Autophagy is a key cellular recycling process necessary for maintaining homeostasis. The protein ATG16L1 serves as a critical regulator and structural component of the autophagy machinery. One of its major roles is to facilitate the formation of the phagophore, a membrane structure that engulfs cytoplasmic material and directs it to lysosomes for degradation.

During SARS-CoV-2 infection, the virus triggers the production of double-membrane vesicles (DMVs), which are essential for viral replication. These vesicles share structural similarities with autophagosomes, and both arise from specialized domains of the endoplasmic reticulum (ER), suggesting that related molecular mechanisms may underlie their biogenesis. Yet, the precise molecular connection between autophagy and viral DMV formation has remained unclear.

Our study demonstrates that ATG16L1 is indispensable for DMV formation in SARS-CoV-2-infected cells. Notably, ATG16L1 variants that fail to support autophagosome formation are similarly defective in promoting DMV assembly. By combining functional analysis, biochemical approaches, and electron microscopy, we delineated the molecular basis of DMV formation.

Together, our findings demonstrate how ATG16L1 facilitates DMV biogenesis during SARS-CoV-2 infection, providing insights into how the virus repurposes host autophagy machinery for its replication. This work highlights potential molecular targets for therapeutic intervention aimed at disrupting viral propagation.