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P43 Cellular protein cyclophilin A is involved in hepatitis B virus replication and its inhibition with DEB025 (alisporivir) or NIM811 demonstrates antiviral activity in vitro

Introduction Cyclophilins are ubiquitously expressed intracellular proteins that have enzymatic activity—peptidyl-prolyl cis-trans isomerase, and are involved in regulation of mitochondrial/cytosolic transport in the cells. DEB025 (Alisporivir) and NIM811 are non-immunosuppressive cyclophilin inhibitors that efficiently block hepatitis C virus replication by targeting host rather than viral proteins, however the potential impact of cyclophillin inhibition on hepatitis B virus (HBV) life cycle is poorly understood. Aim In the present study we employed a panel of liver cell lines to investigate the ability of cyclophilin inhibitors DEB025 (Alisporivir) and NIM811 to affect HBV replication and viral particle secretion from the cells. Method HepG2215 cells, stably transfected with the full HBV genome and supporting the production of both infectious virions and HBsAg particles, and the parent cells (HepG2) were cultured for 7 days (baseline), prior to treatment with DEB025 or NIM811 at 0.25; 1.0 and 5.0 mg/ml. The cells and supernatants were harvested separately at baseline; 6, 24, 48 and 72 h after addition of cyclophilin inhibitors. HBV DNA levels - both intracellular and in culture supernatants—were quantitated by Taqman qPCR (ABI7500). Western blot and ELISA were used to assess intracellular and secreted HBsAg, respectively. PLC/PRF/5 cells, expressing only HBsAg, were also tested. Cyclophilin expression in the cells was silenced by transfection separately with siRNA for cyclophilins A, B, C or D to determine the role of individual cyclophilins in HBV replication. Results Cyclophilin inhibition with either DEB025 or NIM811 significantly reduced cytoplasmic core-particle associated HBV DNA levels in the cells, between 2 and 10-fold as compared with the control cells. The most pronounced reduction of intracellular HBV DNA (by 10-fold at 72 h) was observed with DEB025 5 mg/ml, which was greater than the reduction observed with NIM811. Similarly, DEB025 (at 1 and 5 mg/ml) showed a greater impact in reducing HBV virion secretion in the supernatants, compared with NIM811. HBsAg secretion from the cells was also reduced by up to 50% when compared to controls. Cyclophilin-A expression was markedly reduced after transfection with corresponding siRNA, which led to a rapid decrease of intracellular HBV DNA by 2 logs. HBV-DNA was reduced further when the cyclophillin-A silenced cultures were treated with NIM811 or DEB025. Conclusion These results demonstrate that cyclophilin A is directly involved in HBV replication. Cyclophilin inhibition by DEB025 or NIM811 interferes with HBV replication within liver cells and reduces the secretion of infectious virions and HBsAg particles from the cells, with DEB025 having a greater antiviral activity than NIM811.