Rab27-dependent egress of SARS-CoV-2 via secretory amphisomes

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, hijacks host cellular machinery to replicate and spread. Understanding how SARS-CoV-2 reorganizes host cell architecture to accommodate this is essential for elucidating its pathogenesis and identifying therapeutic targets. While the molecular mechanisms of SARS-CoV-2 entry are well characterized, the pathways governing viral egress remain incompletely understood. Conventional approaches such as transcriptomics and electron microscopy have provided valuable insights but lack the combined spatial and molecular resolution needed to map these processes within intact cells. Here, we apply Ten-fold Robust Expansion Microscopy (TREx) to visualize SARS-CoV-2-induced remodeling of the endolysosomal system in multiciliated cells of primary human airway epithelial tissue. This approach reveals Golgi fragmentation and the formation of enlarged virus-containing organelles. Analysis of endolysosomal markers in Vero E6 cells shows that these structures are positive for CD63, Rab7, and LC3, consistent with amphisome identity. Moreover, pharmacological inhibition of Rab27-dependent amphisome-plasma membrane fusion with Nexinhib20 reduces viral infection, implicating secretory autophagy as a pathway for SARS-CoV-2 egress. These findings establish expansion microscopy as a powerful tool for spatial virology and uncover a Rab27-mediated amphisome fusion mechanism as a druggable route for SARS-CoV-2 release.