Mechanisms of HBx Recognition and Epigenetic Regulation of HBV cccDNA

/ABSTRACT Chronic Hepatitis B Virus (HBV) infection affects nearly 300 million individuals and is a leading cause of hepatocellular carcinoma worldwide. HBV persistence relies on the establishment of 1) the viral covalently closed circular DNA (cccDNA) minichromosome which serves as that viral transcriptional template and 2) the expression of the regulatory protein HBx which prevents cccDNA silencing. Due to limited in vitro and animal models that recapitulate transient events like cccDNA formation early during infection, the mechanisms through which HBx recognizes and regulates cccDNA remain poorly understood. The David lab strives to better understand the molecular mechanisms of chromatin regulation in disease. Towards this end, we have pioneered novel platforms to study HBV biology with the first methods to reconstitutes scarless cccDNA with infectious capacity and full- length HBx protein for biochemical and biophysical studies. We discovered that HBx directly binds to chromatin to induce decompaction in vitro and found that cccDNA histone occupancy is required for HBx expression early during infection. My proposal extends these findings by seeking to elucidate host epigenetic regulation of HBV transcription (Aim 1) and HBx-mediated cccDNA recognition (Aim 2). My working hypothesis is that HBx directly recognizes and decompacts the viral minichromosome to allow host epigenetic machinery to distinctly control viral transcription. We have shown that host histone incorporation onto cccDNA is important for HBV transcription. Importantly, we found that pharmacological disruption of viral chromatin assembly leads to reductions in HBV transcription, antigen secretion, and viral replication in hepatocyte models of infection. The proposed Aim 1 will identify host histone variants and chromatin remodelers that influence HBV transcriptional control. First, I will probe for the presence of active transcription- associated histone variants deposited onto cccDNA during infection using chromatin immunoprecipitation. I will then determine the effect on viral chromatin assembly and transcription of the loss of host epigenetic regulators implicated in cccDNA chromatin regulation using a limited shRNA screen and auxin-inducible degron tagging. In Aim 2, I will use biophysical approaches, such as biolayer interferometry, to define the determinants of HBx- nucleosome binding. I will then investigate changes in viral chromatin compaction due to this interaction through Mg2+ precipitation and MNase protection assays. If validated in vitro, I will expand to testing this phenomenon in cellulo using dCas9 to fluorescently label cccDNA and ATAC-see to measure and visualize host- and cccDNA- specific changes in chromatin accessibility in the absence and presence of HBx. Overall, Aim 2 will reveal new insights into HBx’s role in viral chromatin regulation. Successful completion of this project will reveal mechanisms for how HBx modulates HBV chromatin and uncover novel therapeutic targets for silencing or eradicating cccDNA, improving epigenetic strategies to develop a functional cure for chronic HBV infection. Project Number: 1F31AI197467-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Tracy Biaco | Institution: WEILL MEDICAL COLL OF CORNELL UNIV, NEW YORK, NY | Award Amount: $50,114 | Activity Code: F31 | Study Section: Special Emphasis Panel[ZRG1 F07C-H (20)] View on NIH RePORTER: https://reporter.nih.gov/project-details/1F31AI19746701