Hepatitis B virus protein X promotes survival and loss of hepatocyte identity in a differentiated human liver organoid system

Abstract Hepatitis B virus (HBV) drives hepatocellular carcinoma in part through the activity of its X protein (HBx), yet the mechanisms by which HBx alters hepatocyte function remain incompletely understood. Progress has been limited by the lack of relevant human models that support controlled HBx expression in mature hepatocytes. To address this limitation, here, we use an improved hepatocyte-like organoid (HLO) platform that supports enhanced hepatocyte maturation to investigate HBx function in a differentiated hepatocyte context. Adult stem cell-derived HLOs were differentiated using an optimized protocol to generate hepatocyte-like cells with enhanced maturation and transcriptional similarity to primary liver tissue. HBx function was interrogated using both cognate promoter-driven expression and doxycycline-inducible systems across multiple donor-derived organoid lines. Transcriptomic, pathway, and single-cell imaging analyses were performed to assess the impact of HBx expression on hepatocytes. HBx expression consistently suppressed apoptosis-associated transcripts and reduced expression of core hepatocyte identity genes, including CYP3A4. Pathway analysis revealed downregulation of liver-specific functions, including metabolism, detoxification, complement, and coagulation. At the single-cell level, higher HBx expression was associated with reduced caspase 3/7 activation following apoptotic challenge and decreased hepatocyte marker expression. Functionally, the expression of HBx increased resistance to apoptosis and enhanced the ability of differentiated hepatocyte-like cells to revert to a proliferative state. HBx expression in differentiated human liver organoids reduces apoptosis and impairs hepatocyte identity, consistently across donors and expression systems. Together, these findings support a model in which HBx promotes a survival-permissive state of reduced hepatocyte differentiation. This state may contribute to early HBV-driven tumorigenesis. This HLO platform provides a relevant system to dissect HBV-host interactions and reveals a mechanism by which HBV may prime the liver for malignant transformation. Impact and implications Understanding how HBV promotes hepatocellular carcinoma remains a critical challenge, partly due to the lack of physiologically relevant human derived model systems to study HBx function. Using a differentiated adult human liver organoid system, we show that HBx simultaneously suppresses apoptosis and reduces hepatocyte differentiation, providing a mechanistic framework for how HBV may prime hepatocytes for malignant transformation. These findings are particularly relevant for researchers studying HBV pathogenesis and liver cancer, as well as for clinicians aiming to better understand early disease progression. While further validation in more complex multicellular systems is needed, this platform can support the identification of HBx-targeted therapeutic strategies and guide the development of improved adult human derived models for virus-host interaction studies.