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 IFNβ promotor
 carried out in Huh7 cells (human hepatoma cell line) two regions inducing the IFNβ promotor, further mapping of the responsible regions
IFN = interferon

ORF 3‘ NTR
 further mapping to nt 2406-
2696 of the ORF and to nt
93899616 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 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 IFNβ promotor depends on
RIG-I
 IFNβ 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 IFNβ 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 RIGI 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
 RIGI 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 IFNβ 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 RIGI in a 5‘ ppp dependent manner to activate signalling

Effect of nucleotide composition on IFNβ 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 IFNβ promotor by PU/UC in vivo
 signalling analysis in WT and
RIG-I -/mice
 intravenous administration of HCV
RNA
 induction of hepatic IFNβ 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 IFNβ mRNA levels and
IFNβ 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 IFNβ 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
IFNβ or conditioned media
(supernatant from cells transfected with the indicated RNA species)
 treatment with IFNβ 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
IFNβ or conditioned media treatment with IFNβ 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
 IFNβ 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
Thanks for your attention !