HPV16 recruitment of SMARCAL1 to viral and host replication forks is required for the viral life cycle

High-risk human papillomaviruses (HR HPVs) are responsible for around 5% of the world's cancer burden. Activation and interaction with the host DNA damage response (DDR) promotes the HPV16 life cycle. This study demonstrates a crucial interaction between HPV16 and SMARCAL1, a protein involved in the stabilization of stalled DNA replication forks. SMARCAL1 can complex with E2, is recruited to E1-E2 replicating DNA, and SMARCAL1 knockdown reduces the fidelity of E1-E2 mediated DNA replication in C33a cells but does not alter replication levels. SMARCAL1 is recruited to the HPV16 genome in HPV16-immortalized foreskin keratinocytes (HFK+HPV16), and in situ protein interaction with nascent DNA replication forks (SIRF) assays demonstrated that SMARCAL1 is hyper-recruited to host replication forks in HFK+HPV16 cells. Using COMET, DNA fiber, and cell growth assays it was determined that knockdown of SMARCAL1 increased DNA damage and impaired replication fork progression in HFK+HPV16 cells, ultimately resulting in growth arrest. The viral genome integrates following SMARCAL1 knockdown in HFK+HPV16 cells. Therefore, SMARCAL1 facilitates host and viral DNA replication in HFK+HPV16 cells. Overall, the results demonstrate that HPV16 promotes SMARCAL1 recruitment to viral and host replication forks and is an essential factor for the HPV16 life cycle. The results expand our understanding of DDR proteins that regulate the HPV16 life cycle, and suggest that inhibition of SMARCAL1 function represents a novel anti-viral strategy for the treatment and prevention of HPV infections.