The adeno-associated virus Rep proteins target PP4:SMEK1 by preventing substrate recruitment.

Despite the widespread use of adeno-associated virus (AAV) vectors in gene therapy, their clinical efficacy and large-scale manufacturing remain constrained by an incomplete understanding of the virus-host interactions that govern AAV gene expression and replication. Here, we identify the PP4:SMEK1/2 phosphatase complex as an important regulator of wild-type AAV replication. Binding studies show that the AAV replication proteins engage SMEK1 to negatively influence PP4 activity. Specifically, AAV Rep68 interferes with substrate recruitment to the PP4:SMEK1 complex, resulting in hyperphosphorylation of the PP4 substrates KAP1S824 and RPA2S4/8/33, which in turn enhances viral gene expression and replication. We further uncover a direct interaction between KAP1 and SMEK1, mediated by a MAPP short linear motif that binds the SMEK1 EVH1 domain. Additionally, we identify a multifunctional complex comprising PP4:SMEK1 and PP1:NIPP1 that contributes to KAP1S824 dephosphorylation. These findings reveal a previously unrecognized mechanism by which viruses subvert host phosphatases to promote replication. This mechanistic insight not only advances our understanding of AAV and phosphatase biology but also has the potential to inform strategies for enhancing AAV vector potency.