1. Nature, 24 July 2008; 454(7203):523-527 05.01.2009, Wiebke Albrecht

2. 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 I NOD = nucleotide-binding oligomerization domain PRRs induce type I IFN and proinflammatory cytokines Saito et al. (2008), commentary

3. RIG-I Model of RIG-I activation Yoneyama 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

4. Hepatitis C virus (HCV) HCV life cycle Lindenbach et al. (2005)  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

5. 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 polyprotein Bode et al. (2008)

6. 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

7. ORF3‘ 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

8. 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)

9. 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

10. 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

11. 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

12. 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

13. 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 assay N = N-terminus of RIG-I FL = full lenght RIG-I reporter assay in Huh7 cells, with and without pre-treatement with IFN-β

14. 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

15. 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)

16. 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

17. 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

18. 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

19.  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)

20. 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

21. 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 !