1
Baruch S. Blumberg Institute, Hepatitis B Foundation, 3805 Old Easton Road, Doylestown, PA 18902, USA.
2
Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA.
3
Microbiology and Immunology Graduate Program, Drexel University College of Medicine, 2900 West Queen Lane, Philadelphia, PA 19129, USA.
4
Baruch S. Blumberg Institute, Hepatitis B Foundation, 3805 Old Easton Road, Doylestown, PA 18902, USA. [email protected].
PMID: 32581092 DOI: 10.1128/JVI.00442-20
Abstract
Covalently closed circular (ccc) DNA of hepadnaviruses exists as an episomal minichromosome in the nucleus of infected hepatocyte and serves as the template for the transcription of viral mRNAs. It had been demonstrated by others and us that interferon alpha (IFN-α) treatment of hepatocytes induced a prolonged suppression of human and duck hepatitis B virus cccDNA transcription, which is associated with the reduction of cccDNA-associated histone modifications specifying active transcription (H3K9ac or H3K27ac), but not the histone modifications marking constitutive (H3K9me3) or facultative (H3K27me3) heterochromatin formation. In our efforts to identify IFN-induced cellular proteins that mediate the suppression of cccDNA transcription by the cytokine, we found that down-regulating the expression of signal transducer and activator of transcription 1 (STAT1), structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) or promyelocytic leukemia (PML) protein increased basal level of cccDNA transcription activity and partially attenuated IFN-α suppression of cccDNA transcription. On the contrary, ectopic expression of STAT1, SMCHD1 or PML significantly reduced cccDNA transcription activity. SMCHD1 is a non-canonical SMC family protein and implicated in epigenetic silencing of gene expression. PML is a component of nuclear domain 10 (ND10) and involved in suppressing the replication of many DNA viruses. Mechanistic analyses demonstrated that STAT1, SMCHD1 and PML were recruited to cccDNA minichromosomes and phenocopied the IFN-α-induced post-translational modifications of cccDNA-associated histones. We thus conclude that STAT1, SMCHD1 and PML may partly mediate the suppressive effect of IFN-α on hepadnaviral cccDNA transcription.IMPORTANCE Pegylated IFN-alpha is the only therapeutic regimen that can induce a functional cure of chronic hepatitis B in a small, but significant fraction of treated patients. Understanding the mechanisms underlying the antiviral functions of IFN-α in hepadnaviral infection may reveal molecular targets for development of novel antiviral agents to improve the therapeutic efficacy of IFN-α. By a loss-of-function genetic screening of individual ISGs on hepadnaviral mRNAs transcribed from cccDNA, we found that down-regulating the expression of STAT1, SMCHD1 or PML significantly increased the level of viral RNAs without altering the level of cccDNA. Mechanistic analyses indicated that those cellular proteins are recruited to cccDNA minichromosomes and induce the post-translational modifications of cccDNA-associated histones similar to those induced by IFN-α treatment. We have thus identified three IFN-α-induced cellular proteins that suppress cccDNA transcription and may partly mediate IFN-α silencing of hepadnaviral cccDNA transcription.