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

Adeno-associated virus (AAV) vectors are widely used for in vivo gene therapy, but their clinical effectiveness and scalable production remain limited by gaps in our understanding of AAV biology. In particular, the host-virus interactions that regulate AAV gene expression and replication are not fully defined, yet they hold key insights for engineering improved recombinant AAV (rAAV) vectors. Here, we identify the PP4:SMEK1/2 phosphatase complex as a host restriction factor that suppresses wildtype AAV replication. Active replication and Rep expression correlates with hyperphosphorylation of KAP1S824 and RPA2S4/8/33, known PP4 substrates. Rep proteins directly bind the HEAT/Arm domain of the SMEK1 regulatory subunit of PP4. We further reveal that the KAP1:SMEK1 interaction is mediated by a MAPP short linear motif (SLiM) that interacts with the substrate recruitment groove of the SMEK1 EVH1 domain. Rep68 disrupts this interaction and interferes with substrate recruitment by the PP4:SMEK1/2 complex, leading to substrate hyperphosphorylation. We also uncover a multifunctional complex containing PP4:SMEK1 and PP1:NIPP1, both implicated in KAP1S824 dephosphorylation. Together, these findings reveal a substrate-recruitment interference mechanism by which AAV Rep proteins inhibit host phosphatase activity. This mechanistic insight provides a blueprint for enhancing rAAV vector potency and production in gene therapy applications.