In situ cryo-electron tomography of SARS-CoV-2 replication pore complex and virus assembly

Viruses have evolved distinct strategies to replicate their genomes and assemble their progeny virions in infected cells. Viral proteins often hijack host cellular membranes to facilitate replication and assembly. These processes are tightly coupled and require interactions between viral proteins along with host proteins, which are recruited to assist in viral assembly. Betacoronaviruses have caused previous deadly epidemics, including the ongoing Middle East Respiratory Syndrome (MERS) epidemic. The Coronavirus Disease 2019 (COVID-19) pandemic was caused by the betacoronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Coronaviruses harbor, compared to other RNA viruses, a large RNA genome, which encodes an unusually high number of viral proteins involved in RNA synthesis and RNA-processing. Viral RNA synthesis is associated with replication organelles, which are built from ER membranes and consists of double-membrane vesicles (DMVs) containing pores. Coronavirus replication provides the RNA genome for assembly of progeny virions. The viral budding involves ER-Golgiintermediate compartments (ERGICs), which provide the membrane for assembly of the viral envelope. However, many aspects of viral replication and assembly are unknown, including the function and biogenesis of the replication pore complex inside DMVs. Furthermore, what drives viral assembly and how budding is mediated remains to be elucidated. In this thesis, I investigate the structural aspects of DMV pore formation and viral assembly. In situ cryo-electron tomography (cryo-ET) provides unique structural information, which can be used to build structural models or describe biological processes. I discovered the minimal requirements for DMV pore assembly and showed that the pore structural integrity is essential for DMV biogenesis. I provided new molecular insights into viral budding at the ERGIC and discovered the importance of the envelope protein in this process. Overall, this thesis extends the current knowledge in DMV biogenesis and provides a model for SARS-CoV-2 assembly, where the envelope protein mediates membrane scission and alters membrane curvature at the bud neck. The established SARS-CoV-2 virus-like particle (VLP) system can be used in future experiments to investigate the involvement of host proteins in viral budding and may provide a valuable tool for diagnostics and vaccine development.