Transcript Mécanismes moléculaires d`induction et d`action antivirale

Interaction of HCV with Interferon and the
innate immune response
Eliane F.Meurs
Pasteur Institute, Paris
INTERFERONS
Interferons belong to the family of type II cytokines
( 27 cytokines, 12 receptors).
Interferons are proteins naturally induced by the organism
in response to a situation of stress, such as infection with
bacterial or viral pathogens.
IFNs are involved in the resistance of cells to a viral
infection,in growth regulation and activation of immune
response.
Introduction
INTERFERONS
Identified through their antiviral properties
(Isaacs and Lindenman, 1957)
Influenza Virus
Chicken
chorio-allantoïc
Membranes
Culture medium
Heating, Filtration
reduced viral production
Production of virus
Culture medium contains some
protecting substance: Interferon
Cloned in 1980
Interferon messenger RNA: translation in heterologous cells.
de Maeyer-Guignard J, de Maeyer E, Montagnier L.
A viral inhibitor with the characteristics of mouse interferon is produced by avian and simian cells preincubated
with RNA extracted from interferon-producing mouse cells. Similarly, RNA extracted from interferon-producing
monkey cells induces a monkey interferon-like substance in avian cells and also in a line of simian cells, VERO,
which normally lacks the capacity toproduce its own interferon. In both cases, the RNA effect is inhibited by
treatment of the receptor cells by cycloheximide,but not by actinomycin D. We conclude that interferon messenger
RNA has been translated in the receptor cells. Thus, the production of interferon in heterologous cells can be used
as a sensitive assay of interferon messenger RNA.
Proc Natl Acad Sci U S A. 1972 May;69(5):1203-7
IFN-producing
Monkey cells
Extraction RNA
IFN mRNA translated in the
heterologous VERO cells
Incubation RNA with VERO cells
(cells which cannot make own IFN)
Resistance to viral infection
Inhibited by cycloheximide
Not by actinomycin
The type I IFN system
Bacteria, virus
IFNAR
Activation JAK/STAT
Activation IRF3
Activation NF-B
Induction IRF7
IFNAR
Activation JAK/STAT
Activation IRF3/IRF7
IFN-
subtypes IFN
 some early ISGs
Induction ISGs
Induction IFN 
(Several subtypes)
Amplification phase
Innate immune response
antiviral and
Antiproliferative
state
Activation of adaptive immune response:
-Maturation of DCs (induction CD80,CD86, CD40)
-Stimulation NK
-Stimulation of CTL differentiation
Interferons and their use in therapeutics
The different IFN types
Type I IFNs
9p21p22p23
IFN
IFN
IFN
IFN




1 gene, 35% identity with 
13 gènes, 5 pseudogenes,70 % identity
1 gene, 5 pseudogenes, 55% id. , 29% id. 
1 gene
IFN 
1 gene, 30% homol with ,  or 
(6Mb away from IFN cluster= separated evolution)
IFN  (limitin) 1 gene, 30% id.avec IFN ,  and  (mice)
IFN 
10 genes; 55% identity with , 70% with  (sheep, cattle)
Type III IFNs
19q13,13
1 (IL-29), 2 (IL-28A), 3 (Il-28B) homologies with IFNs 
    and with IL-10 (Only 2 and 3 in mice)
IFN
Type II IFNs
IFN 
12q24,1
1 gene; No identity with other IFNs
Human IFN- genes and proteins
12 distinct IFN-s from 14 genes
IFNA1
IFN-D, IFN-1
IFNA2
IFN-A (IFN-2a), IFN-2 (IFN-2b), IFN-2c
IFN-4a (IFN-76), IFN-4b
IFNA4
IFNA5
IFNA6
IFN-G, IFN-5, IFN-61
IFN-K, IFN-6, IFN-54
IFNA7
IFN-J, IFN-J1, IFN-7
IFNA8
IFNA10
IFNA13
IFN-B2, IFN-B, IFN-8
IFN-C, IFN-10, IFN-1, IFN-6L
IFN-12 ( sequence identical to IFN-1)
IFNA14
IFNA16
IFN-H, IFN-H1, IFN-14
IFN-WA, IFN-16, IFN-O
IFNA17
IFNA21
IFNAP22
IFN-I, IFN-17, IFN-88
IFN-F, IFN-21
IFN-E
IFN-2: predominantly used in clinic
Petska, S review JBC,2007
Therapeutic use of Interferons
IFN-s :
Hairy cell leukemia, malignant melanoma, follicular lymphoma,
genital warts, AIDS-related Kaposi’s sarcoma,laryngeal papillomatosis,
chronic hepatitis C,chronic hepatitis B
Interferon Alfa-2a (Roferon-A; Hoffnung); Interferon Alfa-2b (Intron-A; Schering)
pegylated Alfa-2a (Pegasys; Roche); Pegylated Alfa-2b (Peg-Intron; Schering)
Interferon Alfa-2b+ Ribavirin (Rebetron); Interferon alfacon-1 (Infergen)
Interferon Alfa-2b fused to Albumin (Albinferon)
IFN-:
Multiple sclerosis, Cervical intra-epithelial Neoplasia
Interferon beta-1b (Betaseron); Interferon beta-1a (Avonex); Soluferon (preclinical testing;VPM)
IFN-:
In Phase I studies
Peg-Interferon  (Zymogenetics)
IFN-
Chronic granulomatous disease, severe malignant osteopetrosis
Interferon gamma-1b (Actimmune)
Towards new IFN technologies
-Generate new IFN molecules using gene shuffling technology:
AV
Th1
Binding
Antiprolif.
Brideau-Andersen et al, PNAS, 2007
Maxyalpha in clinical trials (Maxigen)
Interferon treatment and HCV
Monotherapy (Interferon, PegInterferon)
Bitherapy (PegInterferon + Ribavirin)
Adding specific antiviral compounds
20 to 50% SVR
50 to 80 % SVR
can we cure?
Ex: Antiviral effect of Telaprevir (VX-950), an anti-HCV NS3/4A protease
inhibitor ; a 15-day clinical trial
Placebo+ PegIFN
Telaprevir
Telaprevir + PegIFN
Forestier, N., Hepatology,2007
Mechanism(s) of IFN Induction:
IFN INDUCTION
PAMPs (MAMPs)
Bacteria, virus
Pathogen-Associated Molecular Patterns
Microorganism-Associated Molecular Patterns
cell
PRRs
Pattern-recognition receptors
IFN
PAMPs + PRRs
IFN
Adapter proteins
Activation of kinases
Activation of transcription factors
Gene induction
PAMPs
Bacteria
Fungi
LPS
PhospholipoLipoproteins
mannan
Peptidoglycans
GlucuronoxyloGlycolipids
mannan
Flagellins
DNA
Unmethylated CpG
Staph aureus
Trepon.maltophil.
listeria
Candida albicans
Aspergillus fumig.
S.cerevisiae
Pneumocustis car.
Protozoa
Glycosylphosphatidyl
Inositol
Glycoinositolphospholipids
Trypanosoma cruzi
T brucei
Toxoplasma gondii
Leishmania major
Plasmodium falcip.
Viruses
Envelopes
ssRNA
dsRNA
ssDNA
DNA
Influenza
Measles
VSV
Rabies virus
HCMV
HSV-1
RSV
MMTV
WNV
HCV
Toll-like receptors or TLRs
Peptidoglycan
(G+)
Lipoproteins (G-)
Peptidoglycan(G+)
TLR1
LPS(G-)
RSV-F
Flagellins (G+)
TLR4
TLR2
TLR5
Lipoproteins (G+)
mycoplasms
TLR6
TLR10
Not present in mice
Plasma membrane
Endosomes
dsRNA
TLR3
Loxoribine
Imiquimod
ssRNA
TLR7
Imiquidiazoline
Unmethylated CpG
ARNsb viral
Resiquimod
ssRNA
TLR8
TLR9
Not functionnal in mice
Extracellular domains of TLRs : 19-25 tandem copies of LRR
A common intracellular domain : TIR, 200 residus, assembly platform
All TLRs use Myd88 as adapter, exceptTLR3
Signaling pathways activated by TLR 3,4,7,8,9
Expression pattern of TLRs
The 10 human TLRs are expressed in all tissues with various levels
Highest levels
TLR1
TLR2
TLR3
TLR4
TLR5
TLR6
TLR7
TLR8
TLR9
TLR10
kidney, lung, spleen
lung, spleen
Placenta, and ubiquitous
spleen and ubiquitous
ubiquitous
ubiquitous
lung,placenta, spinal cord, spleen
lung, spleen
skeletal muscle, spleen
spleen, thymus
Cytosolic PRRs and PRMs
the NOD-Like Receptors or NLR
the RIG-I-Like Receptors or RLR
the DNA sensors
the NOD-Like Receptors or NLR
NALP7
NALP14
NALP5
NALP12
NALP3
Bacteria, MAMPs,DAMPs
NOD1,2
NALP4
NALP9
NALP11
NALP13
NALP..
NOD9/NLRX1
NF-kB, MAPK
Inflammosome
ROS
IL1
NALP8
NALP1
NALP6
NOD3
NOD9/NLRX1
Antimicrobial defence
NOD2
NOD1
NOD27
CIITA
IPAF
NAIP
Tattoli, I et al, Embo reports 2008
NALP2
the NLR Contain LRR and nucleotide binding domain (NBD)
Cytosolic PRRs: the RIG-I-Like Receptors or RLR
Treatment with IFN
Virus
Human K562 cells
Human K562 cells
(deletion locus IFN type I)
Generation cDNA library
activation IRF3
No IRF3 activation
Poly(I)-poly(C)
Murine L929 cells
Induction IRF-luc reporter
Out of 100 000 cDNA, isolation
of one clone encoding for a
DExD/RNA helicase: RIG-I
Yoneyama et al, 2004
Kato,H et al,July 2005,Immunity
The DExD/H RNA helicase RIG-I/Mda-5/LGP2 family
CARD
RNA helicase
925
RIG-I
DEAD motif: in the ATP-binding domain
23% identity with RIG
35% identity with RIG
MDA5
LGP2
1025
678
31% identity with helicase domain of RIG
41% identity with helicase domain of MDA5
No CARD domain
These RLR are inducible by IFN
Yoneyama et al, J Immunol, 2005
Viruses and dsRNA Induce IFN through the RLRs:
Sensors of non-self RNA
In vitro transcribed RNAs
Infection by NDV, VSV, SeV,
Rabies,Influenza
Measles leader RNA
HCV 5’ and 3 UTR
Poly(I)-poly (C )
Infection by picoRNAvirus
(EMCV, Theiler, Mengo)
activation
RIG-I
Importance of a triphosphate at the 5’
minimun size 21 bp
activation
MDA5
Poly(I)-poly (C )
dsRNA
Other RNAs?
Binding
LGP2
Differential regulation of RIG-I
and MDA5?
LGP2-/- mice:
susceptible to EMCV
Less or highly susceptible to VSV?
Kato, H. Nature 2006
Hornung, V et al, Science,2006
Pichlmaier,A.Science, 2006
Plumet, S. PlosOne, 2007
Venkataraman,T, J Immunol,2007
Kato, Oxford meeting, sept 2007
Detection of structurally distinct RNA species by RIG-I and triggering of signalling
RNA binds to RIG-I C terminal domain CTD;
NMR studies : binding of RNA into a basic cleft
RIG-I
CTD
helicase
dsRNA
5’pppssRNA
CARD
helicase
ATP
Signalling
CTD
CARD
+ polyIpolyC
CTD
helicase
CARD
Abortive
T.Fujita, Oxford ISICR meeting, sept 07
The HCV RNA act as a PAMP to activate RIG
HCV
RNA hel.
RIG-I
3'
CARD
CTD
5’ PPP
5’ PPP
IFN
X
Poly(U/UC)
replicatio
n
T55I
RIG-I
Activation dependent on U/UC region
at its 3’end and of 5’ppp end
CARD
RNA helicase
925
The huh7-5 cells have an endogenous defective RIG-I
which explains, at least in part, their permissivity to HCV
expression
Sumpter,R.J Virol, 2005; Saitoh and Gale,M;Nature 2008
MDA5 binds to long and RIG-I to short dsRNA structures
Atomic Force Microscopy
Kato,H et al, JEM 2008
ATPase MDA5
ATPase RIG-I
long dsRNA
+
-
short dsRNA
+
Activation of RIG-I or MDA5 by RNA VIruses
Genome
VSV
Replicative Intermediates
EMCV ss RNA 8 kB
10 segments
dsRNA
3.9 kB
2.2 kB
1.2 kB
Kato,H et al, JEM 2008
+
+
ds RNA 8 kB
ss RNA, 5’ppp
HCV ss RNA, 5’ppp
Reo
MDA5
ss RNA 11 kB
DI dsRNA 2.2 kB
Flu
RIG-I
+
5’ppp + poly (U/UC)
+
+
+
RIG-I’s mediated antiviral activity requires its ubiquitination through TRIM25
Gack et al, Nature, 2007
Effect of deficiency of RIG-I, MDA5 and LGP2 on viral infections
RIG-I KO: mostly embryonic lethal at 12.5 to 14 days, few mice born alive, die within 3 weeks.
Experiments with isolated cells :
RIG-I essential for induction of IFN by RNA viruses in fibroblasts and DCs
Experiments with RIG-I -/- mice:
highly susceptible to infection with JEV, resistant to infection with EMCV
Kato, H. et al, Immunity, 2005
Kato, H. et al, Nature, 2006
MDA5 KO: mice alive and healthy
Experiments with MDA5 -/- mice:
highly susceptible to infection with EMCV, resistant to JEV
Kato, H. et al, Nature, 2006
LGP2 KO: viable
Experiments with MEFs:
in response to polyIpolyC, the loss of LGP2 increases IFN production
Experiments with LGP2-/- mice:
resistant to VSV infection (in accord with negative regulation of RIGI)
sensitive to EMCV infection ( why?)
Venkataraman, T. J Immunol, 2007
Viral infection
dsRNA
DNA
TLR3
TLR
viral RNA
RIG-I/MDA5
TRIF
MAVS
Facteurs transcription
AP-1
IRF3
CBP/p300
NF-B
IFN-
Bacterial
components
TLR
IPS-1/MAVS/VISA/CARDIF
The adapter between RIG-I/MDA5 and the downstream IFN-inducing kinases
1st identification in 2003: a gene with no known precise function
Large-scale identification and characterization of human genes that activate
NF-B and MAPK signaling Pathways Matsuda,A et al.( 2003) Oncogene 22 3307-3318
Identified in 2005 as RIG-I adapter by
S.Akira (Kawai,T et al, Nature Immunol,29 Aug.2005 ):
High throughput screening
IPS-1 ( IFN- Promoter Stimulator-1)
Z.Chen (Seth,R. et al, Cell, 122,1-14, Aug.2005)
Blast search with domain CARD-like of RIG-I
MAVS (Mitochondrial AntiViral Signaling)
H.B.Shu (Xu,L-G et al, Mol Cell, 19 1-14, Aug. 2005)
Cloning and analysis of the NF-B-inducing ability of the Matsuda gene
VISA (Virus-Induced Signaling Adapter)
J.Tschopp (Meylan,E. et al, Nature, sept 2005)
Blast search with domain CARD-like of RIG-I
CARDIF (CARD adapter Inducing IFN-)
The RIG-I/MDA5-mediated IFN induction pathway
Virus
5’pppRNA, small dsRNA (RIG-I)
long dsRNA (MDA5)
cytosol
CARD
IPS/VISA/MAVS/CARDIF
CARD
TM
CARD PRO
TRAF6
TAB/TAK
IKK
MAPKs
P
P
IKK
IKK
?
Ub
Ub
Ub
Ub
AP1
IB
Mitochondria
TRAF3
TANK
TBK1 IKK
P
P
IRF-3
P50/p65 NF-B
P
AV state
inflammation
IFN-
P
AP1P
P
NF-B
NF-B IRF-3
Nucleus
Pro-inflammatory
cytokines
The IRF3/IRF7-phosphorylating kinases TBK1 and IKK
19
299 305
383
730
Kinase domain
ULD
Coiled-Coil Coiled-Coil
19
299 305
383
717
Kinase domain
ULD
Coiled-Coil Coiled-Coil
TBK1
IKK
Homologous, yet with some differences
-Deletion of TBK1 but not of IKK gene is lethal (Bonnard, M et al, EMBO, 2000; Hemmi, H et al, J Exp Med, 2004 )
-TBK1 more important role than IKK in IFN induction in response to different stimuli. Yet,
double KO cells have complete abolition of IFN induction (Hemmi, H et al, J Exp Med, 2004 )
-Expression of TBK1 is constitutive. IKK is inducible in response to IL-1, PMA
and virus through NF-B and c/EBP (Shimada, T et al, Int.Immunol,1999)
-IKK induces a subset of IFN-induced genes (ADAR-1) through ser708 phosphorylation
of STAT1 (tenOever,B. et al, Science, 2007)
IRF3
Ubiquitous expression;
Localization: inactive in the cytosol, active in the nucleus
TLR3 TLR4
RIG-I/MDA5
IRF3
(Qin et al; Takahasi et al,
Nature Struc Biol;2003)
TBK1 and IKK are the
IRF3-phosphorylating kinases
Activation TBK1/IKK 
Phosphorylation
IRF3
1
427
382
414
SRR (serine rich region)
7 phosphate acceptor sites
-GGAS385S386LENTVDLHIS396NS398HPLS402LT404S405DQYKAYLQD-
essentiels
(S A abolishes
activity)
non essentiels individually
(S A : normal phenotype)
Induction IFN inhibited
when all 5 residues are mutated
Phosphorylation of IRF3 liberates its DNA binding activity
Sharma, S et al, Science 2003; Fitzgerald, K; Nature Immunol, 2003;
Higgs, R and Jefferies,C. 2008
The IRF family
IRF-1
IRF-2
DBD (DNA Binding Domain)
P
IAD (IRF association Domain)
P
Homologies with C-ter of SMADs
C-ter auto-inhibition domain
Proline-rich sequence
IRF-5
IRF-6
P
Phosphorylation site
IRF-8
(ICSBP)
IRF-9
(ISGF3g/p48)
IRFs have distinct roles in the development
and function of immune cells
IRF-4
(PIP)
IRF-3
IRF-7
P
P
Honda, K and Taniguchi, T, review,2006
Viral infection
dsRNA
DNA
TLR3
TLR
viral RNA
RIG-I/MDA5
TRIF
MAVS
Facteurs transcription
AP-1
IRF3
CBP/p300
NF-B
IFN-
Bacterial
components
TLR
OVERALL STRUCTURE OF THE IFN ENHANCEOSOME
PRDIV
PRDI
PRDIII
PRDII
Panne, D et al, Cell,2007
General scheme of IFN induction by viruses and dsRNA
virus
dsRNA
Viral
RNA RIG-I/Mda5
TLR3
TRIF
MAVS
p38MAPK/JNK
AP-1
IKK
NF-B
TBK1/IKK
IRF-3
IFN
CXCl-10
Early ISGs
ISG15,etc…
JAK/STAT
IRF7
(reinduced by IFNs)
All IFN-s
+ ISGs
Including
RIG-I/MDA5/LGP2, TLR3
IFNAR
Induction de gènes par IRF3: consensus AANNGAAA
IRF3 + CBP/p300
c-Jun/ATF2
NF-B
IFN
GAGAAGTGAAAGTG
-104
PRDI-III
PRDIV
PRDII
-55
ISG56
IFN
CAAAGTGAAAGG GGAAAGCCAAAGG
-92
-117
ISG15
IFN
CGGGAAAGGG GAAACCGAAACTGA
-165
-143
IFN1
IRF7
ATGAAGGAAAGCAAAAACAGAAATGGAAAGT
Cell-mediated positive and negative regulation of the IFN induction pathway
Moore, C. and Ting,J. Minireview, Immunity 2008
Gack et al, Nature, 2007
IFN inducing pathways: targets for viral inhibitors
viral RNA
MDA-5
TRIF RIG-I
MAVS
AP-1 NF-B
Capside 3
E3L
NSP3
Reovirus
Vaccinia
Rotavirus
V protein
NS1
Paramyvovirus
Influenza
NS3/4A
HCV
IRF3
vIRF1, vIRF3
HHV8
E1A
Adenovirus
(binds CBP/p300)
CBP/p300
IFN-
E6 (binds IRF3)
NSs
(inhibits TFIIH)
Papillomavirus
Rift valley fever
Bunyavirus
The HCV RNA act as a PAMP to activate RIG…
…yet HCV inhibits the IFN inducing pathway
Activation of IFN
Signaling pathway
NS3/4A
Inhibition phosphorylation IRF3
and of its migration to nucleus
Inhibition of IFN signaling pathway
Foy et al, 2003
Helicase domain
C
NS4A cofactor
Catalytic domain
protease domain
How does HCV NS3/4A interferes
with the IFN inducing pathway?
NS3/4A cleavage site:
P6-P5-P4-P3-P2-P1.....P’1-P’2-P’3-P’4
Consensus sequence:
D- X- X- X- X- C.....S- X- X- L
(E)
(T) (A)
(W/Y)
N from De Francesco R, 2003
Sequence of the NS3/4A-mediated cleavages in HCV polyprotein from different genotypes
Name
H77
HCV-N
JFH1
NZL1
ED43
SA13
6a33
Genotype (GI ) NS3 / 4A
1a (GI:2316097 ) MSADLEVVT STWVLVGG
1b (GI:23957856) MSADLEVVT STWVLVGG
2a (GI:13122261) MQADLEVMT STWVLAGG
3a (GI:514395) MSADLEVTT STWVLLGG
4a (GI:2252489) MSADLEVVT STWVLVGG
5a (GI:3660725) MSADLEVIT STWVLVGG
6a (GI:57791993) MSADLEVIT STWVLVGG
Meurs& Breiman, WJG,2007
NS4A/ 4B
QEFDEMEEC SQHLPYIE
REFDEMEEC ASHLPYIE
EAFDEMEEC ASRAALIE
QQYDEMEEC SQAAPYIE
QQFDEMEEC SKHLPLVE
QQFDEMEEC SASLPYMD
QQFDEMEEC SRHIPYLAE
NS4B/ 5A
ISSECTTPC SGSWLRDI
INEDCSTPC SGSWLRDV
ITEDCPIPC SGSWLRDV
INEDYPSPC SDDWLRTI
INEDCSTPC STPCAESW
IGEDYSTPC DGTWLRAI
VNEDTATPC ATSWLRDV
NS5A/ 5B
reference
ADTEDVVCC SSYSWTGA Kolykhalov et al, 1997
EAGESVVCC SMSYTWTG Beard et al, 1999
EEDDTTVCC SMSYSWTG Kato et al, 2001
SEEQSVVCC SMSYSWTG Sakamoto et al, 1994
SGSEDVVCC SMSYSWTGChamberlain et al, 1997
SDEDSVVCC SMSYSWTGBukh et al, 1998
SDQDDVVCC SMSYSWTGZhou et al, 2004
Is it through its protease activity?
HCV NS3/4A cleaves IPS-1/VISA/MAVS/CARDIF
1
TM
MAVS
CARD PRO
540
508
EREVPCHRPS
Meylan et al (2005) Nature
NS3/4A cleaves also TRIF
dsRNA
TLR3
TRIF
372
N
C
192 DGVSDWSQGCSLRSTGSPAS
326 PILEPVKNPCSVKDQTPLQL
372 PPPPPSSTPCSAHLTPSSLE
1
TM
MAVS
Li, K et al (2005) PNAS
CARD PRO
540
508
EREVPCHRPS
Meylan et al (2005) Nature
-MAVS is delocalized from the mitochondria to the cytosol in cells transfected with
NS3/4A, infected with HCVcc and in liver biopsies from chronic hepatitis patients (Lin et
al, 2006; Loo et al, 2006)
-The BILN 2061 NS3/4A inhibitor can restore the MAVS-mediated IFN induction in cells
Infected with HCVcc (Cheng et al,2006)
MAVS
Cont
MAVS
+ NS3/4A
Lin et al, 2006
Cheng et al,2006
Distinct localizations of IKK and TBK1:
IKK associates with the mitochondria
IKK 
MITO
Merge
TBK1
MITO
Merge
IKK 
IKK 
MAVS
MAVS
Colocalization of Cardif and IKK
IKK 
MAVS
Merge
IKK 
MAVS
Merge
Merge
Merge
Disruption of the IKK/ Cardif complex
localization by expression of NS3/4A
+NS3/4A
Lin et al, J Virol 2006
The RIG-Ipathway
in liver biopsies from HCV-infected patients
17 patients with chronic HCV:
-9 under no treatment
-8 non responders to
treatment
12 controls (other pathologies)
Patient
number
1
2
3
4
5
6
7
8
9
ALT
(IU/L)
78
31
75
93
24
23
70
37
69
10
11
12
13
14
15
16
17
137
42
27
47
47
70
29
36
AST
(IU/L)
29
18
47
54
17
16
34
27
28
Viral load
HCV
(IU/mL)
genotype
5,00E+06
1b
3,45E+05
1b
2,36E+05
4d
2,02E+05
1
2,00E+06
1b
2,00E+05
1a
1,00E+06
4
ND
5,46E+07
1a
76
26
23
32
36
41
27
25
1,12E+05
2,00E+08
2,00E+07
1,20E+07
2,40E+04
5,91E+05
9,36E+06
4,00E+05
dsRNA-activated RNA Helicases
Mitochondrial adapter
Genes analysed
by qRT-PCR
Genes used for normalisation:
TRIM44,HMBS, BC002942
IRF3-phosphorylating kinases
IKK -induced genes
4
1b
1b
1b
1b
1b
1a
1b
Knodell
Index
1+1+1+0
1+1+1+0
3+0+3+0
1+3+4+0
0+1+1+0
NA
1+1+1+0
1+1+3+1
1+1+1+0
Treatment
IFN/Ribavirin
No
No
No
No
No
No
No
No
No
Time after
treatment
3+4+3+3
NA
1+1+3+1
1+1+3+1
1+1+3+0
3+3+3+1
1+1+3+0
1+1+3+1
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
2 years
6 years
9 years
5 months
4 years
7 years
5 years
11 years
RIG-I, MDA5, LGP2
MAVS
IKK, TBK1
IFN, ISG15, CCL3
Analysis of liver biopsies: results 1
NS
NS
9
8
7
6
5
4
3
2
1
Cont
HCV
HCV.NR
IFN-
TBK-1
NS
NS
NS
6
NS
5
4
3
2
1
Cont
HCV
HCV.NR
Relative RNA Expression
NS
Relative RNA Expression x 10 -2
Relative RNA Expression x 10 3
MAVS
8
7
6
NS
NS
5
4
3
2
1
Cont
HCV
RNA expression levels of MAVS, IFN- and TBK-1
are not significantly affected in HCV-infected patients
HCV.NR
Analysis of liver biopsies: results 2
9
8
7
6
5
4
3
2
1
MDA5
P=0.01
P=0.015
NS
Cont
HCV
25
P<0.001
P=0.06
P=0.01
20
15
10
5
HCV.NR
Cont
P=0.005
NS
NS
7
6
5
4
3
2
1
Cont
HCV
12
P=0.016
NS
NS
10
8
6
4
2
Cont
HCV HCV.NR
ISG15
HCV.NR
Relative RNA Expression x 10 -1
Relative RNA Expression
LGP2
HCV HCV.NR
IKK
Relative RNA Expression x 10 -1
RIG-I
Relative RNA Expression
Relative RNA Expression x10 1
HCV interferes in vivo with the dsRNA-RIG-I/ IKK pathway
P<0.001
P<0.001
NS
8
7
6
5
4
3
2
1
The RNA expression levels of
RIG-I, MDA5, LGP2, IKK and
ISG15 are downregulated in
HCV-infected patients.
Cont
HCV HCV.NR
Vilasco et al, Hepatology, 2006
HCV can both provoke and inhibit the IFNinducing pathways
Treatment of
patients with
exogenous IFN
HCV
Activation IRF3
Activation NF-B
IFNAR
Activation JAK/STAT
Induction IRF7
IFNAR
Activation JAK/STAT
Activation IRF3/IRF7
IFN-
subtypes IFN
 some early ISGs
Induction ISGs
Induction IFN 
(Several subtypes)
Amplification phase
antiviral and
Antiproliferative
state
Can HCV affect the response to IFN
and some antiviral mechanisms?
Response of cells to Interferons
THE JAK/STAT SIGNALING PATHWAY
ligand
Receptor
Signal
Transducers and Stat
Activaters of
Transcription
Tyr P
Jak
4 JAKs
7 STATs
STAT1
STAT2
STAT3
STAT4
STAT5A
STAT5B
STAT6
Janus
Activated tyrosine
Kinase
ISG
TYK2
JAK1
JAK2
JAk3
THE IFN RECEPTOR
R1 CHAIN :
- responsible for signal specificity
-long intracellular domain, associates to JAKs
-is phosphorylated on tyrosyl residue
-recruits STAT through their SH2 domains
R2 chain :
-only used for signal transduction
-short intracellular domain
-Recruits JAK
-Allows JAK cross-activation
IFN
R1
R2
Tyr P
IFN- 
fibronectin type III
Il-20
FVIIa
TF
IFN-
Il-10
IL-10R2
Il-22
IL-10R2
IFNAR2
IFNAR1 IFN-R1
IL-10R1
IFN-R2 IFN- R1
IL-10R1
IL-10R2
Il-26 Il-24
IL-10R2
IL-10R2
IL-20R2
IL-20R1
IL-22R1
Il-19
IL-20
IL-24
IL-20R2
IL-20R1
IL-22R1
The cytokine class II receptor superfamily
Kotenko, S and Langer,J. 2004
The different steps of JAK/STAT activation
1
2
IFNAR1
IFNAR1
IFNAR2
Tyk2
Tyr466 y P
Jak1
IFNAR2
Tyk2
Tyk2/Jak1
Y
Stat2
Jak1
Tyk2/Jak1
y P
P
Stat2
Tyk2
Jak1
3
4
Tyk2
Jak1
Tyk2/Jak1
yP
P
Stat1
5
yP
P
Tyk2/Jak1
P
y P
Stat2
Jak/Stat activation by IFN-/
IFNAR1
IFNAR2
Tyk2 Jak1
Tyk2/Jak1
P
P
Stat2
P
P
P
Stat1
Type I IFN activate other Stat complexes
IRF9
P
nucleus
Stat 1:1
Stat 2:1
Stat 1:3
Stat 5:5
Stat 3:3
Stat 2:6 ( in mature B cells)
Stat 4:4 (CD4+ Tcells)
P
ISGF3
P
ISRE
IFN-Stimulated Genes
GGAAANNAAACT
…but those bind GAS or GAS-like elements
Brierly,M and Fish,E. 2005
Jak/Stat1 activation by IFN-
IFN-
IFNR2
R1 R1
IFN-R2
Jak2/Jak1
P
Stat1
P
Type II IFN activate other Stat complexes
P
P
Stat 3:3
Stat 3:1
P
P
nucleus
P
P
GAS
IFN- induced genes
TTNCNNNAA
The Janus kinases (JAK) family
TYK2
JAK1
JAK2
JAk3
-JH: JAK homology domains. Only JH1 has catalytic activity. JH2 has a pseudokinase
domain. JH5 to half of JH4: SH2 domain; JH1-3 and half of JH4: FERM domain (4.1, ezrin,
radixin, moesin): allows stable association with membrane proximal receptors motifs. Jak1
associates with IFNAR2 and IFNGR1; JAK2 with IFNGR2; Tyk with IFNAR1
-Expression in most tissues, except JAK3 (leukocyte)
Jak1 KO mice: die perinatally; tissues defective in response to IL-2, IL-6, IFN and IL-10
Jak2 KO mice: Embryonic lethality (E12.5). Crucial role in erythropoiesis
Jak3 KO mice: severe Combined Immunodeficiency (SCID)-like defects due to physical
link of Jak3 to C, associated to IL-2 family of cytokine receptors
Tyk2 KO mice: modest cytokine defects, type 2 T-cell response.
Tyk2 deficient humans: severe allergic phenotype: impaired antimicrobial defense
The STAT family
STAT1, STAT2, STAT3, STAT4, STATA STATB, STAT6
STAT1
1
N ter
135
Coiled-coil
317
DBD
488
linker
576
SH2
683
TAD
750
Y SSS
Contact
with DNA
Interaction with
Dimer-dimer
regulatory proteins:
interaction
IRF9,c-jun, Nmi-1,…
and stabilisation
P
P
Y701-P:
STAT dimerization
nuclear translocation
DNA binding
Recruitment to YPhosphorylated
Receptors
And other P-STATs
SH2
linker
Stability,
cycling between
cytoplasm
and nucleus
Favor the formation of
STAT1/STAT2 heterodimers
and ISGF3 formation
DNA binding
coiled-coil
S708-P , S727-P, S744-P, :
Maximal activation
Recruitment coactivators:
CBP/p300, MCM5, BRCA1
Physiological importance of STAT1
STAT1-knockout mice: high susceptibility to microbial and viral infections and tumor formation
STAT1- knockout humans: susceptibility microbial and to viral diseases; death in infancy
Kindred B
Kindred A
Wt/m
m/m
STAT1wt
1
N ter
Wt/m
Wt/m
135
BCG
HSV-1
Coiled-coil
317
Wt/m
m/m
DBD
BCG
virus?
linker SH2
488
576
750
603
INFANT 1
1757-1758delAG
TAD
683
L600P
L706SP
INFANT 2
Non conservative substitution
Dupuis et al, Nat Gen.2003
Reich et al. Nature Reviews Immunology 2006)
Schindler,C and Plumlee,C.Sem. In Cell &dvlpmental Biology 2008 in press
conserved Cys
WSXWS
motif
Ig-like
domain
fibronectin type I
D200
class II
class I
GPI
c
EPO-R
PRL-R
IL-9R
IL-4R
IL-2R
IL-5R
IL-3R
GM-CSFR
GH-R IL-7R
CNTF-R
IL-6R
IL-11R
gp130 LIF-R
KH97
The cytokine receptor superfamily
Class I receptors also use the JAK/STAT pathway
IFNAR1
IFNAR2
IFN-R1
IFN-R2
IL-10R1
IL-10R2
TF
etc....
IL-2
IL-10
EPO
IL-4
IL-6
TPO
IL-7
IL-11
IL-3
IL-9
OSM
IL-5
IL-15
LIF
IFN-
Leptin
IL-21
CNTF
G-CSF
GM-CSF
IL-22
IFN-/
IL-12
IL-23
Prolactin
G-H
The majority of the cytokines receptors use different JAK combinations
leading to activation of multiple STATs
Murray, P. J Immunol Review, 2007
IFN
IFN
JAK1/Tyk2
STAT1:2/IRF9
JAK1/JAK2
STAT1:3
STAT2
STAT1:1
STAT1
Combination of IFN and IFN may
enhance AV and immune responses
STAT3
IFN may attenuate IFN activation of Stat
Huh7 cells + IFN with or without IFN
Microarray analysis
Ag presentation (20%)
Immune-related (11%)
Other
unknown
(35%)
Complement activation (6%)
Immune cell recruitment (4%)
Direct antiviral (3%)
Cell growth/apoptosis (12%)
Transcription (9%)
Blatt, L et al, J IFN and cytok.Res.2005
Radeava,S. Biochem J..2004
Regulators of the JAK/STAT pathway
PTP PIAS SOCS
ligand
SOCS:
CD45, PTP, SHP:
receptor
Suppressor of Cytokine Signaling
Jak
STAT
P
Protein tyrosine phosphatases
STAT
P
P
SOCS
PTP1B,
TC-PTP (cytop.),
SHP1, SHP2
STAT4
SH2-containing Tyr Phosphatase
TC-PTP (nuclear),
PIAS1, PIAS3, PIASx, PIASy HDAC
STAT1
STAT3
PIAS:
STAT4
Protein Inhibitor of Activated STATs
SOCS
The family of Suppressor of Cytokines Signalling proteins
-induced by cytokines; also induced by LPS, isoproterenol, statins, cAMP
- act in a negative feedback loop to inhibit cytokine signal transduction
-regulate immune responses and maintain immunological homeostasis
-8 members: SOCS 1- SOCS 7 and CIS
Structure
Acts as pseudosubstrate
Inhibits the JAK’s Tyr kinase activity
(KIR only in SOCS1 and SOCS3)
SOCS1
Recruitment of E3 ubiquitin ligase
Degradation of proteins
Determinant for the target
of each SOCS protein
Yoshimura,A.Nature Reviews Immunology 2007
Function of SOCS
SH2 of SOCS1 bind directly
to IFNAR and IFNGR
and directly to the activation loop of JAK
SH2 of SOCS3 bind directly
to gp130-related cytokine receptors
No good affinity for activation loop of JAK
binds the kinase domain of JAK through
Its KIR domain
Model: SOCS binds the IFN receptor first and then interacts with JAK
Yoshimura,A.Nature Reviews Immunology 2007
Role of SOCS in innate immunity
SOCS1-deficient cells and Socs1-/- mice are resistant to viral infections
Yoshimura,A.Nature Reviews Immunology 2007
HCV infection
Altered expression and activation of STATs and STAT regulators
Expression of HCV proteins inhibits IFN induction through Jak/STAT
Heim et al, J.Virol, 1999
HCV core protein provoques STAT1 degradation, by interacting with STAT1 SH2 domain
Lin,W. Gastroenterol.2005; J Virol, 2006
STAT3 is down-regulated in HCV-infected livers from human patients
and in Huh7 cells bearing the full length HCV replicon
Larrea, E. et al, Gut. 2005
Non-response to AV therapy is associated with obesity and increased SOCS3
in patients with chronic hepatitis C, viral genotype 1
Walsh,M, Gut, 2005
HCV infection in chimpanzees lead to a type I IFN response. However, deficiency in
response to subsequent IFN treatment. Expression of SOCS3 elevated.
Huang, Y et al, Gastroenterol. 2007
The IFN-responsive pathway: Target for viral inhibitors
Viroceptors
(homologs
of soluble receptors)
IFNAR1
Vaccinia
Orthopoxvirus
IFNAR2
T antigen (binds Jak1)
Tyk2
V protein (blocks Stat1 phospho
by Jak1)
High induction of SOCS
Jak1
Stat1
SOCS
P
P
Stat2
IRF9
P
P
Core protein
(binding to STAT1 SH2
and degradation)
nucleus
ISGF3
V protein
(binding and degradation of
STAT)
P
P
ISRE
ISGs
E7
(binding IRF-9)
Murine Polyomavirus
Measles
HCV, influenza
SV5, Mumps
(STAT1)
Hum.parainfluenza 2
(STAT2)
HCV
HPV-16
Antiviral action of some IFN-induced genes
The IFN-induced genes
Virus,
dsRNA
More than 300 genes induced
Activation
NF-B
IRFs AP-1
IFN
Jak1
P
PStats
P
Tyk2
P
ISGs
IFN can inhibit HCV replication
- in subgenomic replicon models 1b (1b:Frese, M. et al, JGV, 2001; 1a: Gu et al, JV, 2003)
-in genomic replicon models (1b:Blight,K. et al, Science,2000 )
-in cellular models infected -with HCVcc JFH1 (Kim,C. JVI,2007)
-in primary hepatocytes infected with HCV serum (Castet et al, J Virol, 2002)
- in human hepatocyte chimeric mice infected with HCVcc 1a and 2a
(Hiraga, N. et al, FEBS, 2007) or with patient serum 1b and 1a (Inoue,K. et al, Hepatol. 2007)
……and in HCV chronically infected patients but efficacity of treatment not 100%
Which IFN-induced gene (s) is (are) involved in the inhibition of HCV?
Some ISGs and role in antiviral defense
Name
OAS
PKR
ISG56/ISG54
ISG15
ISG16
Mx
Viperin
NOXA
Phospholipid scramblase
RIG-I, MDA5
IRF7
Function
RNA degradation
Inhibition initiation translation
Inhibition initiation translation
ISGylation of proteins
Inhibition of apoptosis
Dynamin GTPases, bind viral nucleocapsids
SAM/radical catalysis
BH3-only, apoptosis
Redistribution of phospholipids on Mbs;
Role in Trail-induced apoptosis
Boosts IFN induction
Boosts IFN induction
The discovery of the OAS, 2-5A and PKR in the 70s
IFN-treated cells
Virus- infected
IFN-treated cells
dsRNA
Cell free
Translation system
Inhibition translation
Hunt, T. and Ehrenfled, PNAS 1971
Cell free
Translation system
Inhibition translation
Kerr,I. et al; Nature 1974
Inhibition of translation was dependent on IFN treatment of cells and of
presence of dsRNA, produced during the viral infection
Identification of two IFN-induced dsRNA-activated enzymes:
-Oligoadenylate synthetases
- PKR
Roberts, R. et al; Nature 1976
OAS/RNase L
and control of protein synthesis
through the degradation of RNA
The human OAS family
346
346
OAS1
OAS2
OAS3
364aa
p40
I
p69
342
II
P56-59
OASL
400aa
p46
p30
513aa
219
254aa
(OAS domain but no catalytic function)
Ubl domain homologous to ISG15
683 687aa
727aa
p71
p100
Ubl Ubl
46% (64%)
I
340 400
60% (74%)
II
739
III
1087 aa
49% (66%)
44% (66%)
Rebouillat &Hovanessian, 1999;
Hovanessian & Justessen, 2007
OAS activates upon binding to dsRNA to generate 2’-5-linked
oligoadenylates
dsRNA
OAS1
OAS2
Homotetramer
cytosol,nuclei
Donor substrate
nATP
NTP
NTP
N:
A,U,C,G,I
dA,dU,dG,dC
3’dA
+
+
+
25bp the most efficient, not sequence specific
OAS3
Monomer
Dimer
membranes
Acceptor substrate
ATP
pppA2p5’A
RpA
R:
tRNA
A5’ppp5’A
A5’pppp5’A
NAD+
polyA
ribosomes
Product
pppA(2’p5’A)n + nPPi (from dimers to 30-mers)
pppA2’p5’A2’p5’N +PPii
RpA2’p5’N + PPi
2-5A
PPP 5’
4’
5’
P 5’ 1’A P
A
1’
3’ 2’
4’
3’ 2’
3’ 2’
A
1’
2-5A activates a latent Ribonuclease or RNAse L
2-5A
2
PUG
STYKc
dimerization
RNAse L degrades RNA
Degradation of cellular mRNA, rRNA, and viral RNAs
UU 3’P
UG 3’P
UA 3’P
AU 3’P
2-5A rapidly degraded by 2’-phosphodiesterase and 5’ phosphatase
RNAse L KO Mice: increased sensitivity to RNA viruses (PicoRNAviridae, Reoviridae, Togaviridae,
Paramyxoviridae, Orthomyxoviridae, Flaviviridae, Retroviridae)
Silverman, R. JV, 2007
OAS, Rnase L and genetic predisposition to viral infections or tumors
- OAS1b and susceptibility to flavivirus
-The murine OAS family:
8 OAS1, 1 OAS2, 1 OAS3, 2 OAS-RL
-Inbred laboratory strains susceptible to Flavivirus infection
Wild mice such as Mus musculus domesticus are resistant
The locus of resistance/susceptibility to Flavivirus was mapped
on mouse chromosome 5 at a cluster of OAS genes
Susceptibility to infection is due to a stop codon
in exon 4 in the OAS1b gene leading to a truncated Inactive OAS
( Perelygin, A et al; PNAS, 2002; Mashimo et al, ; PNAS, 2002)
-Rnase L and prostate cancer:
13% of patients with prostate cancer present a mutant allele of RNaseL encoding
for a variant of Rnase L with substitution Arg462Gln
Casey et al, 2002, Nature Genetics
40% of these patients harvour a novel gamma retrovirus (XRMV).
Urisman,A. et al, PLOS Pathog.2006
The OAS/2-5A/RNase L pathway and HCV
-OAS levels are elevated in HCV-infected livers and Rnase L is expressed in
livers (Zhou et al, 2005)
-HCV RNA are susceptible to cleavage by RNase L (in cytoplasmic extracts or
with purified Rnase L)
-HCV RNA ORFs are GC rich. UA and UU dinucleotides were found among the
least abundant dinucleotides in HCV RNA ORFs from 162 isolates. Rnase L
may exert selective pressure on HCV.
(Washenberger et al, Virus res.2007).
RNase L can induce IFN
Transcriptome induced by 2-5A revealed several IFN-stimulated genes
Malathi et al, PNAS,2005
2-5A can induce IFN in RNase L +/+ but not in RNase L -/- cells.
IFN induction in response to 2-5A was greatly reduced in RIG-I and IPS1 -/- cells
RNA cleavage products generated by RNase L are responsible for IFN induction
Free 5’ triphosphorylated or duplex structures is thought to discriminate self
(cellular) from non self (viral) RNA.
Rnase L generates 3’- monophosphoryl groups from self RNA
and from some viral RNA. Activation of RIG-I/Mda5 could be
mediated through the duplex structure of these cleaved
small RNAs.
Malathi et al, Nature,2007
PKR
and control of protein synthesis at
the level of initiation of protein
synthesis
PKR
Protein Kinase dsRNA dependent
1
18
80
107
159
234
272
275
551
V VI
DRBD1
DRBD2
RSKKEAKNAAAKLALEIL
Basic
domain
STKQEAKQLAAKLAYLQIL
Catalytic subdomains I-XI
Recognition motif for
eIF2
dsRNA binding domains
eIF2
eIF2-P
Cytosolic serine/threonine protein kinase:
-activated by dsRNAs
-activated by PACT at the endogenous level
-inhibited by TRBP at the endogenous level
Only two known physiological substrates:
- subunit of protein synthesis initiation factor eIF2
-itself
PKR activation by dsRNA
PKR autophosphorylation occurs
in cis or through the action
of a PKR dimer or
a PKR monomer
Back to back arrangement
of the kinase domain
prevents trans-autophosphorylation
of each monomer within the dimer
Dar et al, Cell, 2005
CONTROL OF INITIATION OF PROTEIN SYNTHESIS BY PKR
e-IF2B
Met-tRNA
GTP
eIF2-GTP
GDP
60S rRNA
mRNA
e-IF2 -GTP-Met tRNA
40S rRNA
Pre-initiation complex
eIF2  -GDP
CONTROL OF INITIATION OF PROTEIN SYNTHESIS BY PKR
e-IF2B
Met-tRNA
stop
eIF2 -P -GDP
eIF2 -P -GDP
60S rRNA
mRNA
e-IF2 -GTP-Met tRNA
PKR
40S rRNA
preinitiation complex
Shut-off of protein synthesis
DOUBLE-STRANDED RNA BINDING DOMAIN (DRBD)
PKR(55-79)
PKR(145-168)
G E G R S K K E A K N A A A K L A V E I L N K E
G T G S T K Q E A K Q L A A K L A Y L Q I L S E
TRBP (97-120)
TRBP (227-250)
G Q G P S K K A A K H K A A E V A L K H L K G G
G S G T S K K L A K R N A A A K M L L R V H T V
Staufen(634-658) G E G N G K K V S K K R A A E K M L V E L Q K L
Staufen(770-794) G T G N S K K L A K R N A A Q A L F E L L E A V
RNAseIII(203-226)G T G S S R R K A E Q A A A E Q A L K K L E L E
PACT(79-102)
PACT(172-195)
VacciniaE3L
G E G T S K K L A K H R A A E A A I N I L K A N
G K G A S K K Q A K R N A A E K F L A K F S N I
A D G K S K R D A K N N A A K L A V D K L L G Y
TRBP and E3L are inhibitors of PKR
PACT is an activator of PKR
The eIF2-KINASE family
ACTIVATION UNDER DIFFERENT CONDITIONS OF STRESS
HRI
GCN2
PKR
PERK
Heme levels
oxydative stress
Heat shock
Amino acid
Deprivation
Ds RNA
ER stress
Misfolded
proteins
Heme,
Hsp90, Hsc70
Uncharged
tRNA
dsRNA
Ire1
motifs
HisRS-related
eIF2-P
eIF2-P
eIF2-P
RE
eIF2-P
DRBD and binding to nucleic acids
-topology ----
-interaction with dsRNA through contact with OH-groups and with phosphates --no
interaction with dsDNA or RNA/DNA
-no recognition of specific sequences
-The DRBD-containing proteins do not bind dsRNA similarly: differents contact sites,
different dsRNA lengths
Inactive
Active
-Non specific binding of PKR to 20 bp dsRNA. No activation
-Specific binding of PKR to 30 bp. Binding of two PKR. Autophosphorylation and active.
The role of dsRNA is to bring 2 or more PKR monomers in close proximity to enhance
dimerization via the kinase domain.(Lemaire,P. et al, JMB,2008)
ISG54/ISG56
and control of protein synthesis at
the level of eIF3
eIF3 and initiation of protein synthesis
Translation CAP dependent:
eIF3 binds 40S and recruits m7GpppRNA via interaction with eIF4F (eIF4E, eIF4A, eIF4G)
Translation CAP independent:
eIF3 (10-12 proteins) binds directly to IRES
Similarity of HCV IRES and eIF4F to anchor the mRNA
Strand near the exit site of the 40S rRNA
HCV does not need eIF4F for viral protein synthesis
S13
40S-eIF3-IRES model 40S-eIF3-eIF4F-mRNA model
Siridechadilok,B.et al, Science, 2005
ISG56 and ISG54 and control of translation
-Belongs to the early ISGs: induced by IFN and rapidly induced by viral stresses
Related (42% sequence conservation); contain TPR motifs
CAP-dependent initiation of translation
ISG56 (2 IRF3 and one NF-B binding sites)
ISG54 (2 IRF3 binding sites only)
Both bind initiation factor eIF3:
ISG54 binds subunit c and e
ISG56 binds subunit e
Both block formation of stable eIF3-ternary complex
In addition, ISG54 interferes with formation of preInitiation complex
ISG56 could inhibit HCV translation
through interaction with eIF3
S13
Wang,C. et al, JVI, 2003
Terenzi,F. et al, JBC, 2006
ISG15
and post-translational modification
of proteins
ISG15
30% homology to Ubiquitin
30% homology to Ubiquitin
LRLRGG
Conjugation to Cysteine
Residues of conjugating
enzymes
UBCH6( UBE2E1) and UBCH8 (UbE2L6)
(HERC5, TRIM25)
(USp18(UBP43),USP2,USP5,USP13,USP14)
Mechanism of action of ISG15
Activation effect: Modification of Components of the
host immune Response
ISG15 and all its conjugated and
deconjugated enzymes are
IFN-induced genes
Targets of ISG15: ≈160 genes
(examples :JAK1, STAT1,PRRs (RIG-I),
IRF3, MxA, PKR, RNase L)
ISG15 can increase IFN induction
(prevents degradation of IRF3)
ISG15 KO mice:increased susceptibility
to infection (flu, Sindbis, HSV-1)
ISGylation of HIV-1 Gag and Tsg101
Inhibits their ubiquitination
(required for virion release)
USP18 KO mice more resistant to viral
Infections (LCMV, VSV)
Sadler, A and Williams, B. Nature reviews, 2008
HCV interferes with multiple cellular pathways
Binds STAT3,binds Smad3
provokes cellular proliferation
Increases mitochondrial Ca2+ uptake
Inhibits IFN induction
Associates with PKR,Grb2,p53
Inhibits ISG induction through
induction of IL-8
Binds and inhibits Rb
Stimulates S phase entry
Role intransformation?
Associates with PKR and PERK
HCV NS5A
Domain I 1-213
Domain II 250-342
Domain III 256-447
447
Zinc binding domains
Amphipathic helix
Anchors NS5A
to the ER
Cluster of hyperphosphorylation
Polyproline cluster
NLS
ISDR
heterogeneity of sequence
associated with sensitivity or
resistance to IFN treatment (genotype 1b)
Enomoto N, N.Engl.J.Med 1996
The maintenance or evolution of the
Domain II or Domain D2
ISDR sequence is linked to its role
-is intrinsically unfolded
In the viral life cycle and the progression
-interacts with NS5B
of liver disease
-contains the ISDR
-contains a PKR-binding domain
-contains a Bcl-2 homology domain
-interacts with GRB2, with the death domain of Myd88
-activates PI3kinase by binding to an SH3 domain of one regulatory subunit
-Provokes induction of IL-8 which then inhibits induction of ISGs
NS5A involved in replication and pathogenesis
VIRAL INHIBITORS OF PKR
Competition for dsRNA
E3L (VV)
 3 and  4 (Réovirus)
NS1 (Influenza)
NSP3 (rotavirus)
TRS1,IRS1 (HCMV)
Degradation
Poliovirus-activated protease
Compartimentalization
EMCV
Sequestration by dsRNA
EBERs (EBV), VA1 (Adénovirus)
NH2
NH2
Direct interaction
p58 IPK (cellular protein
activated by influenza)
Tat (HIV-1)
soluble E2 (HCV)
cIRF2(KSHV)
SM (EBV)
NS5A (HCV)
Us11protein (HSV1)
Competition
With substrate
K3L (Vaccinia)
Substrat
Inhibition of eIf2 phospho
LANA2 (KSHV)
Substrat-P
Activation of phosphatase 1
1 34.5 Kda (HSV1)
HCV E2
Hypervariable region
Signal leader
PePHD (276-287)
TM
1 2
363
HCV 1a/b R S E L S P L L L T T T
18
80
107
79- 90
PKR
48-54
eIF2
159
PKR
1
234
272
KKAVSPLLLTTT
SELSRRS
275
551
V VI
DRBD1
DRBD2
51 79-83
eIF2
KGYID
Taylor et al,1999
Catalytic subdomains I-XI
314
INHIBITION OF PKR BY HCV E2
A cytosolic non-glycosylated domain of E2 binds to and inhibits PKR (and PERK)
Restoration of translation
Fractions
1 2 3 4 5
Glycoprotein 68
p38
E2 glycosylated
top
Bottom
*****
E1
***********
E2
®
ER lumen
PERK Pavio et al. J. Virol. 2003
®

Cytosol
E2
E2 non glycosylated
PKR
AAAAA
Pavio et al. J. Virol. 2002
Is there a correlation with mutations in NS5A ISDR and in E2 PePHD
and with IFN treatment outcome?
4 mutations at least in NS5A ISDR
Genotype 1b
Genotype 1a/b
Sustained virologic response to IFN-
Japan,
Western C.
Genotype 2a
Genotype 3
mutations outside the NS5A ISDR
Genotype 1b
Genotype 1a
Mutations in E2 PePHD
Genotype 1b
Genotype 3a
Genotype 2a/b
yes
Yes, but only a small
number of mutations
yes
no
Sustained virologic response to IFN-
In the carboxy terminal part of NS5A, in the
Variable region 3 and flanking regions
Sustained virologic response to IFN-
no
no
Conflicting reports
Hofmann, W. et al, J Clinical Virol, 2005
inhibition of PKR activity by a pseudosubstrate: the Vaccinia K3L protein
Ser51
314
79-83
human eIF-2
Several binding motifs
for PKR
KGYID
1
vaccinia K3L
33% identity and
68% similarity
with eIF2-
K
88
KGYID
essentiel binding motif
to PKR
1 K
71
K3L322 mutant
KGAID
Stronger PKR inhibitor than K3L
He, cell Death and diff.2006; Garcia et al, Biochimie, 2007
OTHER WAYS TO ESCAPE CONTROL OF TRANSLATION BY PKR
-Alphavirus (Sindbis virus, Semliki Forest Virus): positive ssRNA
Translation of subgenomic 26s RNA efficient in presence of PKR and eIF2-P
hairpin loop structure (DLP) : stalls scanning and allows efficient presentation of
Mtet tRNA to AUGi through an other factor: eIF2A, in the absence of eIF2
Ventoso, I. Genes & Development, 2006)
PKR KO ( e-IF2  functional )
Met tRNA
GTP
PKR +/+ ( e-IF2  non functional )
Met tRNA
Met tRNA
e-IF2  
e-IF2  
AUGi
eIF2A
DLP
DLP
AUGi
scanning
scanning
efficient
DLP
DLP
Met tRNA
GTP
e-IF2  
Met tRNA
Met tRNA
Met tRNA
e-IF2  
AUGi
scanning
efficient
Met tRNA
e-IF2  
AUG
inefficient
AUGi
scanning
Very inefficient
AUG
Very inefficient
How does Interferon work on HCV?
Several ISG are detected in chronic Hepatitis .
This reveals an ongoing response to endogenous IFN and/or viral RNA
(Helbig, K. et al, Hepatol.2005)
Which genes are linked to HCV clearance in vivo?
Decrease of viral levels depends on the interplay of several ISGs
RIG-I, MDA5
: provokes IFN induction
OAS and RNase L
: -provokes degradation of viral RNA
and also sustain IFN induction ( through RIG-I activation)
PKR and ISG56
Viperin
: inhibit initiation of translation on eIF2 and eIF3
: interference with protein assembly at the ER?
STAT1
: sustains IFN induction
ISG15, USP18
: ISGylation, modification of trafficking
NOXA
: apoptose
genes hypothesized to be important in the anti-HCV response
RIG-1
eIF2
ISG16: inhibitor of apoptosis
Ubiquitin ligase
Novel mathematical
method used to explore
association between
decrease in viral titers
and change in gene
expression in HCV
patients following
PegIFN/Ribavirin treatment
Brodsky,L. et al PLoS one, 2006
ISG56
IRF7
Mx1 and 2
OAS 69/71 kDa
OASL
VIPERIN
USP18
Factors involved in the persistence of HCV in the host
-Genetic variability of the virus; lack of fidelity of NS5B (1,5-2x10-3 mutation/ nucleotide/year),
no proofreading (no 3’-5’ exonuclease activity).Generation of escape mutants
-mutations in HVR1 and 2 of E2 (weakens humoral defense)
-mutations in NS3/7A protease (resistance to the new anti-protease VX-950, SCH 503034)
-Host factors:
-cellular immune response weak or defective ;
-Overreaction of cytotoxic T cells and NK cells leading to destruction of infected hepatocytes
And production of inflammatory cytokines provoking liver damages
-Induction of endogenous IFN compromised by inhibition of its induction pathway
through the NS3/4A protease
Why IFN treatment is not 100% efficient on HCV infection?
Interference of the virus with cellular signalling pathways
-NS3/4A prevents sustained induction of endogenous IFN
-through RIG-I/MAVS by cleaving MAVS
-through TLR3/TRIF by cleaving TRIF
-Induction of SOCS proteins interfere with STAT function
-NS5A and cytosolic E2 interfere with PKR
-core interacts with STAT1, provokes its degradation by proteasome
-NS5A provokes induction of IL-8 through NF-B; IL-8 inhibits IFN action