IFN-α inhibits HBV transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccDNA minichromosome
Laura Belloni, … , Maura Dandri, Massimo Levrero
Laura Belloni, … , Maura Dandri, Massimo Levrero
Published January 17, 2012
Citation Information: J Clin Invest. 2012;122(2):529-537. https://doi.org/10.1172/JCI58847.
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Research Article Virology

IFN-α inhibits HBV transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccDNA minichromosome

Abstract

HBV infection remains a leading cause of death worldwide. IFN-α inhibits viral replication in vitro and in vivo, and pegylated IFN-α is a commonly administered treatment for individuals infected with HBV. The HBV genome contains a typical IFN-stimulated response element (ISRE), but the molecular mechanisms by which IFN-α suppresses HBV replication have not been established in relevant experimental systems. Here, we show that IFN-α inhibits HBV replication by decreasing the transcription of pregenomic RNA (pgRNA) and subgenomic RNA from the HBV covalently closed circular DNA (cccDNA) minichromosome, both in cultured cells in which HBV is replicating and in mice whose livers have been repopulated with human hepatocytes and infected with HBV. Administration of IFN-α resulted in cccDNA-bound histone hypoacetylation as well as active recruitment to the cccDNA of transcriptional corepressors. IFN-α treatment also reduced binding of the STAT1 and STAT2 transcription factors to active cccDNA. The inhibitory activity of IFN-α was linked to the IRSE, as IRSE-mutant HBV transcribed less pgRNA and could not be repressed by IFN-α treatment. Our results identify a molecular mechanism whereby IFN-α mediates epigenetic repression of HBV cccDNA transcriptional activity, which may assist in the development of novel effective therapeutics.

Authors

Laura Belloni, Lena Allweiss, Francesca Guerrieri, Natalia Pediconi, Tassilo Volz, Teresa Pollicino, Joerg Petersen, Giovanni Raimondo, Maura Dandri, Massimo Levrero

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Figure 2

IFN-α inhibits cccDNA transcription and HBV replication in chimeric uPA/SCID mice.

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IFN-α inhibits cccDNA transcription and HBV replication in chimeric uPA/...
Uninfected and chronically HBV infected (median, 2 × 108 serum HBV-DNA copies/ml serum) chimeric uPA/SCID mice transplanted with thawed human hepatocytes received daily injections of human IFN-α (1,300 IU/g body weight) for 5 days or saline. (A) HBV viremia (log HBV-DNA copies/ml serum) were determined in individual animals (n = 5) at baseline, shortly before IFN treatment, and 8 hours after the last administration of IFN-α. (B) Representative Northern blot analysis from one IFN-α–treated mouse and one untreated mouse. (C) Steady-state levels of pregenomic RNA (left panel) and preS/S RNAs (right panel). Intrahepatic preS/S RNAs were determined by subtracting pgRNA amounts from total HBV RNAs (pgRNA + preS/S RNA) estimated in the same RNA preparation as described in Methods, and values were normalized using human-specific GAPDH primers. (D) Intrahepatic cccDNA loads in IFN-α–treated (n = 5) and control (saline) HBV-infected mice (n = 5). Real-time qPCR analysis was performed using selective cccDNA primers, and β-globin primers were used to normalize cccDNA copies (median + SD) per human hepatocyte (expressed as human genome equivalents) determined in chimeric livers. (E) Cross-linked chromatin from liver samples of chronically HBV-infected untreated and IFN-α–treated (48 hours) uPA chimeric mice was immunoprecipitated with a relevant control IgG or specific anti-AcH4 antibody. Immunoprecipitated chromatin was analyzed by qPCR as in Figure 1D. All histograms show mean values from 3 independent experiments; bars indicate SD.