GSK3 maintains NSP12 stability to promote bat-origin swine acute diarrhea syndrome coronavirus infection

Bat-origin swine acute diarrhea syndrome coronavirus (SADS-CoV) causes diarrhea and death in piglets and also poses a cross-species transmission risk due to its broad host cell tropism. In the absence of commercial vaccines or therapeutics, identifying the host factors exploited by SADS-CoV may provide potential targets for antiviral therapies. Inhibition of glycogen synthase kinase 3 (GSK3) restricts diverse coronavirus replication by targeting the N protein and disrupting its phosphorylation. Here, we identified GSK3 as a crucial host factor for SADS-CoV RNA synthesis. Both GSK3 deficiency and treatment with a classical GSK3 inhibitor, lithium chloride (LiCl), strongly impaired SADS-CoV dsRNA synthesis. LiCl treatment did not alter electrophoretic mobility or expression of SADS-CoV N protein in N-transfected Huh7 cells but significantly reduced NSP12 protein levels. The absence of GSK3 led to the proteasomal degradation of NSP12 to reduce viral RNA synthesis. However, GSK3 interacted with ubiquitin-specific peptidase 27X (USP27X) and promoted the USP27X-mediated deubiquitination of NSP12, thereby enhancing NSP12 stabilization and facilitating viral RNA synthesis. LiCl not only protected neonatal mice from lethal SADS-CoV challenge but also exhibited a notable broad-spectrum antiviral effect against PEDV and TGEV. Our findings demonstrate that GSK3-mediated NSP12 stabilization is essential for SADS-CoV RNA synthesis, underscoring the potential of GSK3 as a broad-spectrum antiviral target for emerging and re-emerging swine coronaviruses.