title: Hepatitis B virus replication is regulated by the acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones.
abstract: OBJECTIVE
  HBV covalently closed circular DNA (cccDNA), the replicative intermediate responsible for persistent HBV infection of hepatocytes, is the template for transcription of all viral mRNAs. Nuclear cccDNA accumulates as a stable episome organized into minichromosomes by histone and nonhistone proteins. In this study we investigated, by a newly developed sensitive and specific assay, the relationship between viral replication and HBV chromatin assembly, transcription, and interaction with viral and cellular regulatory proteins.
  
  
  METHODS
  To achieve this aim we coupled a quantitative chromatin immunoprecipitation (ChIP) technique to an established method that allows the amplification of virion-encapsidated HBV genomes after transfection of linear HBV DNA into human hepatoma HuH7 cells. The cccDNA-ChIP technique was also applied to study HBV minichromosome transcriptional regulation in liver tissue from HBV-infected patients.
  
  
  RESULTS
  The use of anti-acetyl-H4/-H3 specific antibodies to immunoprecipitate transcriptionally active chromatin revealed that HBV replication is regulated by the acetylation status of the cccDNA-bound H3/H4 histones. Class I histone deacetylases inhibitors induced an evident increase of both cccDNA-bound acetylated H4 and HBV replication. Finally, histones hypoacetylation and histone deacetylase 1 recruitment onto the cccDNA in liver tissue correlated with low HBV viremia in hepatitis B patients.
  
  
  CONCLUSIONS
  We developed a ChIP-based assay to analyze, in vitro and ex vivo, the transcriptional regulation of HBV cccDNA minichromosome. Our results provide new insights on the regulation of HBV replication and identify the enzymatic activities that modulate the acetylation of cccDNA-bound histones as new therapeutic targets for anti-HBV drugs.
authors.affiliation: Laboratory of Gene Expression, Fondazione A. Cesalpino, University of Rome La Sapienza, Rome, Italy.
authors.forename: Teresa
authors.initials: T
authors.lastname: Pollicino
authors.forename: Laura
authors.initials: L
authors.lastname: Belloni
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Raffa
authors.forename: Natalia
authors.initials: N
authors.lastname: Pediconi
authors.forename: Giovanni
authors.initials: G
authors.lastname: Squadrito
authors.forename: Giovanni
authors.initials: G
authors.lastname: Raimondo
authors.forename: Massimo
authors.initials: M
authors.lastname: Levrero
mesh_terms: D000107:Acetylation; D000328:Adult; D000368:Aged; D004270:DNA, Circular / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006515:Hepatitis B virus / Q000502:physiology*; D019694:Hepatitis B, Chronic / Q000378:metabolism; D056572:Histone Deacetylase Inhibitors; D006657:Histones / Q000378:metabolism*; D006801:Humans; D008297:Male; D008875:Middle Aged; D014766:Viremia / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects
chemical_list: D004270:DNA, Circular; D004279:DNA, Viral; D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D006657:Histones