the regulatory hbx protein contributes to evasion from ... · conclusions üspindlin1 is recruited...
TRANSCRIPT
The regulatory HBx protein contributes to evasionfrom intrinsic antiviral response
Hepacivirus et Immunité InnéeInstitut Pasteur
Paris
HBV replication cycle
cccDNA
transcription
translation
Pol
Viral entry
Receptor
encapsidation
ADN-synthesis
ADN+ synthesis
Endoplasmic reticulum
Budding
Secretion
Pregenomic RNA
Natural history of HBV infection
90%
Horizontal transmission(adult infection)
Vertical neonatal transmissionNeonatal (childhood infection)
HBV clearance HBV and HBsAg persistence
Full immuneResponse(NK+ T+B)
asymptomatic or acute hepatitis B
Activation of CD4/8+ T
Chronic active hepatitisCirrhosis, HCC
90% (30%)5-10%
T cell ignoranceExhaustion
asymptomatic chronic hepatitis B
15-40%
Virus outcome: balance between cellular antiviral responses andmechanisms develop by the virus to escape from these antiviralresponses
HBx is essential for viral replication in vivo (Zoulim et al.,1994 )
HBx has pleiotropic activities (signal transduction, cell cycle, apoptosis, transcription)
HBx regulatory protein
HBx
HBVViral HBx is a regulatory protein (17kD)
Bock et al, JMB 2001
HBx is involved in cccDNAtranscriptional regulation
H3/H4 acADNccc
TFTF
TFTF
TF TF
TFP300P300
P300TF
PCAF
PCAFPCAFHBx
HBV cccDNA is organized into chromatin-like structure as a viral minichromosome: up to 20 nucleosomes containing histones et non histone proteins.
HBx is required for the initiation and maintenance of HBV transcription in the setting of infection (Lucifora et al., 2011)
HBx expression correlates with HBV transcription and histone H3 hyperacetylation (Lucifora et al., 2011; Belloni et al., 2009)
Molecular mechanisms involved in HBV cccDNAsilencing in infected hepatocytes
Lise Rivière
HBx increases the level of HBV RNAs
HBV WTHBV X-
PHHdHepaRG
0
100
200
300
400
500
0 5 10 15 20Days p.i.
0
50
100
150
200
0 5 10 15
Days p.i.
Rel
ativ
e H
BV
RN
A le
vel
Rel
ativ
e H
BV
RN
A le
vel
HBV RT-QPCR
HBV wt and X- cccDNAs show different sensitivities to MNase digestion
Infected PHHMNase digestion of cccDNA + Southern Blot
0 0.05 0.1 0.2 1.0
RC
CCC
Long exposure
MonoDiTri
MNase (U/ml):
Micrococcal Nuclease (MNase)
6kb
4kb
3kb
2.5kb
10kb
Silenced HBV X- cccDNA is associated with repressive histone marks
0
2
4
6
8
% in
put
*
% in
put
0
0.2
0.4
0.6 *
*
dHepaRGChIP Q-PCR HBV cccDNA
HBVwt
HBVX-
H3/H4 acH3 K4me3
H3 K9me3
048
1216
% in
put
*
05
10152025
% in
put
01234567*
*
% in
put
HBV WT
HBV X-
histone methyltransferase ?
HBV X-
Histone methyltransferase SETDB1 mediates the deposition of H3K9me3 on the cccDNA
HP1H3 K9me3
HMTH3 K9me3
sh lucsh SETDB1sh suv39H1sh suv39H2
cccDNA%
inpu
t
0
0.5
1.0
1.5
2.0*
ChIP Q-PCRR
elat
ive
SETD
B1
RN
A le
vel
Rel
ativ
e su
v39H
1 R
NA
leve
l
Rel
ativ
e su
v39H
2 R
NA
leve
l *SETDB1
0.0
0.5
1.0
1.5suv39H2
0.0
0.5
1.0
1.5suv39H1
0.0
0.5
1.0
1.5 **RT-QPCR
Control of silencing
HepaRG
HBx restores cccDNA transcription and alleviateschromatin repression
Rela
tive
bind
ing
0
10
20
30
40
50
**
*
0
0.5
1.0
1.5 * *
HBV X-HBV WT
HA (HBx)Tubulin
lenti Mock lenti HA-HBx
00
ChIP qPCRHBV X- + lentivector
Rel
ativ
e H
BV
RN
A le
vel
*
12345
0.5
1.0
1.5
Rel
ativ
e H
BV
RN
A le
vel
HBV transcription (RT-qPCR)
Rela
tive
bind
ing
(Riviere et al., J hepatol 2015)
ü HBx is necessary for viral transcription and replication, in the context ofcellular infection by HBV
üHBx expression correlates with the deposition of activating histonemodifications on HBV cccDNA (histone acetylation, H3K4 me3)
ü In the absence of HBx, repressive epigenetic marks are deposited onHBV cccDNA (H3K9 me3, histone hypoacetylation, recruitment of HP1γ)
üHBV silencing is in part mediated by SETDB1
Conclusion
ü HBV repression can be reverted by the reexpression of HBx
⇒ HBx may counteract an antiviral response that prevents viral transcription via the establishment of repressed chromatin
Ubiquitination of cellular protein(s):
Degradation of cellular substrates or modification of their functions
- ì of virus replication
- Induction of apoptosis
- Transcriptional activity
Working model
HBx
A?B?
DDB1
degradation
Common viral manipulation: subversion of E3 ubiquitin ligase activity
substrate
DDB1 : a core subunit of a Cul4a-based E3 ubiquitin ligase complex.
Cellular factors involved in HBV silencing and counteracted by HBX
Aurélie Ducroux
Spindlin1:
• Associated with meiotic spindles
• SPIN/SSTY family
• Contains three Tudor-like domains
• Histone reader for dual H3 K4me3-R8me2a methylation pattern
•Increases rRNA trancription
•Activator of Wnt/ β-catenin pathway
17 -
25 -34 -
55 -73 -90 -
130 -
43 -
HBx
DDB1
Spindlin 1
Cul4BCul4A
PRMT1
HepG2Tap-tag
kDa
Identification of Spindlin 1 as a newHBx interacting-protein
Results
HBx interacts with endogenous Spindlin1
HEK293 HepG2
Spindlin1 represses HBV transcription in the setting of infection
RT-qPCR
ResultsResultsResults
dHepaRG
Spindlin1 expression represses HBV transcription in the setting of infection
Rel
ativ
eR
NA
leve
lHBV RNA (RT-qPCR)
α−Myc �
0
0,2
0,4
0,6
0,8
1
1,2
1,4
Ctrl Myc-Spin1
Û Û
dHepaRG
HBx decreases recruitment of Spindlin1 to cccDNA
dHepaRG cells
Depletion of Spindlin1 increases H3K4me3 on the cccDNA
dHepaRG cells
ShCtlrShSpin1
Spindlin1 represses the transcription of HSV-1 during infection
A BICP27 RNA (RT-qPCR)
hours after infection:
HSV1 DNA(qPCR)
Spindlin1 RNA (RT-qPCR)
hours after infection:
ShCtlrShSpin1
(Ducroux et al.,Plos Pathogen 2014)
HepaRG cells
Conclusions
üSpindlin1 is recruited on the cccDNA and represses its transcription in thecontext of HBV infection
ü Spindlin1 represses more severely HBV X- virus than wild type virus,suggesting that HBx counteracts Spindlin1 activity on HBV.
üSpindlin1 represses the transcription of Herpes Simplex Virus type 1 in thesetting of infection
è Spindlin1 is a new component of the intrinsic antiviral defense
ü How Spindlin 1 is recruited on the cccDNA?
üHow HBx counteracts spindlin1 activity?
ü Mechanisms involved in cccDNA repression?
Hepatitis B virus X protein identifies the Smc5/6complex as a host restriction factor
Adrien Decorsière1*, Henrik Mueller1†*, Pieter C. van Breugel1†*, Fabien Abdul1*, Laetitia Gerossier2, Rudolf K. Beran3, Christine M. Livingston3, Congrong Niu3, Simon P. Fletcher3, Olivier Hantz2 & Michel Strubin
N AT U R E , VO L 5 3 1 , 2 0 1 6
Indentification of Smc5/6 complex as an HBxinteracting partner
Strategy: Tandem affinity purification assay (TAP)hepG2 cells
Flag-StrepHBX
Smc5/6 complexe is a bona fide substrate of the Cul4/DDB1/HBx complexe
Smc5/6 is a restriction factor for HBV replication and its activity is counteracted by HBx
Nse4 a component of Smc5/6 complex is recruited on cccDNA
Conclusions
ü Smc5/6 complex is a new HBV restriction factor
ü HBx counteracts the repressive activity of Smc5/6 complex via theinduction of its degradation by the CulA/DDB1 ubiquitin ligase
ü How Smc5/6 is recruited on the cccDNA or episomal DNA?
ü Mechanisms involved in cccDNA repression?
Hepacivirus andimmunitéInnéeInstitutPasteur. ParisElianeMeurs
HBVgroupCNeuveutLiseRivièreAurélieDucrouxPierrickMorreauPatrickMaillardSylviePaulous
Yu WeiTianXia
OncogenèseetVirologieMoléculaireMarieAnnickBuendia
U1052INSERM. LyonOlivierHantzLaetitiaGerossier
Acknowledgements
InstitutdegénétiqueHumaineMontpellierMonsef Benkirane
Laboratory ofHepatitis BviruspathogenesisMarie-LouiseMichelSarahDionQiangDeng
LaboratoiredeVirologieetgénétiqueLausanneDidiertronoPriscillaTurelli