Coronaviral nsp6 hijacks ERAD machinery to facilitate lipolysis and supply membrane components for DMV growth.

The positive-strand RNA ( + RNA) viruses extensively remodel cellular endomembranes to facilitate viral replication, with coronaviruses forming a specialized viral replication organelle (RO) known as double-membrane vesicles (DMVs). These DMVs serve as platforms for viral replication and shield viral RNA from host immune recognition. However, the biogenesis, structural organization, and physiological properties of DMVs remain poorly understood. In this study, we demonstrate that the coronavirus non-structural protein 6 (nsp6) anchors DMVs to lipid droplets (LDs), hijacks the endoplasmic reticulum (ER)-associated protein degradation (ERAD) machinery to degrade PLIN2, and redirects fatty acids (FAs) from LDs to DMVs, thereby supplying lipids for DMV growth. Furthermore, nsp6 anchors ERAD-derived vesicles to DMVs, directly refurnishing membrane components for DMV expansion. Disruption of lipolysis or ERAD impairs DMV formation and inhibits coronaviral replication. We further validated the antiviral effects of ERAD inhibition in female mice in vivo. Our findings elucidate the mechanisms and functional significance of virus-induced organelle remodeling and DMV biogenesis. Given the conservation of viral ROs across +RNA viruses, these structures represent a promising and attractive target for the development of broad-spectrum antiviral therapies.