Disruption of ER-mitochondria contact sites by coronavirus replication organelles sustains viral replication via NSP3 stabilization.

Coronaviruses establish infection by reorganizing the host endoplasmic reticulum (ER) to form double-membrane vesicles (DMVs), which function as viral replication platforms. However, the role of other cellular organelles in this process remains incompletely understood. Here, we uncover a self-reinforcing cycle between viral replication organelles and mitochondrial damage that sustains coronavirus replication. We show that DMV formation disrupts ER-mitochondria contact sites (ERMCs), causing mitochondrial damage. This injury initiates a feed-forward mechanism wherein mitochondria release the matrix enzyme ECHS1 into the cytosol. Cytosolic ECHS1 then binds and stabilizes the DMV inducer protein NSP3 by blocking its K963 ubiquitination via the host E3 ligase RBBP6, thereby promoting further DMV formation. Disrupting this cycle, either through enhanced ER-mitochondria tethering or targeted interference with ECHS1-NSP3 binding, effectively suppresses viral replication. Our findings reveal that coronaviruses exploit an inter-organellar feedback loop linking mitochondrial damage to DMV formation, identifying new potential therapeutic targets for inhibition of coronaviral replication.