HBx and epigenetic control of hepatitis B virus cccdna Christine Neuveut Hepacivirus et Immunité Innée Institut Pasteur. Paris
! Inhibition of protein phosphatase 1 (PP1) by HBx : a mechanism for the activation of HBV replication! Role of the viral protein HBx in the epigenetic and transcriptional regulation of HBV cccdna in infected hepatocytes! Cellular mechanisms involved in HBV repression
HBV cccdna Pregenomic RNA 3.5 kb 2.4 kb Cytoplasm Nucleus cccdna ~ ~ ~ ~ ~ ~ ~ ~ ~ 2.1 kb ~ ~ ~ 0.8 kb ~ ~ cccdna: covalently closed circular DNA! template for the transcription of HBV RNAs (including pgrna) cccdna: stable DNA Resistant to antiviral treatment! responsible for viral persistence! responsible for viral rebound upon withdrawal of anti-viral therapy!important to understand the mechanisms regulating the persistence and the transcriptional regulation of cccdna in order to develop new therapeutic approaches.
HBV cccdna Cytoplasm Nucleus cccdna Bock et al, JMB 2001 HBV cccdna is organized into chromatin-like structure as a viral minichromosome: up to 20 nucleosomes containing histones et non histone proteins 4 promoters and 2 enhancers ARN pregenomic 3.5 kb ~ ~ ~ ~ ~ ~ ~ ~ 2.1 kb ~ ~ ~ 0.8 kb ~ ~ 2.4 kb Epigenetic modifications (H3 and H4 acetylation) regulate HBV transcription.
pres1" pres2" The HBx regulatory protein HBV" HBx is essential for viral replication HBx! HBx has pleiotropic activities HBx activates HBV transcription via the assembly of enhancertranscription factors and the regulation of epigenetic events " HBx is recruited to cellular promoters " HBx interacts with CBP/p300 and increases their recruitment to endogenous CREB target genes, which correlates with an increase in gene expression. (Cougot et al., JBC 2007). " HBx is recruited on the HBV cccdna, which correlates with the recruitment of CBP, p300, PCAF and with histone acetylation. (Belloni et al., PNAS 2009).
AIMS: Study the role of CREB in the transcriptional activation of the cccdna by HBx CREB might be involved in viral replication: CRE element is found in the enhancer I and PreS2 promoter! CREB belongs to the basic domain-leucine zipper(bzip) superfamily Burst phase : phosphorylation of CREB + Transcription rate Time (hours)! attenuation :dephosphorylation of ser 133 1 2 3 4 5 P! PP-1! HDAC1! + CREB! H (Canetteri et al., Nat. Struct. Biol. 2003) A c!
The phosphorylation of CREB bound to the cccdna is specifically maintained over time HepAD38! HBV DNA ChIP Q-PCR! CCNA2 ChIP Q-PCR! 0.6! **! 0.9! **! 0.8! 0.5! 0.7!! % input! 0.4! 0.3! % input! 0.6! 0.5! 0.4! 0.2! 0.3! 0.2! 0.1! 0.1! 0! Forskolin(hours):! 0h!1h!3h!6h! 0h!1h!3h!6h! 0! 0h!1h!3h!6h! 0h!1h!3h!6h! -Ab! α pcreb! -Ab! α pcreb! The activity of CREB bound to the cccdna is sustained through prolongation of its phosphorylation.
HBx inhibits PP1 activity In vitro dephosphorylation assay! GST-CREB-P -32 +! PP1! HBx! PP1 + 0.1 µg! 0.5 µg! 1 µg! OA! PP1! PP1! PP1! Incubation time (minute)! 0! 10! 30! 0! 10! 30! 0! 10! 30! 0! 10! 30! 0! 10! 30! % of P 32 phospho -CREB! 100! 50! 30! 100! 100! 100! 100! 100! 30! 100! 97! 94! 100! 100! 100! Autoradiography OA! HA-HBx! PP1-His! +! +! +! Eluate! Lysate! +! +! WB-α PP1! WB-α HA!
HBx cofractionates with PP1 and HDAC1 Gel filtration assay! 293! F"HDAC1( V5"PP1( PP1( HA"HBx( PP2Cγ( U2"B ( complexes! Free! or! small complexes!
Inhibition of PP1 expression restores transcription of the X-deficient virus! HepG2 cells! Fold activation! 1,8" 1,6" 1,4" 1,2" 1" 0,8" 0,6" 0,4" 0,2" 0"! HBV RT-QPCR sirna:! -" α-pp1! α-β-catenin!
Inhibition of PP1 phosphatase activity by HBx: a mechanism for the activation of HBV transcription (Cougot et al., Sci. Signal. 2012) wt HBV! X-deficient HBV! P300/CBP! HAT! HBx! P! AC! HBV transcription " high" P300/CBP! HAT! P! AC! HBV transcription " on" CREB! CREB! - HDAC1! PP1! HBx! P CREB P300/CBP! HAT HBV transcription " high" AC P! HDAC1! PP1! CREB! AC! HBV transcription " low" X
! Inhibition of protein phosphatase 1 (PP1) by HBx : a mechanism for the activation of HBV replication! Role of the viral protein HBx in the epigenetic and transcriptional regulation of HBV cccdna in infected hepatocytes! Cellular mechanims involved in HBV repression
HBx is essential for viral replication in infected hepatocytes Impossible d'afficher l'image. Votre ordinateur manque peut-être de mémoire pour ouvrir l'image ou l'image est endommagée. Redémarrez l'ordinateur, puis ouvrez à nouveau le fichier. Si le x rouge est toujours affiché, vous devrez peut-être supprimer l'image avant de la réinsérer. Infec?on( HepaRG( WT(or(X"(HBV( Secreted(HBeAg(( HBe(Ag((OD) 3 2 1 0 4 6 8 HBV WT HBV X- Days.a/er.infec6on cccdna! transcription! HBeAg! PHH( Primary(human(hepatocytes( (or(heparg(cells( HBe(Ag((OD) 3 2 1 0 4 7 9 Days.a/er.infec6on
HBx is required for HBV transcription in infected hepatocytes HepaRG PHH HBV pgrna. pres1.rna. pres2/s.rna. pgrna. pres1.rna. pres2/s.rna. 4( 6( 8( 4( 6( 8( HBV WT( HBV X-( Relative level of HBV RNA 1 5 0 1 0 0 5 0 0 HBV RT-QPCR HBV.wt HBV.XF Relative level of HBV RNA 10000 8000 6000 4000 2000 0 HBV RT-QPCR HBV.wt HBV.XF 0 5 1 0 1 5 2 0 0 5 10 15 20 Days.a/er.infec6on Days.a/er.infec6on HBx acts at the level of HBV transcription.
Epigenetic modifications and transcription Histone tail acetylation (lysines): Histone acetyltransferases (HAT), histone deacetylases (HDAC) Histone tail methylation (lysines and arginines): Histone methyltransferases (HMT), histones demethylases Transcriptional activation DNA methylation (cytosine in the C-5 position, in CpG dinucleotide context) DNA methyltransferases (DNMT) Transcriptional repression Compaction of chromatin Recruitment of non-histone proteins
HBx expression is associated with increased histone acetylation in infected hepatocytes ChIP Q-PCR! HepaRG. Relative level of histone acetylation (%) 150 100 50 0 HBV(cccDNA. * AcH3 AcH4 150 100 50 0 CCNA2. AcH3 AcH4 HBV.WT. HBV.XF. PHH. Relative level of histone acetylation (%) 150 100 50 0 * AcH3 AcH4 200 150 100 50 0 AcH3 AcH4 HBV(wt( HBV(X"( H3(ac( H3(ac( H3(ac( H3(ac(
HBx oppositely regulates activating and repressive H3 trimethylation marks on cccdna Transcriptional activation me( Transcriptional repression me( 4 9 H3 ARTKQTARKSTGGKAPRKQLATKAARKSA. ChIP Q-PCR! HepaRG( PHH( Relative level of histone methylation (%) Relative level of histone methylation (%) 600 400 200 0 600 400 200 0 HBV( cccdna( * H3 K4 me3 H3 K4 me3 * H3 K9 me3 * H3 K9 me3 HBV(WT( HBV(X"( 200 100 0 300 200 100 0 H3 K4 me3 H3 K4 me3 CCNA2( H3 K9 me3 H3 K9 me3 (negative control) HBV(wt( HBV(X"( H3 K4 me3 H3 K9 me3
H3K9me3 recruits heterochromatin protein 1 (HP1β and γ) on the cccdna in the absence of HBx " H3K9me3 is a docking site for HP1 " In turn, HP1 mediates the recruitment of HMT (Suv39, G9a),.DNMTs to methylate DNA " shutting down of gene expression. HBV.WT. HBV.XF. HepaRG. Relative level of associated protein (%) HBV(cccDNA. 1500 * 1000 500 0 200 150 100 50 0 me me I I CG CG C G CCNA2. HP1 β HP1 γ HP1 β HP1 γ Suv39 DNMTs h HP1 PHH. 1000 500 * 300 200 100 0 HP1 γ 0 HP1 γ
Conclusions (1) ".HBx is necessary for viral transcription and replication, in the context of cellular infection by HBV " In the absence of HBx, repressive epigenetic marks are deposited on HBV cccdna (H3K9 me3, histone hypoacetylation, recruitment of HP1γ) " HBx expression correlates with the deposition of activating histone modifications on HBV cccdna (histone acetylation, H3K4 me3).. HBV X- Recruitment: HDACs, HMTs HP1 HBV WT HP1γ$ H3 K9 me3 PCAF TF H3 K4 me3 HP1γ$ H3 K9 me3 CBP/p300 CBP/p300! HAT! HBx P! HBx H3 K4 me3 PP1! HDAC1! TF CBP/p300 H3 K9 me3 H3 K4 me3 H3 K9 me3 CREB! H3 K4 me3 H3 ac H3 ac H3 ac H3 ac H3 K9 me3 HBV transcription! low! HBV transcription! high!
=>. HBx might overcome an intrinsic cellular response that prevents viral transcription by epigenetic mechanisms HepaRG TR- HBx! Conclusions (2) Lucifora et al.,j. hepatol. 2011
! Inhibition of protein phosphatase 1 (PP1) by HBx : a mechanism for the activation of HBV replication! Role of the viral protein HBx in the epigenetic and transcriptional regulation of HBV cccdna! Cellular mechanims involved in HBV repression
HBx interacting-proteins Flag/HA-IP Analysis of protein interactions (STRING) 200. HDAC1 116,3. 97,4. RUVBL2 66,3. KIAA1967 55,4. 36,4. 31. 21,5. 14,4. 6.!HBx
Role of PML nuclear bodies in the control of HBV infection WT#Virus## (HSV,.HCMV,. ). High(replica?on( Desorganiza?on( PML. Sp100. Degrada6on.or.modifica6on.of. PML.NB.components.by.a.viral. protein.(icp0.of.hsvf1,.. IE1.of.HCMV ). PML#NB# Daxx. Recruitment.of.PML.NB. components. Mutant#virus## (HSV. ICP0. ). Recruitment.of.repressors.(HP1,. HDAC ). Epigene?c(repression( =>(Low(replica?on( (
Depletion of PML increases HBV X- transcription HepaRG cells : HBV WT (MOI 10) RT-QPCR HBV X- (MOI 10) RT-QPCR Relative level of HBV RNA 5000 4000 3000 2000 1000 Relative level of HBV RNA 140 120 100 80 60 40 20 0 HepaRG cells : sh control sh PML 0 sh control sh PML IF PML
FISH:HBV DNA / IF: PML Nuclear localisation of HBV genome and PML nuclear bodies HepAD38 -tet (HBV wt) HBV DNA PML DAPI Merge HBV DNA PML PML DAPI Merge HepG2 HepG2 K6 (HBV X-) HBV DNA
Conclusions " HBx interacts with cellular proteins involved in transcriptional regulation and chromatin remodelling Study whether these proteins are part of the same complex Activity of these proteins/complex in presence or in absence of HBx " PML represses more efficiently the transcription of an HBV virus deficient for the expression of HBx. Study the mechanims of PML repression. Study how HBV bypasses this repression
Acknowledgements Hepacivirus#and#immunité#Innée# Ins:tut#Pasteur.(Paris( Eliane(Meurs( ( Lise%Rivière%% Aurélie%Ducroux.. Stéphanie.Dabo. Nolwen.Jouvenet. Agata.Budkowska.. Patrick.Maillard. Simone.Boccheba.. Oncogenèse#et#Virologie#Moléculaire. Marie(Annick(Buendia( ( Delphine%Cougot... U1052#INSERM.(Lyon( Olivier(Hantz. Fabien.Zoulim. Lucyna.Cova. Soumia.Bayarassou. Ins:tut#de#géné:que#Humaine(( Montpellier# Monsef(Benkirane Unité#de#Régula:on#Epigéné:que( Ins?tut(Pasteur( Chris6an.Muchardt. Eric(Allemand MRCKUniversity#of#Glasgow(( Roger(EvereZ#
Hepatitis B virus pres1" pres2" DNA virus : 3,2 kb!! 2400" 3182/1" HBV" 1600" 800" Structural proteins :! 3 surface glycoproteins (L, M, S)! capsid protein!! Replication:! DNA polymerase-! reverse transcriptase!! Regulatory protein:! HBx!! Secreted antigen: HBe! HBx! HBSP!
Hepatitis B virus replication
Experimental system HepAD38 : HepG2 derived cell line containing HBV genome under the control of tetracycline Total DNA! HepAD38! -Tet +Tet HBV - 3.5 kb - 2.4 kb - 2.1 kb HBV - OC - DS - SS RC DL CCC 10kb 8 kb 6kb 5kb 4kb 3kb 2.5kb Int Int 3,2kb Int 18S Northern blot Southern blot Southern blot Long exposure
HBx, CREB and CBP/p300 are recruited on the cccdna ChIP experiment in HepAD38 cell line producing or not HBV (+/- tetracycline) ChIP Input - Ab α-hbx α-p300 α-cbp cccdna primers: HBV-P23: 1441-1460 HBV-P24: 1889-1869 (Pollicino et al, Gastroenterology, 2004) α-creb
CREB activates HBV transcription HepAD38! HepAD38 +Tet! Forskolin:! 10 µm! HepAD38! HepAD38! sirna! 0h! 1h! 3h! 0h! 1h! 3h! - 3.5 kb! - 3.5 kb HBV! - 3.5 kb! - 2.4 kb! - 2.1 kb! HBV! - 2.4 kb! - 2.1 kb! - 2.4 kb - 2.1 kb 18S! 300! 250! 200! 150! 100! 50! 0! 0.015! 160! 140! 120! 100! 80! 60! 40! 20! 0! 0.03! 0.02! 0.004! % pg RNA 0.016! % pg RNA % pregenomic RNA! 18S! 160! 140! 120! 100! 80! 60! 40! 20! 0! 0.04! sirna:! sirna:! p300! CBP! actin! CREB! Actin! sirna:! HDAC1! Actin!
HBx interacts with PP1 and HDAC1 293 cells:! IP anti-hdac1! -! WB anti-pp1! WB anti-ha! WB anti-hdac1! IP anti-ha! -! WB anti-pp1! WB anti-ha! Inputs! WB anti-ha! WB anti-pp1! Inputs! WB anti-pp1!
HBx inhibits CREB dephosphorylation 293 cells! Induction of CREB phosphorylation by forskoline! forskolin induction:! WB anti-pcreb WB anti- CREB WB anti-pkac mock! HBx! 0! 1! 3! 6! 0! 1! 3! 6! HepG2 Control! HA-HBx! Forskolin (h):" 0! 1! 3! 6! 0! 1! 3! 6! α-pcreb! α-creb! α-actin! Fold induction 14! 12! 10! 8! 6! 4! 2! 0! **!
293 cells! HBx inhibits CREB dephosphorylation mediated by HDAC1! forskolin (hours):! Control! 0 1 3 6 HA-HBx! 0 1 3 6 HDAC1! HDAC1+ HA-HBx! 0 1 3 6 0 1 3 6 α pcreb! α-creb! α -PKA! α -PP1! α -HA(HBx)! Fold induction! 12! 10! 8! 6! 4! 2! 0! *!!HBx interferes with the dephosphorylation process
CREB: dephosphorylation + P! PP-1! HDAC1! + Ac! CREB! H (Canetteri et al., Nat. Struct. Biol. 2003)
HBx inhibits PP1 activity In vitro dephosphorylation assay! GST-CREB-P -32 +! PP1! HBx! PP1 + 0.1 µg! 0.5 µg! 1 µg! OA! PP1! PP1! PP1! Incubation time (minute)! 0! 10! 30! 0! 10! 30! 0! 10! 30! 0! 10! 30! 0! 10! 30! % of P 32 phospho -CREB! 100! 50! 30! 100! 100! 100! 100! 100! 30! 100! 97! 94! 100! 100! 100! Autoradiography OA! HA-HBx! PP1-His! +! +! +! Eluate! Lysate! +! +! WB-α PP1! WB-α HA!
HBx is part of a complex with PP1 and HDAC1. complexes.. free. 75( 50( 37( 20( 15( Flag-HDAC1! V5-PP1! PP1! *( HA-HBx!
HBx is not required for cccdna formation upon infection of hepatocytes Infec?on( WT(or(X"(HBV( Southern(blot((PHH)( RC(DNA( L((DNA( CCC(DNA( RI( Days(pi( HBV(WT( HBV(X"( 2( 4( 8( 13( 17( 2( 4( 8( 13( 17( cccdna! transcription! HBeAg! Primary(human(hepatocytes( (or(heparg(cells( qpcr(hbv(cccdna(/(cellular(dna( (arbitrary(units)( HepaRG(( n. s. 200( n. s. 200( 150( 100( 50( 0( HBV(wt( HBV(X"( 150( 100( 50( 0( PHH( HBV(wt(HBV(X"(
HBx expression is associated with increased histone acetylation in infected hepatocytes Ac Ac Ac Ac Ac Ac Ac H3 ARTKQTARKSTGGKAPRKQLATKAARKSA. H4 SGRGKGGKGLGKGGAKRHRKVLRDNIQGIT. 9 14 18 5 8 12 16 ChIP HepaRG( Relative level of histone acetylation (%) 150 100 50 0 HBV( cccdna( HBV(WT( HBV(X"( 150 100 50 0 CCNA2( (negative control) PHH( Relative level of histone acetylation (%) 100 50 0 * * 100 50 0
Nuclear localisation of HBVwt genome and PML nuclear bodies HepaRG infected by HBV wt at MOI 400 Immuno-Fish ADN HBV PML DAPI Merge
Nuclear association of HBV X- genome with PML HepG2 K6 (HBV X-) HepG2
! Inhibition of protein phosphatase 1 (PP1) by HBx : a mechanism for the activation of HBV replication! Role of the viral protein HBx in the epigenetic and transcriptional regulation of HBV cccdna! Cellular mechanisms involved in HBV repression
CREB activation Burst phase : phosphorylation of CREB Transcription rate attenuation :dephosphorylation of ser 133 Time (hours)! 1 2 3 4 5
HBx does not modulate the stability of HBV RNAs " (HBV(RNAs((RT"qPCR)((HepaRG(cells). 6me.(hours). 6me.(hours). t0: Actinomycin D addition # HBV RNA half life: ~15h for HBV wt and HBV X- HBx acts at the level of HBV transcription.
CREB: dephosphorylation Burst phase : phosphorylation of CREB + Transcription rate Time (hours)! attenuation :dephosphorylation of ser 133 1 2 3 4 5 P! PP-1! HDAC1! + CREB! H A c! (Canetteri et al., Nat. Struct. Biol. 2003)