Date Published: September 12, 2013
Publisher: Public Library of Science
Author(s): Fei Liu, Matthew Campagna, Yonghe Qi, Xuesen Zhao, Fang Guo, Chunxiao Xu, Sichen Li, Wenhui Li, Timothy M. Block, Jinhong Chang, Ju-Tao Guo, Aleem Siddiqui.
Covalently closed circular DNA (cccDNA) of hepadnaviruses exists as an episomal minichromosome in the nucleus of infected hepatocyte and serves as the transcriptional template for viral mRNA synthesis. Elimination of cccDNA is the prerequisite for either a therapeutic cure or immunological resolution of HBV infection. Although accumulating evidence suggests that inflammatory cytokines-mediated cure of virally infected hepatocytes does occur and plays an essential role in the resolution of an acute HBV infection, the molecular mechanism by which the cytokines eliminate cccDNA and/or suppress its transcription remains elusive. This is largely due to the lack of convenient cell culture systems supporting efficient HBV infection and cccDNA formation to allow detailed molecular analyses. In this study, we took the advantage of a chicken hepatoma cell line that supports tetracycline-inducible duck hepatitis B virus (DHBV) replication and established an experimental condition mimicking the virally infected hepatocytes in which DHBV pregenomic (pg) RNA transcription and DNA replication are solely dependent on cccDNA. This cell culture system allowed us to demonstrate that cccDNA transcription required histone deacetylase activity and IFN-α induced a profound and long-lasting suppression of cccDNA transcription, which required protein synthesis and was associated with the reduction of acetylated histone H3 lysine 9 (H3K9) and 27 (H3K27) in cccDNA minichromosomes. Moreover, IFN-α treatment also induced a delayed response that appeared to accelerate the decay of cccDNA. Our studies have thus shed light on the molecular mechanism by which IFN-α noncytolytically controls hepadnavirus infection.
Hepatitis B virus (HBV) is the prototype member of the Hepadnaviridae family and contains a relaxed circular (rc) partially double stranded DNA (3.2 kb in length) genome –. Upon entry into a hepatocyte, the nucleocapsid delivers the genomic rcDNA into the nucleus, where the rcDNA is converted into a covalently closed circular (ccc) DNA. The cccDNA exists as an episomal minichromosome and serves as the template for the transcription of viral RNAs , . Hepadnaviruses replicate their genomes via a protein-primed reverse transcription of pre-genomic (pg) RNA in the cytoplasmic nucleocapsids, which are subsequently enveloped upon synthesis of rcDNA and secreted out of cells as virions , . During the early phase of infection, additional cccDNA are produced from newly synthesized cytoplasmic rcDNA through an intracellular amplification pathway , . These two pathways culminate in the formation of a regulated steady-state population of 5 to 50 cccDNA molecules per infected hepatocyte , , .
Among the many important aims of future HBV research is the development of therapies of finite duration capable of eradicating HBV infection. Accomplishment of this goal requires elimination and/or cure of all the HBV-infected cells in the body. In fact, more than 90% of adulthood HBV infections are resolved by host immune responses via a coordinated kill and cure of infected cells , , . While the virus specific cytolytic T lymphocytes are considered to be the primary force to kill the infected hepatocytes, the cure of the infected hepatocytes is believed to be achieved by the inflammatory cytokine-induced cellular responses –. Considering that cccDNA is the most stable replication intermediate of hepadnaviral DNA, elimination or transcriptional silencing of cccDNA in the infected hepatocytes is essential for the cytokines to cure the HBV-infected cells. Unfortunately, due to the lack of convenient cell culture systems for efficient infection , , the effects of the cytokines on the stability and transcriptional activity of the cccDNA have not been extensively investigated. In recent years, Levrero and colleagues studied HBV transcription in the linear full-length HBV DNA-transfected human hepatoma cells, in which cccDNA molecules were presumably formed by circularization of input linear HBV DNA , . Using this assay, this research group established a role for HBx protein in cccDNA minichromosome transcription and investigated the mechanism by which IFN-α suppresses HBV cccDNA transcription , . However, due to the transient nature of the assay, the long-term effects of the cytokines on cccDNA transcription and maintenance could not be investigated. Using the chicken hepatoma cell-based assay established herein, we investigated the effects and underlying mechanism of IFN-α on DHBV cccDNA transcription and maintenance and revealed the following unique features of hepadnavirus cccDNA metabolism and transcription regulation.