Spiperone targets HBV cccDNA via ER stress induced innate immune activation and epigenetic silencing

Chronic hepatitis B persists due to the stability of nuclear covalently closed circular DNA (cccDNA), which maintains viral transcription despite prolonged antiviral therapy, highlighting the need for strategies that suppress cccDNA via host targeted mechanisms. Here, we identify Spiperone, a clinically approved compound, as a repurposed anti HBV candidate with strong translational potential. Spiperone robustly reduced HBsAg, HBeAg, viral DNA, and pgRNA across HepG2.2.15, HBV infected HepG2 NTCP C4 and HepaRG cells, and multiple in vivo models, including HBV transgenic, hydrodynamic injection, and AAV HBV1.04x models. Notably, intrahepatic cccDNA was significantly diminished. In combination, Spiperone potentiated tenofovir activity, exhibiting synergistic effects, while both intraperitoneal and oral administration reduced antigenemia and viremia. Mechanistically, Spiperone activated the PERK, eIF2a, ATF4 arm of the ER stress response, coupled with mitochondrial perturbation and cytosolic release of oxidized mitochondrial DNA, leading to activation of IFI16, STING, IRF3 signaling. This cascade induced type I interferon and interferon stimulated genes. ChIP qPCR further demonstrated reduced enrichment of activating histone marks on cccDNA, consistent with transcriptional repression. Collectively, these findings position Spiperone as a host directed antiviral that converges ER stress linked innate immunity and epigenetic repression to suppress cccDNA, supporting its advancement in combination strategies toward a functional cure for chronic HBV infection.