Coronavirus Nsp3 Hijacks CLTC to Modulate Autophagosome Nucleation for Promoting DMV Formation and Viral Replication

Double-membrane vesicles (DMVs) are a hallmark of coronavirus replication, yet the host machinery governing their biogenesis remains poorly characterized, largely due to lack of tools for dynamic analysis. Here, we develop a live-cell imaging system using a recombinant virus that enables, for the first time, real-time visualization of DMV formation during authentic coronavirus infection. This system reveals the recruitment of the clathrin heavy chain (CLTC) to DMV assembly sites and demonstrates its essential role in diverse coronaviruses, but not in unrelated viruses. Notably, we define a previously unappreciated role of CLTC in viral replication organelle formation. Mechanistically, CLTC interacts with nonstructural protein 3 (nsp3) and is required for autophagosome nucleation by maintaining the core class III PI3K complex. The resulting CLTC-mediated autophagic precursor membranes are subsequently hijacked by nsp3 to form DMVs. Our study thereby establishes CLTC as a pivotal host factor for coronavirus replication and nominates both CLTC and the autophagosome nucleation pathway as promising antiviral targets.