GSK3A promotes human adenovirus replication and phosphorylates viral L4-22K protein

Human adenovirus B7 (HAdV-B7) is a significant respiratory pathogen in children, associated with substantial morbidity and mortality. Despite its clinical importance, the molecular mechanisms of HAdV-B7-host interaction remain poorly elucidated. In this study, we performed a high-throughput gain-of-function cDNA library screening and identified glycogen synthase kinase 3α (GSK3A) as a key proviral factor facilitating HAdV-B7 replication. The overexpression of GSK3A enhanced viral replication, whereas knockdown or knockout inhibited it. Furthermore, the kinase-active S21A mutant significantly augmented viral replication, whereas the kinase-inactive Y279A and K148A mutants of GSK3A failed to support it, highlighting the importance of its kinase activity on HAdV-B7 replication. Notably, phosphoproteomic and co-immunoprecipitation assays (co-IP) revealed that GSK3A phosphorylates viral L4-22K protein at S78 and S81 residues in a partially kinase-dependent manner. Using structure modeling, protein-protein docking, and truncation assays, we mapped the interaction between GSK3A's kinase domain and the 92-168 aa region of the viral L4-22K protein. In addition, GSK3A acts as a broad-spectrum proviral factor for respiratory HAdV, particularly for Species B, corresponding to a high similarity in L4-22K sequences. As a result, we identified GSK3A as a crucial proviral host factor for HAdV replication and provided a promising avenue for targeting GSK3A in the development of antiviral therapies against HAdV infections.