biostudies-literatureUnknownMohona SNIAID NIH HHSNCI NIH HHSNIGMS NIH HHSNIH HHSAssembly of infectious coronaviruses requires spike (S) protein trafficking by host coatomer, typically via a dibasic signal in the S cytoplasmic tail. However, the human embecoviruses HKU1 and OC43, as well as the model virus MHV, lack this motif. Here we identify a conserved His-x-Asp (HxD) sequence that functions as an unconventional coatomer-binding signal. Structural and biochemical analyses show that the MHV HxD motif engages coatomer subunits through distinct conformations, while cellular imaging demonstrates its role in directing S to assembly sites with the viral M-protein. Disruption of HxD-coatomer interactions impairs S incorporation and provokes compensatory viral adaptations, including emergence of a canonical dibasic motif or mutations in M-protein. Electron microscopy furthbioRxiv : the preprint server for biology2025.10.16.682669https://www.ebi.ac.uk/biostudies/studies/S-EPMC12633080biostudies-literatureAn unconventional HxD motif orchestrates coatomer-dependent coronavirus morphogenesis.PMC12633080P30 CA134274R01 GM150187S10 OD034431P30 GM133893R21 AI176252Klose TQing EDoray BGallagher THasan SSShakya AKDey DAmaro REJennings BCErramilli SKossiakoff AAMohona SSingh SKearns FLJemison KfalseAn unconventional HxD motif orchestrates coatomer-dependent coronavirus morphogenesis.Assembly of infectious coronaviruses requires spike (S) protein trafficking by host coatomer, typically via a dibasic signal in the S cytoplasmic tail. However, the human embecoviruses HKU1 and OC43, as well as the model virus MHV, lack this motif. Here we identify a conserved His-x-Asp (HxD) sequence that functions as an unconventional coatomer-binding signal. Structural and biochemical analyses show that the MHV HxD motif engages coatomer subunits through distinct conformations, while cellular imaging demonstrates its role in directing S to assembly sites with the viral M-protein. Disruption of HxD-coatomer interactions impairs S incorporation and provokes compensatory viral adaptations, including emergence of a canonical dibasic motif or mutations in M-protein. Electron microscopy furth2025-01-01T00:00:00Z2025 Oct2026-06-04T03:21:33.982Z2026-06-04T03:12:42.747ZS-EPMC126330804127999110.1101/2025.10.16.682669