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, Lena Allweiss, Francesca Guerrieri, Natalia Pediconi, Tassilo Volz, Teresa Pollicino, Joerg Petersen, Giovanni Raimondo, Maura Dandri, Massimo Levrero
Laura Belloni, Lena Allweiss, Francesca Guerrieri, Natalia Pediconi, Tassilo Volz, Teresa Pollicino, Joerg Petersen, Giovanni Raimondo, Maura Dandri, Massimo Levrero
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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 1

IFN-α inhibits HBV replication and cccDNA transcription in HCC cells.

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IFN-α inhibits HBV replication and cccDNA transcription in HCC cells. (A...
(A) Left panel: HepG2 cells were transfected with monomeric linear full-length WT HBV adw (genotype A) genomes. HBV core particles were isolated from untreated and IFN-α–treated cells at the indicated time points after transfection. Results are expressed as number of HBV DNA copies per transfected cell. Right panel: Southern blot hybridization. OC, open circular duplex HBV DNA; DS, double-stranded HBV DNA replicative intermediates; SS, single-stranded HBV DNA replicative intermediates. (B) Left panel: cccDNA levels (copies per transfected cell) were analyzed by qPCR with selective cccDNA primers and β-globin primers (DNA sample normalization). Right panel: Southern blot analysis. (C) Left panel: pgRNA levels were analyzed by qPCR using pgRNA selective primers and GAPDH primers (equal loading of each RNA sample). Right panel: Northern blot analysis. pgRNA, HBV pregenomic RNA. (D) Cross-linked chromatin was immunoprecipitated with a relevant control IgG or specific anti-AcH4 antibody and analyzed by qPCR with HBV cccDNA selective primers. Results are expressed as fold induction relative to the untreated cells using the comparative Ct method. (E) HepG2 cells were transfected with monomeric linear full-length WT or HBx mutant HBV genomes (4). Core particles HBV-DNA (left panel) and pgRNA (right panel) results are expressed as in Figure 1, A and C, respectively. All histograms show mean values from 3 independent experiments; bars indicate SD. P values were determined using Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001.