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 pathogen-associated 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 INOD = nucleotide-binding oligomerization domain

PRRs induce type I IFN and proinflammatory cytokines Saito et al. (2008), commentary

RIG-I

Model of RIG-I activationYoneyama et Fujita (2008)

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

Hepatitis C virus (HCV)

HCV life cycleLindenbach et al. (2005)

small enveloped virus (diameter ~50nm)

(+) ssRNA genome

genus: Hepacivirusfamily: 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 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

ORF = open reading frame NTR = non-translated region

HCV polyproteinBode et al. (2008)

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

ORF 3‘ NTR

further mapping to nt 2406-2696 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

nt = nucleotides

Structure of the HCV 3‘ NTR

RI = replication intermediate (- RNA strand)

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 stem-loop structures (~98 nt)

HCV PAMP in the viral 3‘ NTR

reporter assay in Huh7

PU/UC region is sufficient for signal triggering

also shown in Hela cells

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 gel-shift assay)IRF = interferon regulatory factor

ISG = antiviral/interferon-stimulated gene

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

gel-shift assayN = N-terminus of RIG-IFL = full lenght RIG-I

reporter assay in Huh7 cells,

with and without pre-treatement with IFN-β

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

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 hepatic immune responses (2)

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/UC-transfected 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

Thanks for your attention !