The non-vesicular cholesterol transporter GRAMD1C is a pan-coronavirus antiviral target

Coronaviruses (CoVs) rely heavily on host lipid metabolism for efficient replication, but the specific host pathways involved remain incompletely defined. Here, we identify the non-vesicular cholesterol transport protein GRAMD1C as a key host factor required for the replication of multiple coronaviruses across α, β, and δ genera, including TGEV, PEDV, HCoV-229E, SARS-CoV-2, MHV, and PDCoV. Mechanistically, GRAMD1C is recruited to the replication factory through its interaction with the viral nonstructural proteins 3 and 4 (nsp3 and nsp4). Transmission electron microscopy (TEM) analysis revealed that GRAMD1C facilitates the formation of replication double-membrane vesicles (DMVs). Further domain rescue and inhibitor experiments demonstrated that GRAMD1C's cholesterol transport activity is essential for viral replication. Moreover, GRAMD1C-deficient and inhibitor-treated mice exhibited reduced viral replication, underscoring its critical role in vivo. Collectively, our findings expand the current understanding of the importance of non-vesicular cholesterol transport in viral replication and highlight GRAMD1C as a promising broad-spectrum antiviral target.