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Nature, 24 July 2008; 454(7203):523-527
05.01.2009, Wiebke Albrecht
Innate immunity and virus recognition
 innate immunity is characterized by
the use of pattern recognition
receptors (PRRs)
 PRRs recognize pathogenassociated molecular patterns
(PAMPS)
 PRR activation leads to type I
interferon (IFN) and
proinflammatory cytokine production
 3 types:
toll-like receptors (TLRs)
RIG-I-like receptors (RLRs)
NOD-like receptors
RIG-I = retinoic acid-inducible gene I
NOD = nucleotide-binding oligomerization domain
PRRs induce type I IFN and
proinflammatory cytokines
Saito et al. (2008), commentary
RIG-I
 located in the cytoplasm
 consists of a DEXD/H box helicase domain, two CARD-like domains
required for activation of downstream signalling pathways and a C-terminal
domain (CTD) that includes a repressor domain (RD) inhibiting signalling
in the steady state
 recognizes short dsRNA and 5‘ terminal triphosphate RNA (5‘ ppp RNA)
binding of nonself RNA
to RIG-I leads to a
conformational change,
the CARD domains are
exposed and activate
downstream signalling
CARD = caspase-recruiting domain
Model of RIG-I activation
Yoneyama et Fujita (2008)
Hepatitis C virus (HCV)
 small enveloped virus (diameter ~50nm)
 (+) ssRNA genome
 genus: Hepacivirus
family: Flaviviridae
 causes chronic hepatitis, liver cirrhosis
and hepatocellular carcinoma
 six major genotypes are known, differing
in their geographic distribution and their
responsiveness to antiviral therapy
HCV life cycle
Lindenbach et al. (2005)
HCV genome
 genome size: ~9,6 kb
 genome encodes one ORF which is translated as a polyprotein and
subsequently cleaved into ten proteins
 5‘ NTR is a conserved region and consists of four domains, including an IRES
element to direct cap-independent translation
 3‘ NTR consists of 3 domains sufficient for replication
HCV polyprotein
Bode et al. (2008)
ORF = open reading frame
NTR = non-translated region
Identification of HCV PAMP RNA motifs
 luciferase reporter assay with a reporter plasmid containing an IFN-β
promotor
 carried out in Huh7 cells (human hepatoma cell line)
two regions inducing the IFN-β promotor, further mapping of the responsible regions
IFN = interferon
 further mapping to nt 24062696 of the ORF and to nt
9389-9616 of the 3‘ NTR
 deletion of of the 3‘ NTR
but not deletion of the region
nt 2408-2663 attenuated
promotor signalling
 PAMP motifs are typically
conserved, nt 9389-9616
show high conservation
between different HCV starins
ORF
3‘ NTR
nt = nucleotides
Structure of the HCV 3‘ NTR
VR = variable region with
potential secondary
structure (~40 nt)
PU/UC = non-structured
poly (U/UC) tract containing
polyuridine with interspersed
ribocytidine (variable length)
X = highly conserved
segment that formes 3 stemloop structures (~98 nt)
RI = replication intermediate (- RNA strand)
HCV PAMP in the viral 3‘ NTR
 reporter assay in Huh7
 PU/UC region is sufficient for
signal triggering
 also shown in Hela cells
IRF = interferon regulatory factor
ISG = antiviral/interferon-stimulated gene
 full legth 3‘ NTR as well as PU/UC region
stimulated the formation of active IRF3
dimers and expression of ISG56, an IRF3
target gene (shown by immunoblotting)
 PU/UC region forms a complex with RIG-I,
while the X region does not (shown by a gelshift assay)
Induction of IFN-β promotor depends on
RIG-I
 IFN-β promotor induction in
Huh7.5 cells
 Huh7.5 cell lack functional
RIG-I
 cells were refractory to HCV
RNA induced signalling, which
was rescued by
overexpression of WT RIG-I
Involvement of signalling molecules in IFN-β
production
 reporter assays in MEFs (mouse embryonic fibroblasts)
 MDA5 is also a RLR member like RIG-I; MyD88 and TRIF are essential
adaptor protein used by TLR 7/8 and 3 recognizing endosomal RNA
 no reporter induction in RIG-I negative MEF‘s upon RNA stimulation
 lack of MDA5, MyD88 and Trif does not influence signal induction by full
length 3‘ NTR and PU/UC region
HCV PU/UC region co-localizes and
interacts with RIG-I
 FRET analysis
 PU/UC RNA co-localizes and interacts with RIG-I
 RIG-I is the essential PRR that signal innate immune
responses against HCV triggered by the poly (U/UC) region
HCV RNA requires 5‘ ppp for RIG-I binding
and signal triggering
 RIG-I binds to PAMP RNA containing 5‘ terminal triphosphate (5‘ ppp)
 5‘ ppp is required for poly (U/UC) RNA binding by RIG-I and for IFN-β
signalling, but does not mediated binding of RIG-I to the X region; X region
just weakly triggers signalling
reporter assay in
Huh7 cells,
with and without
pre-treatement
with IFN-β
gel-shift assay
N = N-terminus of RIG-I
FL = full lenght RIG-I
Effect of PU/UC or X RNA on RIG-I
activation
 limited trypsin digestion analysis
 upon binding of PAMP RNA the
RIG-I repressor domain (RD) is
displaced and present as a single
fragment
 binding of PU/UC region to RIG-I
rendered the RD fragment
 HCV PU/UC region directs stable
interaction with RIG-I in a 5‘ ppp
dependent manner to activate
signalling
Effect of nucleotide composition on IFN-β
promotor signalling
 replacement of uridine reduced PAMP signalling
 poly-A is also capable to induce signalling, again replacement leads to
reduced signalling
 truncation of the PU/UC region also reduces signalling (not shown)
Effect of nucleotide composition on RIG-I
activation
 poly-A RNA as well as PU/UC bind to RIG-I and lead to displacement of the
RD
 signalling can be triggered by polymeric uridine and
riboadenine motifs serving as PAMP signature within 5‘ ppp RNA
recognized by RIG-I
Induction of IFN-β promotor by PU/UC in
vivo
 signalling analysis in WT and
RIG-I-/- mice
 intravenous administration of HCV
RNA
 induction of hepatic IFN-β mRNA
levels in WT mice, but not RIG-I-/mice, by HCV genome or PU/UC
region
HCV PAMP RNA triggers hepatic immune
responses (1)
 time course studies
 the PU/UC region induced a peak of hepatic IFN-β mRNA levels and
IFN-β serum levels in WT mice, but not in RIG-I-/- mice
HCV PAMP RNA triggers hepatic immune
responses (2)
 the PU/UC region induced also a peak of hepatic RIG-I and ISG56
mRNA levels in WT mice,but not in RIG-I-/- mice
 induction of tissue-wide expression of ISG54 in WT mice suggestingthat
paracrine signalling of IFN-β could play a role in hepatic defenses against
HCV
HCV PAMP RNA triggers paracrine antiviral
effects of the innate immune response
 measurement of HCV production
of infected Huh7.5 cells treated with
IFN-β or conditioned media
(supernatant from cells transfected
with the indicated RNA species)
 treatment with IFN-β or
supernatant from PU/UCtransfected cells induced a immune
response that suppresses HCV
infection
 RIG-I signalling triggered by
PU/UC can induce an antiviral
response through indirect, paracrine
actions of IFN produced from HCV
PAMP signalling
pre-treatment with
IFN- β or conditioned
media
treatment with IFN- β
or conditioned media
48h post infection
Summary
 RIG-I signals innate immune responses against HCV triggered by the poly
(U/UC) region of the 3‘ NTR of the HCV genome or ist replication
intermediate
 IFN-β production is induced in experiments in vitro and in vivo in response
to HCV RNA
 PAMP signature recognized by RIG-I is characterized by polymeric uridine
and riboadenine motifs within 5‘ ppp RNA
 5‘ppp on PAMP RNA is necessary but not sufficient for RIG-I binding
 RIG-I signalling triggered by PU/UC can induce a direct as well as a indirect
antiviral response through paracrine actions of IFN produced from HCV
PAMP signalling
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