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.IMPORTANCEZoonotic transmission of coronaviruses continues to pose a significant threat to human populations. Swine acute diarrhea syndrome coronavirus (SADS-CoV) has been identified as a potentially high-risk emerging coronavirus pathogen that could adversely affect the global economy and human health. However, the replication mechanisms of SADS-CoV remain largely unexplored. Our study demonstrates that GSK3 stabilizes NSP12 by interacting with USP27X, thereby enhancing the synthesis of SADS-CoV dsRNA. LiCl, a GSK3 inhibitor, exhibits broad-spectrum antiviral activity against swine enteric alphacoronaviruses. Our work is distinguished for the first time by the GSK3-USP27X regulatory axis that controls the stability of NSP12 and indicates that GSK3 is a promising therapeutic target for the development of broad-spectrum antiviral drugs against emerging or re-emerging swine coronaviruses.