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Here, we develop a live\u2010cell imaging system using a recombinant virus that enables, for the first time, real\u2010time 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\u00a0maintaining the core class III PI3K complex. The resulting CLTC\u2010mediated 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.<\/jats:p>","DOI":"10.1002\/advs.202521626","type":"journal-article","created":{"date-parts":[[2026,2,21]],"date-time":"2026-02-21T09:20:06Z","timestamp":1771665606000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Coronavirus Nsp3 Hijacks CLTC to Modulate Autophagosome Nucleation for Promoting DMV Formation and Viral Replication"],"prefix":"10.1002","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-7624-1114","authenticated-orcid":false,"given":"Juan","family":"Xu","sequence":"first","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology College of Veterinary Medicine Huazhong Agricultural University  Wuhan P. R. 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