Introduction Cyclophilins are intracellular proteins with enzymatic activity—peptidyl-prolyl-isomerase that plays a major role in the life cycle of Hepatitis C virus. By targeting host cyclophilins Alisporivir (DEB025) exerts potent anti-HCV activity in vitro and in clinical studies. We have recently shown in vitro that cyclophilin inhibition with Alisporivir or NIM811 also interferes with HBV replication, with Alisporivir having a greater effect than NIM811. To elucidate the underlying mechanisms, in the present study we compared in vitro the effects on HBV replication of Alisporivir alone, Alisporivir in combination with a potent antiviral targeting HBV-DNA polymerase, and in cells after selective knockdown of individual cyclophilins.
Methods Stably (HepG2215) and transiently (HUH-7) transfected cells, producing full HBV virions and HBsAg particles, were treated with different Alisporivir concentrations (0.25/1.0/5.0/20 ug/ml) alone, Telbivudine alone, or combinations of Alisporivir and Telbivudine. To determine the involvement of individual cyclophilins, HepG2215 cells were transfected with siRNA-specific for cyclophilin (Cyp) A, C or D and additionally treated with Alisporivir. Cytoplasmic extracts and supernatants were harvested at baseline; 24, 48 and 72 h post-treatment. The kinetics of antiviral activity was assessed by quantitation of intracellular and secreted HBV-DNA (real-time qPCR) and HBsAg levels (ELISA).
Results Alisporivir treatment resulted in dose-dependent reduction of intracellular and secreted HBV-DNA from HepG2215 and HUH-7 cells at all time points, by 70% (p=0.004) and 63% (p<0.001), respectively, compared with untreated controls. The combination of Alisporivir and Telbivudine had greater effects in reducing intracellular (p=0.001) and secreted (p=0.028) HBV-DNA, and >3-fold reduction of HBsAg vs either Alisporivir or Telbivudine alone. CypA, C or D expression was markedly reduced after transfection with corresponding siRNA, which was associated with significant decrease of HBV-DNA and HBsAg levels (p<0.001). Alisporivir treatment of cells silenced for CypA, C or D further reduced HBV-DNA and HBsAg levels, with greater antiviral effects in CypC or CypD silenced cells, compared with CypA silenced cells (p<0.001).
Conclusion These results suggest that Alisporivir interferes with multiple sites of HBV replication and has synergistic antiviral activity with direct antiviral targeting viral DNA polymerase, such as Telbivudine.
Competing interests None declared.
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