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Title: Interaction of HCV with Interferon and the innate immune response Eliane F.Meurs Pasteur Institute, Paris


1
Interaction of HCV with Interferon and the
innate immune responseEliane F.MeursPasteur
Institute, Paris
2
INTERFERONS
Interferons belong to the family of type II
cytokines ( 27 cytokines, 12 receptors). Interfer
ons 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.
3
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
4
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 May69(5)1203-7
Incubation RNA with VERO cells
IFN-producing Monkey cells
Extraction RNA
(cells which cannot make own IFN)
Resistance to viral infection Inhibited by
cycloheximide Not by actinomycin
IFN mRNA translated in the heterologous VERO cells
5
Innate immune response
Activation of adaptive immune response -Maturatio
n of DCs (induction CD80,CD86, CD40) -Stimulation
NK -Stimulation of CTL differentiation
6
Interferons and their use in therapeutics
The different IFN types
Type I IFNs
9p21p22p23
IFN b? 1 gene, 35 identity with ? IFN a
13 gènes, 5 pseudogenes,70 identity IFN ? 1
gene, 5 pseudogenes, 55 id. ?, 29 id. ? IFN ?
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
IFN ?1 (IL-29),??2 (IL-28A), ?3 (Il-28B)
homologies with IFNs a? b? w???? ? and with
IL-10 (Only ?2 and ?3 in mice)
Type II IFNs
IFN ? 1 gene No identity with other IFNs
12q24,1
7
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 IFNA4 IFN-?4a (IFN-?76),
IFN-?4b IFNA5 IFN-?G,
IFN-?5, IFN-?61IFNA6
IFN-?K, IFN-?6, IFN-?54 IFNA7
IFN-?J, IFN-?J1, IFN-?7 IFNA8 IFN-?B2,
IFN-?B, IFN-?8IFNA10
IFN-?C, ?IFN-?10, ?IFN-?1, IFN-?6LIFNA13
IFN-?12 ( sequence identical to IFN-?1) IFNA14
IFN-?H, IFN-?H1, IFN-?14IFNA16 IFN-?WA,
IFN-?16, IFN-?O IFNA17 IFN-?I, IFN-?17,
IFN-?88IFNA21 IFN-?F, IFN-?21IFNAP22
?IFN-?E
Petska, S review JBC,2007
IFN-?2 predominantly used in clinic
8
Therapeutic use of Interferons
IFN-?s
Hairy cell leukemia, malignant melanoma,
follicular lymphoma, genital warts, AIDS-related
Kaposis 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 testingVPM)
IFN-?
In Phase I studies
Peg-Interferon ? (Zymogenetics)
IFN-?
Chronic granulomatous disease, severe malignant
osteopetrosis Interferon gamma-1b (Actimmune)
9
Towards new IFN technologies
  • Generate new IFN molecules using gene shuffling
    technology

Maxyalpha in clinical trials (Maxigen)
Brideau-Andersen et al, PNAS, 2007
10
Interferon treatment and HCV
Monotherapy (Interferon, PegInterferon)
20 to 50 SVR Bitherapy (PegInterferon
Ribavirin) 50 to 80 SVR Adding
specific antiviral compounds 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
11
Mechanism(s) of IFN Induction
IFN INDUCTION
Bacteria, virus
cell
IFN
12
PAMPs
Bacteria
Viruses
Fungi
Protozoa
LPS Lipoproteins Peptidoglycans Glycolipids Flagel
lins DNA Unmethylated CpG
Phospholipo- mannan Glucuronoxylo- mannan
Glycosylphosphatidyl Inositol Glycoinositolphospho
- lipids
Envelopes ssRNA dsRNA ssDNA DNA
Influenza Measles VSV Rabies virus HCMV HSV-1 RSV
MMTV WNV HCV
Trypanosoma cruzi T brucei Toxoplasma
gondii Leishmania major Plasmodium falcip.
Candida albicans Aspergillus fumig. S.cerevisiae P
neumocustis car.
Staph aureus Trepon.maltophil. listeria
13
Toll-like receptors or TLRs
LPS(G-) RSV-F
Lipoproteins (G-) Peptidoglycan(G)
Lipoproteins (G) mycoplasms
Peptidoglycan (G)
Flagellins (G)
TLR1
TLR2
TLR4
TLR5
TLR6
TLR10
Not present in mice
Plasma membrane
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
14
Signaling pathways activated by TLR 3,4,7,8,9
15
Expression pattern of TLRs
The 10 human TLRs are expressed in all tissues
with various levels
Highest levels
TLR1 kidney, lung, spleen
TLR2 lung, spleen TLR3
Placenta, and ubiquitous TLR4 spleen and
ubiquitous TLR5 ubiquitous TLR6 ubiquitous TLR
7 lung,placenta, spinal cord,
spleen TLR8 lung, spleen TLR9 skeletal
muscle, spleen TLR10 spleen, thymus
16
Cytosolic PRRs and PRMs
the NOD-Like Receptors or NLR
the RIG-I-Like Receptors or RLR
the DNA sensors
17
the NOD-Like Receptors or NLR
Bacteria, MAMPs,DAMPs
NOD1,2
NALP..
NOD9/NLRX1
NF-kB, MAPK
ROS
Inflammosome
IL1?
Antimicrobial defence
Tattoli, I et al, Embo reports 2008
the NLR Contain LRR and nucleotide binding domain
(NBD)
18
(No Transcript)
19
The DExD/H RNA helicase RIG-I/Mda-5/LGP2 family
DEAD motif in the ATP-binding domain
678
LGP2
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
20
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)
Poly(I)-poly (C ) dsRNA Other RNAs?
activation
activation
Binding
RIG-I
MDA5
LGP2
Importance of a triphosphate at the 5 minimun
size 21 bp
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
21
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
CARD
dsRNA 5pppssRNA
helicase
CTD
ATP
CTD
helicase
Signalling
CARD
CTD
helicase
polyIpolyC
Abortive
CARD
T.Fujita, Oxford ISICR meeting, sept 07
22
The HCV RNA act as a PAMP to activate RIG
5 PPP
3'
5 PPP
IFN
X
Poly(U/UC)
replication
Activation dependent on U/UC region at its 3end
and of 5ppp end
Sumpter,R.J Virol, 2005 Saitoh and Gale,MNature
2008
23
MDA5 binds to long and RIG-I to short dsRNA
structures
Atomic Force Microscopy
long dsRNA
short dsRNA ATPase MDA5
- ATPase RIG-I -

Kato,H et al, JEM 2008
24
Activation of RIG-I or MDA5 by RNA VIruses
RIG-I
MDA5
Genome
Replicative Intermediates
VSV
ss RNA 11 kB

DI dsRNA 2.2 kB
EMCV
ss RNA 8 kB

ds RNA 8 kB
ss RNA, 5ppp

Flu

5ppp poly (U/UC)
ss RNA, 5ppp
HCV
10 segments dsRNA 3.9 kB 2.2 kB 1.2 kB
Reo


Kato,H et al, JEM 2008
25
RIG-Is mediated antiviral activity requires its
ubiquitination through TRIM25
Gack et al, Nature, 2007
26
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
27
Bacterial components
Viral infection
dsRNA
DNA
TLR
TLR3
TLR
viral RNA
RIG-I/MDA5
TRIF
MAVS
Facteurs transcription
IRF3
AP-1
NF-?B
CBP/p300
IFN-b
28
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-?)

29
The RIG-I/MDA5-mediated IFN induction pathway
Virus
5pppRNA, small dsRNA (RIG-I) long dsRNA (MDA5)
cytosol
CARD
CARD
IPS/VISA/MAVS/CARDIF
TM
PRO
CARD
Mitochondria
TRAF6
TRAF3
TAB/TAK
IKK?
TANK
IKK?
IKK?
MAPKs
IKK?
TBK1
?
Ub
Ub
Ub
P
Ub
IRF-3
I?B?
P
P
AP1
P50/p65
NF-?B
P
AV state inflammation
IFN-?
Pro-inflammatory cytokines
AP1
P
NF-?B
IRF-3
NF-?B
P
Nucleus
30
The IRF3/IRF7-phosphorylating kinases TBK1 and
IKK?
1
9
299
305
383
730
Kinase domain
ULD
Coiled-Coil
Coiled-Coil
TBK1
1
9
299
305
383
717
Kinase domain
ULD
Coiled-Coil
Coiled-Coil
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)
31
IRF3
Ubiquitous expression Localization inactive in
the cytosol, active in the nucleus
TLR3
TLR4
RIG-I/MDA5
Activation TBK1/IKK ?
TBK1 and IKK? are the IRF3-phosphorylating
kinases
IRF3 (Qin et al Takahasi et al, Nature Struc
Biol2003)
Phosphorylation
1
IRF3
427
382
414
SRR (serine rich region)
7 phosphate acceptor sites
-GGAS385S386LENTVDLHIS396NS398HPLS402LT404S405DQYK
AYLQD-
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
32
The IRF family
DBD (DNA Binding Domain)
IRF-1

P
IAD (IRF association Domain) Homologies with
C-ter of SMADs
IRF-2
P
C-ter auto-inhibition domain
IRF-5
Proline-rich sequence
IRF-6
Phosphorylation site
P
IRF-8 (ICSBP)
IRF-9 (ISGF3g/p48)
IRFs have distinct roles in the development and
function of immune cells
P
IRF-3
P
IRF-7
Honda, K and Taniguchi, T, review,2006
33
Bacterial components
Viral infection
dsRNA
DNA
TLR
TLR3
TLR
viral RNA
RIG-I/MDA5
TRIF
MAVS
Facteurs transcription
IRF3
AP-1
NF-?B
CBP/p300
IFN-b
34
OVERALL STRUCTURE OF THE IFN? ENHANCEOSOME
PRDIV
PRDI
PRDIII
PRDII
Panne, D et al, Cell,2007
35
General scheme of IFN induction by viruses and
dsRNA
36
(No Transcript)
37
Cell-mediated positive and negative regulation of
the IFN induction pathway
Moore, C. and Ting,J. Minireview, Immunity 2008
Gack et al, Nature, 2007
38
IFN inducing pathways targets for viral
inhibitors
viral RNA
Capside ?3 Reovirus E3L
Vaccinia NSP3 Rotavirus
MDA-5
V protein Paramyvovirus NS1
Influenza
RIG-I
TRIF
NS3/4A HCV
MAVS
NF-?B
IRF3
AP-1
vIRF1, vIRF3 HHV8
E1A Adenovirus (binds
CBP/p300)
CBP/p300
IFN-b
E6 (binds IRF3) Papillomavirus
NSs (inhibits TFIIH)
Rift valley fever Bunyavirus
39
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
40
How does HCV NS3/4A interferes with the IFN
inducing pathway?
NS3/4A cleavage site P6-P5-P4-P3-P2-P1.....P1-P
2-P3-P4 Consensus sequence D- X- X- X- X-
C.....S- X- X- L (E) (T)
(A) (W/Y)
from De Francesco R, 2003
Sequence of the NS3/4A-mediated cleavages in HCV
polyprotein from different genotypes
Is it through its protease activity?
Meurs Breiman, WJG,2007
41
HCV NS3/4A cleaves IPS-1/VISA/MAVS/CARDIF
MAVS
1
540
TM
CARD
PRO
508
EREVPCHRPS
Meylan et al (2005) Nature
42
(No Transcript)
43
-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)
Lin et al, 2006
Cheng et al,2006
44
Distinct localizations of IKK? and TBK1 IKK?
associates with the mitochondria
Colocalization of Cardif and IKK?
IKK ?
MAVS
Merge
Merge
IKK ?
MITO
IKK ?
MAVS
MITO
Merge
Merge
TBK1
Merge
MAVS
IKK ?
Disruption of the IKK?/ Cardif complex
localization by expression of NS3/4A
MAVS
IKK ?
Merge
NS3/4A
Lin et al, J Virol 2006
45
The RIG-Ipathway in liver biopsies from
HCV-infected patients
Genes used for normalisation TRIM44,HMBS,
BC002942
46
(No Transcript)
47
HCV interferes in vivo with the dsRNA-RIG-I/ IKK?
pathway
Analysis of liver biopsies results 2
The RNA expression levels of RIG-I, MDA5, LGP2,
IKK? and ISG15 are downregulated in HCV-infected
patients.
Relative RNA Expression
Vilasco et al, Hepatology, 2006
48
Treatment of patients with exogenous IFN
HCV can both provoke and inhibit the IFN-inducing
pathways
HCV
Activation IRF3/IRF7
Can HCV affect the response to IFN and some
antiviral mechanisms?
49
Response of cells to Interferons
THE JAK/STAT SIGNALING PATHWAY
ligand
Receptor
Janus Activated tyrosine Kinase
Signal Transducers and Activaters of
Transcription
Jak
Stat
Tyr P
4 JAKs
TYK2 JAK1 JAK2 JAk3
7 STATs
STAT1 STAT2 STAT3 STAT4 STAT5A STAT5B STAT6
ISG
50
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
51
?
IFN-??w
fibronectin type III
Il-19 IL-20 IL-24
Il-20
Il-10
Il-22
Il-26
IFN-?
Il-24
FVIIa
TF
IL-10R2
IL-10R2
IL-10R2
IL-10R2
IL-20R2
IL-20R2
IFNAR2 IFN-gR1 IFN-gR2 IL-10R1 IL-10R2
IL-20R1
IL-20R1
IFNAR1
IL-10R1
IL-22R1
IL-22R1
IFN-? R1
The cytokine class II receptor superfamily
Kotenko, S and Langer,J. 2004
52
The different steps of JAK/STAT activation
53
Jak/Stat activation by IFN-?/?
Type I IFN activate other Stat complexes
Stat 11 Stat 21 Stat 13 Stat 55 Stat 33 Stat
26 ( in mature B cells) Stat 44 (CD4 Tcells)
but those bind GAS or GAS-like elements
Brierly,M and Fish,E. 2005
54
Jak/Stat1 activation by IFN-?
IFN-gR2
IFNgR2
gR1
gR1
Jak2/Jak1
nucleus
Type II IFN activate other Stat complexes
Stat 33 Stat 31
GAS
IFN-g induced genes
TTNCNNNAA
55
TYK2 JAK1 JAK2 JAk3
The Janus kinases (JAK) family
-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
56
The STAT family
STAT1, STAT2, STAT3, STAT4, STATA? STATB, STAT6
STAT1
P
P
Contact with DNA
Stability, cycling between cytoplasm and nucleus
Interaction with regulatory proteins IRF9,c-jun,
Nmi-1,
Y701-P STAT dimerization nuclear
translocation DNA binding
Dimer-dimer interaction and stabilisation
Recruitment to Y- Phosphorylated Receptors And
other P-STATs
SH2
linker
Favor the formation of STAT1/STAT2
heterodimers and ISGF3 formation
S708-P , S727-P, S744-P, Maximal
activation Recruitment coactivators CBP/p300,
MCM5, BRCA1
DNA binding
coiled-coil
57
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
Wt/m
Wt/m
Wt/m
BCG HSV-1
BCG virus?
m/m
m/m
Coiled-coil
N ter
SH2
TAD
DBD
linker
1
135
317
488
576
750
683
STAT1wt
INFANT 1
603
1757-1758delAG
L600P
L706SP
INFANT 2
Non conservative substitution
Dupuis et al, Nat Gen.2003
58
Reich et al. Nature Reviews Immunology 2006)
59
Schindler,C and Plumlee,C.Sem. In Cell
dvlpmental Biology 2008 in press
60
The cytokine receptor superfamily
Class I receptors also use the JAK/STAT pathway
61
The majority of the cytokines receptors use
different JAK combinations leading to activation
of multiple STATs
Murray, P. J Immunol Review, 2007
62
IFN?
IFN?
JAK1/Tyk2
JAK1/JAK2
STAT12/IRF9
STAT13
STAT11
STAT3
STAT1
STAT2
Combination of IFN? and IFN? may enhance AV and
immune responses
IFN? may attenuate IFN? activation of Stat
Huh7 cells IFN? with or without IFN?
Microarray analysis
Ag presentation (20)
Immune-related (11)
Complement activation (6)
Immune cell recruitment (4)
Other unknown (35)
Direct antiviral (3)
Cell growth/apoptosis (12)
Transcription (9)
Blatt, L et al, J IFN and cytok.Res.2005
Radeava,S. Biochem J..2004
63
Regulators of the JAK/STAT pathway

PTP PIAS SOCS
STAT4
64
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
SOCS1
Acts as pseudosubstrate Inhibits the JAKs Tyr
kinase activity (KIR only in SOCS1 and SOCS3)
Recruitment of E3 ubiquitin ligase Degradation of
proteins
Determinant for the target of each SOCS protein
Yoshimura,A.Nature Reviews Immunology 2007
65
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
66
Role of SOCS in innate immunity
SOCS1-deficient cells and Socs1-/- mice are
resistant to viral infections
Yoshimura,A.Nature Reviews Immunology 2007
67
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
68
The IFN-responsive pathway Target for viral
inhibitors
Viroceptors (homologs of soluble receptors)
Vaccinia Orthopoxvirus
IFNAR2
IFNAR1
T antigen (binds Jak1)
Murine Polyomavirus
V protein (blocks Stat1 phospho by Jak1)
Measles
Jak1
Tyk2
SOCS
High induction of SOCS
HCV, influenza
SV5, Mumps (STAT1) Hum.parainfluenza 2 (STAT2)
V protein (binding and degradation of
STAT) Core protein (binding to STAT1 SH2 and
degradation)
IRF9
HCV
nucleus
E7 (binding IRF-9)
HPV-16
ISGF3
ISGs
ISRE
69
Antiviral action of some IFN-induced genes
The IFN-induced genes
Virus, dsRNA
More than 300 genes induced
Activation
NF-kB
IRFs
AP-1
Jak1
P
Stats
P
P
IFN
Tyk2
ISGs
P
70
IFN can inhibit HCV replication
  • in subgenomic replicon models 1b (1bFrese, M.
    et al, JGV, 2001 1a Gu et al, JV, 2003)
  • in genomic replicon models (1bBlight,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?
71
Some ISGs and role in antiviral defense
Name
Function
OAS PKR ISG56/ISG54 ISG15 ISG16 Mx Viperin NOXA P
hospholipid scramblase RIG-I, MDA5 IRF7
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
72
The discovery of the OAS, 2-5A and PKR in the 70s
Virus- infected
IFN-treated cells
IFN-treated cells
dsRNA
Cell free Translation system
Cell free Translation system
Inhibition translation
Inhibition translation
Kerr,I. et al Nature 1974
Hunt, T. and Ehrenfled, PNAS 1971
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
73
OAS/RNase L
and control of protein synthesis through the
degradation of RNA
74
The human OAS family
346
346
Ubl
Ubl
364aa
513aa
p40
P56-59
OAS1
OASL
219
400aa
p46
254aa
p30
342
(OAS domain but no catalytic function) Ubl domain
homologous to ISG15
683
I
II
687aa
p69
OAS2
727aa
p71
46 (64)
340
400
739
I
II
III
OAS3
1087 aa
p100
60 (74)
49 (66)
Rebouillat Hovanessian, 1999 Hovanessian
Justessen, 2007
44 (66)
75
OAS activates upon binding to dsRNA to generate
2-5-linked oligoadenylates
dsRNA
25bp the most efficient, not sequence specific
OAS1
OAS2
OAS3
Homotetramer
Monomer
Dimer
cytosol,nuclei
membranes
ribosomes
Donor substrate Acceptor substrate nATP
ATP NTP
pppA2p5A NTP RpA
Product
pppA(2p5A)n nPPi (from dimers to
30-mers) pppA2p5A2p5N PPii RpA2p5N PPi
2-5A
N A,U,C,G,I dA,dU,dG,dC 3dA
R tRNA A5ppp5A A5pppp5A NAD polyA
A
P
P
5
1
5
A
1
2
3
4
PPP
5
2
3
4
2
3
76
2-5A activates a latent Ribonuclease or RNAse L
2-5A
2
PUG STYKc
dimerization
UU 3P UG 3P UA 3P AU 3P
RNAse L degrades RNA
Degradation of cellular mRNA, rRNA, and viral RNAs
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
77
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
78
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).
79
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
80
PKR
and control of protein synthesis at the level of
initiation of protein synthesis
81
PKR
Protein Kinase dsRNA dependent
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
82
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
83
CONTROL OF INITIATION OF PROTEIN SYNTHESIS BY PKR
e-IF2B
GTP
Met-tRNA
eIF2-GTP
GDP
eIF2 a,b,g -GDP
60S rRNA
mRNA
e-IF2 a,b,g-GTP-Met tRNA
40S rRNA
Pre-initiation complex
84
CONTROL OF INITIATION OF PROTEIN SYNTHESIS BY PKR
e-IF2B
eIF2 a-P,b,g -GDP
Met-tRNA
stop
eIF2 a-P,b,g -GDP
60S rRNA
mRNA
PKR
e-IF2 a,b,g-GTP-Met tRNA
40S rRNA
preinitiation complex
Shut-off of protein synthesis
85
TRBP and E3L are inhibitors of PKR PACT is an
activator of PKR
86
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
Uncharged tRNA
Heme, Hsp90, Hsc70
dsRNA
Ire1 motifs
HisRS-related
RE
eIF2a-P
eIF2a-P
eIF2a-P
eIF2a-P
87
DRBD and binding to nucleic acids
-topology a-b-b-b-a -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)

88
ISG54/ISG56
and control of protein synthesis at the level of
eIF3
89
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
40S-eIF3-IRES model
40S-eIF3-eIF4F-mRNA model
Siridechadilok,B.et al, Science, 2005
90
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
ISG56 (2 IRF3 and one NF-?B binding sites)
ISG54 (2 IRF3 binding sites only)
CAP-dependent initiation of translation
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
pre- Initiation complex
ISG56 could inhibit HCV translation through
interaction with eIF3
Wang,C. et al, JVI, 2003
Terenzi,F. et al, JBC, 2006
91
ISG15
and post-translational modification of proteins
92
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)
93
Activation effect Modification of Components of
the host immune Response
Mechanism of action of ISG15
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 miceincreased 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
94
HCV interferes with multiple cellular pathways
Binds and inhibits Rb Stimulates S phase
entry Role intransformation?
95
HCV NS5A
1-213
Domain I
Domain II
Domain III
250-342
256-447
447
Polyproline cluster
NLS
ISDR heterogeneity of sequence associated
with sensitivity or resistance to IFN treatment
(genotype 1b)
Zinc binding domains
Amphipathic helix Anchors NS5A to the ER
Cluster of hyperphosphorylation
Enomoto N, N.Engl.J.Med 1996
The maintenance or evolution of the ISDR sequence
is linked to its role In the viral life cycle and
the progression of liver disease
Domain II or Domain D2 -is intrinsically
unfolded -interacts with NS5B -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
96
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
Competition With substrate K3L (Vaccinia)
Direct interaction p58 IPK (cellular
protein activated by influenza) Tat
(HIV-1) soluble E2 (HCV) cIRF2(KSHV) SM
(EBV) NS5A (HCV) Us11protein (HSV1)
Substrat
Substrat-P
Inhibition of eIf2 phospho LANA2 (KSHV)
Activation of phosphatase 1 g1 34.5 Kda (HSV1)
97
HCV E2
TM
Signal leader
363
HCV 1a/b R S E L S P L L L T T T PKR K
K A V S P L L L T T T eIF2? S E L S R R S
79- 90
Taylor et al,1999
48-54
551
18
80
107
159
275
272
234
PKR
V
VI
1
DRBD1
DRBD2
Catalytic subdomains I-XI
314
79-83
51
eIF2?
KGYID
98
INHIBITION OF PKR BY
HCV E2 A cytosolic non-glycosylated domain of E2
binds to and inhibits PKR (and PERK)
Restoration of translation
E2 glycosylated


ER lumen
E1
E2


Pavio et al. J. Virol. 2003
PERK
Cytosol
a
E2
AAAAA
E2 non glycosylated
PKR
Pavio et al. J. Virol. 2002
99
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
Sustained virologic response to IFN-?
Japan, yes Western C.
Yes, but only a small number of
mutations
Genotype 1b Genotype 1a/b Genotype 2a Genotype 3
yes no
mutations outside the NS5A ISDR
Sustained virologic response to IFN-?
Genotype 1b
In the carboxy terminal part of NS5A, in
the Variable region 3 and flanking regions
Genotype 1a
Mutations in E2 PePHD
Sustained virologic response to IFN-?
Genotype 1b
no
no
Genotype 3a
Conflicting reports
Genotype 2a/b
Hofmann, W. et al, J Clinical Virol, 2005
100
inhibition of PKR activity by a pseudosubstrate
the Vaccinia K3L protein
Ser51
314
79-83
human eIF-2a
Several binding motifs for PKR
KGYID
1
88
K
vaccinia K3L
33 identity and 68 similarity with eIF2-a
KGYID
essentiel binding motif to PKR
1
71
K
K3L322 mutant
KGAID
Stronger PKR inhibitor than K3L
He, cell Death and diff.2006 Garcia et al,
Biochimie, 2007
101
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)
functional
e-IF2 a
non functional
PKR KO ( )
PKR / ( e-IF2 a? )
Met tRNA
Met tRNA
Met tRNA
GTP
eIF2A
DLP
DLP
e-IF2 a,b, g
e-IF2 a,b, g
AUGi
AUGi
scanning
scanning
efficient
?DLP
?DLP
Met tRNA
Met tRNA
Met tRNA
GTP
Met tRNA
Met tRNA
e-IF2 a,b, g
e-IF2 a,b, g
e-IF2 a,b, g
AUGi
AUG
AUGi
AUG
scanning
scanning
inefficient
Very inefficient
Very inefficient
efficient
102
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
inhibit initiation of translation on eIF2 and
eIF3 Viperin interference
with protein assembly at the ER? STAT1
sustains IFN induction ISG15, USP18
ISGylation, modification of
trafficking NOXA apoptose

103
(No Transcript)
104
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
105
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
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