Bypass of LANA-DNA crosslinks by Polη ensures KSHV genome maintenance and tumor growth

<h2>Summary</h2> Episome maintenance of Kaposi's sarcoma-associated herpesvirus (KSHV) requires LANA protein binding to the viral terminal repeat (TR) DNA. Here, we show that LANA forms cell cycle-dependent DNA-protein crosslinks (DPCs) within the TR. Targeted shRNA screening identified translesional repair DNA polymerase Polη as necessary for the stability of KSHV TR DNA and viral episome maintenance. We found that Polη binds LANA and colocalizes with LANA nuclear bodies. Inhibiting the enzymatic activity of Polη by small molecule PNR-07-02 provided selective cell killing of KSHV⁺ B cells and inhibited tumor progression in mice xenografts. Mechanistically, Polη inhibition results in both single and double-strand breaks at the KSHV TR due to unresolved LANA-DNA crosslinks and replication fork collapse. These findings reveal a new mechanism of viral episome maintenance by DPC coupled to translesion DNA synthesis (TLS) DNA polymerases and identify Polη as a potential target for inhibition of KSHV latent infection in proliferating tumor cells.