title: Hepatitis B virus basal core promoter mutations show lower replication fitness associated with cccDNA acetylation status.
abstract: In chronic hepatitis B virus (HBV) infection, variants with mutations in the basal core promoter (BCP) and precore region predominate and associate with more severe disease forms. Studies on their effect on viral replication remain controversial. Increasing evidence shows that epigenetic modifications of cccDNA regulate HBV replication and disease outcome. Here we determined the transcription and viral replication efficiency of well-defined BCP and precore mutations and their effect on cccDNA epigenetic control. HBV monomers bearing BCP mutations A1762T/G1764A and A1762T/G1764A/C1766T, and precore mutations G1896A, G1899A and G1896A/G1899A, were transfected into HepG2 cells using a plasmid-free approach. Viral RNA transcripts were detected by Northern blot hybridization and RT PCR, DNA replicative intermediates by Southern blotting and RT PCR, and viral release was measured by ELISA. Acetylation of cccDNA-bound histones was assessed by Chromatin ImmunoPrecipitation (ChIP) assay and methylation of cccDNA by bisulfite sequencing. BCP mutations resulted in low viral release, mRNA transcription and pgRNA/cccDNA ratios that paralleled the acetylation of cccDNA-bound H4 histone and inversely correlated with the HDAC1 recruitment onto cccDNA. Independently of the mutations, cccDNA was a target for methylation, accompanied by the upregulation of DNMT1 expression and DNMT1 recruitment onto cccDNA. Our results suggest that BCP mutations decrease viral replication capacity possibly by modulating the acetylation and deacetylation of cccDNA-bound histones while precore mutations do not have a significant effect on viral replication. These data provide evidence that epigenetic factors contribute to the regulation of HBV viral replication.
authors.affiliation: Department of Medicine, Hepatology and Gastroenterology Section, Imperial College, St. Mary's Campus, London, UK. Electronic address: lemonica.koumbi@gmail.com.
authors.forename: Lemonica
authors.initials: L
authors.lastname: Koumbi
authors.affiliation: Department of Pediatric, Gynecologic, Microbiologic, and Biomedical Sciences, University Hospital of Messina, Messina, Italy. Electronic address: tpollicino@unime.it.
authors.forename: Teresa
authors.initials: T
authors.lastname: Pollicino
authors.affiliation: Department of Clinical and Experimental Medicine, University Hospital of Messina, Messina, Italy. Electronic address: giovanni.raimondo@unime.it.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Raimondo
authors.affiliation: Department of Virology, Imperial College, St. Mary's Campus, London, UK. Electronic address: d.stampoulis@ucl.ac.uk.
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Stampoulis
authors.affiliation: Henry Welcome Laboratories, Southampton General Hospital, Southampton, UK. Electronic address: S.I.Khakoo@soton.ac.uk.
authors.forename: Salim
authors.initials: S
authors.lastname: Khakoo
authors.affiliation: University of Nicosia Medical School, Nicosia, Cyprus. Electronic address: karayiannis.p@unic.ac.cy.
authors.forename: Peter
authors.initials: P
authors.lastname: Karayiannis
mesh_terms: D000107:Acetylation; D000076123:DNA (Cytosine-5-)-Methyltransferase 1 / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D004270:DNA, Circular / Q000235:genetics* / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D044127:Epigenesis, Genetic; D056945:Hep G2 Cells; D006515:Hepatitis B virus / Q000235:genetics / Q000378:metabolism; D056284:Histone Deacetylase 1 / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008745:Methylation; D009154:Mutation; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
chemical_list: D004270:DNA, Circular; D004279:DNA, Viral; D006657:Histones; D012333:RNA, Messenger; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; C000619380:DNMT1 protein, human; C110550:HDAC1 protein, human; D056284:Histone Deacetylase 1
keywords: Basal core promoter; Epigenetic; Hepatitis B virus; Precore; Viral replication; cccDNA