Hepatitis B viral DNA integration occurs within three days of infection and is enhanced by ATR inhibition

Chronic hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma, with viral DNA integration into the host genome playing a pivotal role in oncogenesis. While HBV integration has been historically considered an event occurring late in a chronic infection, sensitive assays have detected integrations early infection. This study investigates the specific timing and molecular mechanisms of HBV DNA integration using a replication deficient HBV reporter system (HBV-Zeo) in HepG2-NTCP cells. Infection of this virus followed by positive selection led to cellular colony formation, showing that the input virus is the substrate that undergoes integration. By inducing DNA double-strand breaks via X-ray irradiation at specific timepoints after HBV infection, we observed a 2-3-fold increase in integration frequency when cells are irradiated between 16 and 76 h post-infection. Pharmacological inhibition of DNA repair pathways in this specific time window revealed that suppression of homologous recombination (HR) via ATR inhibitors significantly enhances integration rates (2.4-2.8-fold), while microhomology-mediated end joining (MMEJ) inhibition reduced integration to 17 % of untreated controls. These findings suggest that MMEJ plays a key role in HBV DNA integration occurring within hours of HBV infection. Together, our results advance understanding of HBV-associated hepatocarcinogenesis and may inform therapeutic strategies to disrupt viral integration and mitigate HBV-associated liver cancer risk.