Abstract

After entry, coronaviruses deliver their genomic RNA to the ER-Golgi intermediate compartment to build replication organelles, yet how the RNA first contacts membranes is unclear. Here, we demonstrate that the first transmembrane helix of nsp3 serves as an address code, inserting into the ER via SEC61A1 during translation and is then escorted to the ERGIC by the COPII factors SAR1A and LMAN1. Deletion or proline substitution of key residues in this helix trapped the RNA in the cytosol and crippled viral replication. A 24-residue peptide (Y24) modeled on the helix inhibited viral replication, and AAV9-delivered A20-2 peptide reduced lung viral RNA and pathology in mice. This host-targeted approach offers a potential broad-spectrum strategy against emerging coronaviruses.