An HBV‐Derived Peptide Poly6 as a Novel Candidate for Functional Cure Via IFN‐I–Mediated Epigenetic Regulation of cccDNA

Covalently closed circular DNA (cccDNA) represents the central obstacle to achieving a functional cure for chronic hepatitis B virus (HBV) infection. Poly6, a peptide encoded within the HBV genome, was investigated for antiviral efficacy in hepatocyte-derived cell lines, hydrodynamic injection models, and HBV transgenic mice. Poly6 administration markedly decreased cccDNA, pregenomic RNA, and viral DNA without detectable cytotoxicity. Poly6 also showed synergistic antiviral effects with entecavir. Mechanistic analyzes demonstrated that Poly6 initiates parallel upstream events: mitochondrial stress resulting in oxidized mtDNA release and activation of the STING-IRF3 pathway, and induction of IFI16, a nuclear DNA sensor implicated in interferon regulation. Both signals converged on robust type I interferon (IFN-I) production. The IFN-I response subsequently promoted expression of canonical ISGs, including iNOS, which generated nitric oxide to disrupt nucleocapsid assembly. Concurrently, IFI16, whose abundance was further increased by interferon signaling, amplified IFN-I production and imposed epigenetic silencing of cccDNA through Sp1 sequestration and histone hypoacetylation. Chromatin immunoprecipitation confirmed reduced acetylation of H3K27, H4K5, and H4K12 on cccDNA minichromosomes. These results delineate a unified IFN-I-centered cascade in which Poly6 coordinates complementary antiviral activities, supporting its translational potential as a therapeutic candidate for durable HBV control.