• Abstract talk
  • LS6.005

SARS-CoV2 induced membrane remodelling and assembly revisited in high-pressure frozen and freeze substituted cells


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Pathology, pathogens and diagnostics


  • LS 3: Imaging of large volumes and plastic section tomography
  • LS 6: Pathology, pathogens and diagnostics


Tim Bergner (Ulm / DE), Fabian Zech (Ulm / DE), Maximilian Hirschenberger (Ulm / DE), Konstantin M.J. Sparrer (Ulm / DE), Paul Walther (Ulm / DE), Clarissa Read (Villinger) (Ulm / DE)


Abstract text (incl. figure legends and references)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative of the still ongoing COVID-19 pandemic, is a highly pathogenic β-coronavirus. During the replication of this virus, viral RNA (vRNA) synthesis takes place in double-membrane vesicles (DMVs). The newly synthesized vRNA exit the DMVs, presumably through a molecular pore (1). Virion assembly is initiated by clustering of the nucleocapsid protein complexed with genomic vRNA at the cytoplasmic face of single membrane vesicle (SMV) membranes. This induces membrane curvature and budding of virions into the SMV lumen. Important structural insights into the SARS-CoV2 replication cycle have so far been obtained by EM. Mainly chemically fixed, conventionally dehydrated samples were used for room temperature TEM or frozen hydrated samples were analyzed by cryoTEM.

We analyzed remodeling of cellular structures upon SARS-CoV-2 infection in high-pressure frozen (HPF), freeze substituted (FS) and Epon embedded cells, aiming for high-quality structural preservation and membrane visibility. That is a pre-requisite for scanning transmission electron microscopy (STEM) tomography to visualize the different stages of SARS-CoV2 virion assembly in 3D.

Cells were cultivated on carbon-coated sapphire discs, infected with SARS-CoV-2 for 24h, and inactivated with 4 % PFA for 1 hour at 4°C. Immediately, samples were prepared by HPF, FS (5% H2O, 0.2% OsO4 and 0.1% UA in acetone for 17h) and Epon embedding, as published in (2). For TEM, 70 nm thick sections were collected on glow-discharged grids coated with a carbon reinforced formvar film and imaged with a JEOL JEM-1400 TEM at 120 kV acceleration voltage. For STEM tomography, 700-800 nm thick sections were cut, collected on freshly glow-discharged grids with parallel bars, shortly immersed into a colloidal gold-suspension, and coated with a 5 nm carbon layer. A series of 97 STEM bright-field-images (-72° to +72° with 1.5° increment) was recorded with a JEOL JEM-2100F at 200 kV acceleration voltage. Tomograms were reconstructed using the IMOD software package (3,4).

Through HPF-FS and Epon embedding, membrane profiles were well preserved. The clear membrane visibility allowed characterization of the cytopathological alterations in SARS-CoV-2 infected cells, such as the compartmentation of the cytoplasm. Further, DMVs and SMVs could be well resolved, showing the power of the HPF-FS technique. Both membranes of the DMVs appeared often tightly apposed and showed minimal deformation. Although imaging of numerous DMVs, the postulated nuclear pore connecting the DMV interior with the cytoplasm discovered by cryoTEM (1) was not detected by TEM. In one case, we identified a structure in a STEM tomogram that resembled the molecular pore. However, compared to multiple pores per DMV detected by (1), only a single pore spanned the membranes of the DMV in our study. Furthermore, a detailed view into the virus budding process at SMVs (Fig1) allowed temporal resolution of the assembly process.

We show that HPF-FS allows visualization of structures related to SARS-CoV2 infection comparable to advanced cryoEM techniques, opening the door for further studies. STEM tomography provided a detailed insight into SARS-CoV-2 virion assembly.

Fig1: Virtual STEM section of SARS-CoV2 virion assembly

(1) Wolff et al. (2020) Science

(2) Read et al. (2019) J Virol.

(3) Wieland et al (2021) Histochem Cell Biol.

(4) Kremer et al. (1996) J Struct Biol.

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