The SARS-CoV-2 nucleocapsid protein associates with the replication organelles before viral assembly at the Golgi/ERGIC and lysosome-mediated egress

Scherer, Katharina M.; Mascheroni, Luca; Carnell, George W.; Wunderlich, Lucia C. S.; Makarchuk, Stanislaw; Brockhoff, Marius; Mela, Ioanna; Fernandez-Villegas, Ana; Barysevich, Max; Stewart, Hazel; Sans, Maria Suau; George, Charlotte L.; Lamb, Jacob R.; Kaminski-Schierle, Gabriele S.; Heeney, Jonathan L.; Kaminski, Clemens F.

Despite being the target of extensive research efforts due to the COVID-19 pandemic, relatively little is known about the dynamics of SARS-CoV-2 replication within cells. We investigate and characterise the tightly orchestrated sequence of events during different stages of the infection cycle by visualising the spatiotemporal dynamics of the four structural proteins of SARS-CoV-2 at high resolution. The nucleoprotein is expressed first and accumulates around folded ER membranes in convoluted layers that connect to viral RNA replication foci. We find that of the three transmembrane proteins, the membrane protein appears at the Golgi apparatus/ERGIC before the spike and envelope proteins. Relocation of the lysosome marker LAMP1 towards the assembly compartment and its detection in transport vesicles of viral proteins confirm an important role of lysosomes in SARS-CoV-2 egress. These data provide new insights into the spatiotemporal regulation of SARS-CoV-2 assembly, and refine current understanding of SARS-CoV-2 replication.