The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Membrane (M) protein organizes the assembly and structure of new virions and is essential for virus formation. It interacts with Envelope (E) protein to induce curvature in the membrane, thus allowing the assembly of viral particles and promoting maturation of Spike (S). It also interacts with Nucleocapsid (N) protein to allow genomic RNA stabilization. Recently reported cryo-EM structure of M protein confirms the presence of three transmembrane domains (TMDs) and a homo-dimeric conformation that might allow/promote viral assembly. In this work, we studied M protein membrane insertion by using truncated polypeptides of increasing length. We observed that a TMD1 truncate is sufficient to target the protein to the membrane both in bacterial and eukaryotic cells, and that the following TMDs are inserted into the membrane in a sequential manner. Since the link between structure and function of this protein is still enigmatic, we studied the effect of expressing these truncate polypeptides in cells; revealing that they are able to perform some of the full length protein functions. A TMD1-2 truncate is able to oligomerize and, interestingly, extracellular vesicle purification by ultracentrifugation showed that is also able to form high-yield membrane derived particles. This may indicate that M TMD bundle may play a key role in membrane bending and virion particle formation. Finally, we used TurboID proximity biotinylation to find proximal clients of specific TMDs, revealing potential targets that assist  its folding and biogenesis.

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