Nsp3-Nsp4 FIB milled HEK293F lamella tomography

Coronaviral infection of human host cells induces the formation of double-membrane vesicles (DMVs), which serve as central hubs for genome replication. In SARS-CoV-2, the non-structural proteins Nsp3, Nsp4, and Nsp6 coordinate DMV biogenesis. Nsp3 and Nsp4 form pore-like structures spanning the DMV membranes, and may act as gateways for the export of the synthesized viral RNA to the nucleoprotein resulting in ribonucleoprotein complexes required for virion assembly. This study aims to capture key steps of this replication process using cryo-electron tomography of HEK293 cells overexpressing DMV related proteins, starting with Nsp3 and Nsp4. Towards that we will produce lamellae targeted to regions enriched in GFP-tagged Nsp3. We request beamtime in CM02 to perform tomography experiments on these lamellae to generate a high resolution architecture of the pore complex and gain insights into membrane remodeling events driven by Nsp3 and Nsp4 and various steps of SARS-CoV-2 replication