Rhevir 2005 La Grande Motte 23 Septembre 2005 Inserm Institut national de la santé et de la recherche médicale Patrick Maurel INSERM U632, Hepatic Physiopathology,

Rhevir 2005Rhevir 2005La Grande MotteLa Grande Motte

23 Septembre 2005 23 Septembre 2005

InsermInsermInstitut nationalInstitut nationalde la santé et de la recherche médicalede la santé et de la recherche médicale

Patrick MaurelPatrick Maurel

INSERM U632, Hepatic Physiopathology, Montpellier FranceINSERM U632, Hepatic Physiopathology, Montpellier France

http://www.hepatologyinvitro.org/http://www.hepatologyinvitro.org/

Les récepteurs du virus de l’Hépatite C Les récepteurs du virus de l’Hépatite C dans l’hépatocyte humaindans l’hépatocyte humain

HEPATITIS C VIRUS INFECTIONHEPATITIS C VIRUS INFECTION

• 170-200 million people chronically infected170-200 million people chronically infected• outcome (c.a. 20 years): outcome (c.a. 20 years):

• approximately 20% towards a cirrhosisapproximately 20% towards a cirrhosis• approximately 5% towards a hepatocellular carcinoma (HCC)approximately 5% towards a hepatocellular carcinoma (HCC)

• major spread in Japan occurred in the 1930smajor spread in Japan occurred in the 1930s• major spread in Europe and North America occurred in the 1960smajor spread in Europe and North America occurred in the 1960s• HCC association with HCV: HCC association with HCV:

• >80% in Japan >80% in Japan • around 50% in Europe and North Americaaround 50% in Europe and North America

• HCV infection: currently the major cause of liver transplantationHCV infection: currently the major cause of liver transplantation• No vaccineNo vaccine• Only one current treatment: IFNOnly one current treatment: IFN (50% of non-responders) (50% of non-responders)• New treatments ? (antiprotease, antipolymerase, antihelicase, others)New treatments ? (antiprotease, antipolymerase, antihelicase, others)• no realistic animal model (Chimp) or no realistic animal model (Chimp) or in vitroin vitro model (replicon) model (replicon)

5’-UTR5’-UTR CC E1E1 E2E2 NS2NS2 NS3NS3 NS4A/BNS4A/B NS5A/BNS5A/B 3’-UTR3’-UTR

Poly(U)Poly(U)oror

Poly(A)Poly(A)

ss-RNA HCV genome 9500 ntss-RNA HCV genome 9500 nt5’5’ 3’3’

3’3’ 5’5’(-) strand RNA(-) strand RNA

5’5’ 3’3’

VirusVirus

(+) strand RNA(+) strand RNA

REPLICATIONREPLICATION

LIVERLIVER

(+) strand RNA(+) strand RNA(+) strand RNA(+) strand RNA

(-) strand RNA(-) strand RNA

HCV genome replication

P7P7

C, E1, E2, (+) st RNAC, E1, E2, (+) st RNA

Hepatocytepreparation

Perfusion

anatomopathology

1st surgical resection2nd resection

isolation andculture

Preparation of human hepatocytesPreparation of human hepatocytesfrom liver lobectomiesfrom liver lobectomies

Preparation of human hepatocytesPreparation of human hepatocytesfrom liver lobectomiesfrom liver lobectomies

Surgery departmentSurgery departmentnetwork:network:• Montpellier/Nîmes• Lyon• Toulouse• Bordeaux

L2 laboratoryL2 laboratory

PHENOTYPIC CHARACTERISTICS OFPHENOTYPIC CHARACTERISTICS OFOUR LONG-TERM CULTURE SYSTEMOUR LONG-TERM CULTURE SYSTEM

PHENOTYPIC CHARACTERISTICS OFPHENOTYPIC CHARACTERISTICS OFOUR LONG-TERM CULTURE SYSTEMOUR LONG-TERM CULTURE SYSTEM

Ferrini et al. J Hepatol 2001; Castet et al. J Virol 2002;Pichard-Garcia et al. Methods in Enzymology 2002; Gerbal et al. J Biol Chem 2002;

Biron-Andréani et al. Br J Haematol 2004, Assénat et al. Hepatology 2004

LipoproteinsLipoproteinsLipoproteinsLipoproteins

Plasma proteinsPlasma proteinsPlasma proteinsPlasma proteins

Blood coagulationBlood coagulationFactors (Vit K)Factors (Vit K)

Blood coagulationBlood coagulationFactors (Vit K)Factors (Vit K)

Response toResponse tocytokines (APP)cytokines (APP)

Response toResponse tocytokines (APP)cytokines (APP)

Response toResponse togrowth factorsgrowth factorsResponse toResponse to

growth factorsgrowth factors HCV replicationHCV replicationHCV replicationHCV replication

Expression ofExpression ofC/EBPC/EBP and HNF4 and HNF4

Expression ofExpression ofC/EBPC/EBP and HNF4 and HNF4

Urea synthesisUrea synthesisUrea synthesisUrea synthesisCYPs and DMEsCYPs and DMEs

expressionexpressionCYPs and DMEsCYPs and DMEs

expressionexpression

DrugDrugmetabolismmetabolism

DrugDrugmetabolismmetabolism

Experimental model

Analyses: rTth RT-PCR (Perkin Elmer) / quantitative PCR (Light Cycler,Roche)

HBV (-), HCV(-), HIV(-)

INFECTION

RNA

HCV (+)SERUM

D3

D4 : Washes (3x, WME)

Last day : Washes (3x, PBS)

Genomic form (+)

Replicative form (-)

D0HCV

Treatments± IFN± r-shCD81 (LEL)± anti-CD81 Mabs± r-shLDLR± anti-LDLR Mabs± LDL

LIVERLIVERL3 laboratory

full-length HCVfull-length HCV

EcoRV-KpnIEcoRV-KpnI

1-5821-582

pSP73pSP73

pSP73pSP73

PvuIIPvuIISP6 RNA PolSP6 RNA Pol

EcoRVEcoRVT7 RNA PolT7 RNA Pol

(+) RNA(+) RNA (-) RNA(-) RNA

Synthetic RNA controlsSynthetic RNA controls

(+) strand RNA(+) strand RNAassayassay

(-) strand RNA(-) strand RNAassayassay

Total RNATotal RNA

RR

rTth pol, MnClrTth pol, MnCl22, 70°C, 70°C

5’5’ 3’3’

5’5’3’3’

EGTA, MgClEGTA, MgCl22

FF

Southern blotSouthern blot

RR

rTth pol, MnClrTth pol, MnCl22, 70°C, 70°C

3’3’ 5’5’

3’3’5’5’

EGTA, MgClEGTA, MgCl22

FF

(+) RNA(+) RNA (-) RNA(-) RNA

3232PP 3232PP

3838 342342

cDNAcDNA cDNAcDNA

Analysis of HCV RNAs

rTth: 38 to 342rTth: 38 to 342LC: 68 to 311LC: 68 to 311

L

S26 I1 I2 I3 I4 I5 I6 I8 I10 I12L

S26 I1 I2 I3 I4 I5 I6 I8 I10 I12

RNA (+)

RNA (-)

Liver : FT 147Inoculum : S26 (20 l) (1b; 29.14 Meq/ml)

C Fournier et al. J Virol 1998V Castet et al. J Virol 2002

Detection of HCV (+) and (-) RNA strandsDetection of HCV (+) and (-) RNA strandsby strand-specific rTth RT-PCR agarose gel analysis

Days after infection

C Fournier et al. J Gen Virol 1998

Quantitation of HCV (+) and (-) RNA strandsQuantitation of HCV (+) and (-) RNA strandsby Roche Light-Cycler analysis by Roche Light-Cycler analysis

Serum 42 (1b); culture 172

V Castet et al. J Virol 2002

0

4

8

12

16

30 min day 1 day 3 day 5 (after infection)HC

V c

op

ies/

pla

te (

x 10

6 )

(+) HCV RNA strand

(-) HCV RNA strand

V Castet et al. J Virol 2002

FT147 / S26

MW S26 1000 2500 5000 10 000

(+) st

IFN-a (U/mL)

(-) st

FT161 / S42

500 1500 5000

IFN-a (U/mL)

UT

HC

V c

op

ies/

pla

te (

x 10

6 )

500 1500 5000

IFN-a U/ml

0

10

20

30

UT

(+) st

(-) st

Effect of IFN on HCV replication

L

S26 I1 I2 I3 I4 I5 I6 I8 I10 I12L

S26 I1 I2 I3 I4 I5 I6 I8 I10 I12

(+)

(-)

Receptor candidates:Receptor candidates:

• CD81CD81

• LDLRLDLR

• SR-BISR-BI

• Others...Others...

Receptor candidates:Receptor candidates:

• CD81CD81

• LDLRLDLR

• SR-BISR-BI

• Others...Others...

Virus

5’-UTR5’-UTR CC E1E1 E2E2 NS2NS2 NS3NS3 NS4NS4 NS5NS5 3’-UTR3’-UTR

Poly(U)Poly(U)oror

Poly(A)Poly(A)

ss-RNA HCV genome 9500 ntss-RNA HCV genome 9500 nt5’5’ 3’3’

3’3’ 5’5’

5’5’ 3’3’(+) strand RNA(+) strand RNA

Virus

NN

HepatocyteHepatocyte Receptor ?

?

The mechanism of HCV entry in cellsis not yet fully understood

LDLR: A PUTATIVE CELLULARLDLR: A PUTATIVE CELLULARRECEPTOR OF HCV?RECEPTOR OF HCV?

LDLR: A PUTATIVE CELLULARLDLR: A PUTATIVE CELLULARRECEPTOR OF HCV?RECEPTOR OF HCV?

.

• HCV is associated with LDL in human serum (Thomssen et al.)• LDLR expression in cell lines confers binding to HCV (Monazahian et al.)• LDLR expression in cell lines confers permissivity to HCV (Seipp et al.)• LDLR mediates endocytosis of HCV and other flaviviruses complexed with LDL (Agnello et al.)• Endocytosis of HCV in hLDLR-TG mice (Agnello et al.)

Collaboration with Serono Intl. Geneva

Is LDLR involved in HCV infection of human hepatocytes?

LDLRLDLR

LDLLDL

HCVHCV

LDLLDL

Regulation of cholesterol homeostasisRegulation of cholesterol homeostasisRegulation of cholesterol homeostasisRegulation of cholesterol homeostasis

.

LDLRLDLR

LDLLDL

squalene

Farnesyl-PP

mevalonate

HMG CoA

acetyl CoA

SREBP

Target genesLDLR, squalene synthasefarnesyl PP synthaseHMG CoA reductaseHMG CoA synthaseetc.

SCAPS1P, S2P

pre

From Brown and Goldstein, PNAS 1999

cytosol

nucleus

endoplasmicreticulum

cholesterol, (sterols)cholesterol, (sterols)

.

LDLRLDLR

LDLLDL

SREBP


SREBP

SRE

SCAPS1P, S2P

pre

squalestatin

statins

cytosol

nucleus




squalene

Farnesyl-PP

mevalonate

HMG CoA

acetyl CoA


.

LDLRLDLR

LDLLDL

squalene

Farnesyl-PP

mevalonate

HMG CoA

acetyl CoA

SREBP


SCAPS1P, S2P

pre

From Brown and Goldstein, PNAS 1999

cytosol

nucleus



00

5050

100100

150150

200200

250250

300300

SQ10SQ10 SQ1SQ1 UTUT 25OHC 525OHC 5 25OHC 2025OHC 20

LD

LR

mR

NA

LD

LR

mR

NA

Arb

itra

ry u

nit

sA

rbit

rary

un

its

(µM)(µM)

Functional studies on the LDLR:Functional studies on the LDLR:LDLR mRNA expressionLDLR mRNA expression

Functional studies on the LDLR:Functional studies on the LDLR:LDLR mRNA expressionLDLR mRNA expression

LDL onlyLDL onlyNBNB

SQSQ 25OHC25OHC

Functional studies on the LDLR:Functional studies on the LDLR:LDL traffic (LDL-Bodipy-FL)LDL traffic (LDL-Bodipy-FL)

Functional studies on the LDLR:Functional studies on the LDLR:LDL traffic (LDL-Bodipy-FL)LDL traffic (LDL-Bodipy-FL)

10µM10µM

20µM20µM

40µM40µM

80µM80µM

00

200200

400400

600600

cold-LDLcold-LDL 125125I-LDLI-LDL SQSQ 25OHC25OHC

LD

L e

ntr

y (3

7°C

)L

DL

en

try

(37°

C)

00

8080

160160

cold-LDLcold-LDL SQSQ 25OHC25OHC

LD

L b

ind

ing

(4°

C)

LD

L b

ind

ing

(4°

C)

125125I-LDLI-LDL

Functional studies on the LDLR:Functional studies on the LDLR:LDL traffic (LDL traffic (125125I-LDL)I-LDL)

Functional studies on the LDLR:Functional studies on the LDLR:LDL traffic (LDL traffic (125125I-LDL)I-LDL)

Collaboration with Dr Ronald Barbaras Inserm U563, Toulouse

- strand

+ strand

HDL (µg/ml)

2 20 200

Non treated

LDL (µg/ml)

2 20 200 Non

treated

- strand

+ strand

Effect of LDL and HDL on HCV infectionEffect of LDL and HDL on HCV infectionEffect of LDL and HDL on HCV infectionEffect of LDL and HDL on HCV infection

LDLRLDLR

LDLLDL

HCVHCV

LDLLDL

- strand

+ strand

u-hsLDLR166

0.2 2 20

r-hsLDLR145

0.2 2 20

r-hsLDLR292 (µg/ml)

0.2 2 20 UT

Effect of r-shLDLR on HCV infectionEffect of r-shLDLR on HCV infectionEffect of r-shLDLR on HCV infectionEffect of r-shLDLR on HCV infection

LDLRLDLR

LDLLDL

HCVHCV

LDLLDL


• r-hsLDLR292: full length LDL binding domain (rep 1-7)

• u-hsLDLR166 and r-hsLDLR145: trunckated LDL binding domain (rep 1-4)

0

100

200

300

400

0.2 2 20 0.2 2 20 0.2 2 20 NT

HC

V c

op

y /

GA

PD

H

us166 LDL-R(µg/ml)

rs145 LDL-R(µg/ml)

r s292 LDL-R(µg/ml)

Effect of r-shLDLR on HCV infectionEffect of r-shLDLR on HCV infectionEffect of r-shLDLR on HCV infectionEffect of r-shLDLR on HCV infection

LDLRLDLR

LDLLDL

HCVHCV

LDLLDL


HCV r-hsLDLR292

0 0.5 1 2 4 8 hanalysisat day 5

+ strand

- strand

Treatment with 2 g/ml r-shLDLR292

T0 0.5h 2h 8h non treated

HC

V R

NA

co

pie

s

0

8 000

16 000

nt

2 g/ml r-shLDLR292

T0 8h

GA

PD

H

(cyc

les

)03

9

15

nt

2 g/ml r-shLDLR292

T0 8h

Effect of r-shLDLR292 on HCV infectionEffect of r-shLDLR292 on HCV infectionEffect of r-shLDLR292 on HCV infectionEffect of r-shLDLR292 on HCV infection

LDLRLDLR

LDLLDL

HCVHCV

LDLLDL


+ strand+ strand

La

dd

er

La

dd

er

XX 8

g/m

l8

g

/ml

2

g/m

l2

g

/ml

8

g/m

l8

g

/ml

2

g/m

l2

g

/ml

8

g/m

l8

g

/ml

2

g/m

l2

g

/ml

MAb12.6MAb12.6 MAb28MAb28 MAb29.8MAb29.8

no

n t

rea

ted

n

on

tre

ate

d

no

n i

nfe

cte

d

no

n i

nfe

cte

d

cells infected by HCV(+) serum (S42) cells infected by HCV(+) serum (S42)

La

dd

er

La

dd

er

XX 8

g/m

l8

g

/ml

2

g/m

l2

g

/ml

8

g/m

l8

g

/ml

2

g/m

l2

g

/ml

8

g/m

l8

g

/ml

2

g/m

l2

g

/ml

MAb12.6MAb12.6 MAb28MAb28 MAb29.8MAb29.8

no

n t

rea

ted

n

on

tre

ate

d

no

n i

nfe

cte

d

no

n i

nfe

cte

d

- strand- strand

Effect of mAbs-r-shLDLR on HCV infectionEffect of mAbs-r-shLDLR on HCV infectionEffect of mAbs-r-shLDLR on HCV infectionEffect of mAbs-r-shLDLR on HCV infection

LDLRLDLR

LDLLDL

HCVHCV

LDLLDL


Po

siti

ve s

tran

d

Po

siti

ve s

tran

d

HC

V R

NA

co

pie

sH

CV

RN

A c

op

ies

/10

/106 6

cel

ls

cells

Mab-12Mab-12 Mab-28Mab-28 Mab-29Mab-29 untreateduntreated

4 104 1066

3 103 1066

2 102 1066

101066


LDLRLDLR

LDLLDL

HCVHCV

LDLLDL

Liver : FT 168

S 42 (1b)


0

5

10

15

20

02 0004 0006 0008 000

10 00012 00014 00016 000

I=67%

I=0%

I=92%

I=88%I=66%

I=35%

nt 8 2 8 82 2

MAb12.6 g/ml

MAb28 g/ml

MAb29.8 g/ml

HC

V R

NA

cop

ies

GA

PD

H

(cyc

les

)8 2 8 82 2

MAb12.6 g/ml

MAb28 g/ml

MAb29.8 g/ml

nt


HCV/GAPDHHCV/GAPDH

00

2020

6060

100100

140140

UTUT22 88 22 88 22 88

mAb12mAb12µg/mlµg/ml



LDLRLDLR

LDLLDL

HCVHCV

LDLLDL


LDL-R mRNA expression (arb units)

0

60

120

SQ (10) SQ(1) NT

0

8000

16000

SQ (10) SQ(1) NT

HCV (+) copies/cap S155

Effect of squalestatin on HCV infectionEffect of squalestatin on HCV infectionEffect of squalestatin on HCV infectionEffect of squalestatin on HCV infection

LDLRLDLR

LDLLDL

HCVHCV

LDLLDL

• The model of normal human hepatocytes in primary culture is the

most closely related to the in vivo situation, to study HCV infection

• In this model, IFN exerts a direct blocking effect on HCV RNA replication

• The data obtained with:

• recombinant soluble forms of human LDLR (and CD81)

• specific antibodies to these proteins

suggest that both LDLR and CD81 play a role in the early step of

infection of human hepatocytes by HCV

CONCLUSIONCONCLUSION

AcknowledgementsARNS, ARC

QuickTime™ et un décompresseurPhoto - JPEG sont requis pour visualiser

cette image.

INSERM U632 INSERM U632 Hepatic PhysiopathologyHepatic Physiopathology

Year 2004Year 2004

Absents: E Assenat, D Larrey, P Blanc, D Pageaux

E Rau

let

E Rau

let

JM F

abre

JM F

abre

J Cost

e

J Cost

eC F

ournie

r

C Fourn

ier

V Cas

tet

V Cas

tet

L Pic

hard-G

arci

a

L Pic

hard-G

arci

a

S Molin

a

S Molin

a

Rhevir 2005 La Grande Motte 23 Septembre 2005 Inserm Institut national de la santé et de la recherche médicale Patrick Maurel INSERM U632, Hepatic Physiopathology,

Documents

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