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

## TL;DR

This study reveals how adeno-associated virus (AAV) proteins block a host phosphatase complex to enhance viral replication, offering new insights for improving gene therapy.

## Contribution

The study identifies a novel mechanism by which AAV subverts the PP4:SMEK1 phosphatase complex to promote replication.

## Key findings

- AAV Rep68 blocks substrate recruitment to PP4:SMEK1, causing hyperphosphorylation of KAP1S824 and RPA2S4/8/33.
- A MAPP motif in KAP1 binds SMEK1's EVH1 domain, facilitating their interaction.
- A PP4:SMEK1 and PP1:NIPP1 complex collaborates to dephosphorylate KAP1S824.

## Abstract

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.

Adeno-associated virus (AAV) is a small, harmless virus with a simple single-stranded DNA genome. Its four replication (Rep) proteins act as master regulators of the viral life cycle by taking control of key cellular machinery. Yet, many important interactions between AAV Rep proteins and host cell factors are still not well understood. In this study, we identify the PP4:SMEK1 phosphatase complex as a previously unrecognized obstacle to AAV gene expression and replication. We show that AAV overcomes this barrier by using its Rep proteins to block PP4 activity, resulting in the hyperphosphorylation of host proteins KAP1 and RPA2, necessary for enhanced AAV gene expression and replication. Our findings also reveal that PP4:SMEK1 and PP1:NIPP1 form a large, multifunctional complex that works together to dephosphorylate KAP1. These insights deepen our understanding of how AAV interacts with host cells and may help improve the design and manufacturing of AAV-based gene therapy vectors.