• Abstract talk
  • LS2.004

Cryo-electron tomography of SARS-CoV-2 replication and assembly sites reconstituted in cells


Talk time:
Discussion time:
Location / Stream:


Cryo-electron microscopy and cryo-electron tomography


  • LS 2: Cryo-tomography
  • LS 6: Pathology, pathogens and diagnostics


Liv Zimmermann (Heidelberg / DE), Xiaohan Zhao (Heidelberg / DE), Jana Makroczyova (Heidelberg / DE), Moritz Wachsmuth-Melm (Heidelberg / DE), Vibhu Prasad (Heidelberg / DE), Ralf Bartenschlager (Heidelberg / DE), Petr Chlanda (Heidelberg / DE)


Abstract text (incl. figure legends and references)

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is an enveloped virus with a positive single-stranded RNA (ssRNA) genome. Upon infection, endoplasmic reticulum (ER)-derived double-membrane vesicles (DMVs) serve as replication organelles. Recently a structure spanning the DMVs was identified serving as a putative pore dedicated to the translocation of the newly synthesized viral genome and mRNAs from the DMV lumen into the cytoplasm. The viral membrane (M) and envelope (E) proteins drive virion budding and are responsible for the recruitment of the viral genome packaged by the nucleocapsid (N) protein at the ER-Golgi Intermediate Compartment (ERGIC). However, the exact roles of structural proteins in virus budding are poorly understood and the proteins involved in DMV biogenesis remain to be identified. Here we apply in situ cryo-electron tomography (cryo-ET) to reconstitute the formation of DMVs and virus assembly. To elucidate the DMV pore structure and the role of each structural protein in SARS-CoV-2 budding, we use a transfection-based system allowing us to express different combinations of viral proteins together with a fluorescent marker. Cryo-light microscopy is applied to target transfected cells for cryo-focused ion beam milling and produced lamellae are subjected to cryo-electron tomography. Our data provide information on VLP assembly and the composition and structure of the SARS-CoV-2 DMV pore. In summary, in situ cryo-ET of here established transfection-based system provides an invaluable tool to study SARS-CoV-2 replication and virus assembly at biosafety level 1 condition.

  • © Conventus Congressmanagement & Marketing GmbH