SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography
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Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID19 pandemic, is a highly pathogenic β-coronavirus. As other coronaviruses, SARS-CoV-2 is enveloped, replicates in the cytoplasm and assembles at intracellular membranes. Here, we structurally characterize the viral replication compartment and report critical insights into the budding mechanism of the virus, and the structure of extracellular virions close to their native state by in situ cryo-electron tomography and subtomogram averaging. We directly visualize RNA filaments inside the double membrane vesicles, compartments associated with viral replication. The RNA filaments show a diameter consistent with double-stranded RNA and frequent branching likely representing RNA secondary structures. We report that assembled S trimers in lumenal cisternae do not alone induce membrane bending but laterally reorganize on the envelope during virion assembly. The viral ribonucleoprotein complexes (vRNPs) are accumulated at the curved membrane characteristic for budding sites suggesting that vRNP recruitment is enhanced by membrane curvature. Subtomogram averaging shows that vRNPs are distinct cylindrical assemblies. We propose that the genome is packaged around multiple separate vRNP complexes, thereby allowing incorporation of the unusually large coronavirus genome into the virion while maintaining high steric flexibility between the vRNPs.
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SciScore for 10.1101/2020.06.23.167064: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Cells and viruses: VeroE6, HEK293T and A549 cells were obtained from ATCC and were maintained in Dulbecco’s modified Eagle medium (DMEM) containing 2 mM L-glutamine, non-essential amino acids, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% fetal calf serum (DMEM complete). HEK293Tsuggested: NoneA549suggested: NoneCalu3 cells were a kind gift from Dr. Manfred Frey, Mannheim and were maintained in DMEM complete supplemented with 10 mM sodium pyruvate and a final concentration of 20% FBS. Calu3suggested: NoneA passage 4 working stock of this isolate was generated by passaging the … SciScore for 10.1101/2020.06.23.167064: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Cells and viruses: VeroE6, HEK293T and A549 cells were obtained from ATCC and were maintained in Dulbecco’s modified Eagle medium (DMEM) containing 2 mM L-glutamine, non-essential amino acids, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% fetal calf serum (DMEM complete). HEK293Tsuggested: NoneA549suggested: NoneCalu3 cells were a kind gift from Dr. Manfred Frey, Mannheim and were maintained in DMEM complete supplemented with 10 mM sodium pyruvate and a final concentration of 20% FBS. Calu3suggested: NoneA passage 4 working stock of this isolate was generated by passaging the virus twice in VeroE6 cells. VeroE6suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)Sample processing for cryo-EM: To generate the dataset for this study, 3 independent infections of cells grown on electron microscopy grids were done for A549-ACE2 cells and one infection on VeroE6 and Calu3 cells. A549-ACE2suggested: NoneSoftware and Algorithms Sentences Resources Primers for qPCR were designed using the Primer3 software44: ACE2 (forward) 5’-CACGAAGGTCCTCTGCACAA-3’, ACE2 (reverse) 5’-ATGCTAGGGTCCAGGGTTCT-3, SARS-CoV-2-N (forward) 5’-GCCTCTTCTCGTTCCTCATCAC-3’, SARS-CoV-2-N (reverse) 5’-AGCAGCATCACCGCCATTG-3’, HPRT (forward) 5’-CCTGGCGTCGTGATTAGTG-3’ and HPRT (reverse) 5’-ACACCCTTTCCAAATCCTCAG-3’. Primer3suggested: (Primer3, RRID:SCR_003139)Individual projections were acquired in counting mode using dose fractionation. 10 – 20 individual frames per projections were aligned and summed on-the-fly using the SEMCCD plugin in SerialEM. SerialEMsuggested: (SerialEM, RRID:SCR_017293)Tomogram reconstruction and volume rendering: Tilt series (TS) were aligned and reconstructed with the IMOD software package48. IMODsuggested: (IMOD, RRID:SCR_003297)This denoised tomogram was further processed with Amira 2019.3 (ThermoFisher Scientific) using the Membrane Enhancement Filter with a feature scale of 6.5 nm. Amirasuggested: NoneFor each individual S trimer, the nearest neighboring S trimer on the same virion was identified and the Euclidean distance was calculated using the pandas and numpy software libraries53,54. numpysuggested: (NumPy, RRID:SCR_008633)The RNA filament diameter was measured on the unbinned SIRT-like filtered reconstructed tomogram in FIJI by smoothing the image using gaussian blur (σ = 2 px) and measuring the line profiles perpendicular to RNA filaments using FIJI’s plot profile tool (10 pixels in width, 1.379 Å/pixel). FIJIsuggested: (Fiji, RRID:SCR_002285)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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SciScore for 10.1101/2020.06.23.167064: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Experimental Models: Cell Lines Sentences Resources largely absent in naïve A549 cells (Fig S1A). A549suggested: NoneAlthough ACE2 mRNA levels in A549-ACE2 cells were higher than in Calu3 and VeroE6 cells, VeroE6 and Calu3 cells showed a higher proportion of infected cells at 16 hours post infection (hpi) characterized by antidsRNA staining (Fig. Calu3suggested: BCRJ Cat# 0264, CVCL_0609(A) Tomogram showing an gap between the trimeric density and the intracellular virion of VeroE6 cells infected with … SciScore for 10.1101/2020.06.23.167064: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Experimental Models: Cell Lines Sentences Resources largely absent in naïve A549 cells (Fig S1A). A549suggested: NoneAlthough ACE2 mRNA levels in A549-ACE2 cells were higher than in Calu3 and VeroE6 cells, VeroE6 and Calu3 cells showed a higher proportion of infected cells at 16 hours post infection (hpi) characterized by antidsRNA staining (Fig. Calu3suggested: BCRJ Cat# 0264, CVCL_0609(A) Tomogram showing an gap between the trimeric density and the intracellular virion of VeroE6 cells infected with SARS-CoV-2 at 16 hpi. VeroE6suggested: JCRB Cat# JCRB1819, CVCL_YQ49A549-ACE2 cells, all of which appeared to directly In agreement with our observations in intracellular interact with the cell surface (Fig. 5A). A549-ACE2suggested: NoneResults from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.
Results from OddPub: We did not find a statement about open data. We also did not find a statement about open code. Researchers are encouraged to share open data when possible (see Nature blog).
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