 1966;121(1):190–3. 10.3181/00379727-121-30734 . [DOI] [PubMed] [Google Scholar]
68.Casais R, Thiel V, Siddell SG, Cavanagh D, Britton P. Reverse genetics system for the avian coronavirus infectious bronchitis virus. J Virol. 2001;75(24):12359–69. 10.1128/JVI.75.24.12359-12369.2001 . [DOI] [PMC free article] [PubMed] [Google Scholar]
69.Bosch BJ, van der Zee R, de Haan CA, Rottier PJ. The coronavirus spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex. J Virol. 2003;77(16):8801–11. 10.1128/jvi.77.16.8801-8811.2003 . [DOI] [PMC free article] [PubMed] [Google Scholar]
70.Snijder EJ, van der Meer Y, Zevenhoven-Dobbe J, Onderwater JJ, van der Meulen J, Koerten HK, et al. Ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex. J Virol. 2006;80(12):5927–40. 10.1128/JVI.02501-05 . [DOI] [PMC free article] [PubMed] [Google Scholar]
71.Schonborn J, Oberstrass J, Breyel E, Tittgen J, Schumacher J, Lukacs N. Monoclonal antibodies to double-stranded RNA as probes of RNA structure in crude nucleic acid extracts. Nucleic Acids Res. 1991;19(11):2993–3000. 10.1093/nar/19.11.2993 . [DOI] [PMC free article] [PubMed] [Google Scholar]
72.Widjaja I, Wang C, van Haperen R, Gutierrez-Alvarez J, van Dieren B, Okba NMA, et al. Towards a solution to MERS: protective human monoclonal antibodies targeting different domains and functions of the MERS-coronavirus spike glycoprotein. Emerg Microbes Infect. 2019;8(1):516–30. 10.1080/22221751.2019.1597644 . [DOI] [PMC free article] [PubMed] [Google Scholar]
73.Ginsel LA, Onderwater JJ, Daems WT. Resolution of a gold latensification-elon ascorbic acid developer for Ilford L4 emulsion. Histochemistry. 1979;61(3):343–6. 10.1007/BF00508456 . [DOI] [PubMed] [Google Scholar]
74.Kukulski W, Schorb M, Welsch S, Picco A, Kaksonen M, Briggs JA. Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision. J Cell Biol. 2011;192(1):111–9. 10.1083/jcb.201009037 . [DOI] [PMC free article] [PubMed] [Google Scholar]
75.Kremer JR, Mastronarde DN, McIntosh JR. Computer visualization of three-dimensional image data using IMOD. J Struct Biol. 1996;116(1):71–6. 10.1006/jsbi.1996.0013 . [DOI] [PubMed] [Google Scholar]
76.Lucocq JM, Habermann A, Watt S, Backer JM, Mayhew TM, Griffiths G. A rapid method for assessing the distribution of gold labeling on thin sections. J Histochem Cytochem. 2004;52(8):991–1000. 10.1369/jhc.3A6178.2004 . [DOI] [PubMed] [Google Scholar]

PLoS Biol. doi: 10.1371/journal.pbio.3000715.r001

Decision Letter 0

Roland G Roberts

10 Mar 2020

Dear Dr Bárcena,

Thank you for submitting your manuscript entitled "A unifying structural and functional model of the coronavirus replication organelle: tracking down RNA synthesis" for consideration as a Research Article by PLOS Biology.

Your manuscript has now been evaluated by the PLOS Biology editorial staff, as well as by an academic editor with relevant expertise, and I'm writing to let you know that we would like to send your submission out for external peer review. I should warn you that we're not wholly persuaded of the strength of advance, so we'll be looking for some enthusiasm from the reviewers.

However, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.

Please re-submit your manuscript within two working days, i.e. by Mar 12 2020 11:59PM.

During resubmission, you will be invited to opt-in to posting your pre-review manuscript as a bioRxiv preprint. Visit http://journals.plos.org/plosbiology/s/preprints for full details. If you consent to posting your current manuscript as a preprint, please upload a single Preprint PDF when you re-submit.

Once your full submission is complete, your paper will undergo a series of checks in preparation for peer review. Once your manuscript has passed all checks it will be sent out for review.

Feel free to email us at plosbiology@plos.org if you have any queries relating to your submission.

Kind regards,

Roli Roberts

Roland G Roberts, PhD,

Senior Editor

PLOS Biology

PLoS Biol. doi: 10.1371/journal.pbio.3000715.r002

Decision Letter 1

Roland G Roberts

9 Apr 2020

Dear Dr Bárcena,

Thank you very much for submitting your manuscript "A unifying structural and functional model of the coronavirus replication organelle: tracking down RNA synthesis" for consideration as a Research Article by PLOS Biology. As with all papers reviewed by the journal, yours was evaluated by the PLOS Biology editors as well as by an Academic Editor with relevant expertise and in this case by three independent reviewers. The reviewers appreciated the attention to an important topic.

Based on the reviews, we will probably accept this manuscript for publication, assuming that you will modify the manuscript to address the remaining points raised by the reviewers. Please also make sure to address the Data Policy and other policy-related requests noted at the end of this email.

We expect to receive your revised manuscript within two weeks. Your revisions should address the specific points made by each reviewer. In addition to the remaining revisions and before we will be able to formally accept your manuscript and consider it "in press", we also need to ensure that your article conforms to our guidelines. A member of our team will be in touch shortly with a set of requests. As we can't proceed until these requirements are met, your swift response will help prevent delays to publication.

*Copyediting*

Upon acceptance of your article, your final files will be copyedited and typeset into the final PDF. While you will have an opportunity to review these files as proofs, PLOS will only permit corrections to spelling or significant scientific errors. Therefore, please take this final revision time to assess and make any remaining major changes to your manuscript.

NOTE: If Supporting Information files are included with your article, note that these are not copyedited and will be published as they are submitted. Please ensure that these files are legible and of high quality (at least 300 dpi) in an easily accessible file format. For this reason, please be aware that any references listed in an SI file will not be indexed. For more information, see our Supporting Information guidelines:

https://journals.plos.org/plosbiology/s/supporting-information

*Published Peer Review History*

Please note that you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:

https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

*Early Version*

Please note that an uncorrected proof of your manuscript will be published online ahead of the final version, unless you opted out when submitting your manuscript. If, for any reason, you do not want an earlier version of your manuscript published online, uncheck the box. Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us as soon as possible if you or your institution is planning to press release the article.

*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosbiology/s/submission-guidelines#loc-materials-and-methods

*Submitting Your Revision*

To submit your revision, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' to find your submission record. Your revised submission must include a cover letter, a Response to Reviewers file that provides a detailed response to the reviewers' comments (if applicable), and a track-changes file indicating any changes that you have made to the manuscript.

Please do not hesitate to contact me should you have any questions.

Sincerely,

Roli Roberts

Roland G Roberts, PhD,

Senior Editor

PLOS Biology

------------------------------------------------------------------------

DATA POLICY:

You may be aware of the PLOS Data Policy, which requires that all data be made available without restriction: http://journals.plos.org/plosbiology/s/data-availability. For more information, please also see this editorial: http://dx.doi.org/10.1371/journal.pbio.1001797

Note that we do not require all raw data. Rather, we ask that all individual quantitative observations that underlie the data summarized in the figures and results of your paper be made available in one of the following forms:

1) Supplementary files (e.g., excel). Please ensure that all data files are uploaded as 'Supporting Information' and are invariably referred to (in the manuscript, figure legends, and the Description field when uploading your files) using the following format verbatim: S1 Data, S2 Data, etc. Multiple panels of a single or even several figures can be included as multiple sheets in one excel file that is saved using exactly the following convention: S1_Data.xlsx (using an underscore).

2) Deposition in a publicly available repository. Please also provide the accession code or a reviewer link so that we may view your data before publication.

Regardless of the method selected, please ensure that you provide the individual numerical values that underlie the summary data displayed in the following figure panels as they are needed for readers to assess your analysis and to reproduce it: Figs 1D, 3ACD, 4CD, 5BDE, and S3CDE. NOTE: the numerical data provided should include all replicates AND the way in which the plotted mean and errors were derived (it should not present only the mean/average values).

Please also ensure that figure legends in your manuscript include information on where the underlying data can be found, and ensure your supplemental data file/s has a legend.

Please ensure that your Data Statement in the submission system accurately describes where your data can be found.

------------------------------------------------------------------------

BLOT AND GEL REPORTING REQUIREMENTS:

For manuscripts submitted on or after 1st July 2019, we require the original, uncropped and minimally adjusted images supporting all blot and gel results reported in an article's figures or Supporting Information files. We will require these files before a manuscript can be accepted so please prepare and upload them now. Please carefully read our guidelines for how to prepare and upload this data: https://journals.plos.org/plosbiology/s/figures#loc-blot-and-gel-reporting-requirements

------------------------------------------------------------------------

REVIEWERS' COMMENTS:

Reviewer #1:

This manuscript by Snijder and co-workers entitled "A unifying structural and functional model of the coronavirus replication organelle: tracking down RNA synthesis" describes elegant studies aimed at discerning if double membrane vesicles or other ER sites modified by viral proteins are the site of coronavirus RNA synthesis during viral replication. The authors describe that an early study using immuno-EM revealed that virus-induced double membrane vesicles (DMVs) were the sites of viral RNA synthesis (Gosert et al., 2002 Replication of mouse hepatitis virus takes place at double membrane vesicles, reference 14). With the advancements in EM techniques and particularly EM tomography, researchers appreciated that coronavirus replication induced reorganization of host membranes, generating both convoluted membranes (CM) and DMVs (Knoops et al., SARS-coronavirus replication is supported by a reticulovesicular network of modified ER, reference 15). In addition, the term double membrane spherules (DMS) was introduced to describe membrane rearrangements detected during the replication of infectious bronchitis virus (Maier et al., 2013 IBV generates spherules from zippered ER membranes, reference 29), but it was not clear if these structures were unique to avian coronaviruses, or if they played a role in RNA synthesis. In this study, the authors used radiolabeling of viral RNA and detected the labeled RNA in association with the DMVs. Importantly, they also determined that CMs, DMSs, and DMVs can be visualized for all the coronavirus specimens they studied, including MERS-CoV, suggesting that these structures are generated during the assembly of the viral replication complex and the DMVs necessary for viral RNA synthesis.

The technical work here is tremendous and the images of the viral protein-modified membranes are stunning. However, the manuscript seems have an inordinate focus on the DMSs, potentially implicating these as unique (terminal?) entities. Isn't is possible that the DMS detected at the moment of fixation is in fact on its way to becoming a DMV? Alternatively, some of these DMSs could be unable to progress to DMV status, for reasons that are not yet clear. In the absence of live-cell EM, it is not possible to distinguish these possibilities, but certainly the possibility the DMS to DMV transition could be considered as the authors develop their "unifying structural and functional model" of coronavirus RNA synthesis. Currently, there is a significant emphasis on the DMS in the manuscript, although this structure is not the take home message, and there is no functional role given to these structures, even as potential intermediates. If there is evidence that a DMS can not continue to grow into a DMV, then this should be made clear in the manuscript. Overall, the authors provide important results using state-of-the-art techniques that confirms and extends the early studies that that DMVs are the site of coronavirus RNA synthesis.

Comments for the authors' consideration:

1. Lines 375-378: Consider changing the order of this description to 1) CM and zippered membranes; ii) DMS and iii) DMVs. This would allow you to speculate in a unifying model that coronaviruses modify membranes to promote "zippering", resulting in the formation of spherules that may mature into double membranes vesicles, the site of viral RNA synthesis. This "progression" is still hypothetical because it has not be visualized in real time, but certainly all the data presented here are at least consistent with this unifying model. One of the challenges is that researchers tend to see all of these structures simultaneously, and not a neat progression from one form to another. The discussion lines 410-413 seems to discount a progression model because of a lack of intermediate, but why couldn't a small double membrane spherule progress into a DMV? One could even speculate that the open DMS allows for capturing of a RNA that "seeds" the DMV to become a site of RNA synthesis. It is reasonable to speculate about potential models in the discussion section, and to be open to multiple interpretations of the current findings.

2. Line 381: change "establish" to confirms and extends. This manuscript works very hard at presenting uncertainty or controversy about coronavirus replication sites. However, the questions that arose about the sites of RNA synthesis came about as the techniques to visualize the modified membranes were improved over time, suggesting the potential for more than the originally documented DMVs were involved RNA synthesis. Perhaps this is a stylistic point, but pointing out that the results here confirm and extend earlier studies is important and accurate.

3. The text could be much more concise by focusing on the idea that this work is evaluating all structures in multiple coronaviruses, which has not been done before in one manuscript, and seeking to determine if one or multiple sites are used for viral RNA synthesis. The "controversy" seems vastly over emphasized here.

4. The images in the supplementary section are striking also. If PLoS Biology allows it, try to include all figures in the body of the paper and have only the movie in the supplement.

Reviewer #2:

Coronaviruses have emerged as important zoonotic viruses with major threat to human health. In this paper, the authors characterized the ultra structures of human cells infected by different coronaviruses including MERS, SARS-CoV1, MHV, HCoV-229E and IBV. They have identified three different membranous structures, DMVs, CM and DMS, which have all been observed before in various coronavirus infections. The detection of DMS is new for MERS infection. Importantly, they were able now to provide a more universal picture that all these viruses induce the formation of all these RO structures. By using metabolic labeling of viral RNAs, after shutting down cellular transcription, they were able to demonstrate that DMVs are the likely places for coronavirus RNA synthesis and replication. Somewhat surprisingly, they find no significant roles for CM and DMS in coronavirus RNA synthesis and replication. This conclusion was further supported by detection of viral dsRNAs within DMVs.

The findings are potentially important, especially the metabolic labeling data are quite exciting. I found the manuscript is fun to read with beautiful images. The manuscript requires the following changes.

Fig. 1 shows images after 12 h incubation with many DMSs. What time point are these DMSs first appear?

Fig. 1 and Fig. 6. Please replace the arrowheads with different colors to aid the readers. Gray and black arrowheads could be quite similar on different computer screens.

Fig. 1F: please define what are arrows pointing at?

Fig. 1F: Are the outer DMVs and the ER membranes are continuous membranes or are there gaps between them. It seems that they are continuous based on the presented images here.

Fig. 4B: it would be useful to pinpoint DMVs, since it is very crowded RO is shown here.

Fig. 5. It would be useful to know what % of label was inside versus outside of DMVs? It looks more label is on the outside of DMVs (although very nearby to the DMVs). What is the possible explanation?

Lane 155: based on the findings in this work, I doubt it is correct to call zippered ER and CM as ROs, since no viral RNAs were detected in those structures.

L407: CM and DMS biogenesis does look very abundant at 12 time point in Fig. 1A-B, contradicting that these structures form late during infection. What is the first time point when CM and DMS structures are detected? The time of the vents for the biogenesis of various RO structures is confusing in this work.

Please discuss the origin of DMVs, since the ER origin of CM and DMS is discussed nicely in the discussion part.

I note that DMSs are possibly too small to harbor the large coronavirus RNAs, possibly dsRNA (if that is the template for RNA synthesis), sgRNAs and the replicase complex, while copying the RNA templates. Even the 6x times smaller tombusviruses and nodaviruses form almost that size spherules to support replication as the observed size of DMS in this work.

Is it possible that DMSs are high-density membrane-protein (both viral and host-derived) storage granules to reduce ER stress in the cells?

Do the authors have data on how much percentage of the label is incorporated into genomic versus subgenomic RNA species during metabolic labeling? That would be useful information.

Reviewer #3:

In this manuscript, the authors performed a detailed structural and functional characterization of the replication organelles (ROs) of coronaviruses. Coronavirus ROs are composed of predominantly double membrane vesicles (DMVs). Through comparative analysis, the authors demonstrate that convoluted membranes, zippered ER and double-membrane spherules are conserved across alpha-, beta- and gamma- CoVs and altogether form the viral ROs. Additionally, the authors employed a classical, yet elegant approach that combines ultrastructural analysis with RNA labeling using radiolabeled nucleosides to demonstrate that viral RNA synthesis is associated with double membrane vesicles. This finding provides best available evidence so far that DMVs are the site of viral RNA replication.

This is a timely study that sheds light on the role of the different membranous structures observed during CoV replication, although these structures have been described in several earlier reports. The main point of the present study is the clarification of a long-standing debate by showing that DMVs most likely are the site of viral RNA replication.

Major comment:

The results of this study are clear and the author´s claims are supported by their data. One of the aims was to clarify the apparent differences reported in the ultrastructural architecture of the coronaviral ROs. The authors achieved their goal by showing that the ROs of the different CoVs are essentially the same. One of the limitations is that the authors provide a single snapshot looking at a single time point for the different viruses, often in different cells and using different MOIs. Moreover, the authors do not provide a growth curve of the viruses in the different cell lines. This information would be useful to allow for a direct comparison between the different conditions. The authors could at least comment if the selected time points reflect the initial phase of the viral infection or a later phase.

Additionally, what is the order of appearance of the different structures forming the CoV ROs and is this order conserved among the different CoVs? This could support the idea of a general mechanism used by all CoVs and the regulation of the biogenesis of their ROs. If such information is already present in the literature the author should comment on this point in the discussion.

Minor points:

For the autoradiography experiments time points, MOIs and cell types differ between MERS-CoV and SARS-CoV infections. Are the time points selected by the authors comparable in terms of progression of viral infection of the two viruses in the different experimental conditions? In other words, are the selected time points within the exponential phase of viral replication or at the plateau phase of viral replication? The authors should comment on this.

Line 413 - there is no reference to backup this claim. Since this study does not analyze how the RO architecture changes during infection, the claim that DMV formation precedes the appearance of CMs should come from a previous study. The authors should add the reference of the paper to which they are referring.

FigS2- the figure legend states that Huh7 cells were infected with MERS-CoV while the figure annotation says Vero cells. The authors should clarify.

Line 177 - truncated sentence

PLoS Biol. 2020 Jun 8;18(6):e3000715. doi: 10.1371/journal.pbio.3000715.r003

Author response to Decision Letter 1

7 May 2020

Attachment

Submitted filename: ResponseToReviewers.docx

Click here for additional data file.^{(36KB, docx)}

PLoS Biol. doi: 10.1371/journal.pbio.3000715.r004

Decision Letter 2

Roland G Roberts

14 May 2020

Dear Dr Bárcena,

On behalf of my colleagues and the Academic Editor, Andrea Cimarelli, I am pleased to inform you that we will be delighted to publish your Research Article in PLOS Biology.

The files will now enter our production system. You will receive a copyedited version of the manuscript, along with your figures for a final review. You will be given two business days to review and approve the copyedit. Then, within a week, you will receive a PDF proof of your typeset article. You will have two days to review the PDF and make any final corrections. If there is a chance that you'll be unavailable during the copy editing/proof review period, please provide us with contact details of one of the other authors whom you nominate to handle these stages on your behalf. This will ensure that any requested corrections reach the production department in time for publication.

Early Version

The version of your manuscript submitted at the copyedit stage will be posted online ahead of the final proof version, unless you have already opted out of the process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

PRESS

We frequently collaborate with press offices. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximise its impact. If the press office is planning to promote your findings, we would be grateful if they could coordinate with biologypress@plos.org. If you have not yet opted out of the early version process, we ask that you notify us immediately of any press plans so that we may do so on your behalf.

We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit http://www.plos.org/about/media-inquiries/embargo-policy/.

Thank you again for submitting your manuscript to PLOS Biology and for your support of Open Access publishing. Please do not hesitate to contact me if I can provide any assistance during the production process.

Kind regards,

Vita Usova,

Publishing Editor

PLOS Biology

on behalf of

Roland Roberts,

Senior Editor

PLOS Biology

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Video. Electron tomography of the membrane structures induced in MERS-CoV infection.

(MP4)

Click here for additional data file.^{(9.8MB, mp4)}

S1 Fig. Detection of DMSs in cryo-fixed and FS samples of CoV-infected cells.

(TIF)

Click here for additional data file.^{(6.1MB, tif)}

S1 Text. Autoradiography in electron microscopy.

(DOCX)

Click here for additional data file.^{(60.8KB, docx)}

S1 Data. Relative sizes of DMVs, DMSs, and virions.

DMS, double-membrane spherule; DMV, double-membrane vesicle.

(XLSX)

Click here for additional data file.^{(40.3KB, xlsx)}

S2 Data. Incorporation of radioactive label into RNA with different labeling times in SARS-CoV-infected cells.

SARS-CoV, severe acute respiratory syndrome-coronavirus.

(XLSX)

Click here for additional data file.^{(15.7KB, xlsx)}

S3 Data. Quantitative analysis of the autoradiography signal per subcellular structure in SARS-CoV and MERS-CoV-infected cells.

MERS-CoV, Middle East respiratory syndrome-coronavirus; SARS-CoV, severe acute respiratory syndrome-coronavirus.

(XLSX)

Click here for additional data file.^{(36.5KB, xlsx)}

S4 Data. Autoradiography signal distribution around DMVs in MERS-CoV-infected cells.

DMV, double-membrane vesicle; MERS-CoV, Middle East respiratory syndrome-coronavirus.

(XLSX)

Click here for additional data file.^{(48KB, xlsx)}

S5 Data. Autoradiography signal distribution in CM and DMSs in MERS-CoV-infected cells.

CM, convoluted membranes; DMS, double-membrane spherule; MERS-CoV, Middle East respiratory syndrome-coronavirus.

(XLSX)

Click here for additional data file.^{(45KB, xlsx)}

S6 Data. Analysis of the autoradiography signal distribution around DMVs and DMSs in IBV-infected cells.

DMS, double-membrane spherule; DMV, double-membrane vesicle; IBV, infectious bronchitis virus.

(XLSX)

Click here for additional data file.^{(73.4KB, xlsx)}

Attachment

Submitted filename: ResponseToReviewers.docx

Click here for additional data file.^{(36KB, docx)}

Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

[pbio.3000715.ref001] 1.Romero-Brey I, Bartenschlager R. Membranous replication factories induced by plus-strand RNA viruses. Viruses. 2014;6(7):2826–57. 10.3390/v6072826 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref002] 2.Harak C, Lohmann V. Ultrastructure of the replication sites of positive-strand RNA viruses. Virology. 2015;479–480:418–33. 10.1016/j.virol.2015.02.029 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref003] 3.Nagy PD, Strating JR, van Kuppeveld FJ. Building Viral Replication Organelles: Close Encounters of the Membrane Types. PLoS Pathog. 2016;12(10):e1005912 10.1371/journal.ppat.1005912 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref004] 4.Scutigliani EM, Kikkert M. Interaction of the innate immune system with positive-strand RNA virus replication organelles. Cytokine Growth Factor Rev. 2017;37:17–27. 10.1016/j.cytogfr.2017.05.007 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref005] 5.Schwartz M, Chen J, Janda M, Sullivan M, den Boon J, Ahlquist P. A positive-strand RNA virus replication complex parallels form and function of retrovirus capsids. Mol Cell. 2002;9(3):505–14. 10.1016/s1097-2765(02)00474-4 . [DOI] [PubMed] [Google Scholar]

[pbio.3000715.ref006] 6.Kopek BG, Perkins G, Miller DJ, Ellisman MH, Ahlquist P. Three-dimensional analysis of a viral RNA replication complex reveals a virus-induced mini-organelle. PLoS Biol. 2007;5(9):e220 10.1371/journal.pbio.0050220 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref007] 7.Welsch S, Miller S, Romero-Brey I, Merz A, Bleck CK, Walther P, et al. Composition and three-dimensional architecture of the dengue virus replication and assembly sites. Cell Host Microbe. 2009;5(4):365–75. 10.1016/j.chom.2009.03.007 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref008] 8.Kallio K, Hellstrom K, Balistreri G, Spuul P, Jokitalo E, Ahola T. Template RNA length determines the size of replication complex spherules for Semliki Forest virus. J Virol. 2013;87(16):9125–34. 10.1128/JVI.00660-13 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref009] 9.Fernandez de Castro I, Fernandez JJ, Barajas D, Nagy PD, Risco C. Three-dimensional imaging of the intracellular assembly of a functional viral RNA replicase complex. J Cell Sci. 2017;130(1):260–8. 10.1242/jcs.181586 . [DOI] [PubMed] [Google Scholar]

[pbio.3000715.ref010] 10.Limpens RW, van der Schaar HM, Kumar D, Koster AJ, Snijder EJ, van Kuppeveld FJ, et al. The transformation of enterovirus replication structures: a three-dimensional study of single- and double-membrane compartments. mBio. 2011;2(5). 10.1128/mBio.00166-11 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref011] 11.Belov GA, Nair V, Hansen BT, Hoyt FH, Fischer ER, Ehrenfeld E. Complex dynamic development of poliovirus membranous replication complexes. J Virol. 2012;86(1):302–12. 10.1128/JVI.05937-11 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref012] 12.Doerflinger SY, Cortese M, Romero-Brey I, Menne Z, Tubiana T, Schenk C, et al. Membrane alterations induced by nonstructural proteins of human norovirus. PLoS Pathog. 2017;13(10):e1006705 10.1371/journal.ppat.1006705 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref013] 13.Romero-Brey I, Merz A, Chiramel A, Lee JY, Chlanda P, Haselman U, et al. Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication. PLoS Pathog. 2012;8(12):e1003056 10.1371/journal.ppat.1003056 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref014] 14.Gosert R, Kanjanahaluethai A, Egger D, Bienz K, Baker SC. RNA replication of mouse hepatitis virus takes place at double-membrane vesicles. J Virol. 2002;76(8):3697–708. 10.1128/jvi.76.8.3697-3708.2002 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref015] 15.Knoops K, Kikkert M, Worm SH, Zevenhoven-Dobbe JC, van der Meer Y, Koster AJ, et al. SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum. PLoS Biol. 2008;6(9):e226 10.1371/journal.pbio.0060226 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref016] 16.Knoops K, Barcena M, Limpens RW, Koster AJ, Mommaas AM, Snijder EJ. Ultrastructural characterization of arterivirus replication structures: reshaping the endoplasmic reticulum to accommodate viral RNA synthesis. J Virol. 2012;86(5):2474–87. 10.1128/JVI.06677-11 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref017] 17.Maier HJ, Neuman BW, Bickerton E, Keep SM, Alrashedi H, Hall R, et al. Extensive coronavirus-induced membrane rearrangements are not a determinant of pathogenicity. Sci Rep. 2016;6:27126 10.1038/srep27126 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref018] 18.Zhang W, Chen K, Zhang X, Guo C, Chen Y, Liu X. An integrated analysis of membrane remodeling during porcine reproductive and respiratory syndrome virus replication and assembly. PLoS ONE. 2018;13(7):e0200919 10.1371/journal.pone.0200919 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref019] 19.Weber F, Wagner V, Rasmussen SB, Hartmann R, Paludan SR. Double-stranded RNA is produced by positive-strand RNA viruses and DNA viruses but not in detectable amounts by negative-strand RNA viruses. J Virol. 2006;80(10):5059–64. 10.1128/JVI.80.10.5059-5064.2006 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref020] 20.Ulasli M, Verheije MH, de Haan CA, Reggiori F. Qualitative and quantitative ultrastructural analysis of the membrane rearrangements induced by coronavirus. Cellular Microbiol. 2010;12(6):844–61. 10.1111/j.1462-5822.2010.01437.x . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref021] 21.de Wilde AH, Raj VS, Oudshoorn D, Bestebroer TM, van Nieuwkoop S, Limpens RW, et al. MERS-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin A or interferon-alpha treatment. J Gen Virol. 2013;94(Pt 8):1749–60. 10.1099/vir.0.052910-0 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref022] 22.Angelini MM, Akhlaghpour M, Neuman BW, Buchmeier MJ. Severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles. mBio. 2013;4(4):e00524–13. 10.1128/mBio.00524-13 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref023] 23.Oudshoorn D, Rijs K, Limpens R, Groen K, Koster AJ, Snijder EJ, et al. Expression and Cleavage of Middle East Respiratory Syndrome Coronavirus nsp3-4 Polyprotein Induce the Formation of Double-Membrane Vesicles That Mimic Those Associated with Coronaviral RNA Replication. mBio. 2017;8(6):e01658–17. 10.1128/mBio.01658-17 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref024] 24.Al-Mulla HM, Turrell L, Smith NM, Payne L, Baliji S, Zust R, et al. Competitive fitness in coronaviruses is not correlated with size or number of double-membrane vesicles under reduced-temperature growth conditions. mBio. 2014;5(2):e01107–13. 10.1128/mBio.01107-13 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref025] 25.Lundin A, Dijkman R, Bergstrom T, Kann N, Adamiak B, Hannoun C, et al. Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus. PLoS Pathog. 2014;10(5):e1004166 10.1371/journal.ppat.1004166 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref026] 26.Maier HJ, Hawes PC, Cottam EM, Mantell J, Verkade P, Monaghan P, et al. Infectious bronchitis virus generates spherules from zippered endoplasmic reticulum membranes. mBio. 2013;4(5):e00801–13. 10.1128/mBio.00801-13 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref027] 27.Doyle N, Hawes PC, Simpson J, Adams LH, Maier HJ. The Porcine Deltacoronavirus Replication Organelle Comprises Double-Membrane Vesicles and Zippered Endoplasmic Reticulum with Double-Membrane Spherules. Viruses. 2019;11(11):1030 10.3390/v11111030 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref028] 28.Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus AD, Fouchier RA. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N Engl J Med. 2012;367(19):1814–20. 10.1056/NEJMoa1211721 . [DOI] [PubMed] [Google Scholar]

[pbio.3000715.ref029] 29.van Boheemen S, de Graaf M, Lauber C, Bestebroer TM, Raj VS, Zaki AM, et al. Genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans. mBio. 2012;3(6):e00473–12. 10.1128/mBio.00473-12 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref030] 30.Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, et al. A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med. 2020;382(8):727–733. 10.1056/NEJMoa2001017 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref031] 31.Lu R, Zhao X, Li J, Niu P, Yang B, Wu H, et al. Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet. 2020;395(10224):565–574. 10.1016/S0140-6736(20)30251-8 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref032] 32.Bienz KA. Techniques and applications of autoradiography in the light and electron microscope. Microsc Acta. 1977;79(1):1–22. . [PubMed] [Google Scholar]

[pbio.3000715.ref033] 33.Bozzola JJ, Russell LD. Autoradiography & Radioautography. Electron Microscopy: Principles and Techniques for Biologists. Sudbury (MA):Jones and Bartlett Publishers; 1999. p. 293–308. [Google Scholar]

[pbio.3000715.ref034] 34.Subissi L, Posthuma CC, Collet A, Zevenhoven-Dobbe JC, Gorbalenya AE, Decroly E, et al. One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities. Proc Natl Acad Sci U S A. 2014;111(37):E3900–9. 10.1073/pnas.1323705111 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref035] 35.van den Worm SH, Knoops K, Zevenhoven-Dobbe JC, Beugeling C, van der Meer Y, Mommaas AM, et al. Development and RNA-synthesizing activity of coronavirus replication structures in the absence of protein synthesis. J Virol. 2011;85(11):5669–73. 10.1128/JVI.00403-11 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref036] 36.Muller C, Hardt M, Schwudke D, Neuman BW, Pleschka S, Ziebuhr J. Inhibition of Cytosolic Phospholipase A2alpha Impairs an Early Step of Coronavirus Replication in Cell Culture. J Virol. 2018;92(4):e01463–17. 10.1128/JVI.01463-17 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref037] 37.Ogando NS, Dalebout TJ, Zevenhoven-Dobbe JC, Limpens RW, van der Meer Y, Caly L, et al. SARS-coronavirus-2 replication in Vero E6 cells: replication kinetics, rapid adaptation and cytopathology. J Gen Virol. Forthcoming. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref038] 38.Faas FG, Avramut MC, van den Berg BM, Mommaas AM, Koster AJ, Ravelli RB. Virtual nanoscopy: generation of ultra-large high resolution electron microscopy maps. J Cell Biol. 2012;198(3):457–69. 10.1083/jcb.201201140 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref039] 39.Mayhew TM, Lucocq JM, Griffiths G. Relative labelling index: a novel stereological approach to test for non-random immunogold labelling of organelles and membranes on transmission electron microscopy thin sections. J Microsc. 2002;205(2):153–64. 10.1046/j.0022-2720.2001.00977.x . [DOI] [PubMed] [Google Scholar]

[pbio.3000715.ref040] 40.Tooze J, Tooze S, Warren G. Replication of coronavirus MHV-A59 in sac- cells: determination of the first site of budding of progeny virions. Eur J Cell Biol. 1984;33(2):281–93. . [PubMed] [Google Scholar]

[pbio.3000715.ref041] 41.Goldsmith CS, Tatti KM, Ksiazek TG, Rollin PE, Comer JA, Lee WW, et al. Ultrastructural characterization of SARS coronavirus. Emerg Infect Dis. 2004;10(2):320–6. 10.3201/eid1002.030913 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref042] 42.Stertz S, Reichelt M, Spiegel M, Kuri T, Martinez-Sobrido L, Garcia-Sastre A, et al. The intracellular sites of early replication and budding of SARS-coronavirus. Virology. 2007;361(2):304–15. 10.1016/j.virol.2006.11.027 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref043] 43.Karreman MA, Van Donselaar EG, Agronskaia AV, Verrips CT, Gerritsen HC. Novel contrasting and labeling procedures for correlative microscopy of thawed cryosections. J Histochem Cytochem. 2013;61(3):236–47. 10.1369/0022155412473756 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref044] 44.Klumperman J, Locker JK, Meijer A, Horzinek MC, Geuze HJ, Rottier PJ. Coronavirus M proteins accumulate in the Golgi complex beyond the site of virion budding. J Virol. 1994;68(10):6523–34. . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref045] 45.Ng ML, Tan SH, See EE, Ooi EE, Ling AE. Proliferative growth of SARS coronavirus in Vero E6 cells. J Gen Virol. 2003;84(12):3291–303. 10.1099/vir.0.19505-0 . [DOI] [PubMed] [Google Scholar]

[pbio.3000715.ref046] 46.Becker WB, McIntosh K, Dees JH, Chanock RM. Morphogenesis of avian infectious bronchitis virus and a related human virus (strain 229E). J Virol. 1967;1(5):1019–27. . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref047] 47.Zhou X, Cong Y, Veenendaal T, Klumperman J, Shi D, Mari M, et al. Ultrastructural Characterization of Membrane Rearrangements Induced by Porcine Epidemic Diarrhea Virus Infection. Viruses. 2017;9(9):251 10.3390/v9090251 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref048] 48.Machamer CE, Mentone SA, Rose JK, Farquhar MG. The E1 glycoprotein of an avian coronavirus is targeted to the cis Golgi complex. Proc Natl Acad Sci U S A. 1990;87(18):6944–8. 10.1073/pnas.87.18.6944 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref049] 49.Opstelten DJ, Raamsman MJ, Wolfs K, Horzinek MC, Rottier PJ. Envelope glycoprotein interactions in coronavirus assembly. J Cell Biol. 1995;131(2):339–49. 10.1083/jcb.131.2.339 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref050] 50.Denison MR, Spaan WJ, van der Meer Y, Gibson CA, Sims AC, Prentice E, et al. The putative helicase of the coronavirus mouse hepatitis virus is processed from the replicase gene polyprotein and localizes in complexes that are active in viral RNA synthesis. J Virol. 1999;73(8):6862–71. . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref051] 51.van der Meer Y, Snijder EJ, Dobbe JC, Schleich S, Denison MR, Spaan WJ, et al. Localization of mouse hepatitis virus nonstructural proteins and RNA synthesis indicates a role for late endosomes in viral replication. J Virol. 1999;73(9):7641–57. . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref052] 52.Sims AC, Ostermann J, Denison MR. Mouse hepatitis virus replicase proteins associate with two distinct populations of intracellular membranes. J Virol. 2000;74(12):5647–54. 10.1128/jvi.74.12.5647-5654.2000 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref053] 53.V'Kovski P, Gerber M, Kelly J, Pfaender S, Ebert N, Braga Lagache S, et al. Determination of host proteins composing the microenvironment of coronavirus replicase complexes by proximity-labeling. eLife. 2019;8:e42037 10.7554/eLife.42037 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref054] 54.McBride R, van Zyl M, Fielding BC. The coronavirus nucleocapsid is a multifunctional protein. Viruses. 2014;6(8):2991–3018. 10.3390/v6082991 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref055] 55.Hagemeijer MC, Vonk AM, Monastyrska I, Rottier PJ, de Haan CA. Visualizing coronavirus RNA synthesis in time by using click chemistry. J Virol. 2012;86(10):5808–16. 10.1128/JVI.07207-11 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref056] 56.Beachboard DC, Anderson-Daniels JM, Denison MR. Mutations across murine hepatitis virus nsp4 alter virus fitness and membrane modifications. J Virol. 2015;89(4):2080–9. 10.1128/JVI.02776-14 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref057] 57.Neuman BW. How the double spherules of infectious bronchitis virus impact our understanding of RNA virus replicative organelles. mBio. 2013;4(6):e00987–13. 10.1128/mBio.00987-13 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref058] 58.Hagemeijer MC, Monastyrska I, Griffith J, van der Sluijs P, Voortman J, van Bergen en Henegouwen PM, et al. Membrane rearrangements mediated by coronavirus nonstructural proteins 3 and 4. Virology. 2014;458–459:125–35. 10.1016/j.virol.2014.04.027 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref059] 59.Deng Y, Almsherqi ZA, Ng MM, Kohlwein SD. Do viruses subvert cholesterol homeostasis to induce host cubic membranes? Trends in cell biology. 2010;20(7):371–9. 10.1016/j.tcb.2010.04.001 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref060] 60.Almsherqi ZA, Kohlwein SD, Deng Y. Cubic membranes: a legend beyond the Flatland* of cell membrane organization. J Cell Biol. 2006;173(6):839–44. 10.1083/jcb.200603055 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref061] 61.Paul D, Hoppe S, Saher G, Krijnse-Locker J, Bartenschlager R. Morphological and biochemical characterization of the membranous hepatitis C virus replication compartment. J Virol. 2013;87(19):10612–27. 10.1128/JVI.01370-13 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref062] 62.Melia CE, van der Schaar HM, Lyoo H, Limpens R, Feng Q, Wahedi M, et al. Escaping Host Factor PI4KB Inhibition: Enterovirus Genomic RNA Replication in the Absence of Replication Organelles. Cell Rep. 2017;21(3):587–99. 10.1016/j.celrep.2017.09.068 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref063] 63.Melia CE, van der Schaar HM, de Jong AWM, Lyoo HR, Snijder EJ, Koster AJ, et al. The Origin, Dynamic Morphology, and PI4P-Independent Formation of Encephalomyocarditis Virus Replication Organelles. mBio. 2018;9(2):e00420–18. 10.1128/mBio.00420-18 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref064] 64.van Hemert MJ, van den Worm SH, Knoops K, Mommaas AM, Gorbalenya AE, Snijder EJ. SARS-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro. PLoS Pathog. 2008;4(5):e1000054 10.1371/journal.ppat.1000054 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref065] 65.Kindler E, Gil-Cruz C, Spanier J, Li Y, Wilhelm J, Rabouw HH, et al. Early endonuclease-mediated evasion of RNA sensing ensures efficient coronavirus replication. PLoS Pathog. 2017;13(2):e1006195 10.1371/journal.ppat.1006195 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref066] 66.Thiel V, Ivanov KA, Putics A, Hertzig T, Schelle B, Bayer S, et al. Mechanisms and enzymes involved in SARS coronavirus genome expression. J Gen Virol. 2003;84(Pt 9):2305–15. 10.1099/vir.0.19424-0 . [DOI] [PubMed] [Google Scholar]

[pbio.3000715.ref067] 67.Hamre D, Procknow JJ. A new virus isolated from the human respiratory tract. Proc Soc Exp Biol Med. 1966;121(1):190–3. 10.3181/00379727-121-30734 . [DOI] [PubMed] [Google Scholar]

[pbio.3000715.ref068] 68.Casais R, Thiel V, Siddell SG, Cavanagh D, Britton P. Reverse genetics system for the avian coronavirus infectious bronchitis virus. J Virol. 2001;75(24):12359–69. 10.1128/JVI.75.24.12359-12369.2001 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref069] 69.Bosch BJ, van der Zee R, de Haan CA, Rottier PJ. The coronavirus spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex. J Virol. 2003;77(16):8801–11. 10.1128/jvi.77.16.8801-8811.2003 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref070] 70.Snijder EJ, van der Meer Y, Zevenhoven-Dobbe J, Onderwater JJ, van der Meulen J, Koerten HK, et al. Ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex. J Virol. 2006;80(12):5927–40. 10.1128/JVI.02501-05 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref071] 71.Schonborn J, Oberstrass J, Breyel E, Tittgen J, Schumacher J, Lukacs N. Monoclonal antibodies to double-stranded RNA as probes of RNA structure in crude nucleic acid extracts. Nucleic Acids Res. 1991;19(11):2993–3000. 10.1093/nar/19.11.2993 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref072] 72.Widjaja I, Wang C, van Haperen R, Gutierrez-Alvarez J, van Dieren B, Okba NMA, et al. Towards a solution to MERS: protective human monoclonal antibodies targeting different domains and functions of the MERS-coronavirus spike glycoprotein. Emerg Microbes Infect. 2019;8(1):516–30. 10.1080/22221751.2019.1597644 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref073] 73.Ginsel LA, Onderwater JJ, Daems WT. Resolution of a gold latensification-elon ascorbic acid developer for Ilford L4 emulsion. Histochemistry. 1979;61(3):343–6. 10.1007/BF00508456 . [DOI] [PubMed] [Google Scholar]

[pbio.3000715.ref074] 74.Kukulski W, Schorb M, Welsch S, Picco A, Kaksonen M, Briggs JA. Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision. J Cell Biol. 2011;192(1):111–9. 10.1083/jcb.201009037 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3000715.ref075] 75.Kremer JR, Mastronarde DN, McIntosh JR. Computer visualization of three-dimensional image data using IMOD. J Struct Biol. 1996;116(1):71–6. 10.1006/jsbi.1996.0013 . [DOI] [PubMed] [Google Scholar]

[pbio.3000715.ref076] 76.Lucocq JM, Habermann A, Watt S, Backer JM, Mayhew TM, Griffiths G. A rapid method for assessing the distribution of gold labeling on thin sections. J Histochem Cytochem. 2004;52(8):991–1000. 10.1369/jhc.3A6178.2004 . [DOI] [PubMed] [Google Scholar]

PERMALINK

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

Frank F G A Faas

Abraham J Koster

Montserrat Bárcena

Roles

Abstract

Introduction

Results

MERS-CoV induces a membrane network of modified membranes that contains DMSs

Fig 1. Membrane structures induced by MERS-CoV infection.

Fig 2. Membrane structures induced by gamma-CoV infections.

Diverse CoVs across different genera induce the same RO elements

Fig 3. DMSs are induced by diverse beta- and alpha-CoVs.

CoV RNA synthesis is confined to RO regions

Fig 4. CoV RNA synthesis is confined to RO regions.

DMVs are the primary site of coronaviral RNA synthesis

Fig 5. DMVs are sites of vRNA synthesis.

Fig 6. Newly synthesized vRNA signal does not clearly associate with CM or DMSs.

Fig 7. Metabolic labeling of newly synthesized vRNA in IBV-infected cells and analysis of the autoradiography signal.

Viral markers in the MERS-CoV RO

Fig 8. IEM detection of viral markers in MERS-CoV-infected cells.

Discussion

Materials and methods

Cells, viruses, and infections

Antibodies

Metabolic labeling and label incorporation measurements

Sample preparation for electron microscopy

EM autoradiography samples

IEM

Electron microscopy imaging

Electron tomography

Quantitative analysis of autoradiography samples

Supporting information

Acknowledgments

Abbreviations

Data Availability

Funding Statement

References

Decision Letter 0

Roland G Roberts

Roles

Decision Letter 1

Roland G Roberts

Roles

Author response to Decision Letter 1

Decision Letter 2

Roland G Roberts

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases