Effector pathway of the antiviral effect of interferons in Hepatitis B Virus infection.

Having been used in hepatitis B therapy for more than 20 years, interferon-alpha (IFN-α) is still the only medication that may lead to virus clearance. Although the effect of IFN-α has been widely studied, its definite mode of action is still unclear. In the first part of the thesis, I investigated antiviral effect of murine IFN-α on HBV. mIFN-α restricts HBV replication at the level of HBV-RNA transcription and translation in vitro and in vivo. Different mIFN-α subtypes varied largely in their antiviral efficiency with mIFN-α 4 and 11 showing the strongest anti-HBV effect. Analysis of protein alignment revealed several potential important residues in IFN-α receptor binding region. The second part characterizes the IFN-α signaling pathway. Different HBV replicating hepatoma cell lines were used to examine anti-HBV activity of IFN-α and compared to PHH. IFN-α only elicited antiviral activity in HBV-infected HepaRG cells, but not in HBV-replicating Huh-7 or HepG2 cells. Detailed analysis of the IFN-α signaling pathway revealed that neither Huh-7 nor HepG2 cells responded properly to IFN-α, while HepaRG showed similar induction of IFN-stimulated genes as PHH did. Although the amount of IFN-α/β receptors and phosphorylated STAT (p-STAT) proteins were comparable in all cell lines tested, nuclear translocation of p-STATs was far more efficient in HepaRG cells. We therefore selected HepaRG cells as well as PHH to study IFN-α effects on HBV. The third part describes a novel antiviral mechanism of IFN-α, which affects HBV binding to its host cell. In differentiated HepaRG cells, the supernatant of IFN-α treated cells limited HBV infection. The IFN-α induced factor(s) bound to heparin columns indicating that they interact with heparan sulfate to compete with HBV. Mass spectrometry analysis suggested that complement factor H or fibronectin 1 may be the HBV binding inhibitor. The fourth part focused on the mechanism of noncytopathic viral clearance by IFN-α. In addition to its transcriptional and posttranscriptional effects, IFN-α significantly reduced HBV cccDNA, the template of HBV transcription, in HBV infected PHH and HepaRG cells. Specific 3D-PCR indicated HBV cccDNA sequence alterations. Sequence analysis showed C to U transition of the HBV cccDNA after IFN-α treatment. APOBEC3 family cytidine deaminases A3A and A3G were upregulated upon IFN-α treatment in a time and dose dependent manner. JAK-STAT signaling blockade or HIV-Vif expression proved that IFN-α induced cccDNA deamination by APOBEC3 leads to cccDNA degradation. Subcellular localization analysis and overexpression experiments demonstrated that A3A was the active effector. Treatment with a DNA repair enzyme cocktail corrected all mutations of cccDNA. AP endonuclease incubation reduced cccDNA showing that the base excision pathway was involved. Lacking host genomic DNA deamination upon IFN-α treatment shown suggested that A3A is directed specifically to viral DNA. Since A3A co-localized with HBV core protein in confocal microscopy and interaction was confirmed by co-immunoprecipitation, we propose that A3A utilizes HBc to get access to cccDNA. Taken together, this thesis leads to a better understanding of the antiviral function of IFN-α and opens new options for the development of novel and safe treatments to eradicate HBV and to cure chronic hepatitis B