Integration of Hepatitis B Virus (HBV) DNA: Understanding its Molecular Mechanisms and Consequences

Abstract

HBV infection remains a major global health burden, affecting over 360 million people and causing ~1.1 million deaths annually, mainly due to liver cirrhosis and hepatocellular carcinoma. Its persistence and carcinogenicity are driven by cccDNA and HBV DNA integration. This thesis investigates the molecular mechanisms, clinical dynamics, and functional consequences of HBV DNA integration in chronic infection and HBV-associated carcinogenesis.

Using a replication-defective reporter system, we demonstrated that HBV DNA integration occurs early post-infection and is mediated by host DNA repair pathways. Inhibition of homologous recombination via ATR inhibitors increased integration (2.4-2.8-fold), identifying the ATR-coordinated DNA damage response as a barrier. In contrast, ART558, a POLQ/MMEJ pathway inhibitor, reduced integration to 17% of controls, implicating MMEJ as a dominant pathway, suggesting that targeting MMEJ may block de novo integration during viral replication or hepatocyte turnover.

Clinically, we developed a dd-qinvPCR assay to quantify intrahepatic integration in liver fine-needle aspirates, revealing a median 66.5% reduction over one year, consistent with immune-mediated clearance. This decline was uncoupled from serum biomarkers, highlighting the value of tissue-based measurements for interpreting treatment responses.

At the cellular level, truncated HBx from integration promotes SMC5/6 degradation and retains oncogenic activity. However, integration-driven genomic instability plays a larger role, with increased DNA damage markers (γ-H2AX, p-ATM, 53BP1) and a 3.7-fold higher susceptibility to further integration events. These support a feed-forward model in which integration drives genomic instability and accelerates carcinogenesis.

This work redefines HBV DNA integration as an active, targetable driver of liver cancer, supporting therapeutic strategies that directly inhibit integration and modulate immune responses to disrupt progression to HCC.