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