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Hepatitis B virus basal core promoter mutations show lower replication fitness associated with cccDNA acetylation status

Hepatitis B virus basal core promoter mutations show lower replication fitness associated with cccDNA acetylation status
Hepatitis B virus basal core promoter mutations show lower replication fitness associated with cccDNA acetylation status
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.
0168-1702
150-160
Koumbi, Lemonica
0ddc411b-bffc-4d7d-9433-e2409e92d9b9
Pollicino, Teresa
553d091a-81be-4f41-ac16-c7d8b4a06b69
Raimando, Giovanni
b013aed3-ed78-4738-9250-93400994abd1
Stampoulis, Dimitrios
73a75b4a-d66b-429f-8795-a91dacdbc2d6
Khakoo, Salim
6c16d2f5-ae80-4d9b-9100-6bfb34ad0273
Karayiannis, Peter
f58f783e-c0d0-4123-9238-5d7702284df2
Koumbi, Lemonica
0ddc411b-bffc-4d7d-9433-e2409e92d9b9
Pollicino, Teresa
553d091a-81be-4f41-ac16-c7d8b4a06b69
Raimando, Giovanni
b013aed3-ed78-4738-9250-93400994abd1
Stampoulis, Dimitrios
73a75b4a-d66b-429f-8795-a91dacdbc2d6
Khakoo, Salim
6c16d2f5-ae80-4d9b-9100-6bfb34ad0273
Karayiannis, Peter
f58f783e-c0d0-4123-9238-5d7702284df2

Koumbi, Lemonica, Pollicino, Teresa, Raimando, Giovanni, Stampoulis, Dimitrios, Khakoo, Salim and Karayiannis, Peter (2016) Hepatitis B virus basal core promoter mutations show lower replication fitness associated with cccDNA acetylation status. Virus Research, 220, 150-160. (doi:10.1016/j.virusres.2016.04.022). (PMID:27132039)

Record type: Article

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.

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Accepted/In Press date: 26 April 2016
e-pub ahead of print date: 27 April 2016
Published date: 15 July 2016
Organisations: Clinical & Experimental Sciences

Identifiers

Local EPrints ID: 398154
URI: https://eprints.soton.ac.uk/id/eprint/398154
ISSN: 0168-1702
PURE UUID: 16cdb00e-0e3f-4f8d-90d9-70dc4db56fe6

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Date deposited: 20 Jul 2016 10:53
Last modified: 03 Dec 2019 06:37

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