science.sciencemag.org/cgi/content/full/science.abd3629/DC1

Supplementary Materials for

A molecular pore spans the double membrane of the coronavirus

replication organelle

Georg Wolff, Ronald W. A. L. Limpens, Jessika C. Zevenhoven-Dobbe, Ulrike Laugks,

Shawn Zheng, Anja W. M. de Jong, Roman I. Koning, David A. Agard, Kay Grünewald,

Abraham J. Koster, Eric J. Snijder, Montserrat Bárcena*

*Corresponding author. Email: m.barcena@lumc.nl

Published 6 August 2020 on Science First Release

DOI: 10.1126/science.abd3629

This PDF file includes:

Materials and Methods

Supplementary Text

Figs. S1 to S9

Captions for Movies S1 to S3

References

Other Supplementary Material for this manuscript includes the following:

(available at science.sciencemag.org/cgi/content/full/science.abd3629/DC1)

MDAR Reproducibility Checklist (.pdf)

Movies S1 to S3 (.mp4)

2

Materials and Methods Cell culture, infection and vitrification

17clone1 and Vero E6 cells were grown as described previously (5). R1/4 gold Quantifoil grids (200 mesh, Quantifoil Micro Tools, Jena, Germany) were cleaned overnight by placing them on a chloroform-soaked filter paper in a sealed glass dish. After drying, the grids were glow-discharged and placed in 3.5-cm diameter cell culture dishes (Greiner Bio-One, Frickenhausen, Germany) containing PBS. After UV-sterilization for 30 min in a laminar flow cabinet, PBS was removed and 110,000 17clone1 or Vero E6 cells were seeded per dish and incubated overnight at 37 °C . 17clone1 cells were infected with wild type (wt) MHV (ATTC VR-764) or MHV-Δ2-GFP3 (24). In the MHV-Δ2-GFP3 mutant (generously provided by Mark Denison, Vanderbilt University Medical Centre, Nashville, TN, USA), the nsp2-coding region, which is dispensable for virus replication in cell culture (25), is replaced by the EGFP-coding sequence, yielding a GFP-nsp3 fusion protein (24). Samples were plunge-frozen in liquid ethane at 8 h post-infection (p.i.) (wt MHV) or 10 h p.i. (MHV-Δ2-GFP3) with a Leica EM GP automated plunge-freezer (Leica microsystems, Wetzlar, Germany), after applying 15 s of blotting from the backside of the grid and 1s post-blotting time in conditions of 37 °C and 95% humidity. VeroE6 cells were infected with SARS-CoV-2 isolate BetaCoV/Australia/VIC01/2020 (GeneBank ID: MT007544.1; kindly provided by Leon Caly and Julian Druce, Doherty Institute, Melbourne, Australia (33)) and chemically fixed at 8 h p.i. with 3% PFA in cell culture medium. After 1 h, the original fixative was replaced by 0.5% PFA in cell culture medium and the samples were stored overnight at 4 °C, before being exported from the biosafety level 3 facility of Leiden University Medical Center, the Netherlands. Next, they were incubated in PFA-free medium for 30 min, plunge-frozen in liquid ethane using a Leica EM GP with 10 s blotting, 2 s post-blotting time and chamber conditions of 21 °C and 90% humidity, and stored in liquid nitrogen until further use.

Electron cryo-tomography Plunge-frozen samples were transferred to an Aquilos cryo-focused ion beam

scanning electron microscope (FIB-SEM) (Thermo Fisher Scientific, Hillsboro, OR, USA) and cryo-lamellae were prepared as described (34). For the MHV-Δ2-GFP3 samples, prior to FIB-milling, cryo-fluorescence light microscopy (cryo-FLM) was performed on an upright FLM-system (Axioimager, Zeiss, Oberkochen, Germany) equipped with a cryo-correlative light and EM stage (CMS196, Linkam, Tadworth, UK). The resulting images were integrated in the FIB-milling workflow using MAPS software (Thermo Fisher Scientific) to guide the finding of suitable milling regions (Fig. S8). A total of approximately 200 cryo-lamellae of 120-200 nm thickness were prepared (139 for wt MHV, 32 for MHV-Δ2-GFP3, 23 for SARS-CoV-2), of which about 40% contained the features of interest and were also of a sufficient quality to allow tomographic data collection. Tilt-series acquisition was carried out on FEI Titan Krios (Thermo Fisher Scientific) electron microscopes operated at 300 kV and equipped with a Gatan GIF Quantum energy filter (Gatan, Pleasanton, CA). Movie frames were collected using a Gatan K2 (MHV data) or Gatan K3 (SARS-CoV-2 data). The summit direct electron detection devices were operated in counting mode. Zero-loss imaging with a 20-eV slit width was done with a pixel size of 3.51 Å or 3.37 Å (MHV and SARS-CoV-2 data,

3

respectively) and defocus values ranging from -5 to -8 µm. Bi-directional or dose-symmetric (35) tilt series were collected with FEI Tomo4 and serialEM software, respectively. The total dose applied on the sample was 120-140 e−/Å2, which was distributed over tilt series covering a 100˚-120˚ range with 2˚ or 3˚ tilt increments. Recorded movie frames were aligned with MotionCor2 using 5 x 5 patches (36). The resulting fiducial-free tilt-series were aligned and SART (Simultaneous Algebraic Reconstruction Technique) reconstructed using 9 SART iterations with AreTomo (Zheng and Agard, https://msg.ucsf.edu/software). CTF-estimations were performed with CTFFIND4 (37), while CTF-correction and denoising for visualization and modeling purposes was done with the ctfphaseflip (38) and the nonlinear anisotropic diffusion (39) packages included in IMOD (40), respectively.

Subtomogram averaging Individual DMV-embedded molecular pores were manually picked from 4-times

binned tomograms using the IMODs 3dmod interface. The coordinates were then transferred into Dynamo (41), which was used for particle cropping, alignment and averaging from 2-times binned tomograms (7.02 Å per pixel). For the wt MHV sample, 611 particles (96 pixels box size) were extracted out of the 53 highest quality tomograms (from 33 lamellae), while for the MHV-Δ2-GFP3 sample 154 particles out of 31 tomograms were cropped (from 16 lamellae). The initial alignment allowed full rotational search in all directions and resulted in a rough average that was manually centered onto the central z-axis to establish the shift to be applied to the individual particles. This was followed by a center-refinement step using the 20-fold symmetrized average as a reference to fine align the particles on the central axis. Subsequent alignment steps allowed only azimuthal rotations and limited lateral shifts. For the wt MHV sample, three subsets of random particles were used to generate averages for multi-reference alignment, which resulted in a class of 346 particles that provided the highest resolution and were included in the final averages. The discarded particles likely represent different conformational states of the complex that, nevertheless, appeared to be insufficiently populated to provide further insight at this stage. Two parallel alignments were carried out with the selected particles, with or without applying sixfold symmetry. A tight missing wedge mask that removed all information beyond ± 40˚ in Fourier space was applied to all the particles during alignment. The Fourier shell correlation curves were determined from individually refined half-datasets of the wt MHV and MHV-Δ2-GFP3 subtomogram averages, respectively. Local resolution maps were generated using the ResMap software (42).The correct handedness of the averages was assessed by comparison with an average of aligned ribosome particles originating from our data to a published ribosome structure (43).

3d modeling and model interpretation 3D surface models of the DMVs in the cellular environment were created in Amira

(Thermo Fisher Scientific) and animations in Cinema4D (Maxon Computer, Friedrichsdorf, Germany) software. Model files of ribosomes and pore complexes integrated in these renderings were obtained with Dynamo, in the case of the molecular pores, from the density map and coordinates generated during sub-tomogram averaging, and in the case of ribosomes by template matching with a ribosome average originating from our data.

4

Surface models of the pore complex were generated in UCSF Chimera software (44) from 3d averages low-pass filtered to the resolution achieved by sub-tomogram averaging. The contour level for the surface rendering of the complex was chosen so that the crown would best resemble the appearance of this region in the density map (Fig. 2C, G-I). Molecular weight estimations were done by calculating the (sub)volumes enclosed by the surface models generated in Chimera and dividing them by the average density of globular proteins (45). For the generation of difference maps of the averages from the molecular pores found in cells infected with wt MHV and MHV-Δ2-GFP3, the radial power spectra of the reconstructed volumes were adjusted to each other before subtracting the volumes using the relion_image_handler function in Relion 3.1 (46).

Supplementary Text Number of molecular pores per DMV

To estimate the average number of molecular pores per DMV, 8 tomograms from 6 lamellae were used to count the number of pore complexes present in a total of 30 DMVs. Pores could be found in all the DMVs in the tomograms. The average number of detected pores (4.8 per DMV) was then extrapolated to account for the fact that only part of an average-sized DMV (257 nm in diameter, Fig. S1) is embedded in the thin cryo-lamella used for this quantification (~150 nm thick on average). This resulted in an estimate of ~8 pores per DMV, which can be considered a lower limit to the real number, as it does not take into consideration the missing-wedge effect intrinsic to electron tomography data that hampers the detection of molecular pores in certain orientations of the reconstructed volumes (47).

5

Fig. S1. Overall morphology of the MHV-induced DMVs in cryo-lamellae. (A) Whisker plot of the size distribution of the DMVs as measured from the tomograms at the DMV equators. Most of the DMVs in the cryo-EM samples were clearly spherical, a subset of which was used for the plot. (B-G) Connections between the outer membrane of the DMVs and the ER were occasionally detected in the tomograms. These include the neck-like connections previously documented in resin-embedded specimens (4-6) (B-D, white arrows), and, strikingly, thus far unreported tubular connections reaching up to ~300 nm (D-F, white arrowheads). Some DMVs were clearly interconnected through the ER (C, D) or by a tubular connection (H, black arrowheads). While these different connections were only detected in a subset of DMVs, it should be noted that only a percentage (≤60%) of an average-sized DMV would be contained in the thin cryo-lamellae used in this study (~150 nm thick). These observations were consistent with the previous notion of coronavirus-induced DMVs being largely interconnected as part of a reticulovesicular network (4-6). (I) In some cases, DMVs appeared to fuse together through their outer membrane forming a so-called vesicle packet (4, 31). (B-F, H, I) 7-nm thick tomographic slices. (G) 3D rendering of the tubular connection in (F).

6

Fig. S2. Membrane-membrane interactions in coronaviral DMVs. (A) The outer and inner membranes of MHV-induced DMVs were most commonly separated by a regular spacing of ~4.5 nm. Such regular spacing is likely mediated by the interactions of factors embedded in the inner and outer membrane. (B) In fact, densities crossing the luminal space between the two membranes (boxed area, black arrows) were frequently found in these regions. Notably, the transmembrane proteins nsp3 and nsp4 have been previously implicated in membrane pairing (17-19). Local deviations from the described regular inter-membrane spacing were common and, (C), occasionally, DMVs with largely dissociated membranes and molecular pores at constriction points (black arrowheads) were observed.

7

Fig. S3. Macromolecular features on the outer DMV membrane. (A-C) The outer DMV membrane appeared frequently decorated with a variety of molecular features. A striking recurrent motif observed in the tomographic slices of the reconstructed DMVs were regular arrays (~8 nm spacing) of small globular masses at ~9 nm distance from the outer membrane to which they seemed to connect through a narrow link (boxed areas and insets). While the identity of these different densities could not be ascertained, it is worth noting that numerous viral and host proteins associate with coronavirus replication organelles (48) and that the nsp3 transmembrane protein contains multiple cytosolic domains that have been implicated in interactions with host factors (20, 49).

8

Fig. S4. DMV content. (A-C) DMVs appeared to contain primarily filamentous structures, which could appear as dense points in the tomographic slices when viewed in cross-section (C, white arrowheads). The tomograms revealed relatively straight segments of a length consistent with the persistence length of dsRNA (9), which has been previously detected inside coronavirus-induced DMVs by immuno-EM (4, 5). The dsRNA filaments seemed to be loosely packed inside the DMVs forming what, at the current resolution, appeared as an inextricable mesh of filaments. While ssRNA or protein complexes like the RTC could be contained in the DMV lumen, their presence within this intricate filamentous mesh could not be ascertained. The inner DMV membranes were relatively devoid of distinct associated partners, with only occasional small interacting densities (black arrowheads). (A-C) 7-nm thick tomographic slices. (D) 3D model of the filamentous content inside the DMV shown in (C).

9

Fig. S5. Electron tomography on cryo-lamella from coronavirus-infected cells. Cryo-lamellae generated from cells infected with wt MHV (A, B) and cells infected with SARS-CoV-2 (C, D) that were chemically fixed prior to vitrification. (A, C) Overviews of the cryo-lamellae, in which different structures, such as nucleus, mitochondria (M), the Golgi apparatus (Golgi), lipid droplets (LD), DMVs and virus particles (VPs), could be identified. (B, D) Slices (7 nm thick) of the tomograms reconstructed from tilt-series collected in the indicated regions (white frames in (A) and (C), respectively). Chemical fixation resulted in reduced contrast and produced morphological alterations in diverse organelles. Therefore, the partially distorted morphology of SARS-CoV-2-induced DMVs was likely also a result of fixation. Nonetheless, molecular pores in the DMV membranes could be identified for both samples (white arrowheads).

10

Fig. S6. Subtomogram averaging of the DMV molecular pore. (A) Fourier shell correlation curves from molecular pore particles aligned without imposing symmetry (c1) or upon implying sixfold symmetry (c6). (B) Local resolution map showing lower resolution in the inter-membrane platform relative to the crown of the complex. This suggested that this region has higher structural variability. (C) Plot of the correlation coefficients between unsymmetrized and symmetrized averages, highlighting the sixfold symmetry of the complex. (D) Estimates of the molecular masses of different sub-regions in the complex. (E-G) Slices (0.7 nm thick) throughout the molecular pore averages filtered to the achieved resolution, either parallel (top) or perpendicular (bottom) to the central axis of the complex. Wt, complex in wt MHV-induced DMVs; gfp, complex in MHV-Δ2-GFP3-induced DMVs. Scale bar (E-G), 10 nm.

11

Fig. S7. Cryo-EM imaging of MHV-Δ2-GFP3-infected cells was facilitated by cryo-correlative light microscopy. (A) Fluorescence nsp3-EGFP signal was observed in perinuclear regions of plunge-frozen infected cells, which served as a marker for regions containing virus-induced replication compartments (4, 7). (B) Overlays of the fluorescence signal on the corresponding SEM images guided the selection of suitable milling sites in the cryo-FIB-SEM machine. (C, D) The framed area in (B) as viewed from the FIB-beam direction before (C) and after (D) rough milling of the selected region. (E) Transmission EM overview of the thinned lamella, where regions of interest containing DMVs could be identified. The framed area was used for tilt-series acquisition. (F) A tomographic slice (7 nm thick) from the corresponding tomogram shows DMVs containing molecular pores (white arrowheads). LD, lipid droplet; ER, endoplasmic reticulum.

12

Fig. S8. Gallery of molecular pores from 7 nm thick tomographic slices. (A-Q) Various macromolecular structures appeared to interact with both the cytosolic (top) and the DMV luminal (bottom) interfaces of the molecular pores. Cytosolic interaction partners (black arrowheads) range from (A, B, E) chain-like densities that seem to associate to the complexes prongs to larger assemblies that in some cases (D, G) are reminiscent of helical coronavirus RNPs. DMV luminal interaction partners (white arrows) appeared to vary in mass and shape as well, while they frequently also showed association to the DMV luminal filaments (C, D, I-L) assumed to be viral RNA. (R) In very few instances (less than 5 occurrences in a dataset containing more than 600 molecular pores), pore complexes that appeared to be inverted in the DMV membrane were observed.

13

Fig. S9. RNP assemblies in the cytosol and virions. Tomographic slices of 7 nm thickness showing (A) the abundant presence of cytosolic RNP assemblies in DMV-rich regions. The appearance of these structures is consistent with the helical coronavirus RNPs of ~15 nm in diameter previously described by single-particle analysis (30), and with the RNP observed inside coronavirus particles in tomographic data (C, D) (50). (B) RNP assemblies could associate to smooth-membrane compartments (boxed area) where they appeared to initiate the budding of virions into the lumen (8). (C-D) Budding virus particles containing RNP. Note also the spike protein on newly formed virions (C, black arrowheads).

14

Movie S1. Cryo-ET of MHV-induced ROs. Tomographic reconstructions and 3D segmented model of the region in a MHV-infected cells shown in Fig. 1.

Movie S2. Subtomogram average of the molecular pore present in the DMVs induced by wt MHV. Sections through the sixfold symmetrized volume filtered to the final resolution. The slices (0.7 nm thick) are shown from the cytosolic side and are perpendicular to the central axis of the complex.

Movie S3. Comparison of the wt and mutant Δ2-GFP3 molecular pore. The movie alternates between the central transversal slices through both subtomogram averages, filtered to the resolution of the Δ2-GFP3 reconstruction. Besides the additional densities on the top of the prongs in the mutant complex, conformational variations in the inter-membrane platform become apparent.

References and Notes

1. T. G. Ksiazek, D. Erdman, C. S. Goldsmith, S. R. Zaki, T. Peret, S. Emery, S. Tong, C.

Urbani, J. A. Comer, W. Lim, P. E. Rollin, S. F. Dowell, A. E. Ling, C. D. Humphrey,

W. J. Shieh, J. Guarner, C. D. Paddock, P. Rota, B. Fields, J. DeRisi, J. Y. Yang, N. Cox,

J. M. Hughes, J. W. LeDuc, W. J. Bellini, L. J. Anderson, S. W. Group; SARS Working

Group, A novel coronavirus associated with severe acute respiratory syndrome. N. Engl.

J. Med. 348, 1953–1966 (2003). doi:10.1056/NEJMoa030781 Medline

2. A. M. Zaki, S. van Boheemen, T. M. Bestebroer, A. D. Osterhaus, R. A. Fouchier, Isolation of

a novel coronavirus from a man with pneumonia in Saudi Arabia. N. Engl. J. Med. 367,

1814–1820 (2012). doi:10.1056/NEJMoa1211721 Medline

3. N. Zhu, D. Zhang, W. Wang, X. Li, B. Yang, J. Song, X. Zhao, B. Huang, W. Shi, R. Lu, P.

Niu, F. Zhan, X. Ma, D. Wang, W. Xu, G. Wu, G. F. Gao, W. Tan; China Novel

Coronavirus Investigating and Research Team, A novel coronavirus from patients with

pneumonia in China, 2019. N. Engl. J. Med. 382, 727–733 (2020).

doi:10.1056/NEJMoa2001017 Medline

4. K. Knoops, M. Kikkert, S. H. Worm, J. C. Zevenhoven-Dobbe, Y. van der Meer, A. J. Koster,

A. M. Mommaas, E. J. Snijder, SARS-coronavirus replication is supported by a

reticulovesicular network of modified endoplasmic reticulum. PLOS Biol. 6, e226 (2008).

doi:10.1371/journal.pbio.0060226 Medline

5. E. J. Snijder, R. W. A. L. Limpens, A. H. de Wilde, A. W. M. de Jong, J. C. Zevenhoven-

Dobbe, H. J. Maier, F. F. G. A. Faas, A. J. Koster, M. Bárcena, A unifying structural and

functional model of the coronavirus replication organelle: Tracking down RNA synthesis.

PLOS Biol. 18, e3000715 (2020). doi:10.1371/journal.pbio.3000715 Medline

6. H. J. Maier, P. C. Hawes, E. M. Cottam, J. Mantell, P. Verkade, P. Monaghan, T. Wileman, P.

Britton, Infectious bronchitis virus generates spherules from zippered endoplasmic

reticulum membranes. mBio 4, e00801-13 (2013). doi:10.1128/mBio.00801-13 Medline

7. M. Ulasli, M. H. Verheije, C. A. de Haan, F. Reggiori, Qualitative and quantitative

ultrastructural analysis of the membrane rearrangements induced by coronavirus. Cell.

Microbiol. 12, 844–861 (2010). doi:10.1111/j.1462-5822.2010.01437.x Medline

8. C. A. de Haan, P. J. Rottier, Molecular interactions in the assembly of coronaviruses. Adv.

Virus Res. 64, 165–230 (2005). doi:10.1016/S0065-3527(05)64006-7 Medline

9. J. A. Abels, F. Moreno-Herrero, T. van der Heijden, C. Dekker, N. H. Dekker, Single-

molecule measurements of the persistence length of double-stranded RNA. Biophys. J.

88, 2737–2744 (2005). doi:10.1529/biophysj.104.052811 Medline

10. K. Ding, C. C. Celma, X. Zhang, T. Chang, W. Shen, I. Atanasov, P. Roy, Z. H. Zhou, In situ

structures of rotavirus polymerase in action and mechanism of mRNA transcription and

release. Nat. Commun. 10, 2216 (2019). doi:10.1038/s41467-019-10236-7 Medline

11. K. J. Ertel, D. Benefield, D. Castaño-Diez, J. G. Pennington, M. Horswill, J. A. den Boon, M.

S. Otegui, P. Ahlquist, Cryo-electron tomography reveals novel features of a viral RNA

replication compartment. eLife 6, e25940 (2017). doi:10.7554/eLife.25940 Medline

12. E. J. Snijder, E. Decroly, J. Ziebuhr, The nonstructural proteins directing coronavirus RNA

synthesis and processing. Adv. Virus Res. 96, 59–126 (2016).

doi:10.1016/bs.aivir.2016.08.008 Medline

13. M. Oostra, M. C. Hagemeijer, M. van Gent, C. P. Bekker, E. G. te Lintelo, P. J. Rottier, C. A.

de Haan, Topology and membrane anchoring of the coronavirus replication complex: Not

all hydrophobic domains of nsp3 and nsp6 are membrane spanning. J. Virol. 82, 12392–

12405 (2008). doi:10.1128/JVI.01219-08 Medline

14. M. Oostra, E. G. te Lintelo, M. Deijs, M. H. Verheije, P. J. Rottier, C. A. de Haan,

Localization and membrane topology of coronavirus nonstructural protein 4: Involvement

of the early secretory pathway in replication. J. Virol. 81, 12323–12336 (2007).

doi:10.1128/JVI.01506-07 Medline

15. A. Kanjanahaluethai, Z. Chen, D. Jukneliene, S. C. Baker, Membrane topology of murine

coronavirus replicase nonstructural protein 3. Virology 361, 391–401 (2007).

doi:10.1016/j.virol.2006.12.009 Medline

16. B. W. Neuman, Bioinformatics and functional analyses of coronavirus nonstructural proteins

involved in the formation of replicative organelles. Antiviral Res. 135, 97–107 (2016).

doi:10.1016/j.antiviral.2016.10.005 Medline

17. M. M. Angelini, M. Akhlaghpour, B. W. Neuman, M. J. Buchmeier, Severe acute respiratory

syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane

vesicles. mBio 4, e00524-13 (2013). doi:10.1128/mBio.00524-13 Medline

18. D. Oudshoorn, K. Rijs, R. W. A. L. Limpens, K. Groen, A. J. Koster, E. J. Snijder, M.

Kikkert, M. Bárcena, 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 8, e01658-17 (2017).

doi:10.1128/mBio.01658-17 Medline

19. M. C. Hagemeijer, I. Monastyrska, J. Griffith, P. van der Sluijs, J. Voortman, P. M. van

Bergen en Henegouwen, A. M. Vonk, P. J. Rottier, F. Reggiori, C. A. de Haan,

Membrane rearrangements mediated by coronavirus nonstructural proteins 3 and 4.

Virology 458-459, 125–135 (2014). doi:10.1016/j.virol.2014.04.027 Medline

20. J. Lei, Y. Kusov, R. Hilgenfeld, Nsp3 of coronaviruses: Structures and functions of a large

multi-domain protein. Antiviral Res. 149, 58–74 (2018).

doi:10.1016/j.antiviral.2017.11.001 Medline

21. P. Serrano, M. A. Johnson, M. S. Almeida, R. Horst, T. Herrmann, J. S. Joseph, B. W.

Neuman, V. Subramanian, K. S. Saikatendu, M. J. Buchmeier, R. C. Stevens, P. Kuhn, K.

Wüthrich, Nuclear magnetic resonance structure of the N-terminal domain of

nonstructural protein 3 from the severe acute respiratory syndrome coronavirus. J. Virol.

81, 12049–12060 (2007). doi:10.1128/JVI.00969-07 Medline

22. K. R. Hurst, C. A. Koetzner, P. S. Masters, Characterization of a critical interaction between

the coronavirus nucleocapsid protein and nonstructural protein 3 of the viral replicase-

transcriptase complex. J. Virol. 87, 9159–9172 (2013). doi:10.1128/JVI.01275-13

Medline

23. Y. Cong, M. Ulasli, H. Schepers, M. Mauthe, P. V’Kovski, F. Kriegenburg, V. Thiel, C. A.

M. de Haan, F. Reggiori, Nucleocapsid protein recruitment to replication-transcription

complexes plays a crucial role in coronaviral life cycle. J. Virol. 94, e01925-19 (2020).

doi:10.1128/JVI.01925-19

24. M. C. Freeman, R. L. Graham, X. Lu, C. T. Peek, M. R. Denison, Coronavirus replicase-

reporter fusions provide quantitative analysis of replication and replication complex

formation. J. Virol. 88, 5319–5327 (2014). doi:10.1128/JVI.00021-14 Medline

25. R. L. Graham, A. C. Sims, R. S. Baric, M. R. Denison, The nsp2 proteins of mouse hepatitis

virus and SARS coronavirus are dispensable for viral replication. Adv. Exp. Med. Biol.

581, 67–72 (2006). doi:10.1007/978-0-387-33012-9_10 Medline

26. M. J. Gadlage, J. S. Sparks, D. C. Beachboard, R. G. Cox, J. D. Doyle, C. C. Stobart, M. R.

Denison, Murine hepatitis virus nonstructural protein 4 regulates virus-induced

membrane modifications and replication complex function. J. Virol. 84, 280–290 (2010).

doi:10.1128/JVI.01772-09 Medline

27. R. N. Kirchdoerfer, A. B. Ward, Structure of the SARS-CoV nsp12 polymerase bound to

nsp7 and nsp8 co-factors. Nat. Commun. 10, 2342 (2019). doi:10.1038/s41467-019-

10280-3 Medline

28. M. Sevajol, L. Subissi, E. Decroly, B. Canard, I. Imbert, Insights into RNA synthesis,

capping, and proofreading mechanisms of SARS-coronavirus. Virus Res. 194, 90–99

(2014). doi:10.1016/j.virusres.2014.10.008 Medline

29. L. Subissi, I. Imbert, F. Ferron, A. Collet, B. Coutard, E. Decroly, B. Canard, SARS-CoV

ORF1b-encoded nonstructural proteins 12-16: Replicative enzymes as antiviral targets.

Antiviral Res. 101, 122–130 (2014). doi:10.1016/j.antiviral.2013.11.006 Medline

30. M. Gui, X. Liu, D. Guo, Z. Zhang, C. C. Yin, Y. Chen, Y. Xiang, Electron microscopy

studies of the coronavirus ribonucleoprotein complex. Protein Cell 8, 219–224 (2017).

doi:10.1007/s13238-016-0352-8 Medline

31. N. S. Ogando, T. J. Dalebout, J. C. Zevenhoven-Dobbe, R. W. A. L. Limpens, Y. van der

Meer, L. Caly, J. Druce, J. J. C. de Vries, M. Kikkert, M. Bárcena, I. Sidorov, E. J.

Snijder, SARS-coronavirus-2 replication in Vero E6 cells: Replication kinetics, rapid

adaptation and cytopathology. J. Gen. Virol. 10.1099/jgv.0.001453 (2020).

doi:10.1099/jgv.0.001453 Medline

32. L. Kuo, C. A. Koetzner, P. S. Masters, A key role for the carboxy-terminal tail of the murine

coronavirus nucleocapsid protein in coordination of genome packaging. Virology 494,

100–107 (2016). doi:10.1016/j.virol.2016.04.009 Medline

33. L. Caly, J. Druce, J. Roberts, K. Bond, T. Tran, R. Kostecki, Y. Yoga, W. Naughton, G.

Taiaroa, T. Seemann, M. B. Schultz, B. P. Howden, T. M. Korman, S. R. Lewin, D. A.

Williamson, M. G. Catton, Isolation and rapid sharing of the 2019 novel coronavirus

(SARS-CoV-2) from the first patient diagnosed with COVID-19 in Australia. Med. J.

Aust. 212, 459–462 (2020). doi:10.5694/mja2.50569 Medline

34. G. Wolff, R. W. A. L. Limpens, S. Zheng, E. J. Snijder, D. A. Agard, A. J. Koster, M.

Bárcena, Mind the gap: Micro-expansion joints drastically decrease the bending of FIB-

milled cryo-lamellae. J. Struct. Biol. 208, 107389 (2019). doi:10.1016/j.jsb.2019.09.006

Medline

35. W. J. H. Hagen, W. Wan, J. A. G. Briggs, Implementation of a cryo-electron tomography tilt-

scheme optimized for high resolution subtomogram averaging. J. Struct. Biol. 197, 191–

198 (2017). doi:10.1016/j.jsb.2016.06.007 Medline

36. S. Q. Zheng, E. Palovcak, J. P. Armache, K. A. Verba, Y. Cheng, D. A. Agard, MotionCor2:

Anisotropic correction of beam-induced motion for improved cryo-electron microscopy.

Nat. Methods 14, 331–332 (2017). doi:10.1038/nmeth.4193 Medline

37. A. Rohou, N. Grigorieff, CTFFIND4: Fast and accurate defocus estimation from electron

micrographs. J. Struct. Biol. 192, 216–221 (2015). doi:10.1016/j.jsb.2015.08.008

Medline

38. Q. Xiong, M. K. Morphew, C. L. Schwartz, A. H. Hoenger, D. N. Mastronarde, CTF

determination and correction for low dose tomographic tilt series. J. Struct. Biol. 168,

378–387 (2009). doi:10.1016/j.jsb.2009.08.016 Medline

39. A. S. Frangakis, R. Hegerl, Noise reduction in electron tomographic reconstructions using

nonlinear anisotropic diffusion. J. Struct. Biol. 135, 239–250 (2001).

doi:10.1006/jsbi.2001.4406 Medline

40. J. R. Kremer, D. N. Mastronarde, J. R. McIntosh, Computer visualization of three-

dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996).

doi:10.1006/jsbi.1996.0013 Medline

41. D. Castaño-Díez, M. Kudryashev, M. Arheit, H. Stahlberg, Dynamo: A flexible, user-

friendly development tool for subtomogram averaging of cryo-EM data in high-

performance computing environments. J. Struct. Biol. 178, 139–151 (2012).

doi:10.1016/j.jsb.2011.12.017 Medline

42. A. Kucukelbir, F. J. Sigworth, H. D. Tagare, Quantifying the local resolution of cryo-EM

density maps. Nat. Methods 11, 63–65 (2014). doi:10.1038/nmeth.2727 Medline

43. A. M. Anger, J. P. Armache, O. Berninghausen, M. Habeck, M. Subklewe, D. N. Wilson, R.

Beckmann, Structures of the human and Drosophila 80S ribosome. Nature 497, 80–85

(2013). doi:10.1038/nature12104 Medline

44. E. F. Pettersen, T. D. Goddard, C. C. Huang, G. S. Couch, D. M. Greenblatt, E. C. Meng, T.

E. Ferrin, UCSF Chimera—A visualization system for exploratory research and analysis.

J. Comput. Chem. 25, 1605–1612 (2004). doi:10.1002/jcc.20084 Medline

45. Y. Harpaz, M. Gerstein, C. Chothia, Volume changes on protein folding. Structure 2, 641–

649 (1994). doi:10.1016/S0969-2126(00)00065-4 Medline

46. S. H. Scheres, RELION: Implementation of a Bayesian approach to cryo-EM structure

determination. J. Struct. Biol. 180, 519–530 (2012). doi:10.1016/j.jsb.2012.09.006

Medline

47. V. Lučič, A. Rigort, W. Baumeister, Cryo-electron tomography: The challenge of doing

structural biology in situ. J. Cell Biol. 202, 407–419 (2013). doi:10.1083/jcb.201304193

Medline

48. P. V’kovski, M. Gerber, J. Kelly, S. Pfaender, N. Ebert, S. Braga Lagache, C. Simillion, J.

Portmann, H. Stalder, V. Gaschen, R. Bruggmann, M. H. Stoffel, M. Heller, R. Dijkman,

V. Thiel, Determination of host proteins composing the microenvironment of coronavirus

replicase complexes by proximity-labeling. eLife 8, e42037 (2019).

doi:10.7554/eLife.42037 Medline

49. B. W. Neuman, J. S. Joseph, K. S. Saikatendu, P. Serrano, A. Chatterjee, M. A. Johnson, L.

Liao, J. P. Klaus, J. R. Yates 3rd, K. Wüthrich, R. C. Stevens, M. J. Buchmeier, P. Kuhn,

Proteomics analysis unravels the functional repertoire of coronavirus nonstructural

protein 3. J. Virol. 82, 5279–5294 (2008). doi:10.1128/JVI.02631-07 Medline

50. M. Bárcena, G. T. Oostergetel, W. Bartelink, F. G. Faas, A. Verkleij, P. J. Rottier, A. J.

Koster, B. J. Bosch, Cryo-electron tomography of mouse hepatitis virus: Insights into the

structure of the coronavirion. Proc. Natl. Acad. Sci. U.S.A. 106, 582–587 (2009).

doi:10.1073/pnas.0805270106 Medline