Rhevir 2005 La Grande Motte 23 Septembre 2005 Inserm Institut national de la santé et de la...
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Transcript of Rhevir 2005 La Grande Motte 23 Septembre 2005 Inserm Institut national de la santé et de la...
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
Target genesLDLR, squalene synthasefarnesyl PP synthaseHMG CoA reductaseHMG CoA synthaseetc.
SREBP
SRE
SCAPS1P, S2P
pre
squalestatin
statins
cytosol
nucleus
endoplasmicreticulum
cholesterol, (sterols)cholesterol, (sterols)
Regulation of cholesterol homeostasisRegulation of cholesterol homeostasisRegulation of cholesterol homeostasisRegulation of cholesterol homeostasis
squalene
Farnesyl-PP
mevalonate
HMG CoA
acetyl CoA
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)
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
Collaboration with Serono Intl. Geneva
• 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
Collaboration with Serono Intl. Geneva
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
Collaboration with Serono Intl. Geneva
+ 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
Collaboration with Serono Intl. Geneva
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
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
Liver : FT 168
S 42 (1b)
Collaboration with Serono Intl. Geneva
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
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
HCV/GAPDHHCV/GAPDH
00
2020
6060
100100
140140
UTUT22 88 22 88 22 88
mAb12mAb12µg/mlµg/ml
mAb28mAb28µg/mlµg/ml
mAb29mAb29µg/mlµg/ml
LDLRLDLR
LDLLDL
HCVHCV
LDLLDL
Collaboration with Serono Intl. Geneva
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
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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