LC3C–ATG4D regulatory axis supports coronavirus replication organelle formation independently of canonical autophagy

<jats:title>Abstract</jats:title> <jats:p>Coronaviruses hijack host membranes to assemble ER-derived double-membrane vesicles (DMVs) that shield viral RNA replication from the cell intrinsic surveillance. Although DMVs morphologically resemble autophagosomes, whether and how autophagy factors actively support their biogenesis has remained elusive. Here, we identify a non-canonical requirement for the autophagy protein LC3C in β-coronavirus replication. Loss of LC3s impaired viral RNA replication, whereas genetic ablation of ATG7 did not, indicating that canonical ATG7-dependent lipidation is dispensable in this context. Reconstitution experiments showed that only LC3C substantially restored replication in LC3-deficient cells and that LC3C phospho-mutants, differing in accessibility to ATG4-mediated processing, displayed distinct proviral activities. Additionally, ATG4D, the main protease responsible for maintaining the LC3 non-lipidated pool, is selectively required for viral replication. Both ATG4D and LC3s depletion triggers formation of aberrant DMV-like structures and potently suppresses SARS-CoV-2 replication. Ultrastructural analysis of nsp3–nsp4-induced membranes showed that depletion of LC3s or ATG4 proteases altered DMV abundance and morphology, supporting a role for the LC3C–ATG4D axis in replication organelle biogenesis. These data establish that β-coronaviruses repurpose ATG4D-driven LC3C de-lipidation for non-canonical LC3 recruitment to replication organelles, identifying the lipidation state of LC3 as a molecular determinant of replication organelle biogenesis and efficient viral replication.</jats:p> <jats:sec> <jats:title>Highlights</jats:title> <jats:p>The manuscript shows that β-coronavirus replication depends on LC3 proteins and particularly on LC3C in reconstitution experiments, that this dependency is independent of ATG7-mediated lipidation, and that ATG4D promotes efficient replication and replication organelle morphology. Together, the data support a model in which a non-canonical LC3C–ATG4D pathway contributes to DMV biogenesis and viral RNA replication.</jats:p> </jats:sec>