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 Importance: HPV16 is responsible for the majority of HPV+ cancers, contributing to 54% of cervical cancers and ∼90% of HPV+HNSCC. Integration of viral genomes into host DNA can promote cervical cancer progression and correlates with poor prognosis in HPV-associated HNSCC, where around 70% of HPV+ cancers contain episomal viral genomes. Developing effective antiviral therapies requires a deeper understanding of the interplay between viral replication and host DNA damage response (DDR) pathways. This report demonstrates that SMARCAL1 is essential for HPV16 replication and keratinocyte proliferation and that its depletion leads to replication stress, DNA damage, and viral genome integration. This work underscores the delicate balance between viral exploitation of the host DDR and the risk of genome instability. These insights contribute to the broader understanding of HPV pathogenesis and may inform the development of therapeutic strategies targeting viral replication to prevent disease progression and improve clinical outcomes in HPV-associated cancers.