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

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
Affiliations

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

Eric J Snijder et al. PLoS Biol. .

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Membrane structures induced by MERS-CoV infection.
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.
Fig 2
Fig 2. Membrane structures induced by gamma-CoV infections.
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.
Fig 3
Fig 3. DMSs are induced by diverse beta- and alpha-CoVs.
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 [–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.
Fig 4
Fig 4. CoV RNA synthesis is confined to RO regions.
Newly synthesized vRNA was metabolically labeled by providing tritiated uridine to CoV-infected cells pretreated with actinomycin D to limit host transcription. (A) Analysis of the amount of radioactive label incorporated into RNA as a function of the labeling time in SARS-CoV-infected Vero E6 cells (MOI 10), as measured by scintillation counting on the RNA isolated from the cells (underlying numerical data in S2 Data). The label was provided simultaneously to all the samples at 6 hpi. (B-D) EM detection by autoradiography. (B) Overview of a SARS-CoV-infected Vero E6 cell (MOI 10, 7 hpi, labeled for 20 minutes). Autoradiography grains accumulate in the RO regions. Scale bar, 1 μm. (C, D) Quantification of the autoradiography signal per subcellular structure (see also S3 Data). Labeling densities and RLIs in different subcellular regions of (C) Vero E6 cells infected with SARS-CoV (MOI 10) or (D) Huh7 cells infected with MERS-CoV (MOI 5). Radioactively labeled uridine was provided for the indicated periods of time immediately before fixation at 7 hpi and 12 hpi, respectively. These time points represent, respectively, the middle (SARS-CoV) or late (MERS-CoV) exponential phase of viral replication [21,34]. Control mock-infected cells are excluded from the RLI plots, as RLI comparisons between conditions require the same number of classes (subcellular regions) and these cells lack ROs and virions. CM, convoluted membranes; CoV, coronavirus; cpm, counts per minute; 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; LD, lipid droplet; m, mitochondrion; MERS-CoV, Middle East respiratory syndrome-CoV; MHV, murine hepatitis virus; MOI, multiplicity of infection; N, nucleus; RLI, relative labeling index;; RO, replication organelle; SARS-CoV, severe acute respiratory syndrome-CoV; VCR, virion-containing region; vRNA, viral RNA.
Fig 5
Fig 5. DMVs are sites of vRNA synthesis.
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 (nDMVs = 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.
Fig 6
Fig 6. Newly synthesized vRNA signal does not clearly associate with CM or DMSs.
(A) Overview of a cluster of MERS-CoV-induced membrane modifications in Huh7 cells prepared as described in Fig 5. Some DMSs are boxed in orange, and regions with CM are encircled in blue. In comparison with the densely labeled surrounding DMVs, these regions are relatively devoid of autoradiography signal. (B) The distribution of autoradiography grains on CM was not homogeneous (nCM = 9), and label was predominantly found close to the boundaries of the CM, as expected if the signal arises from the surrounding DMVs. (C-E) Analysis of the label around/on the DMSs (see Materials and methods for selection criteria and details). (C) Enlargements of the DMS areas boxed in (A). Most DMSs were devoid of signal, and those who contained label were close to labeled DMVs (D) (nDMS = 127). (E) The distribution of signal around DMSs shows an increase in the amount of autoradiography grains with the distance from the DMS center, as expected from a random distribution (nDMSs = 58). The underlying numerical data for the plots are in S5 Data. Scale bars, (A) 500 nm, (C) 100 nm. CM, convoluted membranes; DMS, double-membrane spherule; DMV, double-membrane vesicle; vRNA, viral RNA.
Fig 7
Fig 7. Metabolic labeling of newly synthesized vRNA in IBV-infected cells and analysis of the autoradiography signal.
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 (nDMS = 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 (nDMS = 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 (nDMVs = 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.
Fig 8
Fig 8. IEM detection of viral markers in MERS-CoV-infected cells.
(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.
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Comment in

  • Coronavirus replication factories.
    Du Toit A. Du Toit A. Nat Rev Microbiol. 2020 Aug;18(8):411. doi: 10.1038/s41579-020-0406-z. Nat Rev Microbiol. 2020. PMID: 32561857 Free PMC article.

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